Data Reporting in Chromeleon

Technical Note 70 Data Reporting in Chromeleon 1. Introduction One of the most important functions of any Chromatography application is the reporting of processed data. If it is not possible to use the application to report data in accordance with laboratory requirements, it becomes necessary to transcribe or export data to other software applications. This process is time consuming and error prone, and is one of the major bottlenecks in transforming chromatography data into usable information. The Chromeleon Chromatography Magement System (CMS) provides powerful reporting capabilities that allow: Report templates: quick and simple generation of templates that can be applied easily to standard chromatography methods, independent of the number of injections and peaks. Advanced reporting options: including summaries, trend plots and advanced charting. User-defined formulas: easy creation of formulas such as the mathematical operators SUM and AVERAGE, and the logical operators IF and OR. (Formula entry is alogous to Microsoft Excel.) Improved compliance with regulatory rules such as 21 CFR Part 11. These capabilities eble creation of reports that automatically calculate and display the required data and statistics, without the need to use additiol software applications. As such, the Chromeleon CMS allows users to concentrate on the science, not on result calculation, improving laboratory productivity. 2. Traditiol Data Reporting Workflow In many laboratories the traditiol workflow for generating a fil report is as follows: 1. Integrate all chromatograms. 2. Export or transcribe peak results (such as peak area) into a third-party software package (such as Microsoft Excel) for calculation. 3. Enter relevant values such as standard weights and sample weights into the third-party software application. 4. Evaluate calculated results against specifications. 5. Export or transcribe the calculated results into a Certificate of Alysis (C of A) or similar document. 6. Print fil report. Technical Note 70 1

Potential drawbacks of this procedure include: It can cause delays from data acquisition to creation of the fil result. Multiple steps must be repeated if chromatograms need to be reintegrated. Any data transcriptions require error checking and, if errors are found, repetition of steps 2 4 and 5. 2.1. Estimated Data Reporting Time In this example, there are approximately 20 injections, each containing five peaks. The total time required to obtain a result can be calculated using the following assumptions. Table 1. Estimated Data Reporting Time for Traditiol Workflow Step Process Time Taken 1 Integrate and check chromatogram 30 seconds per injection Total = 600 seconds 2 Transcribe peak areas to 10 seconds per peak area third-party application Total = 1000 seconds 3 Enter values such as standard 10 seconds per sample weights and sample weights Total = 200 seconds 4 Evaluate calculated results by 15 seconds per injection checking against specifications Total = 300 seconds 5 Evaluate calculated results 10 seconds per peak area by reviewing transcription Total = 1000 seconds Total: 3100 seconds (51 min 40 s) 5 Transcribe results to C of A 10 seconds per peak result Total = 1000 seconds 6 Review C of A transcription 10 seconds per peak result Total = 1000 seconds Total: 2000 seconds (33 min 20 s) Therefore, the total time required to generate the fil result is 137 minutes (8200 seconds). 2.2. Annual Data Reporting Time This example assumes that an HPLC system runs one, 20-injection sequence each working day. With 250 working days per year, total data processing time per system is 24 days. Reducing this time can significantly improve laboratory productivity. 3. Data Reporting Workflow in CHROMELEON Chromeleon s reporting features eble you to perform all chromatography calculations directly within the application, without the need to export data to thirdparty software. Chromeleon s reporting tool supports flexible formats for any report type, including Certificates of Alysis. The fil results can be printed or exported in Adobe Acrobat Portable Document Format (PDF) for circulation. With all this functiolity, Chromeleon provides a much simpler reporting workflow: 1. Integrate all chromatograms. 2. Enter relevant values such as standard weights and sample weights into Chromeleon. 3. Evaluate calculated results by checking against specification. 4. Print or export (as PDF) fil report. This workflow still requires review of the output. If changes are needed, the new results can be printed or exported as PDF files as soon as the chromatograms are reintegrated. No re-transcription or re-export to a thirdparty application is required. In a typical lab, the integration will probably need to be repeated at least once. This means that steps 1, 2, 4 and 5 must be repeated, adding 48 minutes, 20 seconds to the total time. Also, assuming at least a 5% error rate in transcription, corrections add 100 seconds to the total time (5% from step 2 and 5% from step 5). The total time for steps 1 to 5 is now 102 minutes (6100 seconds). It is assumed that steps 5 and 6 are started only after steps 1 to 4 are completed. Assuming another 5% transcription error here, total time increases by another 100 seconds. 2 Reporting in Chromeleon

