Development and validation of a LC–MS/MS method for the quantification of tenofovir and emtricitabine in seminal plasma

The aims of this study were to describe the blood plasma (BP) and seminal plasma (SP) pharmacokinetics of tenofovir (TFV) in HIV-1-infected men, to assess the role of genetic polymorphism in the variability of TFV transfer into the male genital tract, and to evaluate the impact of TFV SP exposure on seminal plasma HIV load (spVL). Men from the Evarist-ANRS EP 49 study treated with TFV as part of their antiretroviral therapy were included in the study. A total of 248 and 217 TFV BP and SP concentrations from 129 men were available for the analysis. For pharmacogenetic assessment, a total of 121 single nucleotide polymorphisms (SNP) were genotyped. Data were analyzed using a nonlinear mixed-effects modeling approach. TFV pharmacokinetics were best described by a two-compartment model for BP and by an effect compartment with different input and output constants for SP. TFV exposures (area under the concentration-time curve from 0 to 24 h [AUC 0 -24 ]) were higher in SP than in BP (median AUC 0 -24 , 7.01 versus 2.97 mg · liter Ϫ1 · h, respectively). The median (range) SP-to-BP AUC 0 -24 ratio was 2.24 (0.53 to 34.13). After correction for multiple testing, none of the SNPs were significantly associated with the TFV transfer rate constant. The impact of the TFV SP AUC 0 -24 or TFV SP-to-BP AUC 0 -24 ratio on spVL was not significant (P ϭ 0.808 and 0.768, respectively). This is the first population model describing TFV pharmacokinetics in the male genital tract. TFV SP concentrations were higher than BP concentrations. Despite TFV SP exposures being higher than BP exposures, an spVL was detectable for 12.2% of the men.

A simple, short, and rugged LC-MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine was developed and validated. Dried blood spots were prepared with 25 μL of spiked whole blood. A 3 mm punch was extracted with methanol containing labeled internal standards. Ten microliters was injected into the LC-MS/MS using isocratic mobile phase composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (45: 55 v/v) at a flow rate of 0.25 mL/min. The method was validated in the range of 10-2000 ng/mL for all four analytes. The intra-assay accuracy (RE) of the method was -4.73-4.78, 1.35-2.89, -8.89 to -0.49 and - 1.40-1.81 for tenofovir, emtricitabine, elvitegravir and rilpivirine, respectively. The inter-assay accuracy was within ±15% of nominal and precision (CV) was <15%. The hematocrit effect on quantification was nonsignificant at the tested hematocrit levels (35-70%). The dried blood spot method showed good agreement with the plasma method, and hence can be used as an alternative to plasma method.

Rationale: Tenofovir (TFV) is a first-line antiviral agent against hepatitis B virus (HBV)and is recommended for the prevention of mother-to-infant transmission of HBV. To study the distribution of TFV in umbilical cord plasma and amniotic fluid of HBVinfected pregnant women, a rapid and sensitive method for TFV determination was developed and validated. Methods:The quantification method was developed using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The analytes were separated on an Acquity UPLC HSS T3 column under gradient elution with methanol and 0.01% ammonia solution in 10 mM ammonium acetate/water. This is the first reported method for the determination of TFV using alkaline rather than acidic mobile phases.Linearity, accuracy, precision, limit of quantification, specificity and stability were assessed.Results: Detection of TFV was achieved within 4 min. The calibration curves for TFV quantification showed excellent linearity in the range of 1-500 ng/mL. The intraand interbatch precision and accuracy ranged from −4.35% to 6.92%. This method was successfully applied to determination of samples from 50 HBV mono-infected women undergoing tenofovir disoproxil fumarate therapy. The mean concentrations of TFV in the umbilical cord and amniotic fluid samples were 29.2 (4.6-86) and 470.9 (156-902) ng/mL, respectively, which showed a moderate positive correlation (r = 0.5299, P<0.001). Conclusions:A simple, rapid but sensitive bioanalytical method to determine TFV concentration in both umbilical cord plasma and amniotic fluid using LC/MS/MS was developed and applied to HBV-infected women during labor who were undergoing TDF therapy, which will help us understand the efficacy and safety of tenofovir during pregnancy.