A Study of Stability, Robustness and Time Efficiency of a New HPLC and a New Tandem MS

Transcription

1 A Study of Stability, Robustness and Time Efficiency of a New HPLC and a New Tandem MS Jason Lai, Jia Wang, Brad Hart, Pavel Aronov, Kristine Van Natta, Marta Kozak, Jorge Valdivia, Andy Jacobs, Haibo Wang, Erkan Diri Thermo Fisher Scientific, San Jose, CA, USA

2 Overview Purpose: This preliminary study focused on the analytical performance of a new two-channel HPLC and a new Tandem MS in the areas of stability, robustness and time efficiency. Methods: The testing method included analyzing synthetic serum specimens spiked with aplrozolam standard and stable isotopic internal standard. The equipment in use were prototype units of Thermo Scientific Prelude MD HPLC and Thermo Scientific Endura MD MS. The data analysis was performed on Thermo Scientific ClinQuan MD prototype software. Results: A total of 1241 injections were analyzed continuously in 60 hours. The overall analytical performance are summarized as in the following: %RSD of retention time for cross channel in HPLC was 0.94%; %RSD of concentration measurement was 2.02%; %RSD of mass ion ratios of conforming ion to quantifying ion of test compound alprozolam and internal standard were <2% and <4%, respectively. The Prelude MD HPLC with cross channel optimization ensured full utilization of MS, resulting in double sample throughput and reduced total run time by 50%. Syringe pump provided good precision of retention time and low solvent consumption. Endura MD MS provided stable ion ratios for quantitative accuracy. The test results demonstrated that both Prelude MD HPLC and Endura MD MS were stable, robust and efficient. Introduction Stability, robustness and time efficiency of HPLC-tandem MS are three major challenges in laboratories where a large number of samples are analyzed routinely. Here we report on a study with a newly designed two-channel HPLC and a newly designed tandem mass spectrometer to address these three challenges. 2 A Study of Stability, Robustness and Time Efficiency of a New HPLC and a New Tandem MS

3 Methods Sample Preparation Calibration Standards, Internal Standard (IS), QC sample and test sample were spiked with Alprazolam standard in crashed synthetic serum as shown in Table 1 below. TABLE 1. Concentrations of Various Samples Various Samples Concentrations (ng/ml) Calibration Standard (Alprazolam) 1, 2, 5, 10, 25, 50 and 100 Internal Standard (Alprazolam-D5) 5 QC (Alprazolam) Mean 4.63 Test Sample (Alprazolam) Mean Liquid Chromatography (or more generically Separations) The Prelude MD HPLC consists of two separation channels, both of which include an on-line sample cleanup Thermo Scientific TurboFlow column for removing sample matrix, and an analytical column for compound separation. The instrument is capable of crosschannel sequencing for efficient use of the mass spectrometer time. The HPLC parameters for each channel (Channel 1 and Channel 2) were set as in Table 2 below. TABLE 2. HPLC Parameters HPLC Parameters TurboFlow Column Thermo Scientific Cyclone-P (50x0.5mm) Analytical Column Thermo Scientific Accucore aq column (50x2.1mm, 2.6um) Loading Mobile Phase A 10 mm ammonium formate, 0.05% formic acid in water Loading Mobile Phase B 10 mm ammonium formate, 0.05% formic acid in methanol Loading Mobile Phase C Isopropanol:acetonitrile:acetone (45:45:10) Eluting Mobile Phase A 0.05% formic acid in water Eluting Mobile Phase B 0.05% formic acid in methanol Injection Volume 10 ul Thermo Scientific Poster Note PN64240-MSACL-EN 0814S 3

