A Complete Solution for Method Linearity in HPLC and UHPLC

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1 Now sold under the Thermo Scientific brand A Complete Solution for Method Linearity in HPLC and UHPLC Frank Steiner, Fraser McLeod, Tobias Fehrenbach, and Andreas Brunner Dionex Corporation, Germering, Germany Introduction Validation for linearity requires the preparation and analysis of a set of several independently prepared solutions. As an example, according to ICH guidelines, HPLC method linearity is normally based on five concentration levels between 70% and 130% of the nominal concentration, each to be injected three times. The laboratory effort for this process can be substantial, especially if the method is required to quantify several substances simultaneously. To save time, it is common to prepare only one stock solution and subsequently dilute it to the different concentration levels. This procedure means that any error in the stock solution preparation is carried through all subsequent dilutions. In addition, it means that the preparation of the standards differs from the process used in day-to-day operation of the method, thus making comparison of the two processes difficult. This study presents a fully automated gravimetric solution to prepare all solutions for a calibration or linearity test independently and without error-prone manual interference. All solutions prepared are immediately ready for injection onto the UHPLC analysis system. A comprehensive software package establishes the sequence for linearity validation automatically. When the HPLC results are available, linearity according to ICH guidelines is instantaneously calculated by the software, and all results are assessed against specification requirements. This complete solution accelerates the process significantly, excludes typical operator errors that occur during manual sample preparation and result calculation, and produces results of full statistical integrity. Experimental Instrumental Configuration UltiMate 3000 Basic Automated System supporting pressures up to 620 bar at flow rates up to 10 ml/min, consisting of the modules listed in Table 1. Table 1. HPLC Module Configuration and Connection Tubing Pump Sampler Column ACC-3000 Compartment Detector Capillaries LPG-3400SD with 200 µl SpinFlow mixer kit DAD-3000 with semi-analytical flow cell (2.5 µl) Pump Sampler Sampler Column Column Detector mm Viper mm Viper mm Viper

2 UHPLC Method Parameters The UHPLC method for analysis of soft drink ingredients used a core shell (fused core)-based 2.6 µm stationary phase material from Phenomenex. Detailed method parameters are listed in Table 2. Table 2. UHPLC Column and Method Parameters for Soft Drink Analysis Column Phenomenex Kinetex mm, 2.6 µm, C18 Precolumn Phenomenex KrudKatcher, 0.5 µm Injection 20 µl Full-loop injection Volume Temperature 45 C Maximum Pressure 515 bar Data Collection 50 Hz/0.1 s Rate/Rise Time Eluent A Water/0.075% formic acid (ph 3.65) Eluent B CH 3 CN/0.06% formic acid Gradient 0.0 min 7% B 3.7 min 26% B 4.0 min 50% B 5.6 min 50% B 5.7 min 7% B 9.2 min 7% B Instrument Control and Data Handling The UltiMate 3000 system was controlled by Chromeleon Chromatography Data System (CDS) software, version The fully automated sequence generation, data processing, and validation report generation is based on the Chromeleon Extension Pack 2. Sample Preparation All standards for the linearity test of the soft drink analysis method were prepared using automation to the final concentration, without further dilution steps. Solid dosing was performed with the Quantos QB1-L system (Mettler-Toledo, Greifensee, Switzerland) using QH075-B dosing heads (Figure 1). The stacked dosing of all five compounds (acesulfame K, saccharin, caffeine, vanillin, and benzoate) was performed directly into a 100 ml flask. Dosing of the methanol/water 1:1 sample solvent was also performed gravimetrically with the Quantos QB liquid dosing option (Figure 1). After dosing, vials were closed with slotted septa. The samples were then dissolved and homogenized by manual shaking before the vials were placed into the autosampler trays. Figure 1. Quantos QB1-L system for automated solid dosing and gravimetrically controlled liquid dosing. Results and Discussion Preparation of Samples for Linearity Validation Statistically, it is recommended that each concentration level for a linearity test be individually prepared. This has the effect of randomizing potential sources of error (e.g., a possible incorrect weighing for one of the standards). This is a time consuming process, however, so many laboratories prepare a stock solution and then dilute it to the different concentration levels. The advantage of this approach is that it is a fast way of preparing standards, but the major disadvantage is that any error in the stock solution will be carried through to the diluted standards. A better approach is to automate preparation of the standards. This can be achieved using the new Quantos QB1-L system from Mettler-Toledo. The system can automatically dispense and weigh analytes into HPLC vials or volumetric flasks. It can also weigh in the appropriate amount of diluents in order to provide a gravimetric solution. This experiment tested the linearity of five analytes in a soft drink analysis. The Quantos system automatically weighed the correct amount of each analyte, then the correct amount of diluent to provide the concentrations shown in Table 3. The time required to prepare these standards was only 50 min. A manual preparation based on the stock solution approach would take approximately 2 h, or 4 h for the and manual individual preparation of all concentration levels, would require approximately 4 h. 2 Optimization A Complete Solution of the Separation for Method Power Linearity of 1-D in Nano HPLC LC and Analysis UHPLC of Proteomics Samples

