Development and Validation of Methods for Chemotherapy Drugs on the New Prelude SPLC LC-MS/MS System

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1 Development and Validation of Methods for Chemotherapy Drugs on the New Prelude SPLC LC-/ System Kerry Hassell, Dayana Argoti, Sarah J. Fair and Joseph Herman Thermo Fisher Scientific, Franklin, MA, USA

2 Overview Purpose: To verify research methods to quantify a variety of chemotherapeutic drugs on the new Thermo Scientific Prelude sample preparation-liquid chromatography (SPLC) system, coupled to a Thermo Scientific TSQ mass spectrometer. The LC// platform reduces solvent consumption, requires less maintenance, and is easier to use then traditional systems. Methods: Prelude SPLC, Turbulent Flow Chromatography, LC//, chemotherapeutics Results: Methods for the chemotherapeutic drugs,, and Imatinib, were validated using a Prelude SPLC system and TSQ mass spectrometer platform. Introduction Bioanalysis using LC-/ can be difficult due to complex sample preparation and errors in sample handing, which can lead to variability. The use of liquid chromatography-tandem mass spectrometry (LC-/) to quantify chemotherapeutic drugs (i.e., busulfan, methotrexate, imatinib, and docetaxel) is common practice. We demonstrate the application of Prelude SPLC system in developing a faster, more reproducible and lower solvent consuming methods for quantification of chemotherapeutic drugs. Methods Preparation The samples in every method were prepared in human plasma. preparation consisted of protein precipitation with organic solvents that contained internal standard, vortexed, followed by centrifugation. The supernatant was removed and injected into the Prelude SPLC system for analysis. Liquid Chromatography On-line sample clean-up was performed by.x mm Thermo Scientific TurboFlow columns and analytical separation was performed with Thermo Scientific Accucore x2.1mm, 2.6 µm particle size columns. For iminatib, busulfan, and methotrexate, the TurboFlow HTLC-C18 XL column and Accucore PFP analytical column were used. For docetaxel, the TurboFlow HTLC-C8 XL column and Accucore C8 analytical column were used. Mobile phases were (A) 1 mm ammonium formate,.% formic acid in water; (B) 1 mm ammonium formate,.% formic acid in methanol; and (C) 4/4/1 acetonitrile/isopropanol/acetone for iminatib, busulfan, and methotrexate. Docetaxol used acetonitrile for mobile phase B. All run times were 4 minutes or less and multiplexed to 2 minutes per sample. These methods consumed less than 3 ml of mobile phase per injection. Structures of Chemotherapeutic Drugs The Prelude SPLC system use method for on-line clean-up, a 1. The first step (Figure 1A lo load the sample with aqueous onto the TurboFlow column un conditions. Under turbulent flo large molecule (>1 kda) cann the stationary phase and are w while the analytes of interest a the column. Since the majority interferences are from the mat analyte of interest is removed during step 1. Once the sampl from the matrix, the valves sw TurboFlow column is back-flus solvent stored in the loop of th from the previous injection), w analyte of interest from the Tu to the analytical column (Figur step). In order to focus the ana analytical column, the flow from column is teed to a valve with from a second pump. The sep the analytical column provides chromatographic peaks and fu any interferences. The elution switches the second valve so now eluted to the mass spectr loop can be filled with the corr organic solvent for the next sa steps are complete, the valves the loading position where the cleaned and equilibrated for th injection. Mass Spectrometry Detection of eluting analytes w stage quadrupole mass spectr (HESI II) probe in positive ion Data Analysis Quantitation was calculated w Results Accuracy and precision experim separate preparations of calibra day accuracy and precision res for busulfan, 1 2 ng/ml fo methotrexate. The method prec tested. Additionally, accuracy w correlation coefficient values fo linearity throughout all concentr top stability, autosampler stabili effects, were all ~>9%. Data a standard curves for each comp of quantitation (LLOQ) for each Iminatib The improvement in run times r system verses a conventional H phases and columns were used duration of certain steps cannot chromatographic separation ne to how long it takes for solvent sample elution steps are depen steps remain the same. Howev can be reduced. On a conventio on the Prelude SPLC system. T from 1 to 6 seconds. The re minutes). A shorter run time als 2 Development and Validation of Methods for Chemotherapy Drugs on the New Prelude SPLC LC-/ System

