Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV

Abstract

Recently, anti-HIV treatment has achieved high efficacy and tolerability. Nevertheless, few data are available about the intracellular penetration of antiretrovirals, partly due to the technical challenges related to intracellular quantification. This work aimed to validate an ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the simultaneous quantification of maraviroc, nevirapine, rilpivirine, dolutegravir, raltegravir, cobicistat, darunavir, ritonavir, atazanavir, efavirenz, elvitegravir, and etravirine within peripheral blood mononuclear cells (PBMCs) and apply it to samples from patients. PBMCs were isolated by density gradient on cell preparation tubes (CPT). Samples were prepared by addition of internal standards (IS), sonication, centrifugation, and drying. Reconstituted extracts underwent chromatographic separation by reversed phase UHPLC and detection was performed by electrospray ionization and multiple reaction monitoring. Method validation followed FDA and EMA guidelines, showing acceptable accuracy, precision, recovery and IS-normalized matrix effect. The application to 56 samples from patients undergoing antiretroviral treatment provided description of intracellular penetration, showing method eligibility for future studies.

Development and Validation of an Up-to-Date Highly Sensitive UHPLC-MS/MS Method for the Simultaneous Quantification of Current Anti-HIV Nucleoside Analogues in Human Plasma

“…The results of preliminary optimization are summarized in results section. After the completion of the preliminary experiments and optimization of the conditions for sample preparation, the method was fully validated on the basis EMA and FDA guidelines requirements, following the same validation process described in previous works [ 34 ].…”

The Effect of Rifampicin on Darunavir, Ritonavir, and Dolutegravir Exposure within Peripheral Blood Mononuclear Cells: a Dose Escalation Study

“…The evaluation of RIF concentrations was performed only at weeks 4 and 5, since RIF trough concentrations are expected to be undetectable and since its antimicrobial activity is led by its C max . PBMCs were isolated from blood collected with cell preparation tubes (CPT vacutainer; Becton, Dickinson, Franklin Lakes, NJ, USA) based on a separation by density gradient through centrifugation and 2 washing steps as previously described in several works ( 24 , 32 , 33 ); the cell pellets were lysed with water/methanol 30:70 and then shipped on dry ice to the Laboratory of Clinical Pharmacology and Pharmacokinetics of the University of Turin where the analyses were performed.…”

“…The quantifications of DRV, RTV, DTG, and RIF in PBMCs were performed through the application of two previously published validated ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) methods for the quantification of ARVs ( 32 ) and anti-TB drugs ( 24 ), respectively. Cell number in each sample and the mean cellular volume for PBMCs was used to normalize the drug amounts within PBMCs, obtaining concentrations expressed in nanograms per milliliter.…”

Inflammation and intracellular exposure of dolutegravir, darunavir, tenofovir and emtricitabine in people living with HIV

“…performed at the Laboratory of Clinical Pharmacology and Pharmacogenetics, University of Turin at the 'Amedeo di Savoia' hospital using validated and previously published ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methods. [20][21][22] We measured C-reactive protein (CRP), inteleukin-6 (IL-6), soluble CD14 (sCD14) and lipopolysaccharide(LPS) on frozen serum samples collected on the same day as the PK measurement.…”

“…Non-compartmental plasma and IC PK parameters were calculated and reported as ng/mL using mean cell volumes of PBMC to normalize data, as previously described, in order to allow a direct comparison between IC and plasma concentrations, and expressed as geometric means (with 95% confidence intervals [95% CI]). 22 Non-parametric analyses were performed for bivariate correlations (Spearman's rank correlation); all statistical analyses were performed using SPSS Statistics version 23 (IBM, Armonk, NY, USA). Tests were all two-sided, and a P-value of <.05 was considered statistically significant.…”