Analytical and Bioanalytical Chemistry

, Volume 400, Issue 8, pp 2371–2381 | Cite as

Development and validation of a LC-MS/MS method based on a new 96-well Hybrid-SPE-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma

  • Karin Ardjomand-Woelkart
  • Manfred Kollroser
  • Li Li
  • Hartmut Derendorf
  • Veronika Butterweck
  • Rudolf Bauer
Original Paper


A rapid and selective high-throughput HESI-LC-MS/MS method for determining eight cytochrome P450 probe drugs in one-step extraction and single run was developed and validated. The four specific probe substrates midazolam, dextromethorphan, tolbutamide, theophylline and their metabolites 1-hydroxymidazolam, dextrorphan, hydroxyl(methyl)tolbutamide, 1,3-dimethyluric acid, together with the deuterated internal standards, were extracted from rat plasma using a novel 96-well Hybrid-SPE™-precipitation technique. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11 min. The intra- and inter-day precisions for all eight substrates/metabolites were 1.62–12.81% and 2.09–13.02%, respectively, and the relative errors (accuracy) for the eight compounds ranged from −9.62% to 7.48% and −13.84% to 8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool for the evaluation of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2 and 2D6) activities with a cocktail approach in rats to clarify herb–drug interactions. The method can be used as a basic common validated high-throughput analytical assay for in vivo interaction studies.


The new used 96 well Hybrid-SPE™-precipitation plate for plasma extraction.


Hybrid-SPE™ precipitation Validation Phospholipids CYP450 



The Austrian Science Fund (FWF) is thanked for the award of an Erwin-Schroedinger scholarship to K. Ardjomand-Woelkart (J2754-B05).

Supplementary material

216_2010_4618_MOESM1_ESM.pdf (829 kb)
ESM 1 (PDF 828 kb)


  1. 1.
    Lazarou J, Pomeranz BH, Corey PN (1998) Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. JAMA 279:1200–1205CrossRefGoogle Scholar
  2. 2.
    Butterweck V, Derendorf H (2008) Potential of pharmacokinetic profiling for detecting herbal interactions with drugs. Clin Pharmacokinet 47:383–397CrossRefGoogle Scholar
  3. 3.
    Boon H, Jurgens T (2001) Drug interactions with natural health products. Health Canada. Available at: Accessed 9 Apr 2008
  4. 4.
    Zhang S, Song N, Li Q, Fan H, Liu C (2008) Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats. J Chromatogr B 871:78–89CrossRefGoogle Scholar
  5. 5.
    Uchida S, Yamada H, Li YD, Maruyama S, Ohmori Y, Oki T, Watanabe H, Umegaki K, Ohashi K, Yamada S (2006) Effects of Ginkgo biloba extract on pharmacokinetics and pharmacodynamics of tolbutamide and midazolam in healthy volunteers. J Clin Pharmacol 46:1290–1298CrossRefGoogle Scholar
  6. 6.
    Tomalik-Scharte D, Jetter A, Kinzig-Schippers M, Skott A, Sörgel F, Klaassen T, Kasel D, Harlfinger S, Doroshyenko O, Frank D, Kirchheiner J, Bräter M, Richter K, Gramatte T, Fuhr U (2005) Effect of propiverine on cytochrome P450 enzymes: a cocktail interaction study in healthy volunteers. Drug Metab Dispos 33:1859–1866Google Scholar
  7. 7.
    Jerdi MC, Daali Y, Oestreicher MK, Cherkaoui S, Dayer P (2004) A simplified analytical method for a phenotyping cocktail of major CYP450 biotransformation routes. J Pharm Biomed 35:1203–1212CrossRefGoogle Scholar
  8. 8.
    Zhang W, Han F, Guo P, Zhao H, Lin Z, Huang M-Q, Bertelsen K, Weng N (2010) Simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma by LC-MS/MS—a high throughput approach to evaluate drug–drug interactions. J Chromatogr B 878:1169–1177CrossRefGoogle Scholar
  9. 9.
    Chen Y-L, Junga H, Jiang X, Naidong W (2003) Simultaneous determination of theophylline, tolbutamide, mephenytoin, debrisoquin, and dapsone in human plasma using high-speed gradient liquid chromatography/tandem mass spectrometry on a silica-based monolitihic column. J Sep Sci 26:1509–1519CrossRefGoogle Scholar
  10. 10.
    Liu Y, Jiao J, Zhang C, Lou J (2009) A simplifed method to determine five cytochrome P450 probe drugs by HPLC in a single run. Biol Pharm Bull 32:717–720CrossRefGoogle Scholar
  11. 11.
    Yin OQP, Lam SSL, Lo CMY, Chow MSS (2004) Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: application to cytochrome P450 phenotyping studies. Rapid Commun Mass Spectrom 18:2921–2933CrossRefGoogle Scholar
  12. 12.
    Pucci V, Di Palma S, Alfieri A, Monelli F, Monteagudo E (2009) A novel strategy for reducing phospholipids-based matrix effect in LC-ESI-MS bioanalysis by means of HybridSPE. J Pharm Biomed Anal 50:867–871CrossRefGoogle Scholar
  13. 13.
    FDA (2001) Guidance for industry: bioanalytical method validation. US Department of Health and Human Services FDA, RockvilleGoogle Scholar
  14. 14.
    Dams R, Huestis MA, Lambert WE, Murphy CM (2003) Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: influence of ionization type, sample preparation, and biofluid. J Am Soc Mass Spectrom 14:1290–1294CrossRefGoogle Scholar
  15. 15.
    Nagata M, Hidaka M, Sekiya H, Kawano Y, Yamasaki K, Okumura M, Arimori K (2007) Effects of pomegranate juice on human cytochrome P450 2C9 and tolbutamide pharmacokinetics in rats. Drug Metab Dispos 35:302–305CrossRefGoogle Scholar
  16. 16.
    Fuhr U, Jetter A, Kirchheiner J (2007) Appropriate phenotyping procedures for drug metabolizing enzymes and transporters in humans and their simultaneous use in the “cocktail” approach. Clin Pharmacol Ther 81:270–283CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Karin Ardjomand-Woelkart
    • 1
  • Manfred Kollroser
    • 2
  • Li Li
    • 3
  • Hartmut Derendorf
    • 3
  • Veronika Butterweck
    • 3
  • Rudolf Bauer
    • 1
  1. 1.Institute of Pharmaceutical Sciences, Department of PharmacognosyKarl-Franzens-University GrazGrazAustria
  2. 2.Institute of Forensic MedicineMedical University of GrazGrazAustria
  3. 3.Department of Pharmaceutics, College of PharmacyUniversity of FloridaGainesvilleUSA

Personalised recommendations