ASCENT Version User Manual

Transcription

1 ASCENT Version User Manual ASCENT v User Manual 1 Revision X

2 INDIGO BIOAUTOMATION, INC. ASCENT V USER MANUAL Indigo BioAutomation, Inc. 385 City Center Drive, Suite 200 Carmel, IN This manual was written for use with the Indigo BioAutomation ASCENT software. This manual and the software described in it are copyrighted, with all rights reserved. This manual and the software may not be copied, except as otherwise provided in your software license or as expressly permitted in writing by Indigo BioAutomation, Incorporated. All other trademarks and service marks are the property of their respective owners. Copyright 2018 by Indigo BioAutomation, Incorporated. All rights reserved. UNDER NO CIRCUMSTANCES INCLUDING NEGLIGENCE, SHALL INDIGO BIOAUTOMATION, ITS LICENSORS OR THEIR DIRECTORS, OFFICERS, EMPLOYEES OR AGENTS BE LIABLE FOR ANY INCIDENTAL, SPECIAL OR CONSEQUENTIAL DAMAGES (INCLUDING DAMAGES FOR LOSS OF BUSINESS, LOSS OF PROFITS, BUSINESS INTERRUPTION, LOSS OF BUSINESS INFORMATION AND THE LIKE) ARISING OUT OF THE USE OR INABILITY TO USE THE SOFTWARE OR ITS DOCUMENTATION, EVEN IF INDIGO OR AN INDIGO AUTHORIZED REPRESENTATIVE HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES, SOME JURISDICTIONS DO NOT ALLOW THE LIMITATION OR EXCLUSION OF LIABILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES SO THE ABOVE LIMITATION OR EXCLUSION MAY NOT APPLY. SEPTEMBER ASCENT v User Manual 2 Revision X

3 Table of Contents INTRODUCTION... 5 TECHNICAL SUPPORT... 5 SYSTEMS REQUIREMENTS... 5 SYSTEM SECURITY... 5 SPECIAL NOTES... 6 GLOSSARY... 7 ASCENT NAVIGATION ACCESSING ASCENT HOME SCREEN AND MENU ACCESSIONING PREREQUISITES IMPORT THE INSTRUMENT SEQUENCE FILE FIELDS AVAILABLE FOR USER INPUT: BATCH REVIEW BATCH STATUS BATCH SUMMARY STANDARD REVIEW NAVIGATING BATCH REVIEW DATA GRID CHROMATOGRAM DISPLAY QUALITY RULE AND FLAG INFORMATION CALIBRATION PEAK MODIFICATION EXCEPTION BASED REVIEW ENHANCED REVIEW AUTO ANALYSIS MANUAL MODIFICATION REVIEW RESULTS OF INTEREST REVIEW COMPLETING A REVIEW CERTIFICATION DECERTIFICATION RESULTS BATCH DIAGNOSTICS OTHER BATCH FUNCTIONS RESTORING A BATCH REPROCESSING A BATCH ASCENT v User Manual 3 Revision X

4 CANCELING A BATCH DESTROYING A BATCH RE-UPLOADING A BATCH SAMPLE SEARCH AUDITS ARCHIVING AND DATA RETENTION PROFILE USER MANAGEMENT USER SET UP USER PERMISSIONS ASSAY CONFIGURATION COMPOUNDS LOTS AND LEVELS DISPLAY RULES TEST DATA MAINTAIN ASSAY ADDITIONAL ASSAY ACTIONS CONTROL BATCH SETS QC CHARTING CREATING A CBS REVIEWING A CBS CERTIFYING A CBS CBS RESULT FILES DECERTIFYING A CBS DESTROYING A CBS PRODUCTION VOLUME REPORT SUPPORT AND TROUBLESHOOTING APPENDIX A: CUSTOMER PORTAL ACCESSING THE CUSTOMER PORTAL APPENDIX B: CORE QA RULES APPENDIX C: SAMPLES FROM ARCHIVE APPENDIX D: RETENTION TYPE SOURCE ASCENT v User Manual 4 Revision X

5 INTRODUCTION ASCENT is a software service for automating the quantification of compound concentrations from chromatographic (trace) data. ASCENT automatically sweeps data from instruments, and then performs data conversion, peak-picking and integration to produce quantitative results for compounds. After automatically fitting calibrator samples to a calibration curve for use on the remainder of the batch, ASCENT will display quantified values along with advanced QA/QC flagging on a web browser for user review, approval, and certification. The basic workflow of ASCENT is shown in Figure 1: Technical Support Figure 1 ASCENT Workflow For any issues with ASCENT or this manual, please check with a laboratory supervisor first (see SUPPORT and TROUBLESHOOTING) and then contact Indigo BioAutomation Technical Support. The Customer Portal is available from the footer of an ASCENT site webpage or at (click on Support Portal). Systems Requirements To use ASCENT, a user must have a computer with the following. Mozilla Firefox version 52 to 59 or Google Chrome version 57 to 66 installed Min. Screen Resolution: 1280 X 1024 System Security The customer is responsible for maintaining basic logical and physical security systems. ASCENT software relies on both its internal controls (user passwords and manager only functions) and the security measures implemented by the customer. User access and ASCENT v User Manual 5 Revision X

6 software feature access is controlled by the USER MANAGEMENT functions. Individual users of the software will be given named accounts by an ASCENT administrator. Special Notes Important points throughout this manual are highlighted by the ASCENT pinwheel. ASCENT v User Manual 6 Revision X

7 GLOSSARY Accessioning - A tool that facilitates generation of an ASCENT compatible sequence file for a batch. Analyte A compound to be detected and reported in an analytical experiment as native to the sample. Often, the goal of the analytical experiment is to confirm presence and identity, and if present and identified, measure the concentration of the analyte; sometimes referred to as a Target Analyte. Assay A test or analytical experiment to be performed on one or more samples. While an assay may be considered the entire set of operations performed on a sample (e.g. sample preparation, instrument control and acquisition, etc.), ASCENT focuses on the processing, review and reporting of samples. Assay Configuration The set of automated processing, review and reporting instructions for ASCENT to perform on a batch of samples, for the purposes of determining the presence and if present, the concentration, of one or more analytes; often referred to as a processing method or simply, a method. Batch A collection of samples which are considered the logical work product of laboratory analysis; the samples which make up the batch are intended to be associated and travel together through the processing, review, and reporting operations (as defined by the assay configuration). Blank A sample for which the determination of presence and, if present, the concentration, of one or more analytes is the intended goal of the analysis. The blank is expected to have no analytes present (although some may in fact be present), but may have internal standards present which were added as part of the sample preparation process. Calibration A collection of samples (standards) containing a known concentration of one or more compounds, used by ASCENT to assess and establish the state of the system in order to calculate sample results as quantified values. Similarly, a calibration (as visualized as a calibration curve) can also refer to the collection of standard determinations for a particular compound. Chromatogram A trace which has been processed for calculation and display within ASCENT, per the method defined as part of assay configuration. A chromatogram ASCENT v User Manual 7 Revision X

8 represents a chart or graph of the signal intensity vs. time, along with the associated record of processing, if applicable (e.g. display of a peak, availability of a response). A chromatogram is the representation of the processed data artifact resulting from the acquisition record of a sample for a particular compound, and is considered to be characteristic for that compound. Column Headings Data headings in the results table. Compound A specific substance that is tested for in a sample; examples include Morphine and Codeine (as analytes, see above), or Morphine-d6 and Codeine-d6 (as internal standards, see below). Determination - A compound result for a specific sample; e.g. a Morphine result for sample # is a single determination, regardless of the number of chromatograms used for the identification/confirmation and quantification of Morphine. Flags Indicators used to call attention to a particular sample, compound or chromatogram within a batch in order to identify specific conditions or quality metrics as part of review and certification. Internal Standard A compound to be detected and reported in an analytical experiment, but is not native to the sample (e.g. it is usually added to each sample of the batch). An internal standard is often chemically similar to one or more analytes, and is used primarily as a reference for analyte qualification and/or quantification. Normalization Target A compound that is used to determine a response ratio for quantitated calculations, the common selection of which is to specify an analyte s internal standard response. Peak The color-shaded area of the chromatogram that is displayed and calculated within ASCENT as part of the detection and integration process, (visually) representative of the measured amount of a compound in a sample. Qualifier (Qualitative Chromatogram) Chromatogram used to confirm presence and identity of a compound that may be present in the sample; often referred to as a Qual. Quantifier (Quantitative Chromatogram) Chromatogram used to measure the response and/or quantify the amount of a compound that may be present in the sample; often referred to as a Quant or a Quan. ASCENT v User Manual 8 Revision X

9 QC A sample for which the determination of presence and, if present, the concentration, of one or more analytes is the intended goal of the analysis. The QC has a designated Lot and Level which is used to evaluate the system calibration and thus, the state of the system; also referred to as a Quality Control Standard or a QC Standard. A QC has a known concentration of one or more compounds. Response The output from the detection and integration of a peak as present in the chromatogram for a compound (e.g. the area under the curve formed by the peak); a compound s response is indicative of the concentration of the compound in a sample. Sample a discrete analysis request made to (a chromatograph and) a mass spectrometer, which results in the generation of a recorded artifact, typically a file, of analytical data to be processed; also referred to as an Injection or a Run. Standard A sample with a designated Lot and Level which is used to establish a system calibration, visualized as a calibration curve; also referred to as a Calibration Standard or Calibrator. A standard has a known concentration of one or more compounds. Trace - Chart or graph of the signal intensity vs. time; a trace is one representation of the data artifact resulting from the acquisition record of a sample for a particular compound, and is considered to be characteristic for that compound. A trace is normally observed in ASCENT, following processing and reduction, as a chromatogram. Unknown A sample for which the determination of presence and, if present, the concentration, of one or more analytes is the intended goal of the analysis; also referred to as a Specimen. The unknown has no expectation of analytes being present or absent (although some may in fact be present), but may have internal standards present which were added as part of the sample preparation process. ASCENT v User Manual 9 Revision X

10 ASCENT NAVIGATION Accessing ASCENT To log into the ASCENT application: Log into the application using the URL, username and password provided by an ASCENT administrator (see Figure 2) Follow laboratory protocol on password updates (see PROFILE for instructions) Figure 2 ASCENT Login Screen There is a password reset function available from the login screen (red circle, Figure 2). To have reset instructions sent to the address associated with the user account, enter the username and select Reset (red circle, Figure 3). Figure 3 Forgot Password The reset will contain a temporary (expiring) link to reset the password. No password change occurs until the link is used. If the reset is not received, the user has the ASCENT v User Manual 10 Revision X

11 option to have it resent. Otherwise, the user will need to contact an ASCENT administrator who should verify that the address associated with the account is correct. After five incorrect login attempts, the user account will be locked and a message will be sent to the address configured with the user account with the subject Unlock Instructions for ASCENT. Upon clicking the link in the , the user is directed to the ASCENT login page. The user should login with the current password and is not prompted to change the password. If the user does not receive the login instructions, he or she should click the link on the bottom right of the login page (blue circle, Figure 2). The user will be prompted to enter the username for the ASCENT account to receive the instructions again. If problems with user access persist, please submit a request through the customer portal (see SUPPORT AND TROUBLESHOOTING). The Firefox browser can be set to remember passwords. ASCENT does not control or override this behavior. For security purposes, customers may wish to turn off this feature. Home Screen and Menu Upon login, the User is presented with the Batch Status Page (see Batch Status) but other features are available via the menu along the top of the page. The options visible in the menu will be determined by user permissions. The full Menu Bar is shown in Figure 4. Figure 4 Menu Bar At the bottom of the page, there are links to the latest Bulletin, the Support Portal, the Release Notes, and the User Manual (Figure 5). Figure 5 Page Footer ASCENT v User Manual 11 Revision X

12 ACCESSIONING In order for ASCENT to successfully process a batch, the users must supply ASCENT with a sequence file containing a minimal set of information for each sample, such as the name and the type. Accessioning is the function that allows users to create an ASCENT sequence file from an existing instrument sequence file. Prerequisites The Assay Configuration must be complete and promoted to production. The lab employee must know the Lot and Level settings for samples in the batch, if the batch is using standards. ASCENT will utilize the information in the instrument sequence file. However, there are some important practices to keep in mind when generating the instrument sequence file so that it is compatible with ASCENT. Because ASCENT uses a commaseparated-values format, please be sure there are no commas within the data fields in the instrument sequence file. Batch name may contain alphanumeric characters, dashes, and underscores; no spaces or other special characters are allowed. Because the ASCENT data flow relies upon batch name consistency, it is best to adhere to these requirements from the start of the workflow. Import the Instrument Sequence File Begin by selecting the Accessioning link from the ASCENT main menu (Figure 4). When presented with the accessioning page, select the assay from the drop-down list (red circle, Figure 6). Figure 6 Prepare for Accessioning If no instruments have been defined for the assay, ASCENT will return the error in Figure 7. ASCENT v User Manual 12 Revision X

13 Figure 7 No Instruments Drag and drop the instrument sequence file onto the [filename] prompt OR click [filename] to navigate to the file. Duplication of raw data filenames within the sequence file is not allowed. After uploading the user can make certain changes to each sample before generating and downloading the ASCENT sequence file (Figure 8). Fields available for user input: Figure 8 Edit Sequence File Values Batch (This value will become the filename of the generated ASCENT sequence file and batch name in ASCENT. Default is filename of instrument sequence file, but ASCENT v User Manual 13 Revision X

14 name will be checked during file generation to confirm that it is acceptable to ASCENT. User will be provided options to correct if it is not.) Instrument (Choose from the list of instruments configured for the assay.) Sample Name (Do not edit. Values are mapped from corresponding column in instrument sequence file, displayed for reference in specifying sample information. ASCENT sample name is obtained from values acquired in the raw data files.) Type (Supported instrument data system sample types will be mapped to the ASCENT sample types of Blank, QC, Standard, and Unknown. Otherwise, the type will default to Unknown and the user will manually select a different value from the list for each sample as necessary.) Lot and Level for QC and Standard sample types. The list of available lots and levels are specified in the Assay Configuration (available to ASCENT Administrators see Lots and Levels). If the instrument sequence file includes an existing lot and level for each sample, the values must match the lots and levels configured for the assay and they cannot be changed by the user on the Accessioning page. Both Lot and Level columns (case insensitive and may be proceeded by an underscore) must be present in the instrument sequence file with values for each QC and Standard sample. Dilution Factor (Default of 1. Values mapped from corresponding column in instrument sequence file.) Select an instrument name from the list configured for the assay. The Batch Name will be automatically populated from the instrument sequence filename but cannot contain spaces or special characters other than dashes and underscores. The sequence file must be saved to the same location as the raw data for the batch, in order for automated transfer and processing to occur. If using batch subfolders, the sequence file name must match the folder name. Once the sequence file is ready, save the ASCENT sequence file using the ASCENT button at the bottom of the page (red circle, Figure 9). Figure 9 Create Sequence File ASCENT v User Manual 14 Revision X

15 If no instrument name is selected from the list, ASCENT will return an error message (red circle, Figure 10). Figure 10 Field Checking for Instrument Name If there are special characters in the batch name, a popup warning will appear, offering to automatically update the batch name and reminding the user to rename the batch folder (Figure 11). Figure 11 Sequence File Error It is possible to generate the sequence file necessary for ASCENT operation without using the accessioning feature (e.g. it may be programmatically generated from other import sources, such as robotic sample handling worklists). However, generating the sequence file outside of the accessioning feature requires specific and particular requirements in terms of structure and format. For assistance with this activity, it is recommended that you contact Customer Support (see APPENDIX A: CUSTOMER PORTAL for more details). ASCENT v User Manual 15 Revision X

16 BATCH REVIEW All necessary Batch Review steps can be completed using navigation contained within the ASCENT web pages. Navigation between ASCENT pages using the web browser s forward and back buttons is not an intended workflow, and is not supported during certain operations (e.g. moving from a page where a peak modification is in progress is not allowed). Using ASCENT s in-page navigation ensures that the displayed data is synchronized. Batch Status The Batch Status page is the first page presented to the User upon login (Figure 12). To return to the Batch Status Page from any place in the system, click Ascent in the Menu Bar. The Batch Status Page lists all the batches available for action in the system. To begin work with a particular batch, scroll through the list of batches and click on the appropriate batch name hyperlink (red circle, Figure 12), which will open the Batch Summary for that specific batch. When available, an icon will be visible (orange circle, Figure 12) near the batch name to indicate the presence of a Batch Note. Hovering the mouse pointer on the batch note icon will display a pop-up/tool-tip dialog containing the contents of the batch note. Batch Notes can be created, edited and removed on the Batch Summary page. The columns for Reviewer, Certifier and Canceller will contain the first and last name of the user who completed the described operations (e.g. the Reviewer column contains the information of the user who performed the steps for Completing a Review), aiding traceability of the batch. The column for Current Analyst can be used as an aid in determining if a particular batch has been partially reviewed or certified. To search for a specific batch, enter the name of that particular batch into the Batch Name field and click Search (green circle, Figure 12). It is not necessary to fill in the complete batch name; a search may be made on the initial string (e.g. a starts with search). However, it is not a wild card search and will not return results that have that string in the middle or the end of the name. ASCENT v User Manual 16 Revision X

17 In addition to using the Batch Name search, the Filters along the left side of the Batch Status page will assist in locating batches by specific characteristics. Simply select and deselect the checkboxes next to the options in each section to limit the batches visible in the Batch Status page (the page will update automatically; it is not necessary to click Search). The filter selection will remain active for the duration of the login session. To remove the filter and see all available batches, click the Clear button (blue circle, Figure 12). The list of batches can be sorted ascending/descending by clicking on the column headers (e.g. Name, % Flagged, Acquisition Time). Refresh the browser page to update the batch listing without removing search, filter, or sort specified by the user. Figure 12 Batch Status Screen Batches will pass through various statuses as they proceed through the workflow. Available statuses include: Converting: Raw data has been uploaded and is converting (informational no actions available). Processing: Converted data is having peaks picked, quantification performed, and rules applied (informational no actions available). Ready for Review: Data has been processed and can be viewed by all users and reviewed by authorized users. In Review: The batch has been accessed by a user in Review mode (even if no changes have been applied). ASCENT v User Manual 17 Revision X

18 Ready for Certification: A reviewer has completed review of the batch and it is ready for certification by an authorized user. In Certification: The batch has been accessed by a user in Certification (even if no action has been taken). Certified: The batch has been certified and is now visible in Read Only mode. Final reports are available for download. Cancelled: The batch has been cancelled but is available in Read Only mode for reference. Failed: The batch failed during conversion or processing. Information about the failure is available in the Batch Diagnostics page. Batch Summary When a batch is selected from the Batch Status page, the Batch Summary page will appear (Figure 13). This page acts as a central point of orientation for the entire process of batch review and certification. The upper caption region and the various vertical regions of the Batch Summary page are intended to provide information and navigation which is relevant during key stages of review and/or certification operations. Figure 13 Batch Summary Page Caption Region The name of the active batch is displayed in the center of the upper caption region (blue circle, Figure 14). At the right side of this caption region is a link to the main entry point for conventional (e.g. data-grid-based) review activities (red circle, Figure 14). For more information on this review approach, please see Standard Review. ASCENT v User Manual 18 Revision X

