Development and Validation of a UPLC-MS/MS Method for Therapeutic Drug Monitoring, Pharmacokinetic and Stability Studies of First-Line Antituberculosis Drugs in Urine
Abstract
:1. Introduction
2. Results
2.1. Method Validation
2.1.1. Selectivity
2.1.2. Calibration Curves
2.1.3. LLOQ, Precision, and Accuracy
2.1.4. Carry-Over
2.1.5. Matrix Effect
2.1.6. Stability
2.1.7. Dilution Integrity
2.1.8. Application to Clinical Samples
3. Discussion
4. Materials and Methods
4.1. Standards and Reagents
4.2. Instrumentation and Chromatographic Conditions
4.3. Mass Spectrometer Settings
4.4. Stock and Standard Solutions
- RIF, ETH, INH, and PZA: 5.0, 10.0, 25.0, 50.0, 100.0, 250.0, 500.0, and 1000.0 µg/mL.
- 25-D-RIF, RIF-Q, and 3-F-RIF: 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 100.0, and 200.0 µg/mL.
- ETH-IS and RIF-IS: 0.5 µg/mL.
- PZA-IS: 5 µg/mL.
4.5. Preparation of Calibrators, Quality Control Samples and Biological Samples
- RIF, ETH, INH, and PZA: 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 µg/mL.
- 25-D-RIF, RIF-Q, and 3-F-RIF: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 µg/mL.
4.6. Validation
4.6.1. Selectivity
4.6.2. Calibration Curves
4.6.3. LLOQ, Precision, and Accuracy
4.6.4. Carry-Over and Dilution Integrity
4.6.5. Matrix Effect
4.6.6. Stability
4.6.7. Application to Clinical Samples
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Compound | QC Level | Conc. (µg/mL) | Precision (%RSD) | Accuracy (%) | ||
---|---|---|---|---|---|---|
Intra-Day | Inter-Day | Intra-Day | Inter-Day | |||
n = 5 | n = 5 | n = 5 | n = 5 | |||
RIF | LLOQ | 0.5 | 19.4 | 4.7 | 105.9 | 97.3 |
Low | 1 | 11.3 | 8.7 | 108.0 | 91.8 | |
Medium | 10 | 13.2 | 7.2 | 98.5 | 93.0 | |
High | 100 | 10.0 | 11.9 | 99.3 | 111.7 | |
RIF-Q | LLOQ | 0.1 | 15.5 | 12.9 | 115.7 | 99.4 |
Low | 0.25 | 4.2 | 9.4 | 104.9 | 91.0 | |
Medium | 2.5 | 9.2 | 9.2 | 93.2 | 94.5 | |
High | 20 | 6.9 | 4.3 | 107.4 | 114.0 | |
3-F-RIF | LLOQ | 0.1 | 8.9 | 10.5 | 110.5 | 102.2 |
Low | 0.25 | 7.2 | 11.1 | 108.6 | 102.0 | |
Medium | 2.5 | 13.3 | 8.8 | 87.7 | 90.0 | |
High | 20 | 8.3 | 8.9 | 108.6 | 95.8 | |
25-D-RIF | LLOQ | 0.1 | 6.2 | 5.0 | 94.3 | 106.2 |
Low | 0.25 | 12.3 | 6.7 | 101.7 | 99.9 | |
Medium | 2.5 | 9.7 | 6.0 | 101.9 | 104.3 | |
High | 20 | 7.4 | 4.2 | 99.6 | 107.8 | |
INH | LLOQ | 0.5 | 8.2 | 6.7 | 98.1 | 86.4 |
Low | 1 | 10.0 | 8.8 | 104.3 | 97.0 | |
Medium | 10 | 5.5 | 4.5 | 98.8 | 93.8 | |
High | 100 | 3.9 | 2.5 | 107.8 | 100.1 | |
PZA | LLOQ | 0.5 | 18.1 | 8.3 | 95.7 | 87.4 |
Low | 1 | 6.0 | 8.5 | 101.5 | 103.3 | |
Medium | 10 | 6.5 | 1.4 | 97.6 | 104.2 | |
High | 100 | 4.3 | 4.5 | 101.7 | 109.0 | |
ETH | LLOQ | 0.5 | 16.3 | 11.3 | 98.6 | 96.2 |
