Quantitation of intracellular triphosphate metabolites of antiretroviral agents in peripheral blood mononuclear cells (PBMCs) and corresponding cell count determinations: review of current methods and challenges

ABSTRACT

Introduction: Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system and the target cells for human immunodeficiency virus, type 1 (HIV-1) infection. Nucleoside/nucleotide analogs for the treatment of HIV infection are prodrugs that require cellular activation to triphosphate (TP) metabolites for antiviral activity. A reliable method of PBMC isolation and subsequent cell counting, as well as an accurate bioanalytical determination of the TPs in PBMCs are important for understanding the intracellular pharmacokinetic (PK) of the TPs and its correlation with plasma PK, the drug effect, and dose determination.

Areas covered: The authors review the challenges and solutions in PBMC sample collection, sample processing, cell lysis, cell counting methods, analyte extraction, and liquid chromatography/tandem mass spectrometry (LC–MS/MS) quantitative analysis of the nucleoside reverse transcriptase inhibitor-triphosphate (NRTI-TP) metabolites, and analogs.

Expert opinion: Analyzing large numbers of clinical PBMC samples for determination of NRTI-TPs and analogs in PBMCs requires not only a validated LC-MS/MS bioanalytical method but also reliable methods for PBMC isolation, counting, cell lysis, and analyte recovery, and an approach for assessing analyte stability. Furthermore, a simple, consistent, and validated cell counting method often involves DNA quantitation of the PBMCs samples collected from clinical studies.

KEYWORDS:

• NRTI
• NRTI-TP
• LC-MS/MS
• bioanalytical
• intracellular
• PBMC
• DNA quantitation
• cell counting

Article highlights

• Background on the importance of and challenges in the determination of antiretroviral triphosphate metabolites in PBMCs.

• Generally accepted procedures for PBMC sample isolation and processing.

• Generally accepted procedures for cell lysis and the subsequent analyte extraction and DNA extraction.

• Challenges and solutions for LC–MS/MS method validation for quantitation of TFV-DP in PBMCs.

• Cell counting method: hemocytometry, flow cytometry, and DNA standard curve calibration using PBMC standard versus using genomic DNA standard.

• Reliable determination of NRTI-TPs in PBMCs requires not only a validated LC–MS/MS bioanalytical method, but also a validated cell counting method.

Declaration of interest

D Xiao is a senior research scientist of bioanalytical chemistry at Gilead Sciences, Inc. KHJ Ling is the director of bioanalytical chemistry at Gilead Sciences, Inc. T Tarnowski is the senior director of bioanalytical chemistry at Gilead Sciences, Inc. SR Majeed is a clinical pharmacologist at Gilead Sciences, Inc. J Custodio is an associate director and clinical pharmacologist at Gilead Sciences, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was sponsored by Gilead Sciences, Inc.