19 Figure 14 Batch Summary Caption Region Also on the right side of the caption region is a link to open a menu of batch actions. Clicking the stoplight menu icon (green circle, Figure 14) will present a list of available (based on current batch state) actions, such as Standard Review Read Only, Restore, Reprocess, Decertify, Add to Test Date, Cancel and Destroy (Figure 15). Figure 15 Batch Action Menu These same actions can be accomplished on the Batch Status page, using a different interaction mechanism. Additional information on each of these actions can be found in subsequent sections of this manual. Left Region The top section of the left area of the page provides Batch Details, such as the name of the assay configuration used to generate the batch results, the instrument which was identified as the source of the sample data during the accessioning process, and the user with the most recent access to the batch. Clicking the assay configuration (blue circle, Figure 16) will open a separate browser tab, displaying the full details of the assay configuration used to process this batch. If there are differences between the assay configuration used for this particular batch and the assay configuration currently promoted into production (e.g. if this were to be an older batch, using an older version of the assay configuration), the doublearrow link to the left of the assay configuration name (green circle, Figure 16) will be shaded blue, and clicking it will open a separate browser tab, and display the differences between the batch and production assay configurations; if the two assay configurations match, the double-arrow link will be shaded grey to indicate no differences to display. Hovering the ASCENT v User Manual 19 Revision X

20 mouse on the Current user name will provide a small dialog which lists certain elements of the batch history (e.g. when the batch was uploaded to ASCENT, the name of the user who reviewed and/or certified the batch). Directly below those fields is the Import Calibration option. By default, the calibrators used for quantification are those found in the batch itself. Some lab practices, however, may incorporate the use of calibration curves from previous batches for use in subsequent ones. Initially, the option will indicate No imported calibration. To import calibration curves from another batch, use the link to the right of that text (red circle, Figure 16). Figure 16 Import Calibration Option Clicking this stoplight menu icon will display the Import Calibration menu (Figure 17), and allow for user selection of an alternative, or reference, batch from which the calibration will be used with the currently displayed one. The Import Latest link will identify the most recent batch which has been Certified for the same assay and instrument combination, and selecting the link will import the calibration curves; the Import Latest with QCs link will take the same approach, and import the calibration curves as well as the QC samples from the reference batch. If a different reference batch is to be used, the More link will present a dialog of all possible reference batches; the reference batch list includes batches from the same assay and instrument that were Certified in the last seven days and do not themselves utilize an imported calibration. ASCENT v User Manual 20 Revision X

21 Figure 17 Import Calibration Menu Following a successful import, the name of the reference batch will be displayed. The reference batch s calibration curves will be made available to the batch; however, the details of the calibration reference batch (e.g. calibration and QC samples, and their compound results, which make up the calibration) will not be imported into the batch (that is, they will not appear as results in the Data Grid). Calibration flags will be disabled when the target batch has no standards of its own. If the calibration reference batch to be imported does not contain any standards, ASCENT will return an error. If a calibration reference batch is no longer to be utilized, select Remove Calibration from the Import Calibration menu. The calibration reference will be removed, and the batch s own calibration standards will be used. The next section in the left region provides information on the Samples which comprise the batch. The total number of samples in the batch is displayed, along with a breakdown of the sample count by sample type. In cases where one or more of the Standard or QC samples originate from an imported calibration (as above), an orange number will appear next to the sample count, indicating how many of the samples of that type were from the reference batch. If there were any issues in file conversion for any samples, a red indicator will appear to the right of the sample count for the sample type which did not convert. Further information on the samples in the batch may be observed and acted upon by opening the batch sample listing, using the right carat icon (red circle, Figure 18). ASCENT v User Manual 21 Revision X

22 Figure 18 Sample Information Display Link Clicking the icon will reveal a listing of each of the samples in the batch, and display the sample index, filename, sample name, sample type, and dilution factor (green rectangle, Figure 19). If there were any failed samples indicated in the Sample section, a red icon will appear at the left edge of the specific sample row of the affected sample. Figure 19 Sample Information With appropriate Batch Detail User Permissions, and the batch in certain states, the sample name and dilution factor for a particular sample can be modified by clicking on the sample row. More significantly, the underlying data for the sample can be completely replaced by providing an alternative data file/fileset/folder, assembled as a ZIP file. When in edit mode, the sample row will present with a different background, and text edit fields for sample name and dilution factor (red circles, Figure 20) will be visible; the filename field (blue circle, Figure 20) will be replaced with a label which can act as a drag-and-drop target for the replacement s ZIP file, or it may be clicked to ASCENT v User Manual 22 Revision X

23 present a file selection dialog. Edit mode for a sample also allows a sample to be completely removed from the batch by clicking the trash can icon (green circle, Figure 20). To exit edit mode for a sample, prior to making any changes, click the sample row again. Figure 20 Sample Edit Multiple changes may be made to multiple samples prior to accepting the changes and applying them to the batch. When the details in one sample have been queued but not applied, the background color of the field will be goldenrod; hovering on such a queuededit field will display the details of the pending change. After making one or more changes, two buttons at the bottom of the sample list will become enabled. Click Discard Changes (red circle, Figure 21) to revert any changes made back to the initial values; this selection can undo changes to the sample name, dilution factor, and sample data source, as well as reincorporate a sample which had been removed. Click Apply Changes (green circle, Figure 21) to accept the changes and, as necessary, trigger the reprocessing of the batch. Figure 21 Sample Edit Actions A confirmation dialog will be provided in order to confirm that the changes are to be applied and the batch reprocessed click Close to return to Batch Details without applying the changes; all modified results, user edits, and other changes are lost when a batch is reprocessed. When finished, the sample list may be closed by clicking the left carat icon (red circle, Figure 19). ASCENT v User Manual 23 Revision X

24 The last section on the left region, Compounds, provides the number of compounds (e.g. the sum of both analytes and internal standards) which are listed in the assay configuration. Center Region The central area of the page displays the Overview chart also referred to as the Quality Rules graphic which summarizes the breakdown of flagged determinations by flag category, if one or more rules have been enabled for the assay (Figure 22). Below the Overview chart is the Auto Analysis button (red circle, Figure 22). Auto Analysis works in conjunction with a specific quality rule to efficiently address samples which may have been impacted by a prior high concentration sample. For more information on this capability, please see Auto Analysis. Figure 22 Overview Chart and Auto Analysis The Overview chart allows a Reviewer to gauge the state of the batch based on the number of flagged samples and determinations. These flags may be used during review to direct attention to the determinations most in need of investigation. A user may click on one of the bars (or, if the bar is not visible due to relative scaling, the user may click on the number to the right of the bar) to navigate directly to data review of those flagged determinations. For more on review based upon these flags, see Exception Based Review. The Peak Quality/RT, Calibration, and Quality Control categories of flags are particularly significant. Flags generated by these quality rules are indicative of an issue with peak detection and integration (e.g. a peak not detected where one might be expected; a peak ASCENT v User Manual 24 Revision X

25 integrated with a characteristic that is unusual), the calibration curves for the analytes (e.g. a compound who s calibration does not meet an established minimum R2 for the regression equation), or the QC samples for the analytes (e.g. a QC determination whose observed value is not within an appropriate range of the nominal value). Because all subsequent calculations (e.g. a compound s calculated concentration) and/or subsequent evaluations (e.g. various quality rules) are derived from the results of peak processing, along with quantification using the calibration curve and a measure of the system s continued acceptability using the QC samples, it is of key importance that flags relating to peak quality and retention time as well as associated with calibration and QC samples are noted. Clicking on the Peak Quality/RT, Calibration, or Quality Control bars of the Overview plot will invoke a richer set of review options. For more information on this specific review process, see Enhanced Review. Below the Overview chart is an area which may be used to document information or exchange messages regarding a batch, referred to as a Batch Note. When a Batch Note is available, it will be visible in full on the Batch Summary page, along with the name of the user who created/edited it, and the date/time of the last change. Additionally, an icon will be visible on the Batch Status page, which can be hovered upon to display the contents. Batch Notes are intended to be used for temporary, informal information exchanges; thus, the contents of the Batch Note (or any changes to it) will not be recorded in the batch s audit trail, nor in the final archive PDF. Clicking the pencil icon beneath the chart (blue circle, Figure 23) will open the editing box for input. Once information entry is complete, click the Enter key (or otherwise shift focus, e.g. click the mouse, at another location on the page) to save the entry with the batch. Figure 23 Batch Note ASCENT v User Manual 25 Revision X

26 Once a note has been created, it may be edited by clicking inside the text box and entering new/additional information. A note may be removed by clicking the X icon to the right of the Batch Note (blue circle, Figure 24). Figure 24 Batch Note Modification Right Region The top section of the right area of the page provides a History of the batch. Clicking the Audits link (red circle, Figure 25) will present a dialog which displays the audit trail. For a new batch, the Audits dialog will be empty; once review had begun, the dialog will display a record of any user changes, if any are made. Figure 25 History Section Also under the History section heading is a secondary heading for Modifications, under which is a link for Determinations or Calibrations (green circle, Figure 25). These links include a display of the number of determinations and/or calibrations in the batch which have been manually modified by a user. For more information on this, please see Manual Modification Review. ASCENT v User Manual 26 Revision X

27 The next section on the right region provides links to Results of Interest (Figure 26). The Reporting link includes a display of the number of determinations in the batch which have a reportable concentration, per the results of the Reporting Above Limit and/or Reporting Concentration of Interest quality rules. The Repeat and Exclude links includes a display of the number of determinations in the batch which have had their reporting decision changes from Include to one of those two values. Figure 26 Results of Interest Links Hovering on any of the links will display a tooltip with the total number of determinations in the batch, along with a percentage of determinations which have reportable concentrations. For more information on this, please see Results of Interest Review. Further down the right region displays links to any of the available Downloads (Figure 27). Links in this area represent any files available for download (e.g. custom reports, archive PDFs). Figure 27 Downloads Links The last section of the right region contains the buttons used for documenting that a batch has been fully Reviewed and/or Certified. ASCENT v User Manual 27 Revision X

28 Figure 28 Review and Certify For more information on this activity, please see Completing a Review and CERTIFICATION. Standard Review In the caption region of the Batch Summary page, the user may choose to view the results in either Read Only (red circle, Figure 29), after clicking the stoplight menu icon, or Review mode (green circle, Figure 29), using the Standard Review link. Figure 29 Read Only and Review Options Read Only Mode The batch will be opened in read only (non-modifiable) mode when the Standard Review Read Only link is selected OR if the user does not have Review/Certify permissions; also, if a batch is in the certified state, it will only be accessible in read only mode. In read only mode, the user may access the same information as that obtained through the review mode, but will have no ability to modify the data. Review Mode The batch will be opened in review (modifiable) mode when the Standard Review link is selected by a user with Review permissions (selecting this link when the user does not have the necessary permissions, or if the batch is in a certified state, will open the batch in Read Only mode). In concert with this, ASCENT will automatically update the Batch Status page to reflect that the batch is now In Review and the user will be set as the Current Analyst (Figure 30). ASCENT v User Manual 28 Revision X

29 Figure 30 Batch Status - In Review Choosing Review mode will allow the user to: view chromatograms, flags, and results display zoomable chromatograms modify peak integration or calibration record reporting decisions, comments, and codes complete the review and/or (with proper permissions) certify the batch While it is possible for more than one user to access an ASCENT batch in Review mode, it is not a supported activity. Multiple users simultaneously making changes to the same batch, or a single user modifying the same batch from separate browser windows or tabs, may result in data desynchronization, causing an error and potentially requiring the batch to be resubmitted and/or reprocessed. Accessing the same batch in two instances, one in Review mode and one in Read-only mode, is supported. When read-only or review mode is selected through the appropriate link on the Batch Summary page, the Batch Review page will appear (Figure 31). The Batch Review display is broken into three primary regions: Chromatogram display (red rectangle, Figure 31) Quality rule and flag information (blue rectangle, Figure 31) Data grid (green rectangle, Figure 31) A fourth element, a button for accessing Calibration information, is also available in either mode (yellow circle, Figure 31). A button which presents options for peak modification (orange circle, Figure 31) is only visible on the Batch Review page when in review mode. ASCENT v User Manual 29 Revision X

30 Figure 31 Standard Batch Review Page The alignment of chromatograms will be as shown in Figure 31 when displayed on a screen with the minimum supported resolution of 1280 x To return to the Batch Summary page at any time, click the batch name at the top of the page (purple circle, Figure 31). Navigating Batch Review Batch review operations can be managed by use of either the mouse or the keyboard. The Data Grid is used to display textual/tabular information as well as act as the primary navigation mechanism when moving between determinations. That is, the data grid is used to select a particular determination, which then drives the presentation of additional information in the chromatogram display and quality rule and flag information sections. Keyboard navigation is available within ASCENT for speed of review. All fields in the data grid can be accessed by utilizing the arrow keys to navigate and the Enter/Return key to ASCENT v User Manual 30 Revision X

31 activate and deactivate an input field. By pressing the down arrow key while at the bottom of a page, the next page of determinations will automatically load. The mouse can also be used for navigation within ASCENT. This is done by clicking on the rows within the data table to view compound information and using the Next /Last Page arrows at the bottom of the table to progress through the samples. Data Grid The data grid is a standardized display of information that combines input information from the sequence file with results populated by ASCENT (Figure 32). The data grid also provides navigation through the batch result set. The user may go directly to a specific page by typing in the page number or using the forward and backward arrows at the bottom of the grid (red circle, Figure 32). There is also an option to specify the number of rows to be displayed at one time. The user may choose to sort the batch results by compound or type as well as index (green circle, Figure 32). Figure 32 Data Grid Most of these fields are not editable in the grid, although values will be updated based on other review functions such as modifying a peak. The read-only fields are as follows: Index - Number indicating the sequence position of the sample in the batch Sample Name - Name of the sample as defined in the acquired data Compound - Name of the compound as defined in Assay Configuration Type - Sample type (Standard, QC, Blank, or Unknown) Dilution Factor - Raw analyzed concentration is multiplied by this value, taken from the sequence file, to account for sample dilution. Undiluted samples use a value of 1. ASCENT v User Manual 31 Revision X

32 Nominal Conc. - Expected concentration value for the selected compound in the sample obtained from the ASCENT sequence file and specified in Lots and Levels of the Assay Configuration Response - An area-based value determined using the Response Criteria set up in the Assay Configuration. If no peaks are integrated for all chromatograms in the numerator of the response criteria calculation, this field will display N/D (not detected). If no peaks are integrated for all chromatograms in the denominator of the response criteria calculation, this field will display N/C (not calculable). Calc. Conc. The calculated concentration is determined by multiplying the analyzed concentration (calculated from the regression curve as a function of response) by the dilution factor, displayed to three decimal places. If no peaks are integrated for all chromatograms in the numerator of the response criteria calculation, this field will display N/D (not detected). If no peaks are integrated for all chromatograms in the denominator of the response criteria calculation, this field will display N/C (not calculable). Reported Conc. - Calculated concentration formatted without commas, with values greater than the ULOQ displayed as ">ULOQ" and values less than the LLOQ reported as "<LLOQ". ( ULOQ and LLOQ are replaced with the numeric values of these settings in the Assay Configuration. The determination of above or below a limit of quantitation is based upon the analyzed concentration (unmodified by dilution factor). If no peaks are integrated for all chromatograms in the numerator of the response criteria calculation, this field will display N/D (not detected). If no peaks are integrated for all chromatograms in the denominator of the response criteria calculation, this field will display N/C (not calculable). Conc. Deviation (%) - When applicable, the percent deviation between the calculated concentration and the nominal concentration Chrom 1 Name - Name of the chromatogram as defined in Assay Configuration Chrom 1 Area - Area under the integrated peak in Chrom 1 Chrom 1 RT - Retention time (in minutes) of the integrated peak in Chrom 1 Chrom 2 Name - Name of the chromatogram as defined in Assay Configuration Chrom 2 Area - Area under the integrated peak in Chrom 2 Chrom 2 RT - Retention time (in minutes) of the integrated peak in Chrom 2 Chrom 3 Name - Name of the chromatogram as defined in Assay Configuration Chrom 3 Area - Area under the integrated peak in Chrom 3 Chrom 3 RT - Retention time (in minutes) of the integrated peak in Chrom 3 Batch - Name of the batch in which the sample was processed as defined by the ASCENT sequence filename Injection - Number indicating the sequence position of the sample in the batch in which it was originally processed ASCENT v User Manual 32 Revision X

33 Filename - Filename of the raw data for the sample Acquisition Time Time the sample was acquired on the instrument, obtained from the raw data file Flagged - A true/false value indicating if the determination was flagged. There are also three fields in the Data Grid that can accept user input (Figure 33). These fields are included in the default report generated by ASCENT. Report - The Report field is a dropdown menu allowing the user to choose to Include, Exclude, Repeat. Custom result files may use this value to limit content. Comment - The user can record additional information regarding that sample which may be helpful in compound or batch review. Code - Allows the user to input a free text code for laboratory metrics, if desired. Figure 33 Data Grid Input In order for the Report, Comment or Code inputs to be saved, it is necessary to click either the <Enter> or <Tab> keys, or specifically select another cell in the grid by left-clicking on it. Grid Commands Any time during the analysis when the user would like to access the full set of original data records, they may select the symbol on the left hand side at the bottom of the table to Reload the Grid (red circle, Figure 34). Additionally, they may Expand All, or Collapse All grouped sections. ASCENT v User Manual 33 Revision X

34 Figure 34 Reload the Grid Chromatogram Display Depending upon the specific assay method of the laboratory, up to three chromatograms may be displayed for each compound. For purposes of illustration, a typical assay will be described which has two chromatograms for an analyte: Quant (Quantifier - peak area for quantification) Qual (Qualifier - peak area for confirmation of identity/presence) The internal standard is typically defined as a separate compound to allow for a one-tomany relationship between internal standards and analytes. The primary purpose of the internal standard compound is to use its peak area to normalize the quantifier of an analyte; quite often, it is also used for retention time referencing. In the example, the internal standard has one chromatogram, which is labeled IS. Figure 35 and other examples show the IS chromatogram displayed to the right of the analyte chromatograms using the relate command as a macro (see Relate Command). Figure 35 Chromatogram Review Display Chromatograms are displayed with the time axis (and retention time label of an integrated peak) in minutes. Using the Terminal to Display Additional Chromatograms The terminal features are used to display chromatograms of other determinations in the same batch. The user has the ability to review more than one compound at a time for a given sample, compare chromatogram peak shapes between samples, or to display the compound with its internal standard reference in a chromatogram format. ASCENT v User Manual 34 Revision X

35 In order to display additional chromatograms, data commands are accessible from the ASCENT terminal which is activated by pressing the tilde key (~). While in terminal mode, type the command help to display the list of data commands. The terminal has various auto fill capabilities: Type the first letter of the command and press Tab to complete. Type the first letter of the analyte or sample filename (first argument only) and press Tab to complete. Continue pressing Tab to cycle through other analyte or sample filename values automatically. Cycle through previously entered terminal commands by pressing the up and down arrow keys. Terminal commands are case sensitive with regard to analyte names; analyte names must match the names in the data grid. Analyte Command The analyte command can be used to display the chromatogram of another compound in the same sample alongside the currently selected compound. This is done by typing, for example, analyte Hydromorphone to display Hydromorphone s chromatogram(s) (Figure 36). The command analyte clear all will clear the display of additional chromatograms. Figure 36 Analyte Command Sample Command The sample command is used to display the chromatograms for the same compound of a different sample. For example, sample Happy-02 will display the corresponding chromatograms of that particular sample based on which compound is active (Figure 37). This information can be cleared by using the sample clear Happy-02 or sample clear all command. ASCENT v User Manual 35 Revision X