3 Table 3. Concentrations (mg/100 g) of Soft Drink Analytes Prepared by Quantos QB1-L Analyte Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Acesulfame K Saccharin Caffeine Vanillin Benzoate Speeding the Analysis Method Using classic HPLC, a single soft drink analysis may take approximately 30 min. This means that the total run time for the linearity experiment will be 7.5 h (5 calibration levels 3 injections per level 30 min). With UHPLC, however, it is possible to signficantly decrease run time. Figure 2 shows the analysis of all five analytes, with all peaks separated in less than 4 min. This means that all injections can be performed in just 1.25 h. The sequence generation with three injections at five different concentration levels is performed automatically by the chromatography data system, thus reducing labor time. Data Analysis and Reporting of the Method Linearity Validation Once all data has been acquired, it is necessary to calculate the results. In keeping with ICH guidelines, the following values must be reported: correlation coefficient, y-intercept, slope of the regression line, and residual sum of squares. Laboratories must also confirm that the correlation coefficient is within the limits expected of the method (typically 0.999). Performing these calculations can be a time consuming task. Some laboratories use Excel spreadsheets in an effort to speed the process, but even this can be inefficient because users typically manually transcribe values into the spreadsheet, then have another person review the transcription. For this particular analysis, the estimated time for the use of spreadsheets and the associated review step is 2 h. Saccharin Caffeine Vanillin Benzoate Acesulfame K Minutes Figure 2. UHPLC analysis of five soft drink ingredients in less than 5 min. 3

4 The Chromeleon CDS software immediately generates results for all analytes and creates the complete report automatically. All that is required by the operator is to name the peaks and enter their concentrations a process that takes just 5 min. Figure 3 shows the automatically generated report for the analyte saccharin. Figure 3A The combination of the Quantos QB1-L and the Dionex UltiMate 3000 system produced outstanding results for the linearity experiment. Table 4 shows the correlation coefficient for all analytes, each of which has an R 2 value > Table 4. Correlation Coefficients of All Analytes Analyte R 2 Acesulfame K Saccharin Caffeine Vanillin Benzoate Save Time with Transfer to New Process As Table 5 shows, the time to perform the experiment using the traditional process is 13.5 h. This process involves manual sample preparation with individual weighing, HPLC analysis at conventional speed, manual data processing, and report creation. In contrast, the fully automated new UHPLC-based process can complete the required steps in just over 2 h, which corresponds to a more than 6-fold overall productivity increase. Figure 3B Table 5. Time to Perform Linearity Experiment: Traditional vs New Automated Process Step Time Taken (Traditional Process) Time Taken (New Process) Sample Prep 240 min 50 min Analysis 450 min 75 min Results 120 min 5 min Total 810 min 130 min 4 Optimization A Complete Solution of the Separation for Method Power Linearity of 1-D in Nano HPLC LC and Analysis UHPLC of Proteomics Samples

5 Summary The automated Quantos system enables a 4-fold reduction in sample preparation time, relative to manual weighing and liquid dosing. This approach encourages individual weighing for all concentration levels (rather than diluting from a stock solution) yet avoids human error and provides full electronic documentation. Transfer of the analysis to the new UHPLC process can accelerate separation time by factor of 6, and yield chromatograms with excellent peak resolution for the highest accuracy of analytical results. The greatest potential for time saving is in the fully automated data processing and reporting step. Using Chromeleon Extension Pack 2 for ICH-conforming method validation, this task can be performed up to 24 faster than the manual approach. In addition, the new process excludes possible errors associated with transfer of data from the chromatography software to the data evaluation and reporting software. Chromeleon and UltiMate are registered trademarks, and Viper is a trademark of Dionex a Thermo Fisher Scientific Corporation. KrudKatcher is a trademark of Phenomenex, Inc. Speed Simplicity Solutions Dionex Products north America Europe Asia Pacific 1228 Titan Way U.S. / Canada (847) Austria (43) Benelux (31) ; (32) P.O. Box 3603 Denmark (45) France (33) Germany (49) Sunnyvale, CA Ireland (353) Italy (39) Sweden (46) Taiwan (886) south America Switzerland (41) United Kingdom (44) Brazil (55) (408) Australia (61) China (852) India (91) Japan (81) Korea (82) Singapore (65) LPN / Thermo Fisher Corporation, Inc.

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