Development and Validation of an LC/MS/MS Method for the Determination of Tenofovir in Monkey Plasma

Abstract

A simple, specific, sensitive LC/MS/MS method for the quantitation of tenofovir (TFV) in monkey plasma was developed and validated. After the addition of adefovir as an internal standard (IS), methanol was used to produce a protein-free extract. Isocratic chromatographic separation was performed on a reverse-phase Discovery C₁₈ column (4.6×250 mm, 5 μm). The mobile phase consisted of methanol–water–formic acid (20 : 80 : 0.5, v/v/v). Detection of TFV and the IS was achieved with electrospray ionization (ESI)-MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The analytical range was set at 0.005—1.250 μg/ml using a 200 μl plasma sample. The intra- and inter-day precision values were less than 11.4%, and accuracy ranged from 0.4 to 2.9% in all quality control samples. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, recovery, and stability. Due to the high polarity of TFV, the major challenge was to circumvent ion suppression when quantitating the plasma concentration of TFV using the LC/MS/MS method. To avoid ion suppression, sufficient chromatographic separation was the most effective means for the present purposes. Moreover, it was found that the reconstitution solvents of the dried residue had a significant impact on LC peak shapes. The validated method was successfully applied to a bioequivalence study in 6 monkeys after the oral administration of two ester prodrugs of TFV (equivalent to TFV 20 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid TFV determination.