36 Figure 37 Sample Command Relate Command The relate command is a one-way relationship between compounds in which chromatograms of the related compound in the same sample are always displayed with that of the active compound chromatograms. For example, relate Morphine Morphine-D6_IS results in the display of the IS chromatogram next to the analyte chromatograms of all samples in the batch (Figure 38). Figure 38 Relate Command Reset Command The reset command is used to reset the chart pane to its original settings by removing any additional charts the user may have added. The command is simply reset. To return to the display to the default, simply reload the page and any macros will be re-applied. Other Features An ASCENT Administrator can Define Macros in the Assay Configuration to execute the specified terminal commands automatically when any batch of that assay is reviewed (see Define Macros). Using the Reset command will override this setting on a batch by batch basis. ASCENT v User Manual 36 Revision X

37 Quality Rule and Flag Information Directly below the chromatogram display area is a series of rows where flags are shown (Figure 39). The rows are labeled with Sample, Compound, Chrom 1, Chrom 2, and Chrom 3. The latter three options refer to the chromatograms of the given determination and are defined by name in the Data Grid. In Figure 39, Chrom 1 is the Quantifier (Quant) chromatogram and Chrom 2 is the Qualifier (Qual) chromatogram. Chrom 3 will be blank, since a third chromatogram was not defined for the example (if it had been present, a third chromatogram would have been displayed in the left column of the chromatogram display). Flags are not shown for chromatograms displayed using the terminal commands, including the IS compound chromatograms. Therefore, it may be advantageous to review the IS chromatograms before reviewing the related analytes. The compound view order can be set by an Administrator in the assay configuration to support effective review. Figure 39 Flagged Sample Determinations are flagged (Figure 39) as a result of automated QA rule settings, which an Administrator of ASCENT will configure based upon quality assessment appropriate for the specific assay (see APPENDIX B: CORE QA RULES). For additional information about visualizing and prioritizing review operations based upon quality rules, please refer to Exception Based Review. Calibration ASCENT v User Manual 37 Revision X

38 When the Calibration button is clicked (red circle, Figure 40), bringing up the Calibration dialog (Figure 41), the user may access the calibration curves to which the compound samples are being compared for quantification. In order to make changes to the calibration settings, the user must have Calibration permissions. Figure 40 Calibration Figure 41 Batch Calibration Screen The Calibration displays and allows the following actions: The calibration curve displayed is the one selected in the Compounds list on the left hand side of the page (yellow circle, Figure 41). The calibration curve includes the individual data points, the regression curve fit, the regression equation and the R 2 value (coefficient of determination). Weighting of regression is adjustable, as well as origin handling (green circle, Figure ASCENT v User Manual 38 Revision X

39 41). Type or degree of the regression equation is adjustable (green circle, Figure 41). Points on the curve may be dropped (blue circle, Figure 41). Any calibrator with a nominal concentration or response of zero will be excluded from the regression calculation. If this calibrator peak was removed using the Remove Peak function (see Peak Modification) or no peak was detected, Use Record is automatically unchecked for that sample. To apply any of the changes listed above, click the Modify button (orange circle, Figure 41). If a first user is performing a calibration modification (Modify, as above; Import, as below) and a second user attempts to perform a similar action, the second user will receive a lock message and their action will not be performed. By default, the calibrators are those in the batch being reviewed. To import standards from another batch, use the Import Calibration Curve option in the upper left corner of the screen (red circle, Figure 41). Check Include QCs to also import the QC samples from the selected batch. The Reference Batch list includes batches from the same assay and instrument that were Certified in the last seven days and do not themselves utilize an imported calibration. After selecting an available calibration reference batch, click Import (red circle, Figure 41). Following a successful import, the calibration curves will be made available to the batch; however, the details of the calibration reference batch (e.g. calibration and QC samples, and their compound results, which make up the calibration) will not be imported into the batch (that is, they will not appear as results in the Data Grid). Calibration flags will be disabled when the target batch has no standards of its own. If the calibration reference batch to be imported does not contain any standards, ASCENT will return an error. If a calibration reference batch is no longer to be utilized, select None from the Reference Batch list and click Import. The calibration reference will be removed, and the batch s own calibration standards will be used. Peak Modification During peak modification, the Standard Review grid will be visible beneath the Peak Correction dialog, but navigation in the grid is disabled until the modification is ASCENT v User Manual 39 Revision X

40 complete (e.g. to allow changes to certain samples, such as Standards, to propogate into the complete batch). Also, if a first user is performing a peak modification and a second user attempts to perform a similar action on the sample/batch, the second user will receive a lock message and their action will not be performed. Appropriate permissions are necessary for manual peak modification. For some laboratories, this function will not appear at the user level. Refer to User Permissions for additional information on permission assignment to users. To modify one or more peaks associated with a particular determination, select the appropriate row in the data grid and then click the Correct Peak button (red circle, Figure 42) to be presented with the Modify Peak dialog (Figure 43). Figure 42 Peak Modification Alternatively, the chromatogram graphics themselves may be clicked to present the Modify Peak dialog. This action can be used on the chromatograms directly associated with the current determination as well as any chromatogram which has been associated or linked to the current determination. For example, an assay configuration may relate a target analyte to its associated internal standard (see Define Macros). The red frame of Figure 31 includes an example of this style of display. Clicking on either of the two plots in the left column will present the Modify Peak dialog for the analyte, while clicking on the plot in the right column will present the Modify Peak dialog for the related compound (e.g. internal standard). The caption of the Modify Peak dialog will indicate whether the window is displaying the current determination versus a linked or associated compound. ASCENT v User Manual 40 Revision X

41 Figure 43 Modify Peak Dialog The Modify Peak dialog will display a chromatogram plot for each of the peaks defined for that compound in the Assay Configuration. Each chromatogram has independent Zoom Controls (red circle, Figure 43), each zoom-in or zoom-out click control click will affect both the time and intensity scales. Zooming-in will decrease the displayed time range, while maintaining the center; in the intensity range, the y-axis scale will decrease but the zero point will remain fixed. Zooming-out behaves in the opposite manner. The chromatogram may also be zoomed through the use of the mouse wheel (if available); the zoom effect will center on the mouse pointer location on the chromatogram plot, so that a particular region can be the focal point of the zoom. The center Zoom Control (red circle, Figure 43) returns the view to the default zoom and position. The Modify Peak dialog supports a panning action, activated by a left click-and-drag operation. Moving in the horizontal direction will move the chromatogram trace in the time dimension, while moving in the vertical direction will move in the intensity dimension. To support detailed examination of the baseline, a left click-and-drag initiated near the baseline and moving upwards will hold the baseline level and expand the intensity dimension. To support the examination of data which may exceed the displayed intensity range (e.g. a peak is detected, which sets the intensity range to best observe the peak, but there is an adjacent artifact which exceeds that range), a left click-and-drag initiated near the top of the ASCENT v User Manual 41 Revision X

42 trace and moving downwards will increase the y-axis scale (to a maximum degree in which the highest data point of the trace is now visible in the plot). When working in certain peak modification dialogs, this may temporarily reset the y-axis of a particular trace for full scale, although it may have initially been presented with a full scale value which is common to other chromatogram plots in the dialog (e.g. the peak may have been displayed as smaller than full scale). As above, the center Zoom Control (red circle, Figure 43) returns the view to the default zoom and position. Each chromatogram also has independent Peak Modification controls (green circle, Figure 43). To modify a peak which was found, or define a peak which wasn t found, zoom in until the data points are visible, and then click the start and end data point positions of the desired peak (a dotted vertical line will appear at the data point nearest the click, see Figure 44). Click the delimited peak button (blue circle, Figure 44) to remove any chosen data points; clicking near an already selected point will move the vertical delimiter to the new position. Once points have been selected, click the yellow-shaded peak button to integrate the peak using the system calculated baseline (green circle, Figure 44), or the grey-underlined, yellow-shaded peak button to use a designated baseline integration (red circle, Figure 44), e.g. the baseline is drawn as a straight, point-to-point connection between the two selected data points. ASCENT will update the results and flags accordingly. Additionally, any peak which has been manually integrated will have its internal shading displayed in yellow (similar to the peak modification buttons); if selected as the option, peaks will show the designated baseline as a solid grey line (again, as similar to that peak modification button). Figure 44 Peak Zoomed with Peak Limits Defined ASCENT v User Manual 42 Revision X

43 A comparison of the two manual integration approaches is seen in Figure 45: the upper peak was generated using the system calculated baseline, while the lower peak was generated using a designated baseline. Figure 45 Peak following Modification To revert (return) to using the default, initial (system) integration, click the blue-shaded peak button (blue circle, Figure 45). Following this selection, ASCENT will update the results and flags accordingly, and the internal peak shading will be blue (similar to the revert button). In this example, the peak display would return to that observed in Figure 43. The Revert Peak button may be used to restore a removed peak (see below). If the removed peak was a standard or QC, it will be necessary to manually review the calibration to make sure the reverted peak is included. To remove a manually defined or system defined peak, click the button with the peak crossed out (red circle, Figure 45). Following this selection, ASCENT will update the results and flags accordingly; the internal peak shading will also be removed, while an outlined trace of the system defined peak, if available, will be visible (although not used for result calculation). An example of this can be seen in Figure 46. ASCENT v User Manual 43 Revision X

44 Figure 46 Display following Peak Removal When finished with the activities of peak correction or modification, the dialog can be dismissed by clicking the x in the upper right corner of the dialog (green circle, Figure 46). Exception Based Review An assay configuration s automated quality rules and flags can act as a powerful accelerator for the batch review process. For detailed information on the core quality rules and flags, refer to APPENDIX B: CORE QA RULES (additional quality rules and flags can be added to ASCENT; for more information on this option and the submission of a request for professional services, please refer to APPENDIX A: CUSTOMER PORTAL. These quality rules, when appropriately configured and utilized within the scope of the laboratory s overall quality system, can support a review process in which the primary activity is viewing only those results which in some way have triggered a quality rule flag. This approach bases the review process on exceptions to the quality rules, hence the descriptor, exception based review. The exception based review approach can be operated from either the Batch Summary or Batch Review pages: Batch Summary (Figure 13) click one of the bars on the Overview chart. Batch Review (blue circle, Figure 47) click one of the options provided in the dropdown selector. ASCENT v User Manual 44 Revision X

45 Whether viewed through the Overview chart on the Batch Summary page or within the Standard Review data grid, the flags and their categories can bring immediate attention to those results which are relevant to a particular stage or type of batch review. From the data grid, the user can select from a list of various types of flags in order to select which flagged samples and determinations they wish to review. The user may then review a specific subset of determinations and, as required/desired, perform manual modification, select a reporting decision and/or enter a comment or code for the determination. If there are no determinations in the selected category, no chromatograms or flags will display and the data grid will be empty. Figure 47 Filter by Flags Flags are an important review driver. A user can prioritize the order of review based upon the available flagged determinations. The results of applying the ASCENT QA rules have been grouped as follows: Peak Quality/RT Flags: Fit Quality, High Signal No Peak, Dynamic High Signal No Peak, Peak Cluster Fallback, Quant Qual Difference, Relative RT Difference, Shift from Standards Calibration Flags: Concentration Deviation, Int Std CV, Int Std Response Deviation, Minimum R2, Regression Failed, Standards Excluded, Calibrator Missing Result Quality Control Flags: Concentration Deviation, Exceeds Standard Deviation, Negative Control. QC Missing Result Concentration Flags: Carryover Above LLOQ, Carryover Flag Subsequent Samples, Carryover Potential, Dilution Required, Int Std Response, No Intercept, Over Diluted, Present But Below LLOQ, QC Flag Positive Unknowns Contamination Flags: Contamination of Blank Sample Peak Area Flags: Ion Ratio Deviation, Ion Ratio Peak Missing, Peak Area Deviation, Int Std Response ASCENT v User Manual 45 Revision X

46 Other Flags: Custom (e.g. as requested by the client) flags which do not fall into the other categories will be displayed in this group Reporting Flags: Above Limit, Concentration of Interest Enhanced Review As mentioned above, the Peak Quality/RT, Calibration, and Quality Control flags are of particular significance. As such, a set of review operations have been developed which provide a more holistic perspective on the nature and types of considerations which go into these flags. Peak Quality/RT This enhanced review path is entered by clicking on the Peak Quality/RT bar in the Overview chart (red circle, Figure 48). Figure 48 Batch Summary and Overview Chart Clicking on the Peak Quality/RT bar will present the Peak Quality and RT Mosaic (Figure 49). Figure 49 Peak Quality and RT Mosaic ASCENT v User Manual 46 Revision X

47 The Peak Quality and RT Mosaic is a multi-determination view of the batch. The set of determinations which are displayed in the Mosaic may be ordered by either sample or compound, using the sort by selector (red circle, Figure 49). Additionally, the set of determinations which are presented in the Mosaic may be limited to only those of a specific type (e.g. Standards, QCs) using the filter by selector (blue circle, Figure 49). Each determination in the batch which is flagged with one or more of the specified flag category in this case, peak quality and retention time is presented as a Tile. The Tile contains a number of items which allow for quick visual review: sample index and name (red circle, Figure 50), compound name (blue circle, Figure 50), flag abbreviations (green circle, Figure 50) and an indicator of any flags outside of this category (yellow circle, Figure 50). The traces displayed in the Tile are the chromatograms for that determination: the blue trace is Chrom1 (typically, the quantifier) and the green trace is Chrom2 (typically, the qualifier); Chrom3, if specified for the compound, will be represented by a red trace. Chromatograms which have detected and integrated peaks will show a shaded area inside the trace; chromatograms without shading indicate that no peak was detected and integrated. The shading color itself is indicative of a system integrated peak (color hue matches the trace), manual peak integration with system baseline (yellow), or manual peak integration with designated baseline (yellow with a dark line). Figure 50 Peak Quality/RT Tile Details Hovering on the flag abbreviation will bring up a tooltip, which provides the full name of the flag, the calculated value which presented the flag, and the quality rule limits which were used to evaluate the flag (Figure 51). ASCENT v User Manual 47 Revision X

48 Figure 51 Peak Quality/RT Tile and Flag Tooltip If additional investigation is warranted, a Tile may be clicked to bring up a Scope, an expanded view of the determination (Figure 52). Figure 52 Peak Quality/RT Scope The Peak Quality/RT Scope presents a variety of useful information (for details on the visual characteristics of the chromatograms and peaks themselves, refer to the section on Peak Modification): ASCENT v User Manual 48 Revision X

49 Title bar: sample index, file name, and type; sample name; acquisition date/time Compound name and various calculated values (red circle, Figure 52) Peak quality flags and any other flags from other categories (blue circle, Figure 52) If the displayed determination represents a compound which uses another compound as a normalizer (e.g. an analyte which uses an internal standard), a link is provided to bring up the Peak Quality/RT Scope for that related compound (orange circle, Figure 52); any flags which were found on the normalizer are also listed here Data entry fields for report, code and comment (green circle, Figure 52) Up to three chromatograms for the determination, arranged in a vertical format of chromatograms 1, 2 and 3; each chromatogram shows a vertical solid line for the predicted retention time and a dotted line for the actual retention time (if a peak has been detected and integrated for the chromatogram) Peak modification controls, identical in style and use as described for Figure 43 (yellow circle, Figure 52) In order for the Report, Comment or Code inputs to be saved in the data entry fields (green circle, Figure 52), it is necessary to click either the <Enter> or <Tab> keys, or specifically select another input field in the display by left-clicking on it. When successful, a small pinwheel and green check mark will be displayed momentarily in the appropriate data entry field. To return to the Mosaic when the Scope is displayed, click the icon in the navigational control at the bottom of the Scope (red circle, Figure 53). To move to the Scope for an adjacent Tile without returning to the Mosaic, click either of the arrows or determination descriptions (green circles, Figure 53). Figure 53 Peak Quality/RT Scope Navigation Upon returning to the Mosaic, if changes made in the Scope remove the issue(s) which caused the Tile to be present in the Mosaic (e.g. clearing a peak quality flag by manual peak integration), that determination s Tile will be presented in a disabled style (e.g. grey background and subdued content), as an indicator that the determination in question no longer has any flags in this enhanced review category. Remaining in the Mosaic will leave ASCENT v User Manual 49 Revision X

50 the Tile visible; navigating to the Batch Summary page (see below) and then back to the Mosaic will remove the Tile (as it no longer has flags which would cause it to present). As changes are potentially made in the Scope (e.g. manual peak integration), values which are derived from those changes, such as compound responses and concentrations, are automatically re-calculated; as well, Scope changes will also trigger the re-application of the quality rules. To observe the effects of these changes, re-calculations and re-applications via the Overview graph and Batch Summary page, click the name of the batch at the top of Mosaic (red circle, Figure 54). The Overview graph is automatically updated through the link. Figure 54 Peak Quality/RT Mosaic Links It is also possible to navigate from the Peak Quality and RT Mosaic back to the Batch Summary page and Overview graph using the browser s back button. However, using this action will not automatically update the Overview graph; it will be necessary to refresh the display manually. It may be useful at times to see additional information about a batch when working in the Mosaic, Tile and Scope views. To navigate to the Standard Review page (e.g. Data Grid), click the link for Standard Review at the top of the Mosaic (blue circle, Figure 54). Calibration This enhanced review path is entered by clicking on the Calibration bar in the Overview chart (red circle, Figure 55). ASCENT v User Manual 50 Revision X

51 Figure 55 Batch Summary and Overview Chart Clicking on the Calibration bar will present the Calibration Mosaic (Figure 56). Figure 56 Calibration Mosaic The Calibration Mosaic is a multi-compound view of the batch, and can display quality metrics on both analytes and internal standards. The set of analyte calibration curves and internal standard mean plots which are displayed in the Mosaic are presented in the order defined in the assay configuration. Each compound in the batch which is flagged with one or more of the specified flag category in this case, calibration is presented as a Tile. The Tile contains a number of items which allow for quick visual review: compound name (red circle, Figure 57), analyte coefficient of determination (blue circle, Figure 57), and flag abbreviations (green circle, Figure 57). If an internal standard Tile is presented, the response coefficient of variation is provided in place of the coefficient of determination. The graphic displayed in the Tile includes the individual calibrator data points and a curve which illustrates the mathematically determined regression equation (or mean, in the case of an internal standard). Calibrator data points are normally displayed as a blue circle; points which have been excluded from the regression are represented as red squares. ASCENT v User Manual 51 Revision X

52 Figure 57 Calibration Tile Details Hovering on the flag abbreviation will bring up a tooltip, which provides the full name of the flag, the calculated value which presented the flag, and the quality rule limits which were used to evaluate the flag (Figure 58). Figure 58 Calibration Tile and Flag Tooltip If additional investigation is warranted, a Tile may be clicked to bring up a Scope, an expanded view of the analyte s calibration (Figure 59) or internal standard s response distribution. ASCENT v User Manual 52 Revision X

53 Figure 59 Calibration Scope The Calibration Scope for a compound (name indicated in upper left corner) displays and allows the following actions: Flags will be presented for the calibration as a whole (e.g. for analytes, minimum R2, maximum number of standards excluded; for internal standards, maximum CV) on the left side, or individual calibrator determinations (e.g. for analytes, maximum concentration deviation; for internal standards, maximum response deviation) in the lower table, with light-red back-colored text. As with the Tile, hovering on the abbreviations for the determination row flags will provide a tooltip with information on calibration and QC rules, as well as any other rules (e.g. Peak Quality) which may be present for that determination. For analytes, if the associated internal standard has a flag, it will be noted in the last column (black circle, Figure 59. For analytes, the coefficient of determination (as R2 value), and regression equation (red circle, Figure 59); for internal standards, the mean response and the coefficient of variation. For analytes, the calibration curve graphic includes the individual data points, visible as blue circles, and the regression curve; for internal standards, the mean distribution plot. For analytes, weighting of regression is adjustable, as well as origin handling and type or degree of regression (blue circle, Figure 59); for internal standards, a listing of the analytes which utilize this compound. For analytes, points on the curve may be excluded (green circle, Figure 59); an excluded point is visualized on the calibration curve graphic with a red circle. Any calibrator with a nominal concentration or response of zero will be excluded from the regression calculation. If this calibrator peak was removed using the Remove Peak ASCENT v User Manual 53 Revision X