3.1. Estimated Data Reporting Time Using the same sequence as section 2.1, the following data reporting time can be calculated for the Chromeleon workflow. Table 1. Estimated Data Reporting Time for Chromeleon Step Process Time Taken 1 Integrate and check chromatogram 30 seconds per injection Total = 600 seconds 2 Enter values such as standard 10 seconds per sample weights and sample weights Total = 200 seconds 3 Evaluate calculated results 5 seconds per injection by checking against specification Total = 100 seconds 4 Print or export (as a PDF) 0 seconds everything is fil results (e,g, C of A) performed automatically by Chromeleon Total: 900 seconds (15 min) Assuming that the sequence will need reintegrated one time, adding 700 seconds, the total time required to generate the fil result is 27 minutes (1600 seconds). Compare this to 137 minutes for the workflow in section 2.1. 3.2 Annual Data Reporting Time Assuming, as per section 2.2, that each HPLC system runs 250 sequences per year, the total annual data reporting time is 4.7 days. Compared with the traditiol data workflow, that is a saving of 19.3 days an 80% reduction in the total time. Simple productivity calculations such as these show the tangible business benefits to implementation of a Chromeleon Chromatography Data System. Improved data integrity: Even with double-checking, errors in transcribed data may not be detected. This problem is elimited when working with Chromeleon reports. Time and cost savings: Clear reductions in reporting time. Workflow improvements: Automation of repetitive and mundane tasks and software that is more enjoyable to work with increase motivation and free resources to focus on science, not reporting. 4. Powerful Data Reporting with Chromeleon How can Chromeleon offer these kinds of benefits? The standard installation provides several report templates designed to report most types of data, including chromatograms, integration reports and calibration curves as well as standard results such as % w/w, % Label Strength. Chromeleon can compute more than 300 different chromatography values, such as peak area or number of theoretical plates, speeding creation of reports that display the information needed by the laboratory. Approximately 90% of standard chromatography values and results can be automatically reported with the default Chromeleon installation. In addition, Chromeleon supports user-defined formulas, similar to those in Microsoft Excel, making Chromeleon an all-in-one, fully integrated data processing and reporting application. 5. Report Definition File Examples Chromeleon reports, like Excel workbooks, may contain several worksheets that display different types of data and results. All Chromeleon installations come with a set of standardized reports that contain sheets such as Integration, Calibration, Peak Alysis, Summary tables, etc. This following sections provide examples of additiol reports that can be used to save time and increase productivity. 5.1. Summary Table Each default Chromeleon report contains a summary sheet. This is particularly valuable as it displays an instant summary of results over a series of injections. It can be easily modified to show only those injections that are relevant to the user, for example, only standards or only unknowns. The example below summarizes a sequence containing 10 samples. Each sample was injected in duplicate and contained five peaks. The summary requirements were to compare the average amount (in mg/ml) against the peak specification, and to report whether the result passed or failed. Technical Note 70 3

