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 non-nucleoside/nucleotide reverse transcriptase inhibitors (NNRTIs). Analytical separation was achieved on a Hypersil Gold PFP (100×3mm) 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 to 10ng/ml for 14 drugs (9 PIs, 1 NNRTI, 4 NRTIs) and was 150ng/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% to 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.

Keywords: Antiretroviral; Clinical trials; Exactive-MS; High-resolution mass spectrometry; Validation.

Fig. 1

Total ion chromatogram from the Exactive-MS method collected in positive ionization mode. Representative chromatogram of 1000 ng/ml antiretroviral mixture spiked into drug-free human serum.

Fig. 2

Ion spectra of ritonavir generated by Exactive-MS (A) with the inclusion of an in-source collision-induced dissociation scan event for fragment production and detection (B) . Based on the m/z fragment ions monitored, we postulate that the m/z 268 and m/z 296 fragment ions were formed through the cleavage of a keto-isopropyl linkage and an amide bond, respectively. Retention time determination through the application of a mass filter with ± 5 ppm discrimination from theoretical mass of precursor and fragment ions (C).

Fig. 3

Ion spectra of lamivudine generated by Exactive-MS (A) with the inclusion of an in-source collision-induced dissociation scan event for fragment production and detection (B) .The 95 m/z fragment ion was likely formed through the loss of the oxathiol moiety and subsequent loss of a NH₃ constituent. Retention time determination through the application of a mass filter with ± 5 ppm discrimination from theoretical mass of precursor and fragment ions (C).