54 function (see Peak Modification) or no peak was detected, Use Record is automatically unchecked for that sample. To apply any of the changes listed above, click the Modify button (orange circle, Figure 59). If a first user is performing a calibration modification and a second user attempts to perform a similar action, the second user will receive a lock message and their action will not be performed. Individual calibrator determinations may be investigated by clicking in the row of the determination in the table. Such a gesture will present the Peak Quality and RT Scope (Figure 52) for the determination, with all available and permissible user edit options (e.g. manual peak modification; code, comment and reporting decision field entry). For analytes which present a flag on the internal standard, clicking the flag icon will navigate directly to the Peak Quality and RT Scope for the internal standard. For more information on the use of this Scope, please see Peak Quality/RT. Different from the navigational control for the category-presented Peak Quality/RT Scope (Figure 53), when presented via routing from the Calibration Scope, the Peak Quality/RT Scope will present an alternative navigation control (Figure 60). Figure 60 Alternative Peak Quality RT Scope Navigation To move from the Peak Quality/RT Scope back to the Calibration Scope, click the arrow or calibration curve icon in the navigation control at the bottom of the Scope (green circle, Figure 60). To move from the Peak Quality/RT Scope back to the Calibration Mosaic, click the icon in the center of the navigational control (red circle, Figure 60). To return to the Calibration Mosaic when the Calibration Scope is displayed, click the icon in the navigational control at the bottom of the Scope (red circle, Figure 61). To move to the Scope for an adjacent Tile without returning to the Mosaic, click either of the arrows or compound descriptions (green circles, Figure 61). Figure 61 Calibration Scope Navigation ASCENT v User Manual 54 Revision X

55 Upon returning to the Mosaic, if changes made in the Scope remove the issue(s) which caused the Tile to be present in the Mosaic (e.g. clearing a calibration flag by calibrator point removal), that compound s Tile will be presented in a disabled style (e.g. grey background and subdued content), as an indicator that the compound in question no longer has any flags in this enhanced review category. Remaining in the Mosaic will leave the Tile visible; navigating to the Batch Summary page (see below) and then back to the Mosaic will remove the Tile (as it no longer has flags which would cause it to present). As changes are potentially made in the Calibration Scope (e.g. excluding a calibrator point), values which are derived from those changes, such as regression equations and subsequent calculated concentrations, are automatically re-calculated; as well, Scope changes will also trigger the re-application of the quality rules. To observe the effects of these changes, re-calculations and re-applications via the Overview graph and Batch Summary page, click the name of the batch at the top of Mosaic (red circle, Figure 62). The Overview graph is automatically updated through the link. Figure 62 Calibration Mosaic Links It is also possible to navigate from the Calibration Mosaic back to the Batch Summary page and Overview graph using the browser s back button. However, using this action will not automatically update the Overview graph; it will be necessary to refresh the display manually. It is possible to view only those Tiles which are available for just analytes, just internal standards, or all compounds using the Filter selector (green circle, Figure 62). It may be useful at times to see additional information about a batch when working in the Mosaic, Tile and Scope views. To navigate to the Standard Review page (e.g. Data Grid), click the link for Standard Review at the top of the Mosaic (blue circle, Figure 62). Quality Control This enhanced review path is entered by clicking on the Quality Control bar in the Overview chart (red circle, Figure 63). ASCENT v User Manual 55 Revision X

56 Figure 63 Batch Summary and Overview Chart Clicking on the Quality Control bar will present the Calibration Mosaic (Figure 64). Figure 64 Quality Control Mosaic The Quality Control Mosaic is a multi-analyte view of the batch. The set of analyte residual plots displayed in the Mosaic are presented in the order defined in the assay configuration. Each analyte in the batch which is flagged with one or more of the specified flag category in this case, quality control is presented as a Tile. The Tile presents a simplified residual plot: x-axis as calibrated concentration range; y- axis as relative percent difference between a QC determination s nominal and observed concentration. The Tile contains a number of items which allow for quick visual review: compound name (red circle, Figure 65), coefficient of determination (blue circle, Figure 65), and flag abbreviations (green circle, Figure 65). The graphic displayed in the Tile includes the individual QC determinations, with a maximum scale of +/- 50% relative deviation; any QC determinations which differ by more than 50% when using the QC deviation rule will be displayed at the upper or lower edges of the Tile as an arrow (to indicate an off scale value). ASCENT v User Manual 56 Revision X

57 Figure 65 Quality Control Tile Details Hovering on the flag abbreviation will bring up a tooltip, which provides the full name of the flag, the calculated value which presented the flag, and the quality rule limits which were used to evaluate the flag (Figure 66). Figure 66 Quality Control Tile and Flag Tooltip If additional investigation is warranted, a Tile may be clicked to bring up a Scope, an expanded view of the analyte s QC determinations (Figure 67) Figure 67 Quality Control Scope ASCENT v User Manual 57 Revision X

58 The Quality Control Scope for a compound (name indicated in upper left corner) displays and allows the following actions: Flags will be presented for the calibration as a whole (e.g. minimum R2, maximum number of standards excluded) on the left side, or individual QC determinations (e.g. maximum concentration deviation) in the lower table, with light-red back-colored text. As with the Tile, hovering on the abbreviations for the determination row flags will provide a tooltip with information on calibration and QC rules, as well as any other rules (e.g. Peak Quality) which may be present for that determination. If the associated internal standard has a flag, it will be noted in the last column (black circle, Figure 67). The coefficient of determination (as R2 value), and regression equation (red circle, Figure 67). The calibration curve graphic includes the individual data points, visible as blue circles, and the regression curve; the QC determination data points are visible as purple diamonds. Weighting of regression is adjustable, as well as origin handling and type or degree of regression (blue circle, Figure 67). Points on the curve may be excluded (green circle, Figure 67); an excluded point is visualized on the calibration curve graphic with a red circle. Any calibrator with a nominal concentration or response of zero will be excluded from the regression calculation. If this calibrator peak was removed using the Remove Peak function (see Peak Modification) or no peak was detected, Use Record is automatically unchecked for that sample. To apply any of the changes listed above, click the Modify button (orange circle, Figure 67). If a first user is performing a calibration modification and a second user attempts to perform a similar action, the second user will receive a lock message and their action will not be performed. Individual QC determinations may be investigated by clicking in the row of the determination in the table. Such a gesture will present the Peak Quality and RT Scope (Figure 52) for the determination, with all available and permissible user edit options (e.g. manual peak modification; code, comment and reporting decision field entry). For determinations which present a flag on the internal standard, clicking the flag icon will navigate directly to the Peak Quality and RT Scope for the internal standard. For more information on the use of this Scope, please see Peak Quality/RT. Different from the navigational control for the category-presented Peak Quality/RT Scope (Figure 53), when presented via routing from the Quality Control Scope, the Peak Quality/RT Scope ASCENT v User Manual 58 Revision X

59 will present an alternative navigation control (Figure 68). Figure 68 Alternative Peak Quality RT Scope Navigation To move from the Peak Quality/RT Scope back to the Quality Control Scope, click the arrow or calibration curve icon in the navigation control at the bottom of the Scope (green circle, Figure 68). To move from the Peak Quality/RT Scope back to the Quality Control Mosaic, click the icon in the center of the navigational control (red circle, Figure 68). To return to the Quality Control Mosaic when the Quality Control Scope is displayed, click the icon in the navigational control at the bottom of the Scope (red circle, Figure 69). To move to the Scope for an adjacent Tile without returning to the Mosaic, click either of the arrows or compound descriptions (green circles, Figure 69). Figure 69 Quality Control Scope Navigation Upon returning to the Mosaic, if changes made in the Scope remove the issue(s) which caused the Tile to be present in the Mosaic (e.g. clearing a QC deviation flag by modifying the calibration curve), that compound s Tile will be presented in a disabled style (e.g. grey background and subdued content), as an indicator that the compound in question no longer has any flags in this enhanced review category. Remaining in the Mosaic will leave the Tile visible; navigating to the Batch Summary page (see below) and then back to the Mosaic will remove the Tile (as it no longer has flags which would cause it to present). As changes are potentially made in the Quality Control Scope (e.g. excluding a calibrator point), values which are derived from those changes, such as regression equations and subsequent calculated concentrations, are automatically re-calculated; as well, Scope changes will also trigger the re-application of the quality rules. To observe the effects of these changes, re-calculations and re-applications via the Overview graph and Batch Summary page, click the name of the batch at the top of Mosaic (red circle, Figure 70). The Overview graph is automatically updated through the link. ASCENT v User Manual 59 Revision X

60 Figure 70 Quality Control Mosaic Links It is also possible to navigate from the Quality Control Mosaic back to the Batch Summary page and Overview graph using the browser s back button. However, using this action will not automatically update the Overview graph; it will be necessary to refresh the display manually. It may be useful at times to see additional information about a batch when working in the Mosaic, Tile and Scope views. To navigate to the Standard Review page (e.g. Data Grid), click the link for Standard Review at the top of the Mosaic (blue circle, Figure 70). Auto Analysis Results which include certain quality rule flags are often dispositioned in a well characterized way. For example, when following a high concentration sample acquisition, subsequent samples may be adversely affected. One possible side effect is that the subsequent samples may show elevated concentration values, only because of a carryover effect from the high concentration sample. In such instances, the common response is to repeat those samples which may have been impacted by carryover; this is often expressed as a change in the determination s Reporting Decision, e.g. changing from the default Include to a value of Repeat or Exclude. Auto Analysis works in conjunction with a specific quality rule Carryover Potential to efficiently address samples which may have been impacted in such an instance. For more details on the configuration of this quality rule, please see APPENDIX B: CORE QA RULES. The Carryover Potential rule must be active in order to utilize Auto Analysis. ASCENT v User Manual 60 Revision X

61 Figure 71 Auto Analysis Click the Auto Analysis button (red circle, Figure 71) to perform the evaluation using the Carryover Potential rule. The button s style provides an indicator of the applicability of Auto Analysis: when the button is blue with white text, there are Carryover Potential values which are available for consideration. Following automated assessment, a dialog will be presented that indicates which, if any, samples have been potentially impacted by carryover (Figure 72). If any samples are listed, the change in Reporting Decision (as configured in the quality rule) will be displayed. Figure 72 Auto Analysis Dialog To accept this assessment and automatically disposition the results by modifying the Reporting Decision, click Apply All (red circle, Figure 72). To cancel Auto Analysis, click Close (green circle, Figure 72). Following the application of the Auto Analysis actions, the Auto Analysis button (red circle, Figure 71) will change to light grey with dark grey text, illustrating that the action has been taken. ASCENT v User Manual 61 Revision X

62 Manual Modification Review When a batch is (initially) presented in ASCENT as Ready for Review, the batch s results are based exclusively on the assay configuration and automated processing by the system. Following user review, however, it is not uncommon that one or more of the results have been re-calculated based on some type of manual modification. This modification may be done directly on the determination itself (e.g. a compound s quantification peak may have been manually integrated), or to an operation related to the calculation of the determination results (e.g. a compound s calibration curve may have had its mathematical model altered). Manual modification of compound results is frequently considered a primary area for internal and external quality oversight. As an aid in supporting such quality oversight, all manual peak and calibration modifications are recorded as part of a permanent audit trail for the batch (see AUDITS for more information). Additionally, as further aid in visualizing this key quality area, an additional Enhanced Review operation is available, accessed through the Batch Summary page. Determination Modifications When a batch whose results are based on one or more manual peak modifications is displayed on the Batch Summary page, an additional link, Determinations, is available in the History section, under the Modifications secondary heading (red circle, Figure 73). The Determinations link includes a display of the number of determinations in the batch which have been manually modified by a user; hovering on the link will display a tooltip with the total number of determinations in the batch, along with a percentage of determinations which have been manually modified. Figure 73 Batch Summary with Determination Modifications ASCENT v User Manual 62 Revision X

63 This enhanced review path is entered by selecting the Determinations link in the Modifications section (red circle, Figure 73). Clicking on the Determinations link will present the Determination Modifications Mosaic (Figure 74). Figure 74 Determination Modifications Mosaic The Determination Modifications Mosaic is a multi-determination view of the batch. The set of determinations which are displayed in the Mosaic may be ordered by either sample or compound, using the sort by selector (red circle, Figure 74). Additionally, the set of determinations which are presented in the Mosaic may be limited to only those of a specific type (e.g. Standards, QCs) using the filter by selector (blue circle, Figure 74). Each determination in the batch which is currently being calculated and reviewed on the basis of a manual operation in this case, a manual peak modification is presented as a Tile. The Tile contains a number of items which allow for quick visual review: sample index and name (red circle, Figure 75), compound name (blue circle, Figure 75), flag abbreviations (green circle, Figure 75) and an indicator of any flags outside of this category (yellow circle, Figure 75). The traces displayed in the Tile are the chromatograms for that determination: the blue trace is Chrom1 (typically, the quantifier) and the green trace is Chrom2 (typically, the qualifier); Chrom3, if specified for the compound, will be represented by a red trace. Chromatograms which have detected and integrated peaks will show a shaded area inside the trace; chromatograms without shading indicate that no peak is currently integrated. The shading color itself is indicative of a system integrated peak (color hue matches the trace), manual peak integration with system baseline (yellow), or manual peak integration with designated baseline (yellow with a dark line). ASCENT v User Manual 63 Revision X

64 Figure 75 Determination Modifications Tile Details Hovering on the flag abbreviation will bring up a tooltip, which provides the full name of the flag, the calculated value which presented the flag, and the quality rule limits which were used to evaluate the flag. Beyond these visual characteristics, shared with the Peak Quality and RT Mosaic (Figure 49), the Determination Modifications Mosaic includes additional detail which is relevant to its intent. A given determination may be the result of multiple chromatograms/peak in cases where more than one peak is to be presented, the peaks which have not been modified will display with a subdued intensity, to make the manually modified peak details more apparent. This effect is visible in the left Tile of Figure 75 the quantifier (Chrom1) peak was modified, so its blue trace and yellow shading (indicating a manual integration) are displayed at a normal intensity; the qualifier (Chrom2) peak was not modified, so its green trace and green shading (indicating a system integration) are displayed at a subdued intensity. Another relevance highlight is in the display of a vertical line at the location of a manually modified peak s predicted retention time. This indicator can assist in identifying cases where a peak was manually added or a manual shift was required; it may also assist in identifying cases where a potentially reportable peak was manually removed, or a peak was integrated far afield from the predicted retention time. An example of this is visible in the right Tile of Figure 75 the normal intensity display of the blue trace indicates that a manual modification was performed on its peak, and the absence of shading indicates that it was a manual peak removal; the predicted retention time, however, is directly over the apex of what appears to be a well characterized peak, so investigation may be warranted. If additional investigation is warranted, a Tile may be clicked on to bring up a Scope, an expanded view of the determination (Figure 76). The Scope which presents for Determination Modifications is identical in appearance and functionality to the Scope ASCENT v User Manual 64 Revision X

65 which presents from the Peak Quality/RT Mosaic and Tiles. For details on interacting with this Scope, refer to the Peak Quality/RT section of the discussion on Enhanced Review. Figure 76 Determination Modifications Scope As with the Peak Quality/RT enhanced review path, returning to the Mosaic after performing a change in the Scope may result in the Tile presenting in a disabled style (e.g. grey background and subdued content). Different than the Peak Quality/RT path, however, the Determination Modification path is indicative of a manual modification; thus, the only Scope action which will induce this change to a disabled Tile is a revert (return) to the system integrated peak. Effectively, the manual-ness of the peak has been cleared (analogous to making a Scope change, such as a manual integration, which clears a quality rule flag in the Peak Quality/RT path), so the Tile will not be present in a Mosaic refreshed by a return to the Batch Summary page (see below). As changes are potentially made in the Scope (e.g. continued manual peak modification), values which are derived from those changes, such as compound responses and concentrations, are automatically re-calculated; as well, Scope changes will also trigger the re-application of the quality rules. To observe the effects of these changes, re-calculations and re-applications via the Overview graph and Batch Summary page, click the name of the ASCENT v User Manual 65 Revision X

66 batch at the top of the Mosaic (red circle, Figure 77). The Overview graph and the Modifications graph are automatically updated through the link. Figure 77 Determination Modifications Mosaic Links It is also possible to navigate from the Determination Modifications Mosaic back to the Batch Summary page and Overview graph using the browser s back button. However, using this action will not automatically update the graph; it will be necessary to refresh the display manually. It may be useful at times to see additional information about a batch when working in the Mosaic, Tile and Scope views. To navigate to the Standard Review page (e.g. Data Grid), click the link for Standard Review at the top of the Mosaic (blue circle, Figure 77). Calibration Modifications When a batch whose results are based on one or more manual calibration (curve) modifications is displayed on the Batch Summary page, an additional link, Calibrations, is available in the History section, under the Modifications secondary heading (red circle, Figure 78). The Calibrations link includes a display of the number of compound calibrations in the batch which have been manually modified by a user. ASCENT v User Manual 66 Revision X

67 Figure 78 Batch Summary with Calibration Modifications This enhanced review path is entered by selecting the Calibrations link in the Modifications section (red circle, Figure 78). Clicking on the Calibrations link will present the Calibration Modifications Mosaic (Figure 79). Figure 79 Calibration Modifications Mosaic The Calibration Modifications Mosaic is a multi-compound view of the batch, and can display quality metrics on both analytes and internal standards. The set of modified analyte calibration curves and internal standard mean plots which are displayed in the Mosaic are presented in the order defined in the assay configuration. Additionally, the set of compounds which are presented in the Mosaic may be limited to only those of a specific type (e.g. Analyte, Internal Standards) using the filter by selector (red circle, Figure 79). Each compound in the batch which is currently being calculated and reviewed on the basis of a manual operation in this case, some change to the calibration curve methodology - is presented as a Tile. The Tile contains a number of items which allow for quick visual review: compound name (red circle, Figure 80), analyte coefficient of determination (blue circle, Figure 80), and flag abbreviations (green circle, Figure 80). If an internal standard Tile is presented, the response coefficient of variation is provided in place of the coefficient of ASCENT v User Manual 67 Revision X

68 determination. The graphic displayed in the Tile includes the individual calibrator data points and a curve which illustrates the mathematically determined regression equation (or mean, in the case of an internal standard). Calibrator data points are normally displayed as a blue circle; points which have been excluded from the regression are represented as red squares. Figure 80 Calibration Modifications Tile Details Hovering on the flag abbreviation will bring up a tooltip, which provides the full name of the flag, the calculated value which presented the flag, and the quality rule limits which were used to evaluate the flag (Figure 80). Beyond these visual characteristics, shared with the Calibration Mosaic (Figure 56), the Calibration Modifications Mosaic includes additional detail which is relevant to its intent. A given calibration is the result of a variety of assay and user-designated selections. A calibration may have been modified by the selection of an alternative mathematical approach (e.g. origin treatment, weighting, and/or power); the inclusion or exclusion of individual elements of the calibration point set; and/or the modification of the chromatographic peaks which comprise a calibration point. The tooltip includes specialized iconography and descriptions to indicate which of these modifications are in effect for the highlighted compound s modified calibration. ASCENT v User Manual 68 Revision X