Figure 1 shows the summary. It automatically calculates the average value for each set of duplicates, and compares the values against a specification. A summary table is automatically generated for each peak in the processing method. This report format will work for any method, with any number of peaks and any peak mes. Summary of Results Figure 1. Summary of five peaks from 20 injections (10 samples with duplicate injection). 4 Reporting in Chromeleon 5.2. Dissolution Summary Chromeleon provides advanced charting options. This ebles data such as dissolution data to be displayed graphically. In this example, three batches of a compound were tested in a single dissolution bath of six vessels, that is, one batch per two vessels. The report shows the dissolution profile of each vessel, and the average profile of each batch. 5.3. Related Impurities Testing In related impurities testing, a duplicate injection of a sample is typically performed. All unidentified peaks are then integrated and the average result (generally percent area) for each peak is reported. Due to the ture of related impurities testing, the following complications generally arise: The number of peaks that will be detected is unknown. Peaks can be detected in one duplicate but not in another, as they are close to the limit of detection. These complications can make it difficult to automate the calculation. However, it is possible to do this using Chromeleon, using user-defined formulas. As can be seen in Figure 3, the average percent area has been calculated automatically. In this case, if the percent area is below 0.05%, it is reported as <RL (less than the reporting limit). The report will calculate results correctly, independently of the number of peaks. It can also easily be extended to cover methods that require triplicate injections, or duplicate injections of two sample preparations (four injections in total). This report is one of the major time savers for laboratories. The figures in section 3.1 show that data reporting times increase significantly as the number of reported peaks increases. This is not the case with Chromeleon. Companies using these reporting options have realized time savings of up to 98%, mainly because the data reporting step is fully automated. 6. Improved Regulatory Compliance If third-party applications such as Microsoft Excel are used to perform calculations, any such applications must be validated, taking valuable laboratory time. Using Chromeleon to calculate the fil results minimizes validation time. Standard results, such as peak area, require no validation as these are tested during the software development stage (Design Qualification) at Dionex, and during

Figure 2. Dissolution profiles using Chromeleon charting options. Figure 4. Displaying user-defined formulas. Figure 3. Summary of related impurities for a duplicate injection. installation at your site (Operatiol Qualification). Any calculations performed with user-defined formulas should be validated. For example, if a user-defined formula generates a Pass/Fail statement, the formula must be validated to show that it produces the correct statement for the data value generated. Chromeleon contains the following tools that make this process easier: Display and print formula entered (see Figure 4) Tracks history of changes made to formula (Figure 5) Figure 5. Chromeleon History Trail tracks all changes made to reports, including changes made to user-defined formulas. Technical Note 70 5

7. Summary of Benefits Chromeleon provides a suite of powerful reporting options that make it possible to fully automate the complex calculations commonly used in today s laboratories yet impossible to complete in other Chromatography Data Systems. Chromeleon removes the need to calculate results using third-party applications such as Microsoft Excel or to manually transcribe results into separate files. Our examples showed that by using Chromeleon to elimite these steps it is possible to reduce general data reporting times by 80%, and in the case of specialized reporting requirements, such as related impurities, by as much as 98%. Since Chromeleon removes the need to validate third-party software applications, validation costs are also reduced. Regulatory compliance is improved, through Chromeleon s ability to track all changes made to report definition files, even down to the level of changes to user-defined formulas. Chromeleon s flexible reporting tools make it possible to generate results in almost any conceivable format, ebling different departments to work as best suits them. By automating data calculation and reporting Chromeleon not only saves time and increases productivity, it allows scientists to concentrate on the most important laboratory task: the science. Appendices Technical Information A1. Report Definition Files The way in which data is printed (or electronically signed) is defined in a Report Definition File (RDF). These are similar in look and feel to Microsoft Excel spreadsheets. Figure 6 shows the general structure of a report definition file. Figure 6. Main components of a sheet in a report definition file. The specifications of each sheet are as follows: Sheet Name Sheet Size Table 3. Sheet Specifications Any characters can be used. Maximum length is 31 characters. 256 columns 16,384 rows Cell Size Maximum number of characters per cell is 255. 6 Reporting in Chromeleon

