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Optimization in Drug Discovery pp 431–443Cite as

General Guidelines for Setting Up an In Vitro LC/MS/MS Assay

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Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

In this chapter, we will discuss the choice of proper chromatographic and mass spectrometric conditions that are critical for establishing sensitive and robust in vitro and in vivo assays. Liquid chromatography combined with mass spectrometry (LC/MS) has been a primary tool for quantitation of analytes for in vitro and in vivo assays. This is due to the sensitivity and selectivity of modern LC/MS instrumentation and materials which routinely allow detection to the low ng/mL range for many analytes. The preparation of in vitro and in vivo generated samples requires that analytes are efficiently extracted with minimal chemical or enzymatic degradation both before and during LC/MS analysis. Modern chromatographic and MS ionization methods allow quantitation of analytes from small, polar organic molecules to large proteins and peptides over a wide dynamic range. Tandem MS methods via multiple reaction monitoring (MRM) has extended quantitation to low abundance analytes previously requiring radiometric detection. Quantitation software is integral to all MS operating software and will accurately determine concentrations of unknown analyte solutions based on calibration curves generated from known standard solutions.

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References

  1. Chu I, Nomeir AA (2006) Utility of mass spectrometry for in-vitro ADME assays. Curr Drug Metab 7:467–477

    Article  PubMed  CAS  Google Scholar 

  2. Wan H, Holmén AG (2009) High throughput screening of physicochemical properties and in-vitro ADME profiling in drug discovery. Comb Chem High Throughput Screen 12:315–329

    Article  PubMed  CAS  Google Scholar 

  3. Hirtz J (1986) Importance of analytical methods in pharmacokinetic and drug metabolism studies. Biopharm Drug Dispos 7(4):315–326

    Article  PubMed  CAS  Google Scholar 

  4. Han J, Datla R, Chan S, Borchers CH (2009) Mass spectrometry-based technologies for high-throughput metabolomics. Bioanalysis 1(9):1665–1684

    Article  PubMed  CAS  Google Scholar 

  5. Schwartz JC, Cooks GR (1988) Recent developments in tandem mass spectrometry. Spectroscopy 5(1–3):49–63

    Google Scholar 

  6. Dixit V, Hariparsad N, Desai P, Unadkat JD (2007) In-vitro LC-MS cocktail assays to simultaneously determine human cytochrome P40 activities. Biopharm Drug Dispos 28:257–262

    Article  PubMed  CAS  Google Scholar 

  7. Lin T, Pan K, Mordenti J, Pan L (2007) In-vitro assessment of cytochrome P450 inhibition: strategies for increasing LC/MS based assay throughput using a one-point IC50 method and multiplexing high performance liquid chromatography. J Pharm Sci 96(9):2485–2493

    Article  PubMed  CAS  Google Scholar 

  8. Tolonen A, Petsalo A, Turpeinen M, Uusitalo J, Pelkonen O (2007) In-vitro interaction cocktail assay for nine major cytochrome P450 enzymes with 13 probe reactions and a single LC/MSMS run: analytical validation and testing with monoclonal anti-CYP antibodies. J Mass Spectrom 42:960–966

    Article  PubMed  CAS  Google Scholar 

  9. Smalley J, Marino AM, Xin B, Olah T, Balimane PV (2007) Development of a quantitative LC-MS/MS analytical method coupled with turbulent flow chromatography for digoxin for the in vitro P-gp inhibition assay. J Chromatogr B Analyt Technol Biomed Life Sci 845:260–267

    Article  Google Scholar 

  10. Wang Z, Hop CECA, Leung KH, Pang J (2000) Determination of in vitro permeability of drug candidates through caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry. J Mass Spectrom 35:71–76

    Article  PubMed  CAS  Google Scholar 

  11. Caldwell GW, Easlick SM, Gunnet J, Masucci JA, Demarest K (1998) In vitro permeability of eight β-blockers through caco-2 monolayers utilizing liquid chromatography/electrospray ionization mass spectrometry. J Mass Spectrom 33:607–614