69 If additional investigation is warranted, a Tile may be clicked on to bring up a Scope, an expanded view of the calibration (Figure 81). The Scope which presents for Calibration Modifications is largely identical in appearance and functionality to the Scope which presents from the Calibration Mosaic and Tiles. Manually modified elements of a calibration are shaded with a yellow color, similar to the visual effect used to indicate manually modified peaks. For mathematical model changes, the assay configuration setting (as default ) is provided for comparison (red circle, Figure 81). When one of the underlying peaks which is used to prepare a calibration point has been manually integrated, an icon (blue circle, Figure 81) will be displayed near the sample name (for a change to the compound s peaks) or in the IS column (for a change to the compound s normalizer, or internal standard, peaks) For details on interacting with this Scope, refer to the Calibration section of the discussion on Enhanced Review. Figure 81 Calibration Modifications Scope As with the Calibration enhanced review path, returning to the Mosaic after performing a change in the Scope may result in the Tile presenting in a disabled style (e.g. grey background and subdued content). Different than the Calibration path, however, the Calibration Modifications path is indicative of a manual modification; thus, the only Scope action which will induce this change to a disabled Tile is a revert (return) to the system ASCENT v User Manual 69 Revision X

70 integrated calibration. Effectively, the manual-ness of the calibration has been cleared (analogous to making a Scope change, such as a manual operation, which clears a quality rule flag in the Calibration path), so the Tile will not be present in a Mosaic refreshed by a return to the Batch Summary page (see below). As changes are potentially made in the Scope (e.g. continued manual calibration modification), values which are derived from those changes, such as the calibration regression equation and concentrations calculated from that calibration, are automatically re-calculated; as well, Scope changes will also trigger the re-application of the quality rules. To observe the effects of these changes, re-calculations and re-applications via the Overview graph and Batch Summary page, click the name of the batch at the top of the Mosaic (red circle, Figure 82). The Overview graph and the Modifications graph are automatically updated through the link. Figure 82 Calibration Modifications Mosaic Links It is also possible to navigate from the Calibration Modifications Mosaic back to the Batch Summary page and Overview graph using the browser s back button. However, using this action will not automatically update the graph; it will be necessary to refresh the display manually. It may be useful at times to see additional information about a batch when working in the Mosaic, Tile and Scope views. To navigate to the Standard Review page (e.g. Data Grid), click the link for Standard Review at the top of the Mosaic (blue circle, Figure 82). Results of Interest Review Reporting Unknown samples which contain one or more reportable concentrations of analytes are often the primary focus of analytical testing with ASCENT. In order to investigate a batch whose results contain one or more reportable concentrations, based on the Reporting Above Limit quality rule, or results which are deemed a Concentration of Interest by that quality rule, an additional link, Reporting, is available on the Batch Summary page in the ASCENT v User Manual 70 Revision X

71 Results of Interest section (red circle, Figure 83). For more information on the Reporting Above Limit and Concentration of Interest quality rules, please see Reporting in the Quality Rules Appendix. The Reporting link includes a display of the number of determinations in the batch which have a reportable concentration, per the results of the Reporting Above Limit quality rule, or a calculated value within a range of interest, per the results of the Reporting Concentration of Interest rule. Hovering on the link will display a tooltip with the total number of determinations in the batch, along with a percentage of determinations which have reportable concentrations. Figure 83 Batch Summary with Results of Interest This enhanced review path is entered by selecting the Reporting link (red circle, Figure 83). Clicking on the Reporting link will present the Results-Reporting Mosaic (Figure 84). Figure 84 Results-Reporting Mosaic The Results-Reporting Mosaic is a multi-determination view of the batch. The set of determinations which are displayed in the Mosaic may be ordered by either sample or compound, using the sort by selector (red circle, Figure 84). The set of determinations which are displayed in the Mosaic may be set to show only those samples with particular Reporting Decisions, using the filter by selector (green circle, Figure 84). ASCENT v User Manual 71 Revision X

72 Each determination in the batch which is currently being calculated and reviewed as having a reportable concentration using the Reporting Above Limit or Concentration of Interest quality rules is presented as a Tile. As with the Peak Quality and RT Mosaic and Tile, clicking the Results-Reporting Tile will present the Peak Quality and RT Scope. All interactions available with Results-Reporting Tiles and Scopes mirror those of the Peak Quality and RT Tiles and Scopes; for more information, please see Peak Quality/RT. As changes are potentially made in the Scope (e.g. manual peak modification), values which are derived from those changes, such as compound responses and concentrations, are automatically re-calculated; as well, Scope changes will also trigger the re-application of the quality rules. To observe the effects of these changes, recalculations and re-applications via the Overview graph and Batch Summary page, click the name of the batch at the top of the Mosaic (red circle, Figure 85). The Overview graph is automatically updated through the link. Figure 85 Results-Reporting Mosaic Links It is also possible to navigate from the Results of Interest Mosaic back to the Batch Summary page and Overview graph using the browser s back button. However, using this action will not automatically update the graph; it will be necessary to refresh the display manually. It may be useful at times to see additional information about a batch when working in the Mosaic, Tile and Scope views. To navigate to the Standard Review page (e.g. Data Grid), click the link for Standard Review at the top of the Mosaic (blue circle, Figure 85). Repeat / Exclude Part of the review process often involves the dispositioning of samples and determinations by marking them to be repeated or excluded in the final output. In order to investigate a batch whose results contain one or more determinations marked as something other than Include, two additional links, Repeat and Exclude, are available on the Batch Summary page in the Results of Interest section (red circle, Figure 86). ASCENT v User Manual 72 Revision X

73 Hovering on either link will display a tooltip with the total number of determinations in the batch, along with a percentage of determinations which have reportable concentrations. Figure 86 Batch Summary with Results of Interest This enhanced review path is entered by selecting either the Repeat or Exclude link (red circle, Figure 86). Clicking on the Repeat or Exclude link will present the Results- Repeat or Results-Exclude Mosaic, respectively (Figure 87). Figure 87 Results-Repeat Mosaic The Results-Repeat and Result-Exclude Mosaics are multi-determination views of the batch. The set of determinations which are displayed in the Mosaic may be ordered by either sample or compound, using the sort by selector (red circle, Figure 87). Each determination in the batch which is currently marked with the specific reporting decision is presented as a Tile. As with the Peak Quality and RT Mosaic and Tile, clicking the Results-Repeat or Results-Exclude Tile will present the Peak Quality and RT Scope. All interactions available with Results-Reporting Tiles and Scopes mirror those of the Peak Quality and RT Tiles and Scopes; for more information, please see Peak Quality/RT. As changes are potentially made in the Scope (e.g. manual peak modification), values which are derived from those changes, such as compound responses and concentrations, are ASCENT v User Manual 73 Revision X

74 automatically re-calculated; as well, Scope changes will also trigger the re-application of the quality rules. To observe the effects of these changes, re-calculations and re-applications via the Overview graph and Batch Summary page, click the name of the batch at the top of the Mosaic (red circle, Figure 88). The Overview graph is automatically updated through the link. Figure 88 Results-Repeat Mosaic Links It is also possible to navigate from the Results-Repeat or Results-Exclude Mosaics back to the Batch Summary page and Overview graph using the browser s back button. However, using this action will not automatically update the graph; it will be necessary to refresh the display manually. It may be useful at times to see additional information about a batch when working in the Mosaic, Tile and Scope views. To navigate to the Standard Review page (e.g. Data Grid), click the link for Standard Review at the top of the Mosaic (blue circle, Figure 88). Completing a Review After all batch data and results have been reviewed and the user is satisfied with any changes, the batch may be formally marked as being completely reviewed using the Review Complete button on the Batch Summary page (red circle, Figure 89). Figure 89 Review Complete via Batch Summary ASCENT v User Manual 74 Revision X

75 Alternatively, the Review Complete button, visible in the Standard Review mode, can be used (red circle, Figure 90) to indicate that the batch has been reviewed and is ready for certification. Figure 90 Review Complete via Standard Review If the assay has been configured for one step review and certification AND the user has been assigned both review and certify permissions, a user may conduct the review and certify procedure in one step by selecting the Review and Certify button (blue circle, Figure 89 and Figure 90); if the assay is not configured for one step review or the user does not have permission, this button will not be available to the user. For both types of review-complete activities, a verification screen will appear on which the user will be asked to review the changes and type in their password in order to verify that the changes are correct (red circle, Figure 91). Once the password is entered, click the Review Complete button (red circle, Figure 91). If the verification screen is presented after using the Review and Certify option, the button will be labeled as Review and Certify (red circle, Figure 91). ASCENT v User Manual 75 Revision X

76 Figure 91 Review Verification Screen After clicking the Review Complete or Review and Certify button, as provided, a dialog will appear to indicate a successful operation, along with an indication of the number of samples and results for which review or review and certification is now complete (Figure 92). After a few seconds, the dialog will automatically close and return the user to the Batch Summary page; a link is provided to return to the Batch Summary page without delay. Alternatively, a link is provided to return to the Batch Status page. Figure 92 Review Complete ASCENT v User Manual 76 Revision X

77 CERTIFICATION Certification is available following the completion of the batch review. Designated individuals are given access to the Certify function and must re-enter their password to further validate and certify the results that have been reviewed and accepted at this point in the approval process. Batches available for certification will be shown in the Batch Status page and when selected for certification will display as In Certification (Figure 93). Figure 93 Batch In Certification Once the Batch has been selected by an authorized certifier by, the certifier has the opportunity to review all aspects of the batch before certifying and can make further changes, if they have the permission to do so. For example, they must have permission to Modify calibration or Integrate peaks to have the ability to do so. After all batch data and results have been reviewed and the user is satisfied with any changes, the batch may be formally marked as being completely certified using the Certify button on the Batch Summary page (red circle, Figure 94). Figure 94 Certify via Batch Summary ASCENT v User Manual 77 Revision X

78 Alternatively, the Certify button, visible in the Standard Review mode, can be used (red circle, Figure 95) to indicate that the batch has been reviewed and is ready for certification Figure 95 Certify via Standard Review For both types of certification activities, a verification screen will appear on which the user will be asked to review the changes and type in their password in order to verify that the changes are correct (red circle, Figure 96). Once the password is entered, click the Certify button (red circle, Figure 96). Figure 96 Certify Verification Screen After clicking the Certify button, a dialog will appear to indicate a successful operation, along with an indication of the number of samples and results for which certification is now complete (Figure 97). After a few seconds, the dialog will automatically close and return the user to the Batch Summary page; a link is provided to return to the Batch ASCENT v User Manual 78 Revision X

79 Summary page without delay. Alternatively, a link is provided to return to the Batch Status page. Figure 97 Certification Complete ASCENT v User Manual 79 Revision X

80 DECERTIFICATION Once certified, the batch is placed in a read-only format. To accept additional changes, the batch must first be decertified. If the user has permission to do so, select the certified batch of interest by checking the box near the batch name (blue circle, Figure 98) and then click the Decertify button on the Batch Status page (red circle, Figure 98). This may also be accomplished using the Batch Action menu on the Batch Summary page. Figure 98 Decertify a Batch A confirmation dialog will present; clicking the OK button will decertify the batch, while clicking the Cancel button will not, and return to the Batch Status page. Following decertification, the batch will be placed into the Ready for Certification state. ASCENT v User Manual 80 Revision X

81 RESULTS A PDF-formatted archive will generate in the background for each batch upon certification. While it is generating, a message will be displayed (red circle, Figure 99) in the Downloads section of the Batch Summary page. If reports have been configured (e.g. a CSV file intended for automated LIS transcription), there will be link(s) to generate and download those as well. Reports, if configured, will be available prior to certification. Figure 99 Summary Page - Post-Certification Once generation is complete, a download link will be displayed (Figure 100). Segments of a sample archive are attached as APPENDIX C: SAMPLES FROM ARCHIVE. Archives are available for download for 30 days, either from the Batch Summary page while the batch remains active or from the Archives page (see ARCHIVING AND DATA RETENTION). If archive storage services have been purchased, the user will have the ability to retrieve the archive from the Archives page during the retention period contracted by the customer. Figure 100 Downloads Ready ASCENT v User Manual 81 Revision X

82 With appropriate configuration of the Upload Client service, the results file(s) and/or the archive file can be automatically copied from the ASCENT system to a local drive, after an authorized user certifies the batch (see CERTIFICATION). For more information on this enabling this capability for laboratory use, please contact Customer Support (see APPENDIX A: CUSTOMER PORTAL). ASCENT v User Manual 82 Revision X

83 BATCH DIAGNOSTICS If a Batch has a status of Failed, the Batch Diagnostics may help identify the cause and allow users to successfully re-upload the batch. To review the diagnostics, type or paste the name of the batch into the box and click Search (Figure 101). Batch Name is case-sensitive for searching. Figure 101 Batch Diagnostics Scroll through the list of samples, specifically the Status column, to see if any Failed to convert. If so, the batch should be re-uploaded without the problematic sample(s) (see Re-Uploading a Batch). ASCENT v User Manual 83 Revision X

84 OTHER BATCH FUNCTIONS Based on the results of the Batch Diagnostics or the user s observation of the batch results, the user may need to utilize one of the following batch functions. Many of these functions can also be executed using the Batch Actions menu on the Batch Summary page. Restoring a Batch If a batch has had manual edits performed (e.g. modifications to peak integration), it is possible to return the batch to its original, automatically processed state. While similar to Reprocessing a Batch, the Restore operation will regenerate the result set using the assay configuration which was last used to process, or reprocess, the batch. This differs from reprocessing a batch, in that a Reprocess action would regenerate the results using the assay configuration which has been most recently promoted for current production use (see Reprocessing a Batch). If an assay configuration has changed since the batch was last processed, and it would not be appropriate to use the latest assay configuration for regenerating that batch (e.g. a compound has been renamed), then the Restore operation can be utilized. A batch can be restored at any time prior to certification. The user must have Reprocess batch permissions. All modified results, user edits, and other changes are lost when a batch is restored. Restoring a batch does not remove audit trail information. Audit trails are maintained for all committed data, and entries are made for restoring the batch. To restore a batch, select the batch of interest by checking the box near the batch name (blue circle, Figure 102) and then click the Restore button (red circle, Figure 102) on the Batch Status page. Figure 102 Restore a Batch A confirmation dialog will present; clicking the OK button will restore the batch, while clicking the Cancel button will not, and return to the Batch Status page. Following the ASCENT v User Manual 84 Revision X

85 initiation of restoring, the batch will be placed into the Processing state; when finished with restoring, the batch will be placed into the Ready for Review state. Reprocessing a Batch If an assay configuration has changed and the new settings (e.g. peak processing parameters, compound view order, QA rule thresholds) should be applied to the existing batch, the batch will need to be reprocessed. Also, if a batch processing failure occurs due to a reason other than sample conversion failure, a batch marked as Failed on the Batch Status page can be reprocessed. While similar to Restoring a Batch, the Reprocess operation will regenerate the result set using the assay configuration which has been most recently promoted to production. This differs from restoring a batch, in that a Restore action would regenerate the results using the assay configuration which has been most recently used to generate the existing batch result set (see Restoring a Batch). A batch can be reprocessed at any time prior to certification. The user must have Reprocess batch permissions. All modified results, user edits, and other changes are lost when a batch is processed. Reprocessing a batch does not remove audit trail information. Audit trails are maintained for all committed data, and entries are made for reprocessing the batch. To reprocess a batch, select the batch of interest by checking the box near the batch name (blue circle, Figure 103) and then click the Reprocess button (red circle, Figure 103) on the Batch Status page. Figure 103 Reprocess a Batch A confirmation dialog will present; clicking the OK button will reprocess the batch, while clicking the Cancel button will not, and return to the Batch Status page. Following the ASCENT v User Manual 85 Revision X

86 initiation of reprocessing, the batch will be placed into the Processing state; when finished with reprocessing, the batch will be placed into the Ready for Review state. Canceling a Batch A user with Cancel permissions may cancel a batch as needed. Batches are typically cancelled if there is a known issue with the batch that has necessitated reinjection or reconfiguration of the assay and/or batch before proceeding. If a batch is not going to proceed any further in the review process in ASCENT (nor results be reported) but needs to be temporarily retained for reference, then the batch should be cancelled. Otherwise, it should be destroyed. Cancelled batches may not be Restored, Reprocessed or Certified. No further changes may be made to a Cancelled batch, and no archive is generated. To cancel a batch, select the batch of interest by checking the box near the batch name (blue circle, Figure 104) and then click the Cancel button (red circle, Figure 104) on the Batch Status page. Figure 104 Cancel a Batch A confirmation dialog will present; clicking the OK button will cancel the batch, while clicking the Cancel button will not, and return to the Batch Status page. Following the cancellation, the batch will be placed into the Cancelled state, and will only be available in a read-only mode. Destroying a Batch ASCENT Administrators have the ability to destroy batches from the Batch Status page. Batches are typically destroyed when there is a fundamental issue with the assay and/or batch which make them unusable from a laboratory processing, review and reporting perspective. Addressing these issues will often necessitate significant changes, the reacquisition of the samples, and/or the re-uploading of the batch (see below). ASCENT v User Manual 86 Revision X

87 DESTROYING A BATCH IS PERMANENT. ONCE DONE, A BATCH WILL NO LONGER BE ACCESSIBLE. To destroy a batch, select the batch of interest by checking the box near the batch name (blue circle, Figure 105) and then click the Destroy button (red circle, Figure 105) on the Batch Status page. Figure 105 Destroy a Batch A confirmation dialog will present; clicking the OK button will destroy the batch, while clicking the Cancel button will not, and return to the Batch Status page. Following the destruction, the batch will be removed from the Batch Status page. Re-Uploading a Batch It may be necessary to re-upload a batch if there is a problematic (e.g. corrupt) sample data file and the batch fails. ASCENT requires batch names to be unique, so in order to reupload a batch, the user must choose one of two approaches: Upload using a different batch name (e.g. edit ASCENT sequence filename and batch folder name) Destroy the original (Failed) batch and then re-upload using the same batch name. Please note that destroying a batch must be done by an Administrator user. (See Destroy) In either case the sequence file may need to be modified to remove the reference to the problematic samples or generated again from Accessioning. (Values of ascentid in the ASCENT sequence file must be unique.) Then return the batch to the Dealer or Upload Client poll folder. ASCENT v User Manual 87 Revision X

88 SAMPLE SEARCH The Sample Search utility can be used to locate a particular sample. Type or paste the sample name into the search box (blue circle, Figure 106), select a date range for sample acquisition time, and then click Search (yellow circle, Figure 106). If there is more than one sample that meets the search criteria (e.g. a sample which may have been repeated in a subsequent batch, to address a quality issue/flag or a need for a different dilution), they will all be listed in the search result table. It is not necessary to fill in the complete sample name; a search may be made on the initial string (e.g. a starts with search). However, it is not a wild card search and will not return results that have that string in the middle or the end of the name. As an aid in locating repeated samples, it may be of value to use a common sample name coupled with a suffix of some type for the repeated sample (e.g. Sample-1 and Sample-1-repeat, where entering Sample-1 in the search box would then present both samples in the search result table). Figure 106 Sample Search Sample Name is case-sensitive for searching. In the search result table, the batch containing the sample will be presented as a link that displays the first sample of the batch (red circle, Figure 106). Be sure to note the Index to find the specific sample within the batch (green circle, Figure 106). ASCENT v User Manual 88 Revision X

89 AUDITS The Audit function can be used to view the activity on either a batch or an assay. Type or paste the assay or batch name to review the audit trail for that item (Figure 107). Figure 107 Assay Audit It is possible that there may be more than one result for a batch search, if the batch was destroyed and then re-uploaded (Figure 108). The audit trail for a batch is available even after the batch itself is no longer accessible from the Batch Status page. Figure 108 Batch Audit ASCENT v User Manual 89 Revision X

