Development and validation of a multiplex UHPLC-MS/MS assay with stable isotopic internal standards for the monitoring of the plasma concentrations of the antiretroviral drugs bictegravir, cabotegravir, doravirine, and rilpivirine in people living with HIV

Abstract

The widespread use of highly active antiretroviral treatments has dramatically changed the prognosis of people living with HIV (PLWH). However, such treatments have to be taken lifelong raising issues regarding the maintenance of both therapeutic effectiveness and long-term tolerability. Recently approved or investigational antiretroviral drugs present considerable advantages, allowing once daily oral dosage along with activity against resistant variants (eg, bictegravir and doravirine) and also parenteral intramuscular administration that facilitates treatment adherence (eg, long-acting injectable formulations such as cabotegravir and rilpivirine). Still, there remains a risk of insufficient or exaggerated circulating exposure due to absorption issues, abnormal elimination, drug-drug interactions, and others. In this context, a multiplex ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) bioassay has been developed for the monitoring of plasma levels of bictegravir, cabotegravir, doravirine, and rilpivirine in PLWH. A simple and convenient protein precipitation was performed followed by direct injection of the supernatant into the UHPLC-MS/MS system. The four analytes were eluted in less than 3 minutes using a reversed-phase chromatography method coupled with triple quadrupole mass spectrometry detection. This bioassay was fully validated following international guidelines and achieved good performances in terms of trueness (94.7%-107.5%), repeatability (2.6%-11%), and intermediate precision (3.0%-11.2%) over the clinically relevant concentration ranges (from 30 to 9000 ng/mL for bictegravir, cabotegravir, and doravirine and from 10 to 1800 ng/mL for rilpivirine). This sensitive, accurate, and rapid UHPLC-MS/MS assay is currently applied in our laboratory for routine therapeutic drug monitoring of the oral drugs bictegravir and doravirine and is also intended to be applied for the monitoring of cabotegravir/rilpivirine levels in plasma from PLWH receiving once monthly or every 2-month intramuscular injection of these long-acting antiretroviral drugs.

Keywords: UHPLC-MS/MS; antiretroviral therapy; long-acting injectables; pharmacokinetics; therapeutic drug monitoring.

Figure 1

Chemical structures of the analyzed antiretroviral drugs

Figure 2

Ultrahigh‐performance liquid chromatography coupled to tandem mass spectrometry (UHPLC‐MS/MS) separation of a calibration sample containing the four antiretroviral drugs, at the concentration of 10 000 ng/mL for all the analytes except for rilpivirine (2000 ng/mL). Calibration sample was prepared as described in Section 2 [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Qualitative assessment of matrix effect. Overlaid ultrahigh‐performance liquid chromatography coupled to tandem mass spectrometry (UHPLC‐MS/MS) profiles obtained from seven blank plasma extracts during postcolumn infusion of a solution containing the four analytes, as described in Section 2.7.2. Chromatographic peaks obtained during experiments were superimposed for interpretation [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Accuracy profiles over the validated domain in human plasma of the five comedications and the two metabolites. Trueness (red solid line), upper and lower β‐expectation tolerance intervals (β = 80%) (black solid lines) and acceptance limits (λ = ±30%, green dotted lines) are shown [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Chromatographic profile of a plasma from one HIV‐infected individual receiving bictegravir 50 mg once daily (A) and from another patient receiving doravirine 100 mg once daily (B) [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Steady‐state pharmacokinetic profile of bictegravir when administered at 50 mg once daily. Continuous green line represents the population median prediction. Dark green and light green shaded area represent the 50% and 90% prediction interval, respectively. Bictegravir plasma concentrations obtained from our therapeutic drug monitoring (TDM) service in patients included in the Swiss HIV Cohort Study has been superimposed (black points) [Colour figure can be viewed at wileyonlinelibrary.com]