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.

 Table 1: The Drugs of COVID-19 Calibrator 

GS 5734/Remdesivir     Favipiravir        Lopinavir    
GS 441524
(active metabolite of Remdesivir)
Azithromycin Nafamostat
Ritonavir Chloroquine Hydroxychloroquine

 

 

 

 

 

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.  

Table 2: Binary gradient conditions

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 3: MRMs and corresponding ISTD 

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

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.

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

Level I, order no. 0268

Level II, order no. 0269
Analyte Conc.
[µg/l]   

CV [%]
Interassay

CV [%]
Intraassay
Conc. 
[µg/l]
CV [%]
Interassay
CV [%]
Intraassay
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 5: Upper and lower limit of quantification

Analyte

LLOQ
[µg/l]

ULOQ
[µg/l]

Remdesivir

20.8

12000

GS 441524

11.8

400

Lopinavir

104

24000

Ritonavir

12.5

24000

Favipiravir

418

40000

Chloroquine

4.97

2000

Hydroxychloroquine

251

4500

Nafamostat

1.72

1000

Azithromycin

11.20

2000

Ordering Information

Products Order No. 

3PLUS1® Multilevel Plasma Calibrator Set COVID-19 Drugs

92055

MassCheck® COVID-19 Drugs Plasma Controls

0268, 0269
Tuning Mix 92043
Extraction Buffer 92005

Internal Standard Mix Anti-HIV Drugs

92844
Precipitation Reagent 92012
Dilution Buffer 1 92007
Dilution Buffer 2 92008
MassTox® TDM MasterColumn® A 92110
Mobile Phase 1 92001
Mobile Phase 2 92002

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