Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV
Abstract
:1. Introduction
2. Results and Discussion
2.1. Calibration Curve and Dilution Integrity
2.2. Specificity and Selectivity
2.3. Accuracy and Precision
2.4. Lowest Limit of Quantification (LLOQ) and Limit of Detection (LOD)
2.5. Recovery
2.6. Matrix Effect
2.7. Carry-Over
2.8. Stability
2.9. Testing of Patients’ Samples
3. Materials and Methods
3.1. Chemicals
3.2. Stock Solutions, Standards, and Quality Controls
3.3. Chromatographic Conditions
3.4. Mass Spectrometry Conditions
3.5. PBMC Isolation
3.6. STDs, QCs, and Patient Samples Extraction
3.7. Specificity and Selectivity
3.8. Accuracy, Precision, Calibration, and Limit of Quantification
3.9. Recovery
3.10. Stability
3.11. Matrix Effect
3.12. Carry-Over
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Drugs | RT (min) | Highest Spiking Solution (ng/mL) | STD9/ULOQ (ng/Sample) | STD1/LLOQ (ng/Sample) | LOD (ng/Sample) | R2 | [M-H] + (m/z) | Dwell (ms) | Entrance Voltage (V) | FIRST Trace (m/z) | Collision Energy First Ion Trace (eV) | Second Trace (m/z) | Collision Energy Second Ion Trace (eV) |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
MVC | 2.15 | 100 | 10 | 0.039 | 0.019 | 0.996 | 514.4 | 30 | 33 | 117.2 | −69 | 389.3 | −26 |
NVP | 2.35 | 1000 | 100 | 0.390 | 0.195 | 0.997 | 267.1 | 30 | 25 | 80.2 | −56 | 226.1 | −32 |
RPV | 4.08 | 100 | 10 | 0.039 | 0.019 | 0.998 | 367.3 | 30 | 45 | 128.2 | −83 | 195.2 | −48 |
DTG | 5.32 | 100 | 10 | 0.039 | 0.019 | 0.998 | 420.3 | 25 | 30 | 127.1 | −46 | 277.1 | −34 |
RAL | 5.60 | 100 | 10 | 0.039 | 0.019 | 0.996 | 445.2 | 25 | 26 | 109.1 | −41 | 361.1 | −25 |
COBI | 7.90 | 100 | 10 | 0.039 | 0.019 | 0.996 | 776.4 | 25 | 15 | 98.1 | −82 | 606.2 | −34 |
DRV | 8.15 | 1000 | 100 | 0.390 | 0.195 | 0.998 | 548.3 | 25 | 15 | 69.1 | −66 | 392.2 | −18 |
ATV | 9.30 | 100 | 10 | 0.039 | 0.019 | 0.999 | 705.4 | 30 | 10 | 144.2 | −59 | 168.15 | −58 |
EFV * | 11.21 | 1000 | 100 | 0.390 | 0.195 | 0.997 | 314.1 | 25 | −29 | 68.9 | 48 | 244.1 | 22 |
RTV | 11.44 | 100 | 10 | 0.039 | 0.019 | 0.996 | 721.4 | 25 | 27 | 140.1 | −95 | 296.15 | −29 |
ELV | 11.64 | 100 | 10 | 0.039 | 0.019 | 0.996 | 448.2 | 25 | 30 | 143.0 | −61 | 344.0 | −42 |
ETV | 11.76 | 100 | 10 | 0.039 | 0.019 | 0.997 | 435.1 | 25 | 51 | 77.1 | −110 | 144.1 | −55 |
13C6-RPV | 3.67 | - | 1 | 1 | 1 | - | 373.3 | 30 | 39 | 182.2 | −50 | 195.2 | −50 |