Low | 1 | 7.2 | 8.8 | 107.9 | 108.9 | |
Medium | 10 | 9.4 | 14.3 | 91.2 | 106.2 | |
High | 100 | 3.9 | 7.1 | 104.7 | 109.1 |
Compound | QC Level | Conc. [mg/L] | IS-Normalized MF (n = 6) | %RSD | |
---|---|---|---|---|---|
Mean | SD | ||||
RIF | Low | 1 | 1.27 | 0.06 | 5.0 |
High | 100 | 1.06 | 0.14 | 13.1 | |
RIF-Q | Low | 0.25 | 0.43 | 0.19 | 45.2 |
High | 20 | 0.62 | 0.32 | 51.9 | |
3-F-RIF | Low | 0.25 | 0.51 | 0.06 | 11.1 |
High | 20 | 0.66 | 0.10 | 14.5 | |
25-D-RIF | Low | 0.25 | 1.74 | 0.16 | 9.2 |
High | 20 | 1.55 | 0.20 | 12.6 | |
INH | Low | 1 | 0.37 | 0.05 | 12.6 |
High | 100 | 0.45 | 0.08 | 18.3 | |
PZA | Low | 1 | 1.16 | 0.19 | 16.1 |
High | 100 | 0.70 | 0.15 | 21.7 | |
ETH | Low | 1 | 0.85 | 0.11 | 12.8 |
High | 100 | 0.83 | 0.06 | 7.2 |
Compound | QC Level | Bench-Top Stability at RT | Autosampler (12 h, 15 °C) | Autosampler (24 h, 15 °C) | Working Solution Stability (−20 °C) | Short-Term Stability (24 h, −20 °C) | Long-Term Stability (30 Days, −20 °C) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 h | 4 h | 1 Month | 8 Months | pH 4 | pH 5 | pH 6 | pH 7 | pH 8 | pH 4 | pH 5 | pH 6 | pH 7 | pH 8 | ||||
RIF | Low | 98 | 90 | 90 | 92 | 103 | 95 | 74 | 81 | 87 | 86 | 90 | 83 | 105 | 115 | 110 | 101 |
High | 101 | 96 | 99 | 100 | 101 | 94 | 85 | 87 | 90 | 88 | 89 | 96 | 103 | 100 | 95 | 100 | |
RIF-Q | Low | 69 | 39 | 84 | 39 | 101 | 127 | 80 | 81 | 92 | 76 | 90 | 55 | 55 | 64 | 54 | 46 |
High | 87 | 79 | 92 | 96 | 98 | 88 | 119 | 97 | 98 | 119 | 126 | 55 | 27 | 29 | 50 | 80 | |
3-F-RIF | Low | 97 | 61 | 63 | 30 | 97 | 61 | 60 | 58 | 54 | 50 | 64 | 24 | 39 | 70 | 83 | 77 |
High | 91 | 88 | 97 | 92 | 100 | 78 | 119 | 97 | 98 | 119 | 126 | 11 | 20 | 84 | 116 | 104 | |
25-D-RIF | Low | 111 | 119 | 120 | 123 | 104 | 182 | 102 | 112 | 120 | 117 | 114 | 92 | 90 | 87 | 101 | 98 |
High | 95 | 111 | 106 | 123 | 102 | 168 | 103 | 112 | 108 | 115 | 107 | 90 | 110 | 110 | 142 | 137 | |
INH | Low | 99 | 99 | 98 | 91 | 110 | 124 | 75 | 92 | 96 | 97 | 85 | 44 | 47 | 85 | 86 | 77 |
High | 94 | 94 | 102 | 100 | 109 | 115 | 93 | 103 | 107 | 114 | 105 | 88 | 96 | 98 | 114 | 95 | |
PZA | Low | 111 | 104 | 105 | 98 | 105 | 107 | 98 | 90 | 94 | 100 | 79 | 102 | 95 | 102 | 110 | 91 |
High | 102 | 106 | 109 | 109 | 99 | 112 | 106 | 103 | 112 | 110 | 133 | 88 | 78 | 87 | 94 | 111 | |
ETH | Low | 112 | 108 | 115 | 114 | 104 | - | 111 | 108 | 113 | 114 | 102 | 105 | 107 | 105 | 104 | 101 |
High | 112 | 114 | 111 | 105 | 98 | - | 110 | 104 | 108 | 97 | 104 | 99 | 111 | 101 | 114 | 103 |
Compound | Initial Conc. [mg/L] | 1 h | 8 h | 24 h | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
pH 4 | pH 5 | pH 6 | pH 7 | pH 8 | pH 4 | pH 5 | pH 6 | pH 7 | pH 8 | pH 4 | pH 5 | pH 6 | pH 7 | pH 8 | ||
