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Licensed Unlicensed Requires Authentication Published by De Gruyter September 21, 2011

Direct and fast determination of antiretroviral drugs by automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry in human plasma

Therese Koal, Martin Sibum, Emile Koster, Klaus Resch and Volkhard Kaever

Abstract

Background: In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma® system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis.

Methods: The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-μL sample volume, which was only diluted with 200μL of H2O (containing 500ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up.

Results: The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6min, subsequent analyses 3.3min between injections) and has been validated in terms of the limit of detection (LOD, 2–70ng/mL), lower limit of quantification (LLOQ, 78–156ng/mL), linearity (R2, 0.9971–0.9989), linear concentration range (from LLOQ to 10,000ng/mL), intra- and inter-day precision (<13.5% at LLOQ, <7.5% at high concentrations), proficiency testing accuracy (78–127%), laboratory internal accuracy (86–113%), recovery (60–110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability.

Conclusion: Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.


Corresponding author: Dr. Therese Koal, Medical School Hannover, Institute of Pharmacology, Carl-Neuberg-Straße 1, 30625 Hannover, Germany Phone: +49-511-5324098, Fax: +49-511-5328798,

References

1. Koal T, Deters M, Casetta B, Kaever V. Simultaneous determination of four immunosuppressants by means of high speed and robust on-line solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry. J Chromatogr B 2004; 805:215–22.10.1016/j.jchromb.2004.02.040Search in Google Scholar

2. Deters M, Kirchner G, Resch K, Kaever V. Simultaneous quantification of sirolimus, everolimus, tacrolimus, and cyclosporine by liquid chromatography-mass spectrometry (LC-MS). Clin Chem Lab Med 2002; 40:285–92.10.1515/CCLM.2002.045Search in Google Scholar

3. Poquette MA, Lensmeyer GL, Doran TC. Effective use of liquid chromatography-mass spectrometry (LC/MS) in the routine clinical laboratory for monitoring sirolimus, tacrolimus, and cyclosporine. Ther Drug Monit 2005; 27:144–50.10.1097/01.ftd.0000148451.35811.74Search in Google Scholar

4. Volosov A, Napoli KL, Soldin SJ. Simultaneous simple and fast quantification of three major immunosuppressants by liquid chromatography-tandem mass spectrometry. Clin Biochem 2001; 34:285–90.10.1016/S0009-9120(01)00235-1Search in Google Scholar

5. Rakhmanina NY, van den Anker J, Soldin SJ. Therapeutic drug monitoring of antiretroviral therapy. AIDS Patient Care STDS 2004; 18:7–14.10.1089/108729104322740866Search in Google Scholar

6. Boffito M, Acosta E, Burger D, Fletcher CV, Flexner C, Garaffo R, et al. Current status and future prospects of therapeutic drug monitoring and applied clinical pharmacology in antiretroviral therapy. Antivir Ther 2005; 10:375–92.10.1177/135965350501000307Search in Google Scholar

7. Koal T, Burhenne H, Römling R, Svoboda M, Kaever V. Quantification of antiretroviral drugs in dried blood spot samples by means of liquid chromatography-tandem mass spectrometry. Rapid Commun Mass Spectrom 2005; 19:2995–3001.10.1002/rcm.2158Search in Google Scholar

8. Rentsch KM. Sensitive and specific determination of eight antiretroviral agents in plasma by high-performance liquid chromatography-mass spectrometry. J Chromatogr B 2003; 788:339–50.10.1016/S1570-0232(03)00039-4Search in Google Scholar

9. Volosov A, Alexander C, Ting L, Soldin SJ. Simple rapid method for quantification of antiretrovirals by liquid chromatography-tandem mass-spectrometry. Clin Biochem 2002; 35:99–103.10.1016/S0009-9120(02)00286-2Search in Google Scholar

10. Shoup RE, Ren X, Johnson AP, Gray RA, Evarts S, Gill L, et al. Simultaneous determination of six protease/reverse transcriptase inhibitors in human plasma utilizing LC/MS/MS. Curr Separations 1999; 18:17–22.Search in Google Scholar

11. Egge-Jacobsen W, Unger M, Niemann CU, Baluom M, Hirai S, Benet LZ, et al. Automated, fast, and sensitive quantification of drugs in human plasma by LC/MS-MS. Ther Drug Monit 2004; 26:546–62.10.1097/00007691-200410000-00014Search in Google Scholar PubMed

12. Dasgupta A, Wells A, Chow L. Effect of heating human sera at a temperature necessary to deactivate human immunodeficiency virus on measurements of free phenytoin, free valproic acid, and free carbamazepine concentrations. Ther Drug Monit 1999; 21:421–5.10.1097/00007691-199908000-00007Search in Google Scholar PubMed

13. Food and Drug Administration. Guidance for industry, bioanalytical method validation. US Department of Health and Human Services, www.fda.gov/cder/Guidance/4252fnl.pdf.Search in Google Scholar

14. Kappelhoff BS, Crommentuyn KM, de Maat MM, Mulder JW, Huitema AD, Beijnen JH. Practical guidelines to interpret plasma concentrations of antiretroviral drugs. Clin Pharmacokinet 2004; 43:845–53.10.2165/00003088-200443130-00002Search in Google Scholar PubMed

15. Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003; 49:1041–4.10.1373/49.7.1041Search in Google Scholar PubMed

16. Taylor PJ. Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry. Clin Biochem 2005; 38:328–34.10.1016/j.clinbiochem.2004.11.007Search in Google Scholar PubMed

Received: 2005-10-10
Accepted: 2005-12-14
Published Online: 2011-9-21
Published in Print: 2006-3-1

©2006 by Walter de Gruyter Berlin New York

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