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

Introduction

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.

GS 5734/Remdesivir   Favipiravir     Lopinavir    

GS 441524
(active metabolite of Remdesivir)

Azithromycin Nafamostat
Ritonavir Chloroquine Hydroxychloroquine

Table 1: The Drugs of COVID-19 Calibrator

Materials and Methods

Sample Prep

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.

LC-MS/MS-Parameter

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 

Time

Mobile Phase 1 (92001)

Mobile Phase 2 (92002)

0.00 min

100%

0

0.50 min

100 %

0

0.51 min

50 %

50 %

1.50 min

50 %

50 %

1.51 min

0

100 %

2.50 min

0

100 %

2.51 min

100 %

0

3.50 min

100 %

0

Table 2: Binary gradient conditions

Analyte

Corresponding ISTD MRM MRM
Remdesivir ISTD 1 (520 -> 117) 603 -> 402
GS 441524 ISTD 1 (520 -> 117) 292 -> 147
Lopinavir ISTD 3 (637 -> 163) 629 -> 155
Ritonavir ISTD 2 (727 -> 146) 721 -> 140
Favipiravir ISTD 1 (520 -> 117) 158 -> 85
Chloroquine ISTD 1 (520 -> 117) 320 -> 247
Hydroxychloroquine ISTD 1 (520 -> 117) 336 -> 158
Nafamostat ISTD 1 (520 -> 117) 348 -> 162
Azithromycin ISTD 1 (520 -> 117) 749 -> 158

Table 3: MRMs and corresponding ISTD 

Results

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.

Conclusion

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.

Analyte Conc.
[μg/l]   

CV
Interassay

CV
Intraassay
Conc. 
[µg/l]
CV
Interassay
CV
Intraassay
 

Level I, order no. 0268

Level II, order no. 0269
Remdesivir 699 2 % 3 % 1987 3 % 4 %
GS 441524 35.5 2 % 3 % 70 4 % 5 %
Lopinavir 1083 1 % 2 % 4928 2 % 3 %
Ritonavir 779 1 % 1 % 4887 3 % 3 %
Favipiravir 2612 2 % 4 % 7872 4 % 5 %
Chloroquine 113 5 % 7 % 340 6 % 7 %
Hydroxychloroquine 464 5 % 5 % 922 5 % 6 %
Nafamostat 46.6 5 % 5 % 159 3 % 4 %
Azithromycin 158 3 % 3 % 361 2 % 2 %

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

Analyte LLOQ
[µg/l]
ULOQ
[µg/l]
Azithromycin 1120 2000
Chloroquine 4.97 2000
Favipiravir 418 40000
Lopinavir 104 24000
Nafamostat 1.72 1000
Hydroxychloroquin 251 4500
GS 441524 11.8 400
Remdesivir 20.8 12000
Ritonavir 12.5 24000

Table 5: Upper and lower limit of quantification

Ordering Information

Order No.  Products
92055

3PLUS1® Multilevel Plasma Calibrator Set COVID-19 Drugs

0268, 0269

MassCheck® COVID-19 Drugs Plasma Controls

92043

Tuning Mix COVID-19 Drugs

92005 Extraction Buffer
92844

Internal Standard Mix Anti-HIV Drugs

92012 Precipitation Reagent
92007 Dilution Buffer 1
92008 Dilution Buffer 2
92110 MassTox® TDM MasterColumn® A
92001 Mobile Phase 1
92002 Mobile Phase 2

* Products mentioned in this App Note are not available in all countries.