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 |
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 |
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 |
* For research use only and not for diagnostic purposes. Products mentioned in this App Note are also not available in all countries.
Last Update 14th of August 2020