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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


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,


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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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