After incubation at 37.5 °C | ||||||||||||||||
RIF | 25 | 82 | 94 | 96 | 88 | 88 | 57 | 75 | 82 | 86 | 80 | 30 | 55 | 66 | 67 | 68 |
INH | 50 | 88 | 89 | 92 | 90 | 87 | 79 | 84 | 92 | 94 | 81 | 79 | 78 | 86 | 86 | 76 |
PZA | 50 | 86 | 90 | 93 | 90 | 88 | 92 | 92 | 93 | 94 | 86 | 92 | 90 | 91 | 89 | 86 |
ETH | 50 | 88 | 85 | 92 | 100 | 90 | 89 | 93 | 94 | 87 | 99 | 89 | 97 | 98 | 92 | 92 |
After incubation at 20.5 °C | ||||||||||||||||
RIF | 25 | 79 | 87 | 99 | 89 | 87 | 76 | 82 | 86 | 81 | 81 | 58 | 71 | 78 | 77 | 71 |
INH | 50 | 86 | 86 | 90 | 91 | 87 | 79 | 77 | 92 | 88 | 91 | 79 | 74 | 87 | 90 | 79 |
PZA | 50 | 95 | 101 | 99 | 103 | 103 | 90 | 85 | 88 | 87 | 85 | 92 | 87 | 85 | 93 | 85 |
ETH | 50 | 86 | 98 | 87 | 98 | 99 | 97 | 87 | 87 | 92 | 94 | 91 | 88 | 96 | 104 | 91 |
Compound | Conc. (µg/mL) (Mean ± SD) |
---|---|
RIF | 17.9 ± 7.8 |
RIF-Q | 1.9 ± 0.8 |
3-F-RIF | 1.3 ± 1.3 |
25-D-RIF | 4.1 ± 2.5 |
INH | 48.7 ± 22.7 |
PZA | 54.3 ± 19.5 |
ETH | 391.7 ± 200.3 |
Analyte | Precursor Ion (m/z) | Fragment Ion (m/z) | Collision Energy (V) |
---|---|---|---|
RIF | 824.3 | 792.2 | 25 |
398.7 | 37 | ||
RIF-Q | 822.2 | 790.2 | 25 |
397.6 | 37 | ||
3-F-RIF | 727.2 | 667.1 | 17 |
641.2 | 71 | ||
25-D-RIF | 782.3 | 750.2 | 17 |
399.7 | 33 | ||
INH | 138.0 | 121.0 | 19 |
79.0 | 39 | ||
PZA | 124.0 | 64.0 | 7 |
81.0 | 23 | ||
ETH | 205.1 | 116.1 | 21 |
145 | 11 | ||
RIF-IS | 827.3 | 795.3 | 25 |
151.1 | 37 | ||
PZA-IS | 128.0 | 84.0 | 25 |
99.9 | 9 | ||
ETH-IS | 209.2 | 120.0 | 21 |
149.2 | 9 |
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Abouzid, M.; Kosicka-Noworzyń, K.; Karaźniewicz-Łada, M.; Rao, P.; Modi, N.; Xie, Y.L.; Heysell, S.K.; Główka, A.; Kagan, L. Development and Validation of a UPLC-MS/MS Method for Therapeutic Drug Monitoring, Pharmacokinetic and Stability Studies of First-Line Antituberculosis Drugs in Urine. Molecules 2024, 29, 337. https://doi.org/10.3390/molecules29020337
Abouzid M, Kosicka-Noworzyń K, Karaźniewicz-Łada M, Rao P, Modi N, Xie YL, Heysell SK, Główka A, Kagan L. Development and Validation of a UPLC-MS/MS Method for Therapeutic Drug Monitoring, Pharmacokinetic and Stability Studies of First-Line Antituberculosis Drugs in Urine. Molecules. 2024; 29(2):337. https://doi.org/10.3390/molecules29020337
Chicago/Turabian StyleAbouzid, Mohamed, Katarzyna Kosicka-Noworzyń, Marta Karaźniewicz-Łada, Prakruti Rao, Nisha Modi, Yingda L. Xie, Scott K. Heysell, Anna Główka, and Leonid Kagan. 2024. "Development and Validation of a UPLC-MS/MS Method for Therapeutic Drug Monitoring, Pharmacokinetic and Stability Studies of First-Line Antituberculosis Drugs in Urine" Molecules 29, no. 2: 337. https://doi.org/10.3390/molecules29020337