90 ARCHIVING AND DATA RETENTION ASCENT will keep batches available in the Batch Status page for viewing and reprocessing based on the retention policies for compound results per instrument. As the retention thresholds are exceeded, batches will be purged, oldest first, if they are Failed, Cancelled, or Certified with Archive. Batches in other statuses will remain available. Once a batch has been purged from the Batch Status page, the archive must be accessed from the Archives menu. The Archives menu allows users to retrieve a batch archive as long as it is retained by ASCENT. The batch archive begins generating upon certification, but will not be immediately available. Once it is available in the Batch Summary page, it will also be available on the Archives page. By default, archives are purged 30 days after they are generated unless the customer has purchased archive storage. To view an archive for a batch, first search for a batch (Figure 109). You may search by Batch Name, Assay Name, and/or a range of Date Certified. Note: the criteria are cumulative, so if searching by Batch Name, be sure the date range includes the date the batch was certified. Figure 109 Batch Archive Search If there are no results for the search, ASCENT will return the message in Figure 110. Figure 110 No Archive Results Otherwise, ASCENT will present the results as shown in Figure 111. ASCENT v User Manual 90 Revision X

91 Figure 111 Batch Search Results Clicking on the archive link will initiate a download of the archive as shown in Figure 112. Figure 112 Downloading Archive If the archive is in long term archive storage, it may take several hours to retrieve the file. If so, the following informational message will be displayed upon clicking the archive link until the file is available (the message does not provide a real-time status and will be the same even if it is nearly finished). You can navigate away from and return to the page as necessary to check the status. It is not necessary to stay on the page. Already restoring the report for batch [batchname] from long-term storage. This will take 3-5 hours. Refresh this page to check the retrieval status. If the archive has been purged due to expiration of storage policies, the following message will be displayed. The report for batch [batchname] no longer exists. It expired per your company s retention policy. ASCENT v User Manual 91 Revision X

92 PROFILE The Profile menu primarily allows a user to change his password. However, it will allow the user to change other contact information as well (Figure 113). Enter a new password in both fields and click Submit. Immediately log out and then login to ASCENT with the new credentials. Figure 113 Edit User Profile addresses should be entered lowercase to prevent case-sensitivity issues. address is required to support user unlock functionality. ASCENT v User Manual 92 Revision X

93 USER MANAGEMENT ASCENT includes capability-based access controls that must be configured by one or more customer-designated administrators. User Administration requires use of both the Users and Permissions menu options. Selecting a different menu option during an edit will result in loss of entered data (data will not be saved). User Set Up Selecting Users from the menu will display the screen (Figure 114) where an Administrator user can verify/edit the current user population. Figure 114 User Management Page ASCENT utilizes three different user types to control user access to ASCENT. Most batch permissions are controlled from the Permissions function. Root User: Can promote and demote user accounts between Users and Administrators. Can promote an Administrator to Root (however, this will demote the current Root to Administrator upon log off). Can edit ALL user accounts. Can download batch files from Batch Diagnostics page. Can Destroy batches (will not be archived). Can configure assays. Administrators: Can edit Users and themselves (not other Administrators or Root). ASCENT v User Manual 93 Revision X

94 Can Destroy batches (will not be archived). Can configure assays. Users: No user administration privileges. Special User Accounts There are some user accounts created by Indigo for ASCENT installation that should not be changed or deleted. The default Root user, which is usually indigo, should not be altered. Similarly, the service accounts used for batch upload and processing, usually beginning with indigo, should not be changed. Changing or deleting these accounts will cause processing to be interrupted. To add a user, an Administrator clicks on the Add User button (red circle, Figure 114) which opens a User Information window (Figure 115). Please complete all fields. Once the fields are filled out, click Submit and the user account will created. addresses should be entered lowercase to prevent case-sensitivity issues. address is required to support user unlock functionality. Editing a User Figure 115 User Information Window Icons to the right of each user account reflect the actions available to the user logged in for managing the user account (red circle, Figure 116). If the user logged in is not Root then there will be fewer available options. Clicking the pencil will bring up the User Information form to be changed and resubmitted. Clicking the Red X to delete the selected user account brings up a confirmation ASCENT v User Manual 94 Revision X

95 dialog. Customer internal policies will determine whether to delete a user account. Audit trails will be maintained even when a user is deleted. Clicking one of the Green Arrows promotes and demotes user accounts between user types. Promoting an Administrator to Root will demote the current user at logoff. There can only be one Root user. The user will be presented with a confirmation dialog if they attempt to promote an Administrator to Root. The answer should be No. User Permissions Figure 116 Create Administrator Account User Permissions are distinct from User Types, and each user must be configured in the Permissions page. Permissions control a user s ability to access and process batches in ASCENT. The administrator is the user responsible for assigning the level of access that individual users will have to ASCENT. From the Menu Bar, the administrator may choose Permissions to add or edit the level of access for individual users. Permissions can be selected individually by checking the appropriate box(es) in each row (Figure 117). Be sure to click Save to preserve the selection(s). ASCENT v User Manual 95 Revision X

96 Figure 117 Setting Permissions No permissions: A user without any other defined access can use the Accessioning function and view Batches in Read Only mode. Modify Permissions: Only a user with this access can edit the permissions of other users. Certify batch: This access is typically combined with the Review batch access at a minimum. However, used on its own, the user must view Batches in Read Only mode before Certifying. Decertify batch: This access simply allows a user to decertify a previously certified batch. Review batch: This access allows a user to review and edit batch results. However, it does not include the ability to modify peaks, calibrate, or certify batches without explicit access to those functions. Integrate peaks: This access must be combined with Review batch access or the user will not be able to get to the Peak Integration screen. Modify calibration: This access must be combined with Review batch access in order to be useful. It allows access to all calibration functions. Without it, the reviewer will only be able to view the calibration settings. Cancel batch: This access allows the user to cancel a processed batch at any time prior to certification. Reprocess batch: This access allows the user to reprocess a batch after processing and prior to certification. Batch Details: This access allows the user to edit sample information and delete samples on the Batch Summary page. ASCENT comes with some preconfigured permission sets that can speed user management. The predefined sets are cumulative. To use a predefined set, select the ASCENT v User Manual 96 Revision X

97 checkbox to the left of the username, click the applicable permission set button(s), manually add additional permissions if desired, and then click Save. Lab Manager includes Certify, Decertify, Review, Integrate peaks, and Modify calibration. Certifier includes Certify, Review, and Integrate peaks. Decertifier includes Decertify. Reviewer includes Review. ASCENT v User Manual 97 Revision X

98 ASSAY CONFIGURATION In ASCENT, an assay configuration describes the complete route from raw data to automatically generated quantification results, which can then be reviewed (and as necessary, modified); other systems may refer to assay configuration as a processing method. The Assay Configuration interface allows an administrator to define compounds, chromatograms, and peak integration parameters, activate and configure QA Rules, enable one-click review & certify, and set default compound display settings in the assay configuration to best fit the needs of the laboratory. Assay Configuration is also used to manage the availability of test data (as either one or more instrument data files, or a designated ASCENT batch) as well as overall assay maintenance. The assay configuration settings are applied to batches during processing, so changing the configuration does not affect batches that have already been certified unless the batch is decertified and reprocessed (this may cause some unexpected behavior of the assay and should be done with care). There are six tabs in the interface: Compounds this is where the compounds and internal standards are named, and their chromatograms defined. It also includes the peak integration and calibration settings. Lots/Levels this is where the Lots and Levels for quality control materials (calibration standards and Quality Control samples) are defined. Display this tab includes the one-click review/certify setting as well as default display settings for compounds (the commands that are included in the console interface). It is also where instruments are linked to each assay. Rules this tab contains the QA rules and allows the administrator to turn them on/off and adjust the parameter values. Test Data this tab allows the user to manage the batches of data available to Preview Peak Integration and Generate Batch Comparison. Maintain this is where assay changes to the draft versus productions settings are tracked and comparisons generated. ASCENT expects some of the parameters to conform to certain basic formats or data types. These are listed in red italics in the following sections. To begin assay configuration, select CREATE NEW or if working with an existing assay, select it from the drop-down menu (Figure 118). ASCENT v User Manual 98 Revision X

99 Figure 118 Select An Assay When creating a new assay, please observe the important naming constraints (Figure 119). Compounds Figure 119 New Assay Error Any chemical entity which will be determined in a particular sample is identified as a compound; this includes target analytes as well as internal standards (IS). A compound is principally identified by at least one, and potentially multiple, chromatograms (e.g. quantifier, and possibly qualifier, traces/plots). The relationship between a target analyte and an internal standard, for the purposes of quantification, will also be established within the Compounds tab. Each compound name must be a unique alphanumeric name within the assay configuration and may not contain spaces. ASCENT v User Manual 99 Revision X

100 Figure 120 Compounds To add a compound, type its name in the empty box in the left column and press Enter (red circle, Figure 120). To edit an existing compound, select it from the list. Compounds can also be renamed or deleted by selecting the proper icon. There are far reaching implications to editing the list of compounds once an assay configuration is in use in the production site. Batches already uploaded may not process or reprocess correctly due to a mismatch between expected and present compound names. It will also produce a conflict when attempting to use interbatch regression with batches using different lists of compounds. For this reason, customers are discouraged from making changes to the compound list of an assay once it is in use in the production site. If changes must be made, Indigo suggests using the Save As function to create a copy of the assay configuration as described in Additional Assay Actions. In some cases, Restoring a Batch may be appropriate. When deleting more than one compound, certain browser settings may present a dialog regarding the creation of additional dialogs, as seen below: ASCENT v User Manual 100 Revision X

101 If the option to Prevent this page is selected, the deletion of subsequent compounds will not be possible. If this occurs, the page must be refreshed in order to continue with compound deletion. Compounds will appear on the review page in the order listed in the Compound tab of the assay configuration. Consideration should be made of the most effective order for review when setting up the assay. For example, since the IS are defined as separate compounds and their flags will not show up when reviewing the analyte, it is recommended that reviewers are presented first with the IS compounds. If it is necessary to re-order the compounds, simply edit the Index number before each compound name (red circle, Figure 121). Define Chromatograms Figure 121 Compound Order ASCENT v User Manual 101 Revision X

102 The associated chromatograms must be defined for each compound. The system will allow an unlimited number of chromatograms, but only the first three will be displayed in the review pane. Use the boxes on the right hand side to add chromatograms (Figure 122). Figure 122 Define Chromatograms Different compounds should have at least one unique value in one of these settings: precursor ranges, product ranges, polarities, and activation energies. Assign each chromatogram a name, which must be a unique alphanumeric name within the compound (while the labels Quant and Qual are recommended, the user may choose different names), and then specify the associated values: Precursor Lower and Precursor Upper (positive numbers, lower must be less than the upper): (m/z) precursor range for extraction of chromatogram from raw data (lower value included but upper value excluded from range). Leave blank for singlestage mass spectrometry experiment (e.g. SIM). Product Lower and Product Upper (positive numbers, lower must be less than the upper): (m/z) product range for extraction of chromatogram from raw data (lower value included but upper value excluded from range). For single-stage mass spectrometry experiments (e.g. SIM), enter the monitored m/z range. Activation Energy (number, may be blank): if specified, used to match experimental parameters and differentiate chromatograms acquired in raw data. Enter 40 for activation energy of 40 ev; enter -40 for activation energy of -40 ev. Please note that some instrument raw data files may only supply a magnitude value of activation energy and not the polarity; thus, it may be necessary to remove the polarity for appropriate chromatogram definition. Polarity: (none, positive, negative) used to match experimental parameters and differentiate chromatograms acquired in raw data. To match both positive and negative polarity values, specify none (no filter on polarity). Combine Ions: (enable/disable) in some instrument data files, the detected product ion(s) are recorded as a mass spectrum (e.g. each chromatographic time point may have more than one, or varying, ion values); enabling this feature sums intensities in the specified mass range. ASCENT v User Manual 102 Revision X

103 It is recommended to define all the chromatograms in the assay prior to setting the peak integration properties, as the properties may reference chromatograms for other compounds in the assay. Peak Integration Properties Once the chromatograms have been defined, the peak integration properties must be configured for each compound. The properties have been divided into four groups which are expanded by clicking on the header. Retention Time (Figure 123) Figure 123 Peak Integration, Retention Time Expected RT (positive number): (minutes) expected retention time RT Window Width (positive number): (minutes) retention time window is Expected RT +/- RT Window Width. RT Lower Tolerance and RT Upper Tolerance (positive number): (minutes) lower and upper thresholds on retention time applied if Reference Type Source is Chromatogram. Candidate peak not integrated unless retention time in range from (Expected RT RT Lower Tolerance) to (Expected RT + RT Upper Tolerance). To not use tolerance, set equal to RT Window Width. For Equable peak processing (see below, Advanced Settings), these values are used to establish the integration range of the trace, after application to the expected/predicated retention time. ASCENT v User Manual 103 Revision X

104 Reference Type Source: (Chromatogram, Samples, Expected) Method to determine predicted retention time for the purposes of automated peak processing (ASCENT can address variances in actual retention time by using a prediction mechanism, described below, on a compound by compound and sample by sample basis). Of those that meet peak thresholds, candidate peak for integration is closest to predicted retention time. Chromatogram predicted retention time is observed retention time of peak in RT Reference chromatogram of the same sample plus RT Bias. If no peak is integrated in RT Reference chromatogram, predicted retention time is Expected RT. Samples predicted retention time is median of observed retention times for the same chromatogram in samples specified as RT Reference. If no peaks integrated in RT Reference samples, predicted retention time is Expected RT. Expected value provided as Expected RT is used without modification. For an example application of Reference Type Source, see APPENDIX D: RETENTION TYPE SOURCE. RT Reference (must contain a valid value based on the Reference Type Source): based on Reference Type Source Chromatogram or Samples (Standard, QC, Standard and QC) to reference for determining expected retention time. Not used for Reference Type Source Expected. For Chromatogram, all chromatograms in the assay configuration are listed as options in the format [Compound Name] [Chromatogram Name]. It is valid to reference a chromatogram that itself references another chromatogram. However, care must be taken to avoid circular references (e.g. a compound referencing itself through a series of other references). RT Bias (number): (minutes) if Reference Type Source Chromatogram, retention time bias from referenced chromatogram. Specify negative value if peak elutes before referenced chromatogram. Not used for Reference Type Source Samples or Expected. RT Estimation Width (positive number): (minutes) if Reference Type Source Samples, retention time estimation width used for the initial determination of the actual RT in the batch. Because reference samples are expected to have fewer contaminating peaks, a wider window may be desired for finding the RT before using a narrower window to account for per-sample drift. Once the RT has been found, the RT Window Width will continue to be used on a per sample basis for smaller drifts that might occur due to differences in sample preparation. Not used for Reference Type Source Chromatogram or Expected. ASCENT v User Manual 104 Revision X

105 Peak Thresholds (Figure 124) Figure 124 Peak Integration, Peak Thresholds Absolute Area (positive number): (area) absolute area threshold. Peaks with area below absolute threshold are excluded. Absolute Height (positive number): (height) absolute height threshold. Peaks with height below absolute threshold are excluded. Relative Area (decimal value between 0 and 1): area threshold relative to peak in retention time window with the largest area. Peaks with area below threshold (fraction of largest peak area in retention time window) are excluded. Relative Height (decimal value between 0 and 1): height threshold relative to peak in retention time window with the largest height. Peaks with height below threshold (fraction of largest peak height in retention time window) are excluded. Dynamic Area (decimal value between 0 and 1): multiplier applied to lowest observed peak area in (non-zero nominal amount) Standard samples. Peaks with areas below threshold are excluded. Batch must contain native calibrators for dynamic threshold to be applied (will not apply if only standards in the batch have been imported). Dynamic Height (decimal value between 0 and 1): multiplier applied to lowest observed peak height in (non-zero nominal amount) Standard samples. Peaks with heights below threshold are excluded. Batch must contain native calibrators for dynamic threshold to be applied (will not apply if only standards in the batch have been imported). Signal to Noise Ratio (positive number): (signal to noise ratio) signal to noise ratio threshold. Peaks with computed signal to noise ratio below this threshold are excluded. The signal to noise threshold is applied at different times during different stages of peak detection and integration processing. In some cases, a peak may initially pass this threshold but later in the processing activities be found to be beneath this threshold; this may result in an adjacent peak being found to pass the threshold and be ASCENT v User Manual 105 Revision X

106 selected. One case where this may occur is in the use of non-integer values for the signal to noise ratio threshold. In such cases, it may be valuable to lower the threshold slightly or set it to an integer value. Min. Merge Difference (positive whole number): (number of data points) minimum difference between adjacent peak apexes to merge peaks. Smoothing (Figure 125) Figure 125 Peak Integration, Smoothing Optimal: (enable/disable) enable to apply ASCENT optimal smoothing algorithm to chromatogram. Fixed (positive whole number): (minimum 3, maximum 33) if optimal smoothing is disabled, specified value of smoothing parameter. Min and Max (positive whole numbers, max must be greater than min): (minimum 3, maximum 33) if optimal smoothing is enabled, allowable range of smoothing parameter. Start (positive whole number): (minimum 3, maximum 33) if optimal smoothing is enabled, initial smoothing parameter value used by algorithm to optimize based on chromatogram. ASCENT v User Manual 106 Revision X

107 Advanced Settings (Figure 126) Figure 126 Peak Integration, Advanced Settings First Derivative and Second Derivative (decimal value greater than zero): parameters of the ASCENT peak detection algorithm. These values are multipliers on the chromatogram standard deviation such that smaller values make the peak detection algorithm more sensitive. Smaller deviations from chromatogram baseline are detected as peaks. Peak Probability (decimal value between 0 and 1): parameter of the ASCENT peak detection algorithm. Peaks with computed probability below this threshold are excluded. Saturation Threshold (positive number): (height) parameter of the ASCENT peak detection algorithm. Chromatogram data points with intensity above this threshold are considered saturated and relevant peak detection conditions are applied. Peak Model: (EMG, None, Equable) Method to determine peak area. EMG - an exponentially modified Gaussian (EMG) model is fit to the peak, and area under the curve is determined from the model. EMG may fall back to a Gaussian peak model or Simpson s Rule (trapezoidal) if EMG fails to converge to a solution. Saturated peaks are fit using Simpson s Rule. None - no peak model is used, e.g. Simpson s rule (trapezoidal) method for numerical integration is used to calculate area between smooth chromatogram and global baseline, in the identification and integration to a local maximum as a transient signal with delimits at peak start and end. Equable - trace is processed without seeking a local maximum and without delimiting against peak start and end. Rather, the peak is calculated as a region bounded in the y-dimension by the zero-intensity baseline and the trace intensity, and bounded in the x-dimension by the expected/predicted retention time, minus and plus the Lower RT Tolerance and the Upper RT Tolerance, respectively. ASCENT v User Manual 107 Revision X

