The development and validation of a method using high-resolution mass spectrometry (HRMS) for the qualitative detection of antiretroviral agents in human blood

Abstract

Background Antiretroviral drugs are used for the treatment and prevention of HIV infection. Non-adherence to antiretroviral drug regimens can compromise their clinical efficacy and lead to emergence of drug-resistant HIV. Clinical trials evaluating antiretroviral regimens for HIV treatment and prevention can also be compromised by poor adherence and non-disclosed off-study antiretroviral drug use. This report describes the development and validation of a high throughput, qualitative method for the identification of antiretroviral drugs using high-resolution mass spectrometry (HRMS) for the retrospective assessment of off-study antiretroviral drug use and the determination of potential antiretroviral therapy (ART) non-compliance. Methods Serum standards were prepared that contained 15 antiretroviral drugs: 9 protease inhibitors (PIs), 4 nucleotide/nucleoside reverse transcriptase inhibitors (NRTIs), and 2 nonnucleoside/nucleotide reverse transcriptase inhibitors (NNRTIs). Analytical separation was achieved on a Hypersil Gold PFP (100 × 3 mm) column and the eluent was analyzed using the Thermo Exactive Orbitrap mass spectrometer (Exactive-MS) operated in full scan mode. Limit of identification, signal intensity precision, retention time analysis, selectivity, and carryover studies were conducted. Concordance with liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods was evaluated using remnant plasma samples from a clinical trial. Results The limit of identification ranged from 5-10 ng/ml for 14 drugs (9 PIs, 1 NNRTI, 4 NRTIs) and was 150 ng/ml for 1 NNRTI. Precision studies with high and low control mixtures revealed signal intensity coefficients of variation of 3.0-27.5%. The Exactive-MS method was selective for the compounds of interest. Overall, concordance ranged from 89.1%-100% for the screening of antiretroviral drugs in clinical plasma specimens as compared to LC-MS/MS methods. Conclusion Using the Exactive-MS, we developed and validated a highly selective, robust method for the multiplexed detection of 15 antiretroviral drugs.

Failure to Identify HIV-Infected Individuals in a Clinical Trial Using a Single HIV Rapid Test for Screening

“…15 Samples from HPTN 061 participants were also screened for the presence of antiretroviral (ARV) drugs, including 9 protease inhibitors (PIs), 4 nucleoside/nucleotide reverse transcriptase inhibitors, and 2 non-nucleoside reverse transcriptase inhibitors (NNRTIs). 16 Detection of an NNRTI or PI was considered to be indicative of ART. The individuals who performed testing at the centralized laboratory were blinded to the HIV status of the HPTN 061 participants.…”

Evaluation of a Multidrug Assay for Monitoring Adherence to a Regimen for HIV Preexposure Prophylaxis in a Clinical Study, HIV Prevention Trials Network 073

“…(i) Multidrug assay. Plasma samples were tested for the presence of 20 ARV drugs using a qualitative assay based on HRMS (18). The 20 drugs included 6 NRTIs (abacavir, FTC, lamivudine, stavudine, TFV, and zidovudine), 3 NNRTIs (efavirenz, nevirapine, and rilpivirine), 9 PIs (amprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir), a CCR5 receptor antagonist (maraviroc), and an INSTI (raltegravir).…”

“…The ϳ90% sensitivity thresholds for serum TFV and FTC concentrations in HPTN 066 were 35.5 and 49.1 ng/ml, respectively, for daily dosing and 4.2 and 4.6 ng/ml, respectively, for the 4-dose/week regimen (13). We previously developed a qualitative method based on high-resolution mass spectrometry (HRMS) (18) that is less costly than LC-MS/MS methods. That assay detected 15 or 16 ARV drugs in three drug classes (19)(20)(21).…”

Low-Level Viremia Early in HIV Infection

“…13 This assay uses high resolution mass spectrometry (HRMS) to screen for 15 ARV drugs (non-nucleoside reverse transcriptase inhibitors [NNRTIs], nucleoside reverse transcriptase inhibitors [NRTIs], and protease inhibitors [PIs]). The modified version of the assay is faster and has higher resolution; the lower limit of identification for all 15 ARV drugs is 10 ng/mL.…”