3 iety of chemotherapeutic drugs aration-liquid chromatography ass spectrometer. The uires less maintenance, and is phy, LC//, ulfan,, tem and TSQ mass mplex sample preparation and The use of liquid S) to quantify imatinib, and docetaxel) is relude SPLC system in nt consuming methods for plasma. preparation that contained internal standard, as removed and injected into Thermo Scientific erformed with Thermo olumns. For iminatib, busulfan, n and Accucore PFP analytical -C8 XL column and Accucore.% formic acid in water; ethanol; and (C) 4/4/1 and methotrexate. Docetaxol 4 minutes or less and onsumed less than 3 ml of Drugs usulfan The Prelude SPLC system uses a two column method for on-line clean-up, as shown in Figure 1. The first step (Figure 1A loading step) is to load the sample with aqueous mobile phase onto the TurboFlow column under turbulent flow conditions. Under turbulent flow conditions, large molecule (>1 kda) cannot interact with the stationary phase and are washed to while the analytes of interest are retained on the column. Since the majority of matrix interferences are from the matrix proteins, the analyte of interest is removed from the matrix during step 1. Once the sample is removed from the matrix, the valves switch and the TurboFlow column is back-flushed with organic solvent stored in the loop of the first valve (filled from the previous injection), which elutes the analyte of interest from the TurboFlow column to the analytical column (Figure 1B transfer step). In order to focus the analyte onto the analytical column, the flow from the TurboFlow column is teed to a valve with an aqueous flow from a second pump. The separation step on the analytical column provides gaussian chromatographic peaks and further separates any interferences. The elution step (Figure 1C) switches the second valve so that the sample is now eluted to the mass spectrometer and the loop can be filled with the correct percent of organic solvent for the next sample. Once these steps are complete, the valves are returned to the loading position where the columns can be cleaned and equilibrated for the next sample injection. Results FIGURE 1. Valve positions for On-line Clean up and Analytical Separation. 2 µ L loop 2 µ L loop 2 µ L loop Mass Spectrometry Detection of eluting analytes was done with a Thermo Scientific TSQ Vantage triplestage quadrupole mass spectrometer, equipped with a heated electrospray ionization (HESI II) probe in positive ion mode using selected reaction monitoring (SRM). Data Analysis Quantitation was calculated with Thermo Scientific LCQUAN software. Accuracy and precision experiments were performed for system verification from three separate preparations of calibrators and controls on three different days. Interday and intraday accuracy and precision results were obtained at concentration ranges of 2 2 ng/ml for busulfan, 1 2 ng/ml for imatinib, 1 ng/ml for docetaxel, and 1 7 ng/ml for methotrexate. The method precision had RSD values less than 1% for all compounds tested. Additionally, accuracy was 1% of the theoretical value for all the methods. The correlation coefficient values for all the compounds ranged from.991 to.998, showing linearity throughout all concentrations and analytes. All the analytes passed carryover, bench top stability, autosampler stability, and specificity criterion. Recoveries, including matrix effects, were all ~>9%. Data are summarized in Tables 1 4. Figure 2 depicts representative standard curves for each compound tested. Representative chromatograms at the lower limit of quantitation (LLOQ) for each compound are shown in Figure 3. The improvement in run times resulting from the lower void volumes of the Prelude SPLC system verses a conventional is illustrated in Figure 4 for. The same mobile phases and columns were used for the comparison. When using on-line clean-up, the duration of certain steps cannot be changed because they are dependent on the chromatographic separation needed. The duration of other steps in the process are related to how long it takes for solvent changes to reach the column. The sample clean-up and sample elution steps are dependent on the chromatography, and therefore, the time for those steps remain the same. However, the transfer, column cleaning and re-equilibration steps can be reduced. On a conventional the transfer step was 7 seconds vs. 6 seconds on the Prelude SPLC system. The column clean-up and equilibration steps were reduced from 1 to 6 seconds. The result is a reduction in run time of 29% (:1 minutes to 3:4 minutes). A shorter run time also reduced solvent consumption by 33%. (A) (B) (C) TABLE 1. Method Range, Linearity, Compound name Method Range Imatinib TABLE 2. Intraday Accuracy and P Compound name Low QC Imatinib Intrad (%Difference TABLE 3. Interday Accuracy and P Compound name Low QC 4.76 Imatinib Inter (%Differen TABLE 4. Bench Top Stability, Auto Compound name Bench Top Sta Imatinib FIGURE 2. Standard Curves for Ea System with TSQ Peak Area Ratio (/IS) Peak Area Ratio (/IS) Imatinib Thermo Scientifi c Poster Note PN AS13_M11_KHassel_E 7/13S 3