108 Window Multiplier (positive number greater than or equal to 1, may be left blank): If specified, the chromatogram is truncated to this multiple of the retention time window for processing in ASCENT. Because the primary purpose of this feature is to increase processing performance by reducing unnecessarily large traces, the Window Multiplier will only be applied if the number of chromatogram data points exceeds one thousand. THIS FUNCTION HAS BEEN REPLACED BY LOWER AND UPPER TRACE WIDTH IN COMPUTE VERSION and higher. The value in Lower and Upper Trace Width will be equal to the Window Multiplier * RT Window Width. Lower Trace Width (any positive number greater than zero, may be left blank): Allows a user to winnow the data point collection that makes up a trace to potentially remove artifacts which interfere with appropriate peak detection. For example, a very high signal peak at the beginning or end of the trace for the compound peak of interest may perturb the baseline; the use of the upper or lower trace width limit would allow such a baseline artifact to be excluded from the peak detection process. The lower trace width is applied to the expected/predicted retention time to determine the time segment from which data points for traces are utilized. For example, if the retention time was 1 minute and the Lower Trace Width was 0.25, only data points after 0.75 minutes would be utilized for peak detection (even if data points were available before 0.75 minutes). Upper Trace Width (any positive number greater than zero, may be left blank): Allows a user to winnow the data point collection that makes up a trace to potentially remove artifacts which interfere with appropriate peak detection. For example, a very high signal peak at the beginning or end of the trace for the compound peak of interest may perturb the baseline; the use of the upper or lower trace width limit would allow such a baseline artifact to be excluded from the peak detection process. The upper trace width is applied to the expected/predicted retention time to determine the time segment from which data points for traces are utilized. For example, if the retention time was 1 minute and the Upper Trace Width was 0.25, only data points up to 1.25 minutes would be utilized for peak detection (even if the total range of data points for the trace exceeded 1.25 minutes). Preview Peak Integration This feature requires Compute version or later. After the Peak Integration properties have been set, the user can preview the results using Preview Peak Integration. This function allows the user to immediately preview the result of applying the draft assay settings to a selected sample. Test samples may be imported from Production batches or uploaded directly. This is discussed in the Test Data section. ASCENT v User Manual 108 Revision X

109 Figure 127 Preview Peak Integration To preview the draft settings, select a sample and a chromatogram from the available list in the Preview Peak window and click Integrate Peak (red circle, Figure 127). It may take a moment to generate the preview. Once it is displayed, the user can scroll through the list of calculated values (green circle, Figure 127) to ensure that the configuration is performing as expected. Figure 128 Calibration Settings Calibration (Figure 128) Once the Peak Integration properties have been input, Calibration can be enabled and the settings adjusted. If calibration is enabled (by clicking the slider until it turns green), options include: Degree: (linear, quadratic, cubic) specified degree of regression equation Weighting: (None, 1/X, 1/X^2, 1/Y, 1/Y^2) specified weighting for regression Origin: (Force, Include, Ignore) origin handling for regression If the Weighting is specified to be other than None and the Origin handling is specified to Include, the requested calibrator point of (0,0) will utilize a weighting value of 1 in the regression calculation. ASCENT v User Manual 109 Revision X

110 Response Criteria (Figure 128) The Response Criteria section of the Calibration tab is used to select a chromatogram, or chromatograms, from which the integrated peak area (taken as the response) will be used for quantification. Any chromatogram specified in the assay configuration, listed in the format [Compound Name] - [Chromatogram Name], can be used to provide a Response Basis. When performing quantification using an internal standard, chromatograms may be added to the Normalization Target list for use in relative response calculation. To add a chromatogram for use as either the response basis or the normalization target, click in an empty area in either of the two boxes below the label; a listing of all chromatograms specified in the assay will be available for selection. Multiple chromatograms may be selected by clicking in the empty area below any chromatograms already specified; this may be useful for signal enhancement or isomer analysis. When performing quantification using an external standard approach, select 1 (no normalization) for the normalization target. Response Basis: (numerator) For the specified chromatogram(s), the integrated peak areas are summed. Normalization Target: (denominator) For the specified chromatogram(s), the integrated peak areas are summed. If no chromatograms are specified, then select 1 (no normalization). Lots and Levels Lots and Levels accommodate variability in the solution composition of QCs and Standards. The Lots/Levels tab allows the administrator to define concentration values for specific lots and levels of calibration standards and Quality Control (QC) samples. These values are referenced by ASCENT for calibration curves and rules. The Lots defined in this tab are used in the sequence file to allow ASCENT to assign the appropriate values to standards and QC samples in a batch. There must be at least one Lot and Level for each QC and Standard. A Lot (and by extension a Level) must contain at least one Compound. To create a new Lot, click Create New Lot to bring up a new Lot dialog box (Figure 129). Name the Lot and choose the sample type to which it applies (QC, Standard). Check off the compounds to which the Lot will apply and click Save. Lots can also be edited or deleted by the selecting the proper icon. ASCENT v User Manual 110 Revision X

111 A newly-added compound cannot be added to an existing Lot. Figure 129 Creating a New Lot Next, add a level by clicking on the desired Lot then clicking Create New Level. Enter the name of the level then click Save (Figure 130). Create at least one Level and as many Levels as needed for the assay. Figure 130 Creating a New Level Now the concentration values for the compounds can be entered. Figure 131 is an example of a Standard. If concentration values are constant for a particular lot and level, click the down-carat icon on the right side of the caption bar to utilize a copy-down action (red circle Figure 131) using the upper edit each compound row. Entering values into the edit row and clicking the triangle ( delta ) icon will apply the changes to all fields in that column; clicking the cancel ( X ) icon will discard the changes. Click Save when finished. ASCENT v User Manual 111 Revision X

112 Figure 131 Adding Nominal Concentrations to a Standard Level For QC samples the measured standard deviation for each compound in the lot can be added (Figure 132). These values are referenced by the rule QC Exceeds Standard Deviation and the CONTROL BATCH SETS QC CHARTING capability; if this rule is not used, these values may be left at zero (the CBS capability will still operate with standard deviation values left at zero). Figure 132 Adding Nominal Concentrations to a QC Level The lot names used in a sequence file must match the lot name here exactly (casesensitive) for the correct values to be used when processing the batch. If the list of compounds is changed, a new Lot and Level must be created. Also, the nominal concentration is associated with a sample during Accessioning, so changes to Lots and Levels will not be reflected in batches already uploaded to ASCENT. A new sequence file must be generated and the batch re-uploaded. ASCENT v User Manual 112 Revision X

113 Display This tab aggregates a number of different functions described below (Figure 133). Figure 133 Display Tab Compute Version Ascent utilizes a core Compute engine to process the data. The Compute engine is versioned separately to allow each assay to be tested with the new version before it is used to process production data. The Compute Version setting is the version of the processing engine that will be used to process data for the assay. Refer to the release notes for specific changes between versions of the processing engine. Assay Options When the One Step Review & Certify option is turned on, a user with Review and Certify permissions can perform both functions with one click after reviewing the batch. The decision about whether to enable this option is driven by the customer s SOP. Display Settings Three numeric values can be shown in scientific notation instead of decimal format. Dropdown selectors allow this choice for the Peak Area, Concentration and Response fields of the Data Grid. This section also allows provides a dropdown selector to choose the default Review Method as being organized by Compound, Sample Type or Sample (Index). This will define the initial sorting of results for the Standard Review process. ASCENT v User Manual 113 Revision X

114 Assay Properties This section allows the administrator to edit key-value pairs for assays where customization by Indigo has provided for it (e.g. a custom result file which associates ASCENT assay compound names to those to be used for LIS transfer). For more information on customization, please contact Indigo via the Support Portal (detailed information is contained in APPENDIX A: CUSTOMER PORTAL). Instrument Names Instruments for each assay should be registered here to be available in Accessioning. The assay must be promoted again after adding new instruments. Instrument names cannot contain spaces. Instrument names are required to promote the assay to Production. Define Macros See section on Terminal commands for more information. The macro function allows default terminal commands to be defined for the assay so that they are automatically loaded on review, rather than the reviewer needing to manually enter the terminal commands for each assay. For example, the relate Codeine Codeine-IS command causes the Codeine- IS chromatogram to be displayed alongside the Codeine chromatograms when reviewing each batch. Rules This tab allows the administrator to enable/disable rules and configure tolerances on a per-compound basis. A description of the rule will be on the left, while user entries are in fields on the right (Figure 134). The list of ASCENT core quality rules are described in APPENDIX B: CORE QA RULES. To enable the rule for a compound or chromatogram, click the slider button under Enabled so that it is green. To disable the rule, click the slider so that it is red. Changes made to an individual rule setting are not saved unless the Save button is clicked (red circle, Figure 134). Moving directly to another rule or screen will discard the changes. ASCENT v User Manual 114 Revision X

115 Figure 134 Sample Rules Configuration Each rule may have one or more Compound Attributes, which are defined in the rule description. If a rule must reference a particular chromatogram, there is a drop-down list of available chromatograms to choose from. When a rule references the ULOQ or LLOQ, the ULOQ or LLOQ must be defined for each compound for which the rule is enabled. The entered value will become the global value for the ULOQ or LLOQ for that compound (e.g. changes to the ULOQ or LLOQ value in one rule will automatically propagate to all other rules which use that same value). If quality rule settings are constant for a particular rule, click the down-carat icon on the right side of the caption bar to utilize a copy-down action (blue circle, Figure 134) using the upper edit each compound row. Entering values into the edit row and clicking the triangle ( delta ) icon will apply the changes to all fields in that column; clicking the cancel ( X ) icon will discard the changes. The slider button for the rule s activation can be toggled to a green state for enabled, a red state for disabled, and a grey state, indicating that the rule toggle should not be changed in a copy-down operation. Click Save when finished. Test Data ASCENT v User Manual 115 Revision X

116 To enable the Peak Integration Preview and the Batch Comparison Reports, ASCENT requires test data, which can be managed on the Test Data tab. Figure 135 Add to Test Data There are two types of test data, which are obtained differently: Imported From Production These batches have been processed in ASCENT and identified as good references for Batch Comparison. To add them to the pool of Test Data, select the Batch in the Batch Status page (red circle, Figure 135) and then click Add to Test Data (blue circle, Figure 135). It is possible to add failed batches to Test Data. This may also be accomplished using the Batch Action menu of the Batch Summary page. Uploaded Test Data These batches have not been processed in ASCENT and have not been run through Accessioning. They are useful for testing edge cases in Preview Peak Integration. To upload test data, you will need to create a zip file containing the raw data files (for instructions on creating a zip file, see the Test Data page). Once the file is created, you may drag and drop it onto the page OR click the gray box to browse to the file (red circle, Figure 136). ASCENT v User Manual 116 Revision X

117 Figure 136 Upload Test Data Uploaded Data will need to be converted into ASCENT-specific files before being available to Preview Peak Integration. During conversion, the green dot will be incomplete (red circle, Figure 137) and, if the sample list is expanded by clicking on the name, the conversion status of the individual files will be visible. Figure 137 Converting Uploaded Data Once batches have been successfully uploaded/imported for testing, they can be viewed and managed from the Test Data tab. The green dot indicates that conversion was completed and the samples are available for use. The individual sample list can be viewed by clicking on the batch name to expand the list. Batches may be deleted from the set of test data by clicking on the trash can (red circle, Figure 138). ASCENT v User Manual 117 Revision X

118 Figure 138 Manage Test Data Batches which have been added to the Test Data will display their source as either uploaded by user or imported from production on the right side of the batch identifier. However, only Test Data imported from Production through the Batch Status page can be used in the next section. Maintain Assay This page provides information which relate the Draft settings for an assay configuration (those settings which are displayed in the user interface, as described in the preceding sections) to the Production settings for an assay configuration (those settings which will be used for automated batch processing). This includes the tracking and display of the changes made to the draft assay settings compared with the current production settings (e.g. changes made in the user interface which have not been promoted). As changes are made to the draft settings in the user interface, they are not automatically made available for automated production on future batches; rather, this type of update must be explicitly performed by the user. When a new assay is created, the Production panel will show that it has not yet been promoted (red circle, Figure 139). The Draft panel will reflect the date that the assay was created (green circle, Figure 139). ASCENT v User Manual 118 Revision X

119 Figure 139 Maintain New Assay After an assay is promoted (e.g. the draft settings are moved to production), if any changes are made to the draft Compound settings, a report will become available to show the changes (red circle, Figure 140). Changes made to Compute version, Lots/Levels, Rules, or Display values will not be presented in the report, although they do update the modified date of the draft. When selected, the report will open in a new tab and is available for printing or saving (Figure 141). A report is also available of the current production settings (green circle, Figure 140). It contains the same assay information displayed in the PDF archive (see APPENDIX C: SAMPLES FROM ARCHIVE). Figure 140 Modify Promoted Assay The Compute version is only displayed on the page. Figure 141 Draft Assay Changes Report Batch Comparison To compare the output of processing a batch using the draft assay settings to processing using the production settings, a user may generate a batch comparison report. One or more ASCENT v User Manual 119 Revision X

120 batches must be imported from Production for comparison (Figure 135); directly uploaded files do not contain important information gleaned from the ASCENT sequence file and cannot be selected for comparison reports (Figure 142). Select the name of a batch of test data and click Generate Comparison (red circle, Figure 143). Figure 142 Only Production Data Figure 143 Generate Batch Comparison Depending upon the size of the assay and the test batches, the report may take some time to generate (Figure 144). Do not promote the assay until the report is complete. Figure 144 Report Generation Pending Once the comparison report has generated, it will be available in the report window (red circle, Figure 145). It is a comma-separated values report that can be opened with Excel. Comparison reports may be destroyed by clicking on the trash can (green circle, Figure 145). ASCENT v User Manual 120 Revision X

121 Figure 145 Report Generation Complete Assay Promote and Revert Once the assay is satisfactory, it is ready to be promoted to Production. To do so, simply click on the Promote arrow (red circle, Figure 146). If it is necessary to discard all changes to the Draft assay settings and revert to the Production settings, click the Revert arrow (red circle, Figure 146). The assay configuration pages will now match the production settings. Figure 146 Promote and Revert If new quality rules are deployed, the Promote action must be performed prior to any Revert action (selecting Revert prior to Promote will remove the new rules; if this occurs, refer to APPENDIX A: CUSTOMER PORTAL for assistance). Additional Assay Actions Figure 147 Assay Action Menu There are several additional actions that can be performed on an assay from the drop down menu accessed by clicking the gear icon (red circle, Figure 147). Save As New Assay ASCENT v User Manual 121 Revision X

122 Save As New Assay creates a new assay using a copy of the current promoted assay settings to accelerate the creation of a new similar assay. At present, it does not copy custom QA rules or reports. Please submit a service request through the customer portal to copy deploy customizations. Delete Delete eliminates all the assay configuration parameters visible to the customer. It does not delete custom QA rules or reports, so the assay will reappear in the list until those are removed by Indigo. Please submit a service request through the customer portal to completely eliminate the assay. Check Assay Check Assay allows the user to check the assay for certain types of typographical and logical errors prior to promoting the assay to production. The parameters checked by this function are listed in red italics in the prior sections. Once the assay configuration parameters have been created or edited, a user may use the Check Assay function to confirm that the entries meet the system requirements. If there is an error, the screen in Figure 143 will appear. The results box will display the location of the error, and the corresponding Compound and parameter will be highlighted in red. Once the error is corrected, the assay can again be checked or it will be checked during assay promotion. Figure 148 Check Assay ASCENT v User Manual 122 Revision X

123 Replace Assay From File The input to the Replace Assay From File function is a file created using Save Assay To File function. This file can be used to restore an older assay configuration or to move an assay between test and production sites. The file contains the parameters for all core and custom assay parameters. However, it does NOT contain the custom framework, which must be deployed by Indigo. When creating a NEW assay to use the file settings, the custom framework must be deployed by Indigo (via an order) BEFORE replacing the assay configuration. If it is replaced prior to deploying the custom framework, the custom settings will be discarded. The Replace function will need to be used again after the deployment in order to take effect. The Replace function overwrites the Draft assay settings. Save Assay To File The Save Assay To File function creates a file containing all of the production assay settings. It includes the parameters for custom rules and reports, but not the custom rules/reports framework. It is useful for creating a backup of an assay configuration. It can also be used to move an assay between test and production sites, although assistance from Indigo will be required to ensure the custom framework is present. ASCENT v User Manual 123 Revision X

124 CONTROL BATCH SETS QC CHARTING A control batch set (hereafter referred to as a CBS) is a collection of one or more batches, aggregated together for the purposes of generating a quality control (QC) chart set. These charts are often referred to as Levey-Jennings (LJ) charts, and are used for the periodic monitoring of the performance of quality control samples over a designated time period (e.g. prepared for a calendar month). A QC chart is a graphical representation of the calculated concentration for a given compound s specific lot and level of a quality control sample, plotted against the QC sample s acquisition time. An example of a QC chart is seen in Figure 149: Figure 149 Example QC Chart Creating a CBS To create a CBS, navigate to the Control Batch Set Status page by clicking its top-level navigation link (red circle, Figure 150): Figure 150 ASCENT menu link for CBS Once on the CBS Status page, click the Create button to initiate the process (red circle, Figure 151): ASCENT v User Manual 124 Revision X

125 Figure 151 CBS Status Page Clicking the Create button will present a dialog, which is used to designate the assay (red circle, Figure 152) and instrument (blue circle, Figure 152) for which the QC chart set will be created. A QC chart set consists of one or more QC chart series; one QC chart series will be created for each target analyte (e.g. internal standard compounds will not have a QC chart), with each series consisting of a single QC chart for each specific lot and level combination which are part of the batch results. Thus, a CBS which represents three analytes, each of which has two specified lot/level for QCs, will consist of three QC chart series, one for each analyte, with each QC chart series consisting of two QC charts, one for each lot/level, for a total of six QC charts in the CBS. ASCENT v User Manual 125 Revision X

126 Figure 152 CBS Create Dialog After selecting the assay and instrument name from the dropdown selectors, select a start (green circle, Figure 152) and end date (orange circle, Figure 152) from which to search for batches to make up the CBS. A different month and year can be selected using the selectors above the calendar display (yellow circles, Figure 152). By default, the date selection will be the current calendar month (as indicated by the bold numbers in the calendar display; the bold and highlighted number in the calendar display is the current date). Only certified batches which fall into the specified date range will be used in the creation of the CBS. A valid assay, instrument and date range must be selected to proceed. Once those selections have been made, click the Create button near the bottom (black circle, Figure 152) to generate a new CBS. ASCENT v User Manual 126 Revision X

127 Reviewing a CBS Once a CBS has been created, it is available for review selection from the CBS Status page. A CBS may be opened in either Read Only or Review modes (like a batch, once a CBS is certified, it may only be opened in read only mode). To open a CBS in review mode, click the Review link (red circle, Figure 153); to open the CBS in read only mode, click the Read Only link (blue circle, Figure 153). Figure 153 CBS Review Links CBS Summary page Opening a CBS in either review or read only mode through the links in Figure 153 will lead to the CBS Summary page (Figure 154). Figure 154 CBS Summary Page The title of the CBS Summary display describes the name of the assay and instrument for which the CBS was created, along with the date range used to search for certified batches to aggregate to form the CBS. ASCENT v User Manual 127 Revision X

128 The default Batches tab displays the names and ASCENT date/time stamps (e.g. date and time when the batch was received by ASCENT) for the batches which have been found for use in the CBS. If a batch is to be completely excluded for use in the CBS, it can be deselected by unchecking the box to the left of its name (red circle, Figure 154). A previously deselected batch may be reselected for inclusion by checking the box. After making changes of either type, click the Reprocess button (blue circle, Figure 154) to regenerate the CBS with these choices applied to this updated CBS. The Production Changes tab of the CBS Summary will indicate any changes to the potential list of batches which make up the CBS since the last time the CBS was accessed. For example, if a batch which makes up the CBS is decertified, the Production Changes display will indicate the situation (Figure 155). If no changes to the list of batches which make up the CBS are present, the message This control batch set is up-to-date with production batches will be displayed. Figure 155 CBS Production Changes When a change is displayed, the CBS is not automatically regenerated. Rather, the option of creating a new CBS is initiated by clicking the Recreate button (red circle, ASCENT v User Manual 128 Revision X

