Validation and clinical application of a method to quantify efavirenz in cervicovaginal secretions from flocked swabs using liquid chromatography tandem mass spectrometry

Abstract

Background : A liquid chromatography tandem mass spectrometry method to quantify drugs in dried cervicovaginal secretions from flocked swabs was developed and validated using the antiretroviral efavirenz as an example. Methods: Cervicovaginal swabs (CVS) were prepared by submerging flocked swabs in efavirenz-spiked plasma matrix. Time to full saturation, weight uniformity, recovery and room temperature stability were evaluated. Chromatographic separation was on a reverse-phase C18 column by gradient elution using 1mM ammonium acetate in water/acetonitrile at 400 µL/min. Detection and quantification were on a TSQ Quantum Access triple quadrupole mass spectrometer operated in negative ionisation mode. The method was used to quantify efavirenz in CVS samples from human immunodeficiency virus (HIV)-positive women in the VADICT study (NCT03284645). A total of 98 samples (35 paired intensive CVS and DBS pharmacokinetic samples, 14 paired sparse CVS and DBS samples) from 19 participants were available for this analysis. Results: Swabs were fully saturated within 15 seconds, absorbing 128 µL of plasma matrix with coefficient of variation (%CV) below 1.3%. The method was linear with a weighting factor (1/X) in the range of 25-10000 ng/mL with inter- and intra-day precision (% CV) of 7.69-14.9%, and accuracy (% bias) of 99.1-105.3%. Mean recovery of efavirenz from CVS was 83.8% (%CV, 11.2) with no significant matrix effect. Efavirenz remained stable in swabs for at least 35 days after drying and storage at room temperature. Median (range) CVS efavirenz AUC 0-24h was 16370 ng*h/mL (5803-22088), C max was 1618 ng/mL (610-2438) at a T max of 8.0 h (8.0-12), and C min was 399 ng/mL (110-981). Efavirenz CVS:plasma AUC 0-24h ratio was 0.41 (0.20-0.59). Conclusions: Further application of this method will improve our understanding of the pharmacology of other therapeutics in the female genital tract, including in low- and middle-income countries.

Pharmacogenetics of efavirenz exposure in cervicovaginal fluid during pregnancy and postpartum

“…The understanding of antiretroviral pharmacokinetics in the female genital tract (FGT) is of growing interest in the HIV prevention field because of its importance in designing more effective therapeutic options to reduce serodiscordant and mother-to-child HIV transmission [1]. Although a number of clinical studies that focus on quantifying antiretroviral drugs in the FGT have been reported in the literature [2][3][4][5][6]. ARV characterization in this compartment has not yet been well understood.…”

“…To circumvent these challenges, , cervicovaginal fluids (CVF are used as an acceptable surrogate for vaginal tissue concentrations of ARVs [20]. In our laboratory, we recently validated a minimally invasive LC-MS/MS method to quantify the HIV drug efavirenz in cervicovaginal fluid collected on flock swabs [6], which improved our ability to conduct intensive clinical pharmacokinetic studies with CVF.…”