Organizing data into separate sheets makes evaluating and interpreting large amounts of data much easier. It also provides more flexibility when printing and exporting. In a typical example, one sheet might display the chromatogram and results sample by sample, while another sheet displays the calibration data and curves for the sequence. It is also possible create sheets that display a summary of the results. In fact, the number of sheets that be created within a report definition file is limited only by your computer memory. A2. Report Definition File Objects Table 3 shows the types of Chromatographic objects that can be inserted within each sheet of a report definition file. These objects can be used in any combition. Each of these objects is described in more detail below. A2.1. Chromatogram The chromatogram offers a number of different display and formatting options. In essence every single part of the chromatogram can be customized to show data that is relevant to the alysis (see Figure 7). Object Chromatogram Calibration Plot Spectra Plot Mass Spectra Plot 3D Field Plot Trend Plot Fraction Tray Plot Inject Tray Plot Chart Description Freely movable and scaleable representation of the chromatogram Freely movable and scaleable representation of the calibration plot Freely movable and scaleable representation of the spectra plot, showing a 2D representation of the UV spectra from any point in the chromatogram. Freely movable and scaleable representation of the mass spectrometry plot. Freely movable and scaleable representation of the 3D plot. Freely movable and scaleable representation of the trend plot, showing any chromatogram result against either injection time or sample number. Freely movable and scaleable representation of the fraction tray. Freely movable and scaleable representation of the inject tray. Freely movable and scaleable charting object. Chromeleon Report Variable Display of a single result or value associated with the data. Chromeleon Report Table Free Text Table 3. Report Definition File Objects User-Defined Formula Display of a result or value set associated with the data. A free-text entry. A user-defined formula similar to the formulas available in Microsoft Excel. Technical Note 70 7

A B C D Figure 7. Examples of different chromatogram options. (A) Peak tangents and peak width displays. (B) Baseline zoom to view smaller peaks or to check integration. (C) Overlaid chromatograms. (D) Fraction collection display fractions are marked by green shading. A2.2 Calibration Plot The calibration plot shows the individual calibration points and the best fit line for a specific peak. It can also show the calibration confidence levels (see Figure 8). A2.3 Spectra Plot The spectra plot shows a UV spectrum from any point in the chromatogram and is only available for data acquired using a Diode Array Detector (see Figure 9). 8 Reporting in Chromeleon

A B Figure 8. Examples of calibration plot display options.(a) Default calibration plot. (B) Calibration plot with 95% confidence limits. Limits of 95, 98, 99, 99.7, 99.8, 99.9, and 99.99 can be shown. A B C D Figure 9. Examples of spectra plot display options. (A) Standard spectra plot showing the UV spectrum at the peak apex. (B) Standard spectra plot showing the UV spectrum at the peak apex. (C) Spectra plot and first derivative plot. (D) Spectra plot overlaid with spectral library hit plot. Technical Note 70 9

A2.4. 3D Field Plot The 3D field plot shows all data from a diode array acquisition, that is, the sigl from all acquired wavelengths at all retention times. As such, this plot has two main display options as seen in Figure 10. A2.5. Mass Spectra Plot The mass spectra plot shows spectra from any point in the chromatogram using data acquired from a mass spectrometer (see Figure 11). A B Figure 10. Display options for 3D field plots. (A) ISO plot. This is a contour plot of the 3D field. (B) 3D plot. The 3D field is displayed from a user-defined perspective. A B Figure 11. Display options for Mass Spectra Plots. (A) Profile mode. This provides the true mass response for each peak. (B) Centroid mode. This bunches the mass response to provide single m/z values for the peak. This example shows an overlay of three spectra from the same peak: 10% peak height at the front, peak apex, and 10% peak height at the tail. 10 Reporting in Chromeleon

A2.6. Trend Plot The trend plot in Chromeleon can display data in two main ways. As a trend of data against injection time or sample number, with the mean and standard deviations calculated based on the values in the trend. As a trend of data against injection time or sample number, with a user-defined mean and standard deviation. This can be used as a control chart, for example. Figure 12 shows an example of the main two display options. A2.7. Inject Tray Plot The inject tray plot shows a graphical representation of the injection tray. It is available in two main modes, the plot of a standard autosampler tray and the plot of an injection tray for a fraction collection instrument. Figure 13 shows an example of the two display modes. A B Figure 12. Display options for trend plots. (A) Trend plot with mean and standard deviation calculated from data values. (B) Trend plot with mean and standard deviation manually defined. This is generally used as a control chart. A B Figure 13. Display options for inject tray plots. (A) Inject tray plot for a standard autosampler tray. The active sample appears in green. (B) Inject tray plot for a fraction collection instrument. The inject tray is shown in the main part of the plot. The position of the tray within the fraction collection instrument is a darker shade of gray. All other settings are as for view (A). Technical Note 70 11

