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 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 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 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 AUC0-24h was 16370 ng*h/mL (5803-22088), Cmax was 1618 ng/mL (610-2438) at a Tmax of 8.0 h (8.0-12), and Cmin was 399 ng/mL (110-981). Efavirenz CVS:plasma AUC0-24 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.
Highlights
Heterosexual transmission through the genital mucosa and mother-to-child transmission, mainly during delivery, are major routes of human immunodeficiency virus (HIV) acquisition in Sub-Saharan Africa
Liquid chromatography tandem mass spectrometry conditions Representative chromatograms are presented in Figure 1 showing efavirenz at the lower limit of quantification (LLOQ), LQC and a patient Cervicovaginal swabs (CVS)
To facilitate intensive pharmacokinetic studies to better understand the pharmacology of therapeutics in the female genital tract, we developed and validated a simple, accurate and precise method for drug quantification in cervicovaginal fluid collected with flocked swab
Summary
Heterosexual transmission through the genital mucosa and mother-to-child transmission, mainly during delivery, are major routes of human immunodeficiency virus (HIV) acquisition in Sub-Saharan Africa. HIV shedding in the female genital tract has been shown to increase the risk of mother-to-child transmission during delivery[2]. A proper understanding of ARV pharmacokinetics in the female genital tract is crucial in developing effective pre-exposure prophylaxis (PrEP) and prevention of mother-to-child transmission interventions. 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 matrix. The method was used to quantify efavirenz in CVS samples from human immunodeficiency virus (HIV)-positive women in the VADICT study (NCT03284645). Mean recovery of efavirenz from article can be found at the end of the article
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