    Article  PubMed  CAS  Google Scholar 

  12. Soglia JR, Contillo LG, Kalgutkar AS, Zhao S, Hop CE, Boyd JG, Cole MJ (2006) A semiquantitative method for the determination of reactive metabolite conjugate levels in vitro utilizing liquid chromatography-tandem mass spectrometry and novel quaternary ammonium glutathione analogues. Chem Res Toxicol 19:480–490

    Article  PubMed  CAS  Google Scholar 

  13. Chen M, Howe D, Leduc B, Kerr S, Williams DA (2007) Identification and characterization of two chloramphenicol glucuronides from the in vitro glucuronidation of chloramphenicol in human liver microsomes. Xenobiotica 37(9):954–971

    Article  PubMed  CAS  Google Scholar 

  14. Bradshaw HB, Rimmerman N, Hu SSJ, Benton VM, Stuart JM, Masuda K, Cravatt BF, O’Dell DK, Walker JM (2009) The endocannabinoids anandamide is a precursor for the signaling lipid N-arachidonoyl glycine by two distinct pathways. BMC Biochem 10:14

    Article  PubMed  Google Scholar 

  15. Lindqvist A, Hilke S, Skoglund E (2004) Generic three-column parallel LC-MS/MS system for high-throughput in vitro screens. J Chromatogr A 1058:121–126

    PubMed  CAS  Google Scholar 

  16. Castro-Perez J, Plumb R, Liang L, Yang E (2005) A high-throughput liquid chromatography/tandem mass spectrometry method for screening glutathione conjugates using exact mass neutral loss acquisition. Rapid Commun Mass Spectrom 19:798–804

    Article  PubMed  CAS  Google Scholar 

  17. Luippold AH, Arnhold T, Jörg W, Krüger B, Süssmuth RD (2011) Application of a rapid and integrated analysis system (RIAS) as a high throughput processing tool for in vitro ADME samples by liquid chromatography/tandem mass spectrometry. J Biomol Screen 16:370–377

    Article  PubMed  CAS  Google Scholar 

  18. Luippold AH, Arnhold T, Jörg W, Süssmuth RD (2010) An integrated platform for fully automated high-throughput LC-MS/MS analysis of in vitro metabolic stability assay samples. Int J Mass Spectrom 296:1–9

    Article  CAS  Google Scholar 

  19. Drexler DM, Edinger KJ, Mongillo JJ (2007) Improvements to the sample manipulation design of a LEAP CTC HTS PAL autosampler used for high-throughput qualitative and quantitative liquid chromatography-mass spectrometry assays. J Assoc Lab Autom 12:152–156

    Article  CAS  Google Scholar 

  20. Drexler D, Barlow DJ, Falk P, Cantone J, Hernandez D, Ranasinghe A, Sanders M, Warrack B, McPhee F (2006) Development of an on-line automated sample clean-up method and liquid chromatography-tandem mass spectrometry analysis: application in an in vitro proteolytic assay. Anal Bioanal Chem 384:1145–1154

    Article  PubMed  CAS  Google Scholar 

  21. Shin S, Fung H (2011) Evaluation of an LC-MS/MS assay for 15N-nitrite for cellular studies of L-arginine action. J Pharm Biomed Anal 56:1127–1131

    Article  PubMed  CAS  Google Scholar 

  22. Cox JM, Troutt JS, Knierman MD, Siegel RW, Qian Y, Ackerman BL, Konrad RJ (2012) Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation. Anal Biochem 430:130–137

    Article  PubMed  CAS  Google Scholar 

  23. Otten JN, Hingorani GP, Hartley SD, Kragerud SD, Franklin RB (2011) An in vitro, high throughput, seven CYP cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS. Drug Metab Lett 5:17–24

    Article  PubMed  CAS  Google Scholar 

  24. Grosse CM, Davis IM, Arrendale RF, Jersey J, Amin J (1994) Determination of remifentanil in human blood by liquid-liquid extraction and capillary GC-HRMS-SIM using a deuterated internal standard. J Pharm Biomed Anal 12(2):195–203

