Quantitative Analysis of Isoniazid and Its Four Primary Metabolites in Plasma of Tuberculosis Patients Using LC-MS/MS
Abstract
:1. Introduction
2. Results and Discussion
2.1. Method Optimization for LC and MS Conditions
2.2. Method Validations
2.2.1. Selectivity, Linearity, and Carryover
2.2.2. Accuracy and Precision
2.2.3. Matrix Effects and Recovery
2.2.4. Stability
2.3. Application to Clinical Research
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. Preparation of Standards and Quality Controls Samples
3.3. Sample Preparation
3.4. Liquid Chromatography–Tandem Mass Spectrometry
3.5. Method Validation
3.5.1. Selectivity and Carryover
3.5.2. Linearity and Lower Limit of Quantification
3.5.3. Accuracy and Precision
3.5.4. Matrix Effect and Recovery
3.5.5. Stability
3.6. Clinical Application
3.7. Determination of NAT2 Genotype
3.8. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Analytes | Transition (m/z) | DP (V) | CE (eV) | Calibration Range (ng/mL) | Correlation Coefficient | Internal Standard |
---|---|---|---|---|---|---|
INH | 138 → 121 | 50 | 30 | 80–10,000 | 0.9970 | Phenacetin |
AcINH | 180 → 121 | 60 | 30 | 80–10,000 | 0.9958 | Phenacetin |
INA | 124 → 80 | 40 | 25 | 80–10,000 | 0.9947 | Phenacetin |
HZ | 237.2 → 120 | 80 | 25 | 1–30 | 0.9975 | Sulfamethazine |
AcHZ | 177 → 118 | 50 | 19 | 40–1200 | 0.9993 | Sulfamethazine |
Analytes | Nominal Conc. (ng/mL) | Intra-Day (n = 5) | Inter-Day (n = 3) * | ||
---|---|---|---|---|---|
RSD (%) | Accuracy (%) | RSD (%) | Accuracy (%) | ||
INH | 80 | 4.41 | 104.43 | 4.78 | 104.34 |
240 | 3.92 | 107.78 | 5.29 | 103.84 | |
2000 | 5.21 | 106.16 | 3.01 | 103.10 | |
8000 | 7.93 | 105.84 | 3.30 | 101.69 | |
AcINH | 80 | 3.34 | 92.85 | 7.10 | 97.46 |
240 | 11.57 | 95.38 | 4.67 | 98.74 | |
2000 | 3.73 | 99.10 | 4.93 | 99.25 | |
8000 | 4.59 | 97.20 | 5.43 | 99.89 | |
INA | 80 | 13.43 | 102.07 | 8.51 | 108.78 |
240 | 10.54 | 91.63 | 9.69 | 103.01 | |
2000 | 9.31 | 96.00 | 2.87 | 97.89 | |
8000 | 8.74 | 100.94 | 8.95 | 100.59 | |
HZ | 1 | 5.58 | 112.00 | 9.36 | 106.26 |
3 | 1.86 | 109.00 | 3.73 | 107.74 | |
12 | 6.28 | 98.10 | 6.49 | 100.36 | |
24 | 6.90 | 100.32 | 2.49 | 102.13 | |
AcHZ | 40 | 6.54 | 106.00 | 9.19 | 99.19 |
120 | 1.12 | 114.00 | 8.22 | 106.07 | |
480 | 11.30 | 97.68 | 5.32 | 100.26 | |
960 | 9.00 | 100.30 | 5.60 | 100.67 |
Analytes | Nominal Conc. (ng/mL) | Recovery (n = 6) | Matrix Effect (n = 6) | ||
---|---|---|---|---|---|
Mean (%) ± SD | RSD (%) | Mean (%) ± SD | RSD (%) | ||
INH | 240 | 92.38 ± 3.56 | 3.86 | 36.42 ± 2.55 | 7.01 |
8000 | 96.92 ± 1.97 | 2.03 | 43.46 ± 3.08 | 7.09 | |
AcINH | 240 | 110.76 ± 3.05 | 2.75 | 53.88 ± 1.70 | 3.15 |
8000 | 96.65 ± 1.23 | 1.27 | 54.08 ± 2.95 | 5.45 | |
INA | 240 | 66.94 ± 6.27 | 9.36 | 40.65 ± 1.67 | 4.12 |
8000 | 78.17 ± 0.84 | 1.07 | 39.68 ± 2.97 | 7.50 | |
HZ | 1.5 | 109.67 ± 7.65 | 6.98 | NA | NA |
24 | 70.35 ± 1.36 | 1.94 | NA | NA | |
AcHZ | 120 | 105.12 ± 2.42 | 2.30 | NA | NA |
960 | 112.19 ± 1.64 | 1.46 | NA | NA |
Analytes | Nominal Conc. (ng/mL) | Stability (%) | ||
---|---|---|---|---|
Bench-Top at RT (4 h) | Autosampler at 4 °C (*) | Freeze–Thaw (3 Cycles) | ||
INH | 240 | 103.82 | 104.46 | 77.36 |
8000 | 93.84 | 93.55 | 92.83 | |
AcINH | 240 | 94.10 | 97.44 | 97.97 |
8000 | 95.76 | 98.33 | 106.54 | |
INA | 240 | 92.93 | 92.98 | 79.44 |
8000 | 108.23 | 92.86 | 113.96 | |
HZ | 3 | 86.65 | 91.07 | 87.13 |
24 | 97.77 | 83.83 | 86.83 | |
AcHZ | 120 | 113.67 | 111.33 | 112.33 |
960 | 112.00 | 101.00 | 107.00 |
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Ky Anh, N.; My Tung, P.; Kim, M.J.; Phuoc Long, N.; Cho, Y.-S.; Kim, D.-H.; Shin, J.-G. Quantitative Analysis of Isoniazid and Its Four Primary Metabolites in Plasma of Tuberculosis Patients Using LC-MS/MS. Molecules 2022, 27, 8607. https://doi.org/10.3390/molecules27238607
Ky Anh N, My Tung P, Kim MJ, Phuoc Long N, Cho Y-S, Kim D-H, Shin J-G. Quantitative Analysis of Isoniazid and Its Four Primary Metabolites in Plasma of Tuberculosis Patients Using LC-MS/MS. Molecules. 2022; 27(23):8607. https://doi.org/10.3390/molecules27238607
Chicago/Turabian StyleKy Anh, Nguyen, Pham My Tung, Min Jung Kim, Nguyen Phuoc Long, Yong-Soon Cho, Dong-Hyun Kim, and Jae-Gook Shin. 2022. "Quantitative Analysis of Isoniazid and Its Four Primary Metabolites in Plasma of Tuberculosis Patients Using LC-MS/MS" Molecules 27, no. 23: 8607. https://doi.org/10.3390/molecules27238607