Multiplexed therapeutic drug monitoring (TDM) of antiviral drugs by LC-MS/MS

Abstract

Background: Therapeutic drug monitoring (TDM) can be a useful tool in the clinical management of anti-hepatitis C virus (anti-HCV) drugs. Methods for the determination of various types of anti-HCV drugs in biological samples are, therefore, needed for clinical laboratories.

Objective: In this work, employing the LC-MS/MS approach, we aimed to develop a multiplexed method for identification of the following anti-HCV drugs: Ribavirin (RBV), Boceprevir (BOC), Telaprevir (TVR), Simeprevir (SIM), Daclatasvir (DAC), Sofosbuvir (SOF) and its metabolite GS 331007 (SOFM) in liquid plasma and in dried plasma spots (DPSs).

Method: A single-step extractive-deproteinization was employed for both liquid plasma and DPSs. Reverse-phase liquid chromatography coupled with MRM detection was developed for multiplexed drug detection and quantification.

Results: Sensitivities (expressed as LOQ) were 10 (±1.2), 10 (±4.9), 10 (±4.4), 10 (±4.4), 10 (±6.4), 10 (±3.4), 10 (±6.4) ng/ml for RBV, SOFM, SOF, DAC, BOC, TVR, and SIM, respectively; accuracy (expressed as BIAS%) was <10% for all drugs; reproducibility (intra- and inter-day CV%) was <10% for all drugs; dynamic range was 10-10,000 ng/ml for all drugs.

Conclusions: A novel, simple, rapid and robust LC-MS/MS multiplex assay for the TDM of various anti-HCV drugs that are currently in the clinic was successfully developed. Application to DPS samples enabled TDM to be used for outpatients as well.

Keywords: Directly acting antiviral drugs (DAAs); Dried plasma spots (DPS); Hepatitis C; Hepatitis C virus (HCV); Ribavirin; Therapeutic drug monitoring (TDM).

Fig. 1

Chemical structures. Different chemical structures of the seven analytes determined with the proposed method.

Fig. 2

Ion extraction chromatograms (XICs) recorded in MRM mode in a calibration sample analysis. XICs (intensity vs time) for RBV, TVR, BOC, DAC, SIM, SOF and SOFM. Blue and red traces are relative to the quantifier and the qualifier transitions, respectively (see Table 1). Retention times are signaled by a maximum in intensity in both of them. The quantifier/qualifier area ratio (±10%) at analyte retention time is a typical qualifying parameter for each analyte.

Fig. 3

Superimposition of XICs recorded in MRM mode in four DPS samples analysis. Superimposed XICs traces (intensity vs time) recorded in case of DPS analyses of samples from patients receiving different combined antiviral therapies. A. RBV (blue and red traces, t_r = 0.8 min), IS (green and grey traces, t_r = 0.8 min), SOFM (light blue and pink traces, t_r = 1.3 min) and SOF (dark green and purple, t_r = 2.3 min). B. RBV (blue and red traces, t_r = 0.8 min), IS (green and grey traces, t_r = 0.8 min), DAC (light blue and pink traces, t_r = 2.3 min) and SIM (dark green and purple, t_r = 3.1 min). C. RBV (blue and red traces, t_r = 0.8 min), IS (green and grey traces, t_r = 0.8 min), and BOC (light blue and pink traces, t_r = 2.5 min). D. RBV (blue and red traces, t_r = 0.8 min), IS (green and grey traces, t_r = 0.8 min), and TVR (light blue and pink traces, t_r = 2.7 min).

Fig. 4

Post-column infusion experiment. Superimposition of the quantifier transition XICs (Y% axis vs time) obtained in post-column infusion conditions (red traces) to typical XICs (blue traces) for RBV, TVR, DAC, SOF, BOC, SIM and SOFM.

Fig. 5

Liquid plasma vs DPS sample method comparison. Passing-Bablok regression of results obtained on 30 liquid plasma and corresponding DPS samples for RBV, TVR, DAC, SOF, BOC, SIM and SOFM, as indicated. Parameters of the linear correlation, reported in each box, indicate stringent correlation of data obtained with the two methods for all drugs.

Fig. 6

Time-course of the drug levels (measured both in liquid plasma and DPS samples) in a patient under therapy. Time course of drug trough concentrations in plasma samples from four different patients receiving different combined INF-free antiviral therapies. Concentration of the drugs (ng/ml) are plotted vs time (days) since the beginning of therapy. TDM was performed every other week for three months. A. Blue diamond = liquid plasma RBV, purple cross = DPS RBV, red square = liquid plasma SOF, light blue star = DPS SOF, green triangle = liquid plasma SOFM and orange circle = DPS SOFM concentration respectively. B. Blue diamond = liquid plasma RBV, red square = DPS RBV, green triangle = liquid plasma DAC, purple cross = DPS DAC, light blue star = liquid plasma SIM and orange circle = DPS SIM concentration respectively. C. Blue diamond = liquid plasma RBV, red square = DPS RBV, green triangle = liquid plasma BOC, purple cross = DPS BOC concentration respectively. D. Blue diamond = liquid plasma RBV, red square = DPS RBV, green triangle = liquid plasma TVR, purple cross = DPS TVR concentration respectively.

Supplementary Fig. S1

Superimposition of TVR ion extraction chromatograms (XICs) recorded before and after sample degradation. Superimposition of quantifier TVR XICs obtained before (blue) and after (red) sample degradation occurred in a non-acidified plasma kept overnight at RT.