alexa Development and Validation of High Performance Liquid C
ISSN: 2157-7064

Journal of Chromatography & Separation Techniques
Open Access

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Research Article

Development and Validation of High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS) Method for Determination of Tenofovir in Small Volumes of Human Plasma

Mulubwa M1, Rheeders M1, Du Plessis L2, Grobler A2 and Viljoen M1*

1Centre of Excellence for Pharmaceutical Sciences (Pharmacen), Division of Pharmacology, North-West University, Potchefstroom 2520, South Africa

2DST/NWU Preclinical Drug Development Platform (PCDDP), North-West University, Potchefstroom 2520, South Africa

*Corresponding Author:
Michelle Viljoen
Centre of Excellence for Pharmaceutical Sciences (Pharmacen)
Division of Pharmacology
North- West University
Private Bag X6001
Potchefstroom 2520, South Africa
Tel: +27182992232
Fax: +27182992232
E-mail: [email protected]

Received date: October 01, 2015; Accepted date: October 21, 2015; Published date: November 10, 2015

Citation: Mulubwa M, Rheeders M, Du Plessis L, Grobler A, Viljoen M (2015) Development and Validation of High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS) Method for Determination of Tenofovir in Small Volumes of Human Plasma. J Chromatogr Sep Tech 6:300. doi:10.4172/2157-7064.1000300

Copyright: © 2015 Mulubwa M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

Abstract

Determination of plasma tenofovir (TFV) concentrations in small plasma volumes and in a short time period is most desirable in a clinical setting. The HPLC-MS/MS method was developed and validated for the determination of TFV. Plasma sample volumes of 10 μL were extracted by protein precipitation. Cimetidine, used as internal standard (ISTD) and TFV were separated on a C18 (Phenomenex Kinetex TM 30 mm × 2.1 mm, 2.6 μm) reversed phase column with a pre-column. The gradient mobile phase consisted of 10 mmol/L ammonium acetate in water and acetonitrile/methanol (50:50, v/v). TFV and ISTD retentiontimes were 0.27 and 0.90 minutes, respectively, with a run time of 2 minutes. Transition of the parent to the product ion of TFV (m/z 288.072→176.038) and ISTD (m/z 253.13→158.987) were monitored in positive ionization mode. Calibration curves were linear (average r2, 0.9958) over a TFV concentration range of 12.5-600 ng/mL. The mean recovery was 96.9%. Accuracy, inter and intra-assay of three quality controls and lower limit of quantification (12.5 ng/mL) were within the acceptable limit of <15% relative standard error. TFV was stable at studied conditions and neither matrix effect nor significant carry-over was observed. A distinct, reliable and robust method for determination of TFV in a small volume (10 μL) of human plasma was developed, validated and incorporated to determine the plasma TFV levels in 30 HIV-infected women on antiretroviral treatment.

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