13C,2H5-DTG | 5.32 | - | 1 | 1 | 1 | - | 426.3 | 25 | 40 | 133.2 | −50 | 277.1 | −35 |
QX | 6.48 | - | 0.8 | 0.8 | 0.8 | - | 313.1 | 25 | 44 | 78.1 | −72 | 246.2 | −43 |
13C6-DRV | 8.15 | - | 10 | 10 | 10 | - | 554.3 | 25 | 16 | 113.1 | −31 | 398.2 | −20 |
2H6-ATV | 9.21 | - | 1 | 1 | 1 | - | 711.5 | 30 | 39 | 147.2 | −61 | 338.2 | −41 |
13C6-EFV * | 11.17 | - | 10 | 10 | 10 | - | 320.1 | 25 | −24 | 68.9 | 61 | 250.1 | 23 |
13C,2H3-RTV | 11.35 | - | 0.4 | 0.4 | 0.4 | - | 725.3 | 25 | 8 | 201.1 | −57 | 272.2 | −41 |
13C6-ETV | 11.72 | - | 1 | 1 | 1 | - | 441.2 | 25 | 45 | 77.1 | −110 | 150.2 | −55 |
Drugs | QC High | QC Medium | QC Low | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Spiking Sol. Conc. (ng/mL) | Final Amount per Sample (ng) | Accuracy % | Imprecision RSD% | Spiking Sol. Conc. (ng/mL) | Final Amount per Sample (ng) | Accuracy % | Imprecision RSD% | Spiking Sol. Conc. (ng/mL) | Final Amount per Sample (ng) | Accuracy % | Imprecision RSD% | ||||
Intra-Day % | Inter-Day % | Intra-Day % | Inter-Day % | Intra-Day % | Inter-Day % | ||||||||||
MVC | 80 | 8 | 95.6 | 5.3 | 11.5 | 10 | 1 | 95.5 | 3.4 | 14.7 | 1 | 0.1 | 92.5 | 6.0 | 7.6 |
NVP | 800 | 80 | 96.1 | 4.0 | 10.0 | 100 | 10 | 104.8 | 1.9 | 12.0 | 10 | 1 | 87.7 | 5.8 | 9.2 |
RPV | 80 | 8 | 103.7 | 2.8 | 5.2 | 10 | 1 | 87.4 | 4.4 | 3.2 | 1 | 0.1 | 89.7 | 4.5 | 10.6 |
DTG | 80 | 8 | 98.6 | 2.9 | 7.6 | 10 | 1 | 85.1 | 3.4 | 13.8 | 1 | 0.1 | 93.4 | 7.2 | 6.8 |
RAL | 80 | 8 | 106.3 | 4.1 | 6.0 | 10 | 1 | 90.8 | 4.3 | 9.9 | 1 | 0.1 | 92.8 | 5.9 | 9.5 |
COBI | 80 | 8 | 94.7 | 7.8 | 8.5 | 10 | 1 | 86.9 | 7.7 | 12.0 | 1 | 0.1 | 95.2 | 1.5 | 9.3 |
DRV | 800 | 80 | 101.4 | 1.8 | 3.7 | 100 | 10 | 87.4 | 3.0 | 9.5 | 10 | 1 | 88.1 | 3.3 | 12.0 |
ATV | 80 | 8 | 103.7 | 2.7 | 5.2 | 10 | 1 | 92.7 | 3.4 | 12.9 | 1 | 0.1 | 94.4 | 6.1 | 9.3 |
EFV | 800 | 80 | 103.1 | 1.1 | 3.7 | 100 | 10 | 90.8 | 3.1 | 5.7 | 10 | 1 | 88.1 | 4.4 | 4.0 |
RTV | 80 | 8 | 111.5 | 12.6 | 9.5 | 10 | 1 | 93.5 | 12.1 | 9.7 | 1 | 0.1 | 94.4 | 9.9 | 8.2 |
ELV | 80 | 8 | 100.4 | 4.7 | 8.4 | 10 | 1 | 95.5 | 3.0 | 5.3 | 1 | 0.1 | 98.9 | 8.7 | 7.9 |
ETV | 80 | 8 | 104.1 | 1.8 | 4.0 | 10 | 1 | 90.1 | 4.5 | 9.5 | 1 | 0.1 | 94.3 | 5.5 | 5.9 |
Drugs | Recovery (RSD) | Matrix Effect (RSD) | IS-Normalized Matrix Effect (RSD) |
---|---|---|---|
MVC | 89.2% (6.1%) | 4.6% (6.7%) | 13.9% (9.1%) |
NVP | 89.5% (2.6%) | −10.1% (5.9%) | −2.4% (10.5%) |
