METHOD ARTICLE Validation and clinical application of a method to quantify efavirenz in cervicovaginal secretions from flocked swabs using liquid chromatography tandem mass spectrometry [version 2; peer review: 1 approved, 1 approved with reservations] Adeniyi Olagunju 1,2 , Jacinta Nwogu 3 , Oluwasegun Eniayewu 2,4 , Shakir Atoyebi 1,2 , Alieu Amara 1 , John Kpamor 5 , Oluseye Bolaji 2 , Ebunoluwa Adejuyigbe 6 , Andrew Owen 1 , Saye Khoo 1 1 Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, UK 2 Department of Pharmaceutical Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria 3 Department of Pharmaceutical Chemistry, University of Ibadan, Ibadan, Nigeria 4 Department of Pharmaceutical and Medicinal Chemistry, University of Ilorin, Ilorin, Nigeria 5 Federal Medical Centre, Makurdi, Nigeria 6 Department of Paediatrics and Child Health, Obafemi Awolowo University, Ile-Ife, Nigeria First published: 24 Sep 2021, 6:246 https://doi.org/10.12688/wellcomeopenres.17202.1 Latest published: 07 Apr 2022, 6:246 https://doi.org/10.12688/wellcomeopenres.17202.2 v2 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%. Open Peer Review Approval Status 1 2 version 2 (revision) 07 Apr 2022 version 1 24 Sep 2021 view view Eva Choong , University of Lausanne, Lausanne, Switzerland 1. Mario Vincenzo Russo , University of Molise, Campobasso, Italy 2. Any reports and responses or comments on the article can be found at the end of the article. Page 1 of 17 Wellcome Open Research 2022, 6:246 Last updated: 09 APR 2022