129 Figure 155). The use of the Recreate button will completely overwrite the full QC chart set, including any manual changes made to the analytes QC chart series. The History tab of the CBS Summary (Figure 156) will describe any changes made to the CBS from its time of original creation through any review and certification activities. Items noted here would include the inclusion and exclusion of batches, certification and decertification of the CBS, and the inclusion and exclusion of specific determinations from a specific QC chart of a QC chart series. Destroying or recreating a CBS will clear its history; reprocessing a CBS will be added to the history. Figure 156 CBS History CBS (QC) Chart review The individual quality control charts which make up a CBS can be reviewed by clicking the View Charts link on the CBS Summary page (red circle, Figure 157). Figure 157 CBS Chart Review Link ASCENT v User Manual 129 Revision X

130 The QC chart review display is broken in four primary regions: Analyte and lot/level selectors (red rectangle, Figure 158) QC chart metrics (blue rectangle, Figure 158) QC chart display (green rectangle, Figure 158) QC measurements (yellow rectangle, Figure 158) A specific analyte may be selected using the left dropdown selector; this selector also supports text searching by typing the first few characters of an analyte name. A specific lot/level may be selected using the right dropdown selector. The available lot/level combinations are drawn from the batches and QC samples themselves; each unique lot/level value combination will create a new QC chart for the analyte s QC chart series. For example, if the lot/level values change within the selected date range (e.g. a new concentration of QC standard is introduced mid-month), separate QC charts will be created, even if the name of the lot/level doesn t change. The QC chart metrics include the target mean and standard deviation (SD) of the QC sample, as provided by the lot/level specification in the assay configuration (if a standard deviation is not provided, the QC chart can still be generated, and the target SD will be set to zero). The coefficient of variation (CV) is calculated as the quotient of the target SD and target mean, expressed as a percentage. The QC chart metrics also include the calculated mean, standard deviation, and coefficient of variation for the concentration values drawn from the QC samples of the designated lot and level. The number of points which make up the calculated mean/sd/cv are noted, and if any points are excluded (see below), the field will indicate number of points used and the number of points originally found. The QC chart display is an x/y plot, with the calculated concentration plotted on the y- axis, and the date/time of acquisition of the determination represented on the x-axis. A solid, horizontal blue line is drawn at the target mean, and additional horizontal marker dotted lines are drawn at one (green), two (yellow) and three (red) standard deviations above and below the mean. ASCENT v User Manual 130 Revision X

131 Figure 158 QC Chart Review The QC charts can be modified by the inclusion and exclusion of one or more determination measurements. To remove a determination, uncheck the box in the Included column (orange circle, Figure 158); when finished, click the Modify button (black circle, Figure 153). The QC chart will then display that point as a red square as opposed to the blue circle (red circle, Figure 159), and the calculated mean/sd/cv will reflect the removal of the point; also, the number of points field in the QC metrics will be updated (blue circle, Figure 159). An excluded determination may be re-included by checking the box in the Included column (green circle, Figure 159); again, when finished, click the Modify button to update the chart display and the metrics. ASCENT v User Manual 131 Revision X

132 Figure 159 QC Chart Modification Certifying a CBS Once the review activities for a CBS are completed, the CBS may be certified. The act of certification has two primary effects: first, the CBS is now locked in a read only mode and while it can be viewed, it cannot be modified; second, a PDF archive report will be generated, consisting of the QC chart metrics and display for each of the QC chart series for the entire QC chart set. To certify a CBS, navigate to one of the QC charts and click the Certify button near the bottom of the page (red circle, Figure 160). Figure 160 CBS Certification Action ASCENT v User Manual 132 Revision X

133 The CBS history will be displayed, along with a field to enter the user s password. After entering the password, click the Certify button to finish the certification process (red circle, Figure 161). Figure 161 CBS Certification Confirmation CBS Result Files Two types of output files are available for a CBS: a tabular report as a CSV file, and a graphical report as a PDF file. This CSV is importable into other applications, such as Microsoft TM Excel TM, for the purposes of further evaluation. The CSV file can be generated at any time after the CBS has been created, but the PDF file is only available following CBS certification. To download either the CSV or the PDF file, use the links on the left side of the CBS Summary page (red circle, Figure 162). Figure 162 CBS Result File Links Decertifying a CBS If it becomes necessary to make changes to a CBS which has been certified, it must first be decertified. Decertifying a CBS can be done from the CBS Status page. Select the certified CBS of interest by checking the box (red circle, Figure 163) and then click ASCENT v User Manual 133 Revision X

134 the Decertify button (blue circle, Figure 163). A confirmation dialog will present; clicking the OK button will decertify the CBS, while clicking the Cancel button will not, and return to the CBS Status page. Figure 163 CBS Decertification Destroying a CBS If a CBS is no longer needed (e.g. it was generated as a temporary and/or intermediate view of the assay/instrument performance; the QC charts have been filed), it can be removed from the system. Destroying a CBS can be done from the CBS Status page. Select the CBS of interest by checking the box (red circle, Figure 164) and then click the Destroy button (blue circle, Figure 164). A confirmation dialog will present; clicking the OK button will destroy the CBS, while clicking the Cancel button will not, and return to the CBS Status page. Figure 164 CBS Destruction ASCENT v User Manual 134 Revision X

135 Production Volume Report A report of the volume of data flowing into the ASCENT system is available by clicking the white Indigo pinwheel icon in the footer of any web page (red circle, Figure 165). Figure 165 Production Volume Report Link Clicking the link will generate and navigate to the Production Volume Report web page. This page displays the site name, and allows the data volume to be expressed by clicking the drop-down selector and choosing Chromatograms or Samples as the basis (red circle, Figure 166). Figure 166 Production Volume Report The report table lists the number of basis items (chromatograms or samples) which have been uploaded to ASCENT and which have reached the Ready to Review state. Quantities are provided for today, the calendar month, the calendar quarter, and the calendar year. Quantities are listed as a total for the ASCENT site, as well as listed individually by instrument name (as listed in any ASCENT assay which has had data uploaded during the time periods listed). ASCENT v User Manual 135 Revision X

136 SUPPORT AND TROUBLESHOOTING Lab Managers serve a front-line role in providing support and troubleshooting for the internal users of the ASCENT software. To obtain support for issues from Indigo BioAutomation, please visit the online Customer Portal. Detailed information is found in APPENDIX A: CUSTOMER PORTAL. ASCENT v User Manual 136 Revision X

137 APPENDIX A: CUSTOMER PORTAL Accessing the Customer Portal A link to the customer support portal can be found in the footer region of each ASCENT web page (red circle, Figure 167). Figure 167 Support Portal Link A link is also available at the top of the Indigo BioAutomation home page (red circle, Figure 168). Figure 168 Support Portal Link - The web address of the main support portal page is For information on establishing an account, and entering and monitoring support cases, please refer to the orientation video: Indigo Video - Support Portal ASCENT v User Manual 137 Revision X

138 APPENDIX B: CORE QA RULES For a given sample in a batch, the presence or quantity of a compound is determined from your method. In ASCENT, this is referred to as a determination. For each compound, ASCENT s core quality rules are Applied To a specified type of sample, at the level of each chromatogram or determination. If a sample meets the Condition of a given rule, ASCENT will Then Flag either a chromatogram, determination, or sample with the ASCENT Flag Code (concise text indicating which rule was triggered; green italicized text indicates real numerical values or calculated values that are part of the flag). Most quality rules have a Compound Attribute as part of the logic, initially set to a default value. For example, the Contamination rule is applied to Blank samples. If the calculated concentration is x% of the LLOQ, that determination is flagged with the code Blank >= x% LLOQ. The compound attribute x is configurable. Calibration The following rules can be tailored to your lab requirements to alert users if a Standard sample or the overall calibration curve fails: ASCENT Flag Code Concentration Deviation z % (> x %) Int Std CV z (> x) Response Deviation z % (> x %) R2 z (< x) * Regression Failed z Standards excluded (> x) Applied To Condition Then Flag Compound Attribute [x} Standard Samples Standard Samples Standard Samples Standard Samples Standard Samples Standard Samples If absolute value of concentration deviation is > x % If the coefficient of variation of non-zero response values is > x If absolute value of response deviation is > x % If the calibration regression equation s R2 value is < x If regression could not be performed, If > x Standards are excluded from the regression, That determination (z = concentration deviation) All standard determinations (z = CV) That determination (z = response deviation) All standard Determinations (z = regression equation R2 value) All determinations All standard determinations Standard Concentration Deviation Threshold- Maximum acceptable deviation from the nominal concentration (%) Maximum CV Value maximum acceptable value for coefficient of variation (as percentage) Int Std Response Deviation Threshold maximum acceptable deviation from mean Minimum R2 Value Minimum acceptable value for R2 (coefficient of determination) N/A Maximum Excluded Standards - Maximum acceptable ASCENT v User Manual 138 Revision X

139 Calibrator Missing Result No Peak No Normalizer No Intercept Standard Samples If nominal concentration is > 0, and the response is not >0 If nominal concentration is > 0 and the concentration is not calculable (z = number of excluded standards) That determination number of standards excluded from regression N/A * The Minimum R2 rule should not be used for single-point calibration quantification. Carryover The following rules flag when a sample result may be due to contamination from a previous sample: ASCENT Flag Code > ULOQ z (> ULOQ) Possible carryover Carryover Potential z Applied To Condition Then Flag Compound Attribute [x] All Samples If the analyzed That ULOQ Upper limit of except concentration is > determination quantification Standards ULOQ, (z = analyzed All Samples Unknown Samples If the analyzed concentration is > ULOQ If the analyzed concentration is > ULOQ or ULOC, or if analyzed concentration is not calculable concentration) The determination for the next x samples with analyzed concentration >= LLOQ The determination for the next x samples with analyzed concentration >= LLOQ or LLOC, until a sample precedes it with an analyzed concentration < LLOQ or LLOC (z = index of sample initiating carryover) Subsequent Sample Count - Number of subsequent samples to flag for potential carryover ULOQ Upper limit of quantification LLOQ Lower limit of quantification LLOC Lower limit of carryover LLOQ Lower limit of quantification ULOC Upper limit of carryover ULOQ Upper limit of quantification Subsequent Sample count number of subsequent samples to evaluate for carryover Upper Carryover Impact Percentage maximum analyzed concentration attributable to carryover impact ASCENT v User Manual 139 Revision X

140 Concentration A concentration rule flags when the analyzed concentration, or the on-column value, is outside the limits of quantitation. In ASCENT, analyzed concentration is defined as the calculated concentration divided by the dilution factor. The limits of quantitation are defined for each compound. ASCENT Flag Code Dilution Required z (> ULOQ) Int Std Response z% (> x% or < y%) No Intercept Over-Diluted z (< LLOQ) < LLOQ z (< LLOQ) QC Flag Positive Unknowns Applied To Condition Then Flag Compound Attribute [x] Unknown ULOQ Upper limit of Samples quantification All Samples (Excluding Blank Samples) All determinations Unknown Samples Unknown Samples Unknown Samples (which follow a flagged QC sample) If the analyzed concentration is > ULOQ, If the relative chromatogram peak area is above (x) or below (y) the peak area percentage limits around the mean relative chromatogram peak area. (mean relative peak area is calculated from the Standard samples in which the peak is integrated), In the case of a quadratic or cubic regression equation, if the calculated concentration is undefined for the response, If the dilution factor is > 1 and analyzed concentration > 0 and < LLOQ, If a peak is integrated and the calculated concentration is < LLOQ, If a QC sample from the same batch and which precedes the unknown sample is flagged and the calculated concentration of that compound is LLOQ, That determination (z = analyzed concentration) That determination (z = percentage of mean peak area) That determination That determination (z = analyzed concentration) That determination (z = analyzed concentration) That determination Relative Chromatogram chromatogram used to normalize response Lower Peak Area Percentage minimum acceptable percentage of peak area (%) Upper Peak Area Percentage maximum acceptable percentage of peak area (%) Quantifier Chromatogram used for calculation LLOQ Lower limit of quantification LLOQ Lower limit of quantification Quantifier Chromatogram used for calculation LLOQ Lower limit of quantification ASCENT v User Manual 140 Revision X

141 Contamination The following rule can be used to alert users if a Blank does not meet the acceptable criteria: ASCENT Flag Code Blank z ( > x % * LLOQ) Applied To Condition Then Flag Compound Attribute [x] Blank Samples If calculated concentration is x % of the LLOQ, That determination (z = calculated concentration) LLOQ Lower limit of quantification Blank Contamination Limit LLOQ Percentage - Maximum acceptable fraction of LLOQ (%). Ion Ratio If applicable for the compound, the following rule verifies consistent ion ratio across all samples in the batch, using the mean of the Standard samples as reference: ASCENT Flag Code Ion Ratio z % (y, m n) Ion Ratio Peak Missing Applied To Condition Then Flag Compound Attribute [x] All Samples (Excluding Blank Samples) All Samples If the absolute value of the ion ratio deviation is x % and the calculated concentration LLOQ, (Ion ratio is defined as the ratio of Qualifier peak area to Quantifier peak area. Deviation is calculated from the mean ion ratio of the Standard samples with both Qualifier and Quantifier peaks integrated). If Qualitative chromatogram peak is integrated but no Quantitative chromatogram peak is integrated, That determination (z = ion ratio deviation; y = ion ratio; (m n) = acceptable ion ratio range That determination Ion Ratio Deviation Threshold - Maximum acceptable deviation from the mean ion ratio of Standards (%) Qualifier chromatogram used for calculation LLOQ Lower limit of quantification Qualifier chromatogram used for calculation Quantifier Chromatogram used for calculation ASCENT v User Manual 141 Revision X

142 Peak Area The following rule verifies peak area is consistent across all samples in the batch, using the mean of the Standard samples as reference: ASCENT Flag Code NAME Area Deviation z % (> x %) Int Std Response z% (> x% or < y%) Applied To Condition Then Flag Compound Attribute [x] All Samples If the absolute value of (Excluding the Quantifier peak Blank area deviation is x, Samples) All Samples (Excluding Blank Samples) If the quantifier peak area is above (x) or below (y) the peak area percentage limits around the mean quantifier peak area. (mean quantifier peak area is calculated from the Standard samples in which the peak is integrated), That determination (NAME = name of chromatogram; z = deviation) That determination (z = percentage of mean peak area) Quantifier Chromatogram used for calculation IS Area Deviation Threshold - Maximum acceptable deviation from the mean peak area of standards (%). Quantifier Chromatogram used for calculation Lower Peak Area Percentage minimum acceptable percentage of peak area (%) Upper Peak Area Percentage maximum acceptable percentage of peak area (%) ASCENT v User Manual 142 Revision X

143 Peak Quality The following rules alert users if ASCENT detects a potential problem with a chromatogram (e.g., poor fit to the peak model or missed peak): ASCENT Flag Code Fit Quality z % (< x %) High Signal, No Peak z (> x) High Signal, No Peak z (> b% * x) High Signal, No Peak z (> x) Applied To Condition Then Flag Compound Attribute [x] Each If a peak is integrated That Fit Quality Threshold Chromatogram with fit quality < x, chromatogram - Minimum acceptable (z = fit quality) peak fit quality. Each Chromatogram Each Chromatogram If no peak is integrated and the signal within the retention time tolerance > x, If no peak is integrated and the signal within the retention time tolerance > x That chromatogram (z = maximum intensity within the retention time tolerance) That chromatogram (z = maximum intensity within the retention time tolerance) Maximum Signal Without Peak Integrated - Maximum acceptable signal without an integrated peak. Dynamic High Signal No Peak Threshold Multiplier for lowest observed value of maximum signal intensity, applied to lowest observed peak are in Standard samples Maximum Signal Without Peak Integrated - Maximum acceptable signal without an integrated peak. Peak Cluster Fallback Each Chromatogram If a scaled peak model cannot be fit to a cluster That chromatogram (provide unscaled peak for review) N/A ASCENT v User Manual 143 Revision X

144 QC The following rules can be used to alert users if a QC does not meet the acceptable criteria: ASCENT Flag Code Concentration Deviation z % (> x %) Standard Deviation z (> x SD) Negative QC z (> x% * LLOQ) QC Missing Result No Peak No Normalizer No Intercept Applied To Condition Then Flag Compound Attribute [x] QC Samples If absolute value of concentration deviation is x %, QC Samples Negative QC Samples QC Samples If the calculated concentration is x standard deviations from the nominal concentration, If the calculated concentration is > x % * LLOQ, If nominal concentration is > 0, and the response is not >0 If nominal concentration is > 0 and the concentration is not calculable That determination (z = concentration deviation) That determination (z = (calculated concentration nominal concentration) / standard deviation That determination (z = calculated concentration) That determination QC Concentration Deviation Threshold - Maximum acceptable deviation from the nominal concentrations (%). Maximum Standard Deviations - Maximum acceptable number of standard deviations from the nominal concentration. LLOQ Lower limit of quantification Negative QC Limit LLOQ Percentage - Maximum acceptable fraction of LLOQ (%). NA ASCENT v User Manual 144 Revision X

145 Reporting The following rule can be used to alert users if a determination has a reportable concentration: ASCENT Flag Code Calculated Above Reporting Limit z ( x) Concentration of Interest z ( x and y) Applied To Condition Then Flag Compound Attribute [x] Unknown Samples Unknown Samples If the calculated concentration is x, using LLOR (or LLOQ, if LLOR is not specified) If the calculated concentration is x, using LLOI (or LLOQ, if LLOI is not specified) and the calculated concentration is y, using ULOI That determination (z = calculated concentration) That determination (z = calculated concentration) LLOQ Lower limit of quantitation LLOR Lower limit of reporting (e.g. cutoff) LLOI Lower limit of interest LLOQ Lower limit of quantitation ULOI Upper limit of reporting ASCENT v User Manual 145 Revision X

146 Retention Time The following rules tailored by your lab requirements to alert users if a sample or determination does not meet your defined criteria for RT: ASCENT Flag Code Quant-Qual RT z min (> x min) Relative RT z % (> x %) RT Shift z min (> x min) Applied To Condition Then Flag Compound Attribute [x] All Samples If the absolute value of That Qualifier (Excluding the difference between determination chromatogram used Blank the Quant and Qual (z = absolute for calculation Samples) retention times is > x value of the RT Quantifier minutes, difference) Chromatogram used for calculation Maximum Quant Qual RT Difference - Maximum acceptable RT difference between Quant and All Samples (Excluding Blank Samples) All Samples (Excluding Blank Samples) If the absolute value of the relative retention time deviation is x%, (Deviation is calculated from the mean relative RT of the Standard samples. Relative RT is calculated as the ratio of Quant RT to relative chromatogram RT of the determination.) If the absolute value of the difference between the Quant RT of the determination and the mean Quant RT derived from the Standards > x minutes, That determination (z = relative RT deviation) That determination (z = absolute value of the RT difference) Qual (in minutes). Relative RT Deviation Threshold - Maximum acceptable deviation from the mean relative RT of Standards (%). Standards samples excluded from the curve are also excluded from the mean relative RT. Quantifier Chromatogram used for calculation Relative Chromatogram Chromatogram used for calculation Maximum RT Shift From Standards - Maximum acceptable Quant RT shift from Standards (in minutes). Quantifier Chromatogram used for calculation ASCENT v User Manual 146 Revision X

147 APPENDIX C: SAMPLES FROM ARCHIVE These images do not represent a complete archive, but simply a snapshot of the types of data captured: compound calibration, sample results, audit trails, and assay configuration parameters. The archive does not contain any raw data. ASCENT v User Manual 147 Revision X

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