A2.8. Fraction Tray Plot The fraction tray plot shows the location of fractions collected. It is linked to the inject tray plot. That is, if the inject tray plot shows that the active sample is position 7, the fraction tray plot shows all fractions collected from this injection. As such, there is only one display mode, as shown in Figure 14. Type 2D 3D Area Table 5. Chart Types Bar Line Step Combition Pie Horizontal Bar Figure 14. Display option for fraction tray plots. A. Fraction Tray Plot. A. This plot displays the location of all fractions collected. The areas marked in yellow show the location of fractions collected from the active sample. This plot also shows the position of the fraction tray within the fraction collection instrument (in a darker shade of grey). Cluster Bar Hi Lo Gantt A2.9. Chart The charting option provides more advanced charting than is available through the trend plot. For example, Figure 15 shows a bar chart of instrument usage data. Table 5 shows all available chart types. Bubble Doughnut Contour Surface XY (Scatter) XYZ (Scatter) Polar Radar Figure 15. Using a bar chart to display instrument utilization data. 12 Reporting in Chromeleon

A2.10. Chromeleon Report Variables A Chromeleon report variable shows a single Chromeleon value or result, such as sequence me or sample me. There are other 300 variables available from several different categories such as Peak Results (area, height, etc.) and Peak Calibration (slope, R 2, etc.). Numeric variables can be modified using four mathematical operators: plus, minus, multiply and divide. They can be combined with absolute values (Figure 16) or with other variables. Note: Summary tables are particularly useful since they evaluate data for an entire sequence in a single table, saving time that would be required to review each individual integration report. In addition, summary tables can be filtered to contain only appropriate results. The filter can include or exclude samples according to their type, me, comments, or other fields, then sort the results as required. Figure 17 shows an example filter and sort settings for a summary table. Figure 16. Dividing the Relative Retention Time variable by 100 to generate an absolute value, rather than a percentage value. Figure 17. Defining the data set to be shown in a summary table. A2.11. Chromeleon Report Table A Chromeleon report table displays a collection of chromatography results. For example, an integration table for a chromatogram will display variables such as peak area and height for all peaks. The two main table types are: Integration table. This displays the peak results for each chromatogram. Summary table. This displays results for each injection. A2.12. User-Defined Formula Chromeleon supports user-defined formulas that are alogous to those used in Microsoft Excel, to perform calculations that are not included as standard Chromeleon calculations. Table 6 shows the available functions. Technical Note 70 13

