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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 I (92007; ratio 1:5) or Dilution Buffer II (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 A (92001) |
Mobile Phase B (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
|
Analyt |
Corresponding ISTD MRM |
MRM |
|
Remdesivir |
ISTD 2 (520/117) |
603 -> 402 |
|
GS 441524 |
ISTD 2 (520/117) |
292 -> 147 |
|
Lopinavir |
ISTD 2 (520/117) |
629 -> 155 |
|
Ritonavir |
ISTD 16 (637/163) |
721 -> 140 |
|
Favipiravir |
ISTD 2 (520/117) |
158 -> 85 |
|
Chloroquine |
ISTD 2 (520/117) |
320 -> 247 |
|
Hydroxychloroquine |
ISTD 2 (520/117) |
336 158 |
|
Nafamostat |
ISTD 2 (520/117) |
348 -> 162 |
|
Azithromycin |
ISTD 12 (727/146) |
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 [%][%] |
Conc. [µg/l] |
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 |
ULOQ |
|
Azithromycin |
11,20 |
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 |
Ordering Information
| Products | Order No. |
|
3PLUS1® Multilevel Plasma Calibrator Set COVID-19 Drugs |
92055 |
|
MassCheck® COVID-19 Drugs Plasma Controls |
0268, 0269 |
| Extraction Buffer | 92005 |
|
Internal Standard Mix Anti-HIV Drugs |
92844 |
| Precipitation Reagent | 92012 |
| Dilution Buffer I | 92007 |
| Dilution Buffer II | 92008 |
| MassTox® TDM MasterColumn® A | 92110 |
| Mobile Phase A | 92001 |
| Mobile Phase B | 92002 |