4 FIGURE 1. Valve positions for On-line Clean up and Analytical Separation. 2 µ L loop (A) TABLE 1. Method Range, Linearity, and Recovery. Compound name Method Range (ng/ml) Linearity (r 2 ) Recovery Imatinib FIGURE 3. Representative Tested Using a Prelude SP Busul TFC column 2 µ L loop (B) TABLE 2. Intraday Accuracy and Precision. Intraday Accuracy Intraday Precision (%RSD) Compound name (%Difference from Theoretical) Low QC Mid QC High QC Low QC Mid QC High QC Imatinib TABLE 3. Interday Accuracy and Precision e (C) Interday Accuracy Interday Precision (%RSD) Compound name (%Difference from Theoretical) Low QC Mid QC High QC Low QC Mid QC High QC µ L loop Imatinib FIGURE 4. Comparison of SPLC System with TSQ hermo Scientific TSQ Vantage triplewith a heated electrospray ionization ted reaction monitoring (SRM). tific LCQUAN software. TABLE 4. Bench Top Stability, Autosampler Stability, and Selectivity. Compound name Bench Top Stability Autosampler Stability Selectivity (% of LOQ) Imatinib N/A Conventional Prelude SPLC med for system verification from three on three different days. Interday and intrad at concentration ranges of 2 2 ng/ml ng/ml for docetaxel, and 1 7 ng/ml for lues less than 1% for all compounds eoretical value for all the methods. The s ranged from.991 to.998, showing es. All the analytes passed carryover, bench criterion. Recoveries, including matrix Tables 1 4. Figure 2 depicts representative resentative chromatograms at the lower limit own in Figure 3. lower void volumes of the Prelude SPLC in Figure 4 for. The same mobile on. When using on-line clean-up, the ause they are dependent on the n of other steps in the process are related the column. The sample clean-up and atography, and therefore, the time for those lumn cleaning and re-equilibration steps nsfer step was 7 seconds vs. 6 seconds up and equilibration steps were reduced in run time of 29% (:1 minutes to 3:4 t consumption by 33%. FIGURE 2. Standard Curves for Each Compound Tested Using a Prelude SPLC System with TSQ Peak Area Ratio (/IS) Peak Area Ratio (/IS) Imatinib Discussion LC/ methods for these c liquid extraction and longer liquid-liquid extraction are ti disposal point of view. On-lin and variability of the method Prelude SPLC system reduc allows for higher sample thr capabilities of the Prelude S the solvent consumption, le The Prelude SPLC system w down time, and operating co sample clean-up prior to HP designed to deliver the volu single push of the piston. Th seals and check valves bec several hundred if not thous required on traditional therefore, syringe pumps ar Prelude SPLC system syrin the Prelude SPLC system h mobile phases possible. The reduced, resulting in shorter 4 Development and Validation of Methods for Chemotherapy Drugs on the New Prelude SPLC LC-/ System

