Measurement of intracellular ribavirin mono-, di- and triphosphate using solid phase extraction and LC-MS/MS quantification

Abstract

Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-(13)C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection.

Keywords: Analytical methods; Clinical pharmacology; Dried blood spots; Intracellular pharmacology; LC–MS/MS; Ribavirin triphosphate.

Figure 1

Cross-Validation Data: 240 samples (6 runs) total were extracted then run on both the Quantum and Vantage. Vantage results are on the Y-axis and Quantum results are on the X-axis in pmol/sample

Figure 2

LLOQ chromatograph for RBV. Top panel is analyte, bottom the corresponding internal standard. Y-axis is relative abundance to 100%. Retention time was ∼3.0 for both RBV and RBV-IS.

Figure 3

Blank-IS chromatograph for RBV. Top panel is analyte, bottom panel is the corresponding internal standard. Y-axis is relative abundance scaled to LLOQ response. Expected retention time for RBV/RBV-IS is ∼3.0 minutes.

Figure 4

Extracted clinical research sample chromatograph for RBV in hPBMC. Top panel is the analyte and bottom is corresponding IS. RBV-TP retention time is ∼3.0 minutes. This sample had a RBV-TP concentration of 3.14 pmol/10⁶ cells.

Figure 5

Extracted clinical research sample chromatograph for RBV-TP in RBC. Top panel is the analyte and bottom is corresponding IS. RBV-TP retention time is ∼3.0 minutes. This sample had a RBV-TP concentration of 34.2 pmol/10⁶ cells.

Figure 6

Extracted clinical research sample chromatograph for RBV-TP from DBS RBC lysate at week 35 post initial RBV dose. RBV-TP retention time is at ∼2.8 minutes and the sample was quantified at 1044 pmol/punch.

Figure 7

Effect of different storage conditions on RBV-TP from DBS lysate when stored for 196 to 310 days. The Y-axis represents the percent difference from storage at −80°C and the X-axis is storage time in days. Triangles=-20°C storage, squares=4°C storage and diamonds=room temperature storage.

Figure 8

Paired finger stick and pipetted blood samples. Y-axis represents finger stick and X-axis is pipetted venous blood in pmol/punch. R² =0.89 and Y=0.902x after performing regression analysis.

Figure 9

Paired RBC to DBS analysis. DBS in pmol/punch is on the Y-axis and RBC in pmol/million cells is shown on the X-axis. R²=0.95 and y=8.66x after performing regression analysis.