A Novel HPLC-MS/MS Method for the Intracellular Quantification of the Active Triphosphate Metabolite of Remdesivir: GS-443902
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Standards and Internal Quality Control Samples
2.3. PBMCs Isolation
2.4. Sample Preparation
2.5. HPLC-MS/MS Settings
2.6. Method Validation
2.6.1. Specificity and Selectivity
2.6.2. Accuracy, Precision, Calibration, Sensitivity, Dilution Integrity
2.6.3. Recovery
2.6.4. Adsorption and Stability
2.6.5. Matrix Effect
2.6.6. Carry-Over
2.6.7. Application and Statistical Analysis
2.6.8. Incurred Sample Reanalysis
3. Results
3.1. Calibration Curve Linearity and Dilution Integrity
3.2. Specificity and Selectivity
3.3. LLOQ and LOD
3.4. Adsorption and Stability
3.5. Recovery and Matrix Effect
3.6. Carry-Over
3.7. Testing of Patients’ Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
SARS-CoV-2 | Severe Acute Respiratory Syndrome Coronavirus Type 2 |
WHO | World Health Organization |
RDV | Remdesivir |
PK | Pharmacokinetics |
PBMCs | peripheral blood mononuclear cells |
HPLC-MS/MS | High-Performance Liquid Chromatography coupled with tandem Mass Spectrometry |
STD 9 | highest calibrator standard |
RSD | relative standard deviation |
IS | Internal Standard |
QC | Quality Control |
LLOQ | Lower Limit of Quantitation |
RT | retention time |
LOD | limit of detection |
STD1 | lowest calibrator standard |
REC | Recovery |
ME | matrix effect |
TFV-d6 | 2 H6-tenofovir diphosphate |
SPE | solid phase extraction |
TDF | Tenofovir disoproxil fumarate |
TAF | Tenofovir alafenamide |
ACN | Acetonitrile |
MeOH | Methanol |
DMSO | dimethyl sulfoxide |
MCV | mean cell volume |
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GS-443902 Concentration in the Calibrating Solution [ng/mL] | GS-443902 Amount in the PBMC Sample [ng] | |
---|---|---|
STD 1 | 3.125 | 0.3125 |
STD 2 | 6.25 | 0.625 |
STD 3 | 12.5 | 1.25 |
STD 4 | 25 | 2.5 |
STD 5 | 50 | 5 |
STD 6 | 100 | 10 |
STD 7 | 200 | 20 |
STD 8 | 400 | 40 |
STD 9 | 800 | 80 |
QC H | 640 | 64 |
QC M | 160 | 16 |
QC L | 40 | 4 |
General Detector Settings | ||||
Drying Gas Temperature [°C] | 130 | |||
HSID Temperature [°C] | 300 | |||
Nebulizer Gas [L/h] | 350 | |||
ElectroSpray V1 Negative [kV] | −4.8 | |||
Source Temperature [°C] | 350 | |||
Multipole 1 RF | 370 | |||
Collision Pressure | 420 | |||
Ionization | ESI- | |||
Analyte-Specific Parameters | ||||
GS-443902 | 2H6-TNF-DP (IS) | |||
Primary | Secondary | Primary | Secondary | |
Ion Trace (m/z) | 529.9 > 158.9 | 529.9 > 431.9 | 452.0 > 176.9 | 452.0 > 354.9 |
Collision Energy | 43 | 30 | 31 | 28 |
Entrance voltage | −29 | −39 | −23 | −23 |
Collision Cell Lens 2 | 100 | 120 | 96 | 92 |
Recovery Mean% (RSD%) | Matrix Effect Mean% (RSD%) | Extraction Efficiency Mean% (RSD%) | IS-nREC Mean% (RSD%) | IS-nME Mean% (RSD%) | IS-nEE Mean% (RSD%) | ||
---|---|---|---|---|---|---|---|
GS-443902 | H | 51.2 (21.3) | 53.7 (17.) | 95.0 (8.1) | 119.0 (3.7) | 113.3 (7.1) | 101.2 (5.1) |
M | 85.8 (14.6) | 67.3 (15.4) | 101.9 (11.2) | 114.0 (11.4) | 112.9 (9.2) | 98.8 (1.7) | |
L | 119.5 (13.7) | 114.6 (15.3) | 105.8 (18.2) | 114.0 (3.4) | 111.7 (11.9) | 98.9 (2.9) |
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Palermiti, A.; De Nicolò, A.; Antonucci, M.; Soloperto, S.; Billi, M.; Manca, A.; Cusato, J.; Menegatti, G.; Lamorde, M.; Calcagno, A.; et al. A Novel HPLC-MS/MS Method for the Intracellular Quantification of the Active Triphosphate Metabolite of Remdesivir: GS-443902. J. Xenobiot. 2025, 15, 107. https://doi.org/10.3390/jox15040107
Palermiti A, De Nicolò A, Antonucci M, Soloperto S, Billi M, Manca A, Cusato J, Menegatti G, Lamorde M, Calcagno A, et al. A Novel HPLC-MS/MS Method for the Intracellular Quantification of the Active Triphosphate Metabolite of Remdesivir: GS-443902. Journal of Xenobiotics. 2025; 15(4):107. https://doi.org/10.3390/jox15040107
Chicago/Turabian StylePalermiti, Alice, Amedeo De Nicolò, Miriam Antonucci, Sara Soloperto, Martina Billi, Alessandra Manca, Jessica Cusato, Giorgia Menegatti, Mohammed Lamorde, Andrea Calcagno, and et al. 2025. "A Novel HPLC-MS/MS Method for the Intracellular Quantification of the Active Triphosphate Metabolite of Remdesivir: GS-443902" Journal of Xenobiotics 15, no. 4: 107. https://doi.org/10.3390/jox15040107
APA StylePalermiti, A., De Nicolò, A., Antonucci, M., Soloperto, S., Billi, M., Manca, A., Cusato, J., Menegatti, G., Lamorde, M., Calcagno, A., Waitt, C., & D’Avolio, A. (2025). A Novel HPLC-MS/MS Method for the Intracellular Quantification of the Active Triphosphate Metabolite of Remdesivir: GS-443902. Journal of Xenobiotics, 15(4), 107. https://doi.org/10.3390/jox15040107