Table 6. Functions ABS ACOS ACOSH ADDRESS AND ASC ASIN ASINH ATAN ATAN2 ATANH AVERAGE CEILING CHAR CHOOSE CLEAN CODE COLUMN COLUMNS CONCATENATE COS COSH COUNT COUNTA COUNTIF DATE DATEVALUE DAY DAYS360 DBCS ERRORTYPE EVEN EXACT EXP FACT FALSE FIND FINDB FIXED FLOOR HLOOKUP HOUR IF INDEX INDIRECT INT ISBLANK ISERR ISERROR ISLOGICAL ISNA ISNONTEXT ISNUMBER ISREF ISTEXT LEFT LEFTB LEN LENB LN LOG LOG10 LOOKUP LOWER MATCH MAX MID MIDB MIN MINUTE MOD MONTH N NA NOT NOW ODD OFFSET OR PI PRODUCT PROPER RAND REPLACE REPLACEB REPT RIGHT RIGHTB ROUND ROUNDDOWN ROUNDUP ROW ROWS SEARCH SEARCHB SECOND SIGN SIN SINH SQRT STDEV STDEVP SUBSTITUTE SUM SUMIF SUMSQ T TAN TANH TEXT TIME TIMEVALUE TODAY TRIM TRUE TRUNC TYPE UPPER VALUE VAR VARP VLOOKUP WEEKEND YEAR A3. Autorepeat Areas Autorepeat areas expand the power of the Chromeleon reports. Any area of a sheet can be defined as an autorepeat area to be printed repeatedly for each set of channel, sample or peak information. For example, a summary table might report values for a specific peak in all samples. If there is more than one peak, it can be time consuming to create a new summary table for each peak. Instead, the summary table can be defined as an autorepeat area that automatically reprints for each peak in the method. The following autorepeat area options are available. A3.1. Print and Repeat Channels This option applies if more than one data channel is acquired, for example, one channel at 230 nm and one channel at 254 nm. Chromeleon can autorepeat an area for all channels, or for specific channels. Figure 18 shows an automatically-generated two-page chromatogram and integration report for a sample with two data channels. A3.2. Repeat Samples This option prints information about different samples on the same page. For example, it can print all chromatograms of the same replicates on the same page. (See Figure 19.) For example, if it is necessary to display the maximum value from a set of results the MAX function can be used. Detailed explations for each function are available in the Chromeleon online help system. In all cases they operate in the same way as the equivalent functions in Microsoft Excel. 14 Reporting in Chromeleon

Figure 18. Automatically repeated chromatogram and integration report for a sample with two data channels. Figure 19. Automatically repeated chromatograms of the same replicates. Technical Note 70 15

A3.3. Print and Repeat Peaks This option applies if there is more than one peak in the chromatogram. Chromeleon can repeat an area of the report for all peaks, or for specific peaks. Figure 20 shows an automatically-generated report that shows the calibration plot for all peaks, with the associated calibration data. A4. Print Settings Each sheet in a report (e.g. integration sheet, summary sheet, etc.) has different printing requirements. For example, an integration report is generally printed for each chromatogram, whereas a summary report is printed only once. Chromeleon makes it simple to define when each sheet of a report should be printed. The main settings are: Table 7. Print Settings Do not print Print for every sample Print under certain conditions Used if sheet shows data for information only Generally used for sheets such as an integration report Generally used for sheets such as a summary report. Print under certain conditions provides several the options shown in Figure 21. Figure 20. Automatically repeated calibration plot for each peak in the chromatogram. A3.4. Benefits of the Autorepeat Function The autorepeat area function makes it possible to create a single report template that will be applicable to almost all methods. It does not matter if the method uses one data channel or several, or alyzes only one compound or several. The report will automatically adjust based on the data collected to show all the relevant results. Other benefits are: Reduced administrative costs: create and apply a single template for multiple methods. Reduced error: ensure that all data is reported correctly. Easier review (users become quickly used to locating important data settings) Figure 21. Printing conditions for sheets. These options provide very fine control over the conditions in which a sheet is printed. For example, a calibration sheet might print only for standards, or a Summary Sheet might print only for the last sample in the sequence. 16 Reporting in Chromeleon

Adobe and Acrobat are reegistered trademarks of Adobe Corporation, Microsoft is a registered trademark of Microsoft Corporation, and Chromeleon is a registered trademarks of Dionex Corporation. Passion. Power. Productivity. Dionex Corporation north America Europe Asia Pacific 1228 Titan Way P.O. Box 3603 U.S. (847) 295-7500 Cada (905) 844-9650 Austria (43) 1 616 51 25 Benelux (31) 20 683 9768 (32) 3 353 4294 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Australia (61) 2 9420 5233 Chi (852) 2428 3282 India (91) 22 28475235 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190 Sunnyvale, CA Ireland (353) 1 885 1700 Italy (39) 02 51 62 1267 Switzerland (41) 62 205 9966 South America LPN 1925 04/07 94088-3603 United Kingdom (44) 1276 691722 Brazil (55) 11 3731 5140 (408) 737-0700 www.dionex.com 2007 Dionex Corporation Technical Note 70 17