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Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Published on: September 26, 2011
A validated new method for nevirapine quantitation in human plasma via high-performance liquid chromatography
Courtney F Silverthorn1, Teresa L Parsons
1Division of Clinical Pharmacology, Department of Medicine, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Osler 527, Baltimore, MD 21287-5554, USA.
Abstract:
A fully validated and clinically relevant assay was developed for the assessment of nevirapine concentrations in neonate blood plasma samples. Solid-phase extraction with an acid-base wash series was used to prepare subject samples for analysis. Samples were separated by high performance liquid chromatography and detected at 280 nm on a C8 reverse-phase column in an isocratic mobile phase. The retention times of nevirapine and its internal standard were 5.0 and 6.9 min, respectively. The method was validated by assessment of accuracy and precision (statistical values <15%), specificity, and stability. The assay was linear in the range 25-10,000 ng/mL (r2 > 0.996) and the average recovery was 93% (n = 18). The lower limit of quantification (relative standard deviation <20%) was determined to be 25 ng/mL for 50 microL of plasma, allowing detection of as little as 1.25 ng of nevirapine in a sample. This value represents an increase in sensitivity of up to 30-fold over previously published methods.