RPV | 83.2% (3.0%) | −5.0% (11.6%) | 2.8% (4.7%) |
DTG | 92.8% (3.8%) | −11.1% (7.2%) | 0.1% (4.7%) |
RAL | 91.2% (2.2%) | −15.7% (5.4%) | −5.3% (4.9%) |
COBI | 77.6% (3.0%) | 31.2% (9.1%) | 19.8% (7.5%) |
DRV | 84.6% (2.9%) | −15.0% (4.3%) | −1.0% (12.9%) |
ATV | 80.3% (2.9%) | 9.3% (6.0%) | −0.1% (4.2%) |
EFV | 86.6% (2.4%) | −4.0% (4.8%) | −0.6% (1.7%) |
RTV | 85.3% (2.7%) | −8.1% (7.7%) | −7.2% (8.0%) |
ELV | 87.3% (4.3%) | −27.4% (10.3%) | −10.1% (10.5) |
ETV | 79.7% (3.1%) | −17.6% (6.0%) | 4.5% (6.6%) |
Median Trough Concentrations (IQR) | ||||
---|---|---|---|---|
Drugs | n | Plasma (ng/mL) | PBMC (ng/mL) | PBMC/Plasma Ratio |
MVC | 1 | 68 (na *) | 163 (na) | 2.40 (na) |
RPV | 1 | 80 (na) | 399 (na) | 4.98 (na) |
DTG | 34 | 913 (616–1392) | 270 (156–450) | 0.26 (0.14–0.53) |
RAL | 2 | 138 (136–na) | 43 (40–na) | 0.31 (0.29–na) |
COBI | 17 | 49 (6–142) | 204 (63–686) | 3.52 (3.15–4.48) |
DRV | 31 | 2389 (1523–3963) | 935 (581–1642) | 0.34 (0.23–0.57) |
ATV | 21 | 778 (424–1547) | 1429 (552–3255) | 1.53 (1.01–2.72) |
RTV | 29 | 82 (47–389) | 497 (278–845) | 3.81 (2.07–6.70) |
ELV | 1 | 228 (na) | 1641 (na) | 7.20 (na) |
ETV | 5 | 514 (78–763) | 2137 (500–3126) | 5.51 (3.01–6.03) |
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De Nicolò, A.; Ianniello, A.; Ferrara, M.; Avataneo, V.; Cusato, J.; Antonucci, M.; De Vivo, E.; Waitt, C.; Calcagno, A.; Trentalange, A.; et al. Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV. Pharmaceuticals 2021, 14, 12. https://doi.org/10.3390/ph14010012
De Nicolò A, Ianniello A, Ferrara M, Avataneo V, Cusato J, Antonucci M, De Vivo E, Waitt C, Calcagno A, Trentalange A, et al. Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV. Pharmaceuticals. 2021; 14(1):12. https://doi.org/10.3390/ph14010012
Chicago/Turabian StyleDe Nicolò, Amedeo, Alice Ianniello, Micol Ferrara, Valeria Avataneo, Jessica Cusato, Miriam Antonucci, Elisa De Vivo, Catriona Waitt, Andrea Calcagno, Alice Trentalange, and et al. 2021. "Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV" Pharmaceuticals 14, no. 1: 12. https://doi.org/10.3390/ph14010012
APA StyleDe Nicolò, A., Ianniello, A., Ferrara, M., Avataneo, V., Cusato, J., Antonucci, M., De Vivo, E., Waitt, C., Calcagno, A., Trentalange, A., Muccioli, G., Bonora, S., Di Perri, G., & D'Avolio, A. (2021). Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV. Pharmaceuticals, 14(1), 12. https://doi.org/10.3390/ph14010012