Therapeutic Drug Monitoring of COVID-19 Drugs with Components of a Commercial Assay

Researchers are working furiously to test a variety of potential drugs to find a suitable treatment for COVID-19, the disease caused by SARS-CoV-2. Dose tolerance and dose response exploration studies are usually required that demands for a great analysis. Pharmacokinetic and pharmacodynamic studies are also often involved in these phases. But also later,should one of the drugs be approved, TDM may be a requirement. To help laboratories with this challenge, we describe a protocol* that allows the determination of 9 COVID-19 drug candidates and metabolites in a short run time on the basis of MassTox® TDM Series A*, a modular system that allows the determination of more than 200 drugs.

Table 1: The Drugs of COVID-19 Calibrator

The sample preparation was performed in line with the MassTox Series A protocol (Chromsystems). In brief: 50 μl of sample, reconstituted 3PLUS1® calibrator (order. No 92055) or MassCheck® control (order. no. 0268, 0269), containing 9 drugs and metabolites (see table 1), was pipetted into a 1.5 ml reaction vial. 25 μl of Extraction Buffer (order. no 92005) was added and briefl y mixed. 250 μl of a mixture (Internal Standard Mix Anti-HIV drugs, order no. 92844, and Precipitation Reagent, order no. 92012, at a 1:16 ratio), was added and vortexed for 30 s and centrifuged for 5 min at 15000 g. The supernatant was then diluted, depending on sensitivity of the LC-MS/MS-system, with Dilution Buffer 1 (92007; ratio 1:5) or Dilution Buffer 2 (92008; ratio 1:20). The substances were separated on MasterColumn A® (order no. 92110) according to the protocol below and detected with electrospray ionisation in positive ion mode with a Sciex 6500 Citrine mass spectrometer. MRM transitions and internal standard assignments are found in table 3.

Injection volume: 10-20 µl
Column and autosampler temperature: ambient
Flow rate: 0.6 ml/min
Gradient: binary
Column: MasterColumn® A (order. no 92110)
Analysis time: 3.5 min 

Table 2: Binary gradient conditions

Table 3: MRMs and corresponding ISTD 

The lower limits of quantification for COVID-19 drugs were between 1.7 and 418 μg/l, upper limits of quantification were in a range of 400 to 40000 μg/l (see table 5). Interassay and intraassay precision were determined between 1 and 7 % (see table 4). All drugs could be analysed with a short run time of 3.5 min (fig.1).

Figure 1: Chromatogram of COVID-19 drugs with components of MassTox® TDM Series A.

We demonstrate with this protocol that MassTox® TDM Series A can be employed for the determination of 9 COVID-19 drug candidates/metabolites in serum/plasma. Laboratories that have already MassTox® TDM Series A in operation can simply use the same chromatographic set up, sample prep and MasterColumn® A. All you need are a few additional components from MassTox® TDM Series A.

Table 4: Inter- and intraassay data at different concentrations of COVID-19 drugs.

Table 5: Upper and lower limit of quantification