4 Mass Spectrometry The Endura MD MS is a tandem MS equipping with the neutral blocker that keeps ion optics cleaner, and the advanced electronics that maintains mass stability. Tandem MS was operated in selected reaction monitor (SRM) mode with heated electrospray ionization (HESI) probe in positive ion mode using the following transition parameters in Table 3. TABLE 3. Tandem MS Parameters Compound Alprazolam Alprazolam-D5 IS Precursor Product (m/z) (m/z) Type Confirming Ion Confirming Ion Quantifying Ion Confirming Ion Confirming Ion Quantifying Ion Data Analysis The test results of LC-MS analysis were processed using ClinQuan MD prototype software. Results HPLC Solvent Consumption This HPLC instrument uses a low amount of solvents for each injection, and Table 4 below shows the solvent consumption for each injection in this study. TABLE 4. Low Solvent Consumption Solvent Loading Mobile Phase A Loading Mobile Phase B Loading Mobile Phase C Eluting Mobile Phase A Eluting Mobile Phase B Usage per Injection 2.83 ml 1.40 ml 1.00 ml 1.87 ml 0.53 ml 4 A Study of Stability, Robustness and Time Efficiency of a New HPLC and a New Tandem MS

5 Improving Productivity; Maximizing MS Utilization and Throughput This HPLC includes automation of online sample cleanup using TurboFlow Technology, and removes tedious hand-on manual steps. The use of two HPLC channel separations can minimize MS idle time while waiting for chromatographic elution without data acquisition in a typical single channel HPLC-MS analysis. A total of 1241 injections were analyzed with 621 samples and 620 samples running on channel 1 and 2, respectively. Figure 1 depicts the schematic process of cross channel sequencing of tow parallel HPLC separations. FIGURE 1. Cross channel sequencing for two parallel HPLC separations with series MS detection increase MS utilization HPLC Performance: Stability, Precision and Robustness This HPLC instrument uses syringe pumps instead of conventional piston pumps, which provides stability and precision in chromatographic separation. A total of 1241 crashed synthetic samples spiked with alprazolam standard and isotopic internal standard were analyzed continuously in 60 hours, with an additional 25 QC samples inserted. The measurements of retention time showed a precision of %RSD at 0.94% as shown in Figure 2. All QC values were within 2 standard deviations. Thermo Scientific Poster Note PN64240-MSACL-EN 0814S 5

6 FIGURE 2. Retention Time Plot of 1241 injections from two HPLC channels ; Average RT = min, %RSD = 0.94% Tandem MS Performance: Stability, Precision and Robustness This Tandem MS provides excellent stability of quantitative measurement. The %RSD of concentration measurement of these 1241 injections was 2.02% As shown in Figure 3 All QC values were within 2 standard deviations. FIGURE 3. Concentration Plot of 1241 injections; Average Concentration = ng/ml, %RSD = 2.02% 6 A Study of Stability, Robustness and Time Efficiency of a New HPLC and a New Tandem MS

7 Stability of Mass Ion Ratios The ion ratio of confirming ion to quantifying ion demonstrated an excellent stability over 1241 injections as shown in the Table 5 below. TABLE 5. Stability of Mass Ion Ratios Compound Ion Ratio Average %RSD Alprazolam m/z to m/z m/z to m/z Alprazolam-D5 IS m/z to m/z m/z to m/z Conclusion This preliminary study demonstrated analytical performance of Prelude MD HPLC and Endura MD MS are stable, robust and timeefficient for LC-Tandem MS analysis of this sample set of 1241 injections and a continuous run time of 60 hours. % RSD of retention time in cross channel separations: 0.94% % RSD of concentrations: 2.02% % RSD of mass ion ratios of alprazolam: <2% % RSD of mass ion ratios of apprazolam-d5 IS: <4% Thermo Scientific Poster Note PN64240-MSACL-EN 0814S 7

8 FIGURE 4. Prelude MD HPLC (Left) and Endura MD MS (Right) For in vitro diagnostic use. Not available in all countries Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientific, San Jose, CA USA is ISO Certified. Africa Australia Austria Belgium Canada China (free call domestic) Denmark Europe-Other Finland France Germany India Italy Japan Latin America Middle East Netherlands New Zealand Norway Russia/CIS Singapore Spain Sweden Switzerland UK USA PN64240-EN 0814S