    Article  PubMed  CAS  Google Scholar 

  25. Cañas B, Piñeiro C, Calvo E, López-Ferrer D, Gallardo JM (2007) Trends in sample preparation for classical and second generation proteomics. J Chromatogr A 1153:235–258

    Article  PubMed  Google Scholar 

  26. Shi T, Su D, Liu T, Tang K, Camp DG, Qian W, Smith RD (2012) Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics. Proteomics 12:1074–1092

    Article  PubMed  CAS  Google Scholar 

  27. Badman ER, Beardsley RL, Liang Z, Bansal S (2010) Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns. J Chromatogr B Analyt Technol Biomed Life Sci 878:2303–2313

    Article  Google Scholar 

  28. Gama MR, Silva R, Collins CH, Bottoli CBG (2012) Hydrophilic interaction chromatography. Trends Anal Chem 37:44–60

    Article  Google Scholar 

  29. Meira GR, Vega JR (2010) Band broadening in GPC/SEC. In: Cazes J (ed) Encyclopedia of chromatography, vol 1. CRC Press, Boca Raton, FL, pp 147–156

    Google Scholar 

  30. Burgess K, Creek D, Dewsbury P, Cook K, Barrett MP (2011) Semi-targeted analysis of metabolites using capillary-flow ion chromatography coupled to high resolution mass spectrometry. Rapid Commun Mass Spectrom 25:3447–3452

    Article  PubMed  CAS  Google Scholar 

  31. Kane MA, Folias AE, Wang C, Napoli JL (2008) Quantitative profiling of endogenous retinoic acid in vivo and in vitro by tandem mass spectrometry. Anal Chem 80:1702–1708

    Article  PubMed  CAS  Google Scholar 

  32. Strege MA (1999) High-performance liquid chromatographic-electrospray ionization mass spectrometric analyses for the integration of natural products with modern high-throughput screening. J Chromatogr B Biomed Sci Appl 725(1):67–78

    Article  PubMed  CAS  Google Scholar 

  33. Lee H (2005) Pharmaceutical applications of liquid chromatography coupled with mass spectrometry. J Liq Chrom Relat Tech 28:1161–1202

    Article  CAS  Google Scholar 

  34. Woodget BW, Cooper D (1987) Analytical standards and calibration curves from samples and standards. Wiley, London, pp 109–145

    Google Scholar 

  35. Shou WZ, Weng N (2005) Simple means to alleviate sensitivity loss by trifluoroacetic acid (TFA) mobile phases in the hydrophilic interaction chromatography-electrospray tandem mass spectrometric (HILIC-ESI/MS/MS) bioanalysis of basic compounds. J Chromatogr B Analyt Technol Biomed Life Sci 825(2):186–192

    Article  PubMed  CAS  Google Scholar 

  36. Hoffman D (2009) Statistical considerations for assessment of bioanalytical incurred sample reproducibility. AAPS J 11(3):570–580

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The views expressed here are solely those of the author and do not reflect the opinions of Janssen Research & Development, LLC.

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Authors and Affiliations

  1. CREATe Analytical Sciences, Janssen Research & Development, LLC, Spring House, PA, USA

    John A. Masucci & Gary W. Caldwell

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  1. John A. Masucci
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  2. Gary W. Caldwell
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Editor information

Editors and Affiliations

  1. CREATe Core Science Technologies, Janssen Research & Development, LLC, Spring House, Pennsylvania, USA

    Gary W. Caldwell

  2. CREATe Core Science Technologies, Janssen Research & Development, LLC, Spring House, Pennsylvania, USA

    Zhengyin Yan

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Masucci, J.A., Caldwell, G.W. (2014). General Guidelines for Setting Up an In Vitro LC/MS/MS Assay. In: Caldwell, G., Yan, Z. (eds) Optimization in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-742-6_25

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  • DOI: https://doi.org/10.1007/978-1-62703-742-6_25

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-741-9

  • Online ISBN: 978-1-62703-742-6

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