5 arity (r 2 ) Recovery FIGURE 3. Representative Chromatograms at the LOQ for Each Compound Tested Using a Prelude SPLC System with TSQ Intraday Precision (%RSD) Low QC Mid QC High QC Imatinib Interday Precision (%RSD) 2 Low QC Mid QC High QC FIGURE 4. Comparison of the Method Run Time for on a Prelude SPLC System with TSQ to that of a Conventional System and Selectivity. pler Stability Selectivity (% of LOQ) N/A sted Using a Prelude SPLC Conventional Prelude SPLC Discussion Transfer Transfer Elution Elution Column Column Time (sec) LC/ methods for these chemotherapeutic drugs have been done previously with liquidliquid extraction and longer methods (7-1 minutes) 1-3. Off-line techniques such as liquid-liquid extraction are time consuming and costly from both a material and disposal point of view. On-line sample clean-up lowers the sample preparation duration and variability of the methods by and minimizing sample handling. Utilizing the new Prelude SPLC system reduced the run time to under 4 minutes. The shorter method time allows for higher sample throughput, which is increased further by the multiplexing capabilities of the Prelude SPLC system. The shorter run time also dramatically reduced the solvent consumption, leading to further reductions operating cost. The Prelude SPLC system was specifically designed to reduce instrument maintenance, down time, and operating costs for high-throughput LC// applications that require sample clean-up prior to analysis. The Prelude SPLC system utilizes syringe pumps designed to deliver the volume of mobile phase required for each sample analysis with a single push of the piston. This pump design greatly reduces the wear and tear on pump seals and check valves because the pistons in dual piston reciprocating pumps can move several hundred if not thousands of times per sample run. The majority of maintenance required on traditional pumps results from the wear of the seals and check valves; therefore, syringe pumps are more robust than traditional pumps. In addition, the Prelude SPLC system syringe pumps have no need for pulse dampers. The result is that the Prelude SPLC system has extremely low dead volumes, making rapid changes in mobile phases possible. The time required for many of the steps in a method to occur are reduced, resulting in shorter run times and lower solvent costs for equivalent methods Prelude SPLC System & TSQ En Cross-validation of chemotherape coupled to the new Thermo Scien spectrometry platform. Conclusion Chemotherapeutic research with method durations of 4 m The Prelude SPLC system h systems, resulting in sample time also results in reduced c The Prelude SPLC system u the need for pulse dampener pumps, does not need propo check valves. The result: far and down time. References 1. Lambert, W.E., et al. Anal C 2. Haouala, A. et al. J. Chrom. 3. Panderi, I. J. Chrom. B. 2 Acknowledgemen I would like to thank everyone in the providing support and troubleshoot For res All trademarks are the prop Thermo Scientifi c Poster Note PN AS13_M11_KHassel_E 7/13S

6 LOQ for Each Compound S Prelude SPLC System & TSQ Vantage for on a Prelude nal System olumn lean-up Column Prelude SPLC System & TSQ Endura Cross-validation of chemotherapeutics will be repeated on the Prelude SPLC system coupled to the new Thermo Scientific TSQ Endura triple-stage quadrupole mass spectrometry platform (sec) have been done previously with liquidinutes) 1-3. Off-line techniques such as ly from both a material and ers the sample preparation duration mple handling. Utilizing the new r 4 minutes. The shorter method time sed further by the multiplexing r run time also dramatically reduced ns operating cost. d to reduce instrument maintenance, LC// applications that require e SPLC system utilizes syringe pumps uired for each sample analysis with a reduces the wear and tear on pump piston reciprocating pumps can move le run. The majority of maintenance wear of the seals and check valves; itional pumps. In addition, the for pulse dampers. The result is that olumes, making rapid changes in of the steps in a method to occur are lvent costs for equivalent methods Conclusion Chemotherapeutic research methods were validated on the new Prelude SPLC with method durations of 4 minutes or less. The Prelude SPLC system has lower void volumes than conventional systems, resulting in sample run times that are 2-3% shorter. The reduced run time also results in reduced cost due to lower consumption of mobile phases. The Prelude SPLC system uses a single syringe fill per sample, which removes the need for pulse dampeners, reduces the mechanical wear and tear on the pumps, does not need proportioning valves, and removes the need for active check valves. The result: far less maintenance is required, further reducing cost and down time. References 1. Lambert, W.E., et al. Anal Chem. 2, 77, Haouala, A. et al. J. Chrom. B. 29, 877, Panderi, I. J. Chrom. B. 29, 877, Acknowledgements I would like to thank everyone in the Prelude research and development team for providing support and troubleshooting in allowing this work to be accomplished. For research use only. Not for use in diagnostic procedures. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. 6 Development and Validation of Methods for Chemotherapy Drugs on the New Prelude SPLC LC-/ System

7 213 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, Inc. and its subsidiaries. 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 91:28 Certified. Africa-Other Australia Austria Belgium Canada China Denmark Europe-Other Finland/Norway/Sweden France Germany India Italy Japan Latin America Middle East Netherlands New Zealand Russia/CIS South Africa Spain Switzerland UK USA AS13_M11_KHassel_E 6/13S

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