Simultaneous Quantification of Propylthiouracil and Its N-β-d Glucuronide by HPLC-MS/MS: Application to a Metabolic Study
Abstract
:1. Introduction
2. Results and Discussion
2.1. HPLC-MS/MS Conditions and Method Optimization
2.2. Assay Validation
2.2.1. Selectivity
2.2.2. Sensitivity
2.2.3. Calibration Curve and Linearity
2.2.4. Carry-Over
2.2.5. Accuracy and Precision
2.2.6. Recovery and Matrix Effect
2.2.7. Stability
2.3. Application to In Vitro Metabolism Study
2.3.1. Formation of PTU-GLU
2.3.2. Inhibitory Study
2.3.3. Enzymatic Kinetics Study
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. Instrumental Conditions of HPLC-MS/MS
3.3. Calibration Standards and Quality Control (QC) Samples
3.4. Biological Sample Preparation
3.5. Assay Validation
3.6. In Vitro Metabolism Study
3.6.1. Assays of PTU-GLU Determination
3.6.2. Enzymatic Inhibition Study
3.6.3. Kinetic Study
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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PTU | PTU-GLU | MTU(IS) | |
---|---|---|---|
MRM Transition m/z (Q1-Q3) | 169.2 > 58.05 | 345.2 > 169.2 | 141.00 > 58.00 |
MS Ionization | ESI mode | ||
Q1 (V) | 12.0 | 13.0 | 10.0 |
Q2 (V) | 22.0 | 14.0 | 22.0 |
Q3 (V) | 20.0 | 17.0 | 19.0 |
Column | ZORBAX Extend-C18 column (2.1 × 50 mm, 1.8 μm) | ||
Column Temperature | 35 °C | ||
Run Time | 4 min | ||
Mobile Phase | Phase A: water (0.1% formic acid) Phase B: methanol/acetonitrile (0.1% formic acid) (2:1, v/v) | ||
Flow | 0.1 mL/min (40:60 = A:B) | ||
Injection Volume | 1 µL | ||
Retention Time | 1.66 min | 1.50 min | 1.40 min |
PTU | PTU-GLU | ||||||||
---|---|---|---|---|---|---|---|---|---|
0.1 µM | 0.5 µM | 5 µM | 25 µM | 0.1 µM | 0.5 µM | 5 µM | 25 µM | ||
Intra-Day Assay (n = 6) | Measured a | 0.107 ± 0.0054 | 0.574 ± 0.038 | 5.60 ± 0.62 | 24.9 ± 0.5 | 0.0907 ± 0.0085 | 0.575 ± 0.018 | 5.50 ± 0.30 | 26.3 ± 0.9 |
%Bias | 7.58% | 14.8% | 11.3% | −0.485% | −9.33% | 14.9% | 10.0% | 5.05% | |
%RSD | 5.02% | 6.61% | 9.42% | 9.87% | 9.38% | 7.33% | 5.42% | 3.35% | |
Inter-Day Assay (n = 6) | Measured a | 0.106 ± 0.0079 | 0.526 ± 0.51 | 5.40 ± 0.73 | 24.9 ± 2.5 | 0.0890 ± 0.0085 | 0.547 ± 0.040 | 5.74 ± 0.35 | 28.0 ± 1.0 |
%Bias | 6.35% | 5.21% | 8.02% | −0.225% | −11.0% | 9.47% | 14.7% | 11.9% | |
%RSD | 7.44% | 5.46% | 11.2% | 2.12% | 6.26% | 3.19% | 6.12% | 3.65% |
PTU | PTU-GLU | ||||||
---|---|---|---|---|---|---|---|
0.5 µM | 5 µM | 25 µM | 0.5 µM | 5 µM | 25 µM | ||
Recovery (n = 6) | Measured a | 101.7% ± 4.2% | 115.1% ± 14.0% | 114.2% ± 1.7% | 114.6% ± 12.1% | 98.2% ± 8.6% | 106.1% ± 0.6% |
%RSD | 4.1% | 12.2% | 1.5% | 10.6% | 8.8% | 0.6% | |
Matrix Effect (n = 6) | Measured b | 100% ± 9.7% | 86.0% ± 11.8% | 100% ± 12.2% | 99.3 ± 8.6% | 109% ± 6.9% | 87.6% ± 4.1% |
%RSD | 9.7% | 12.9% | 12.2% | 8.7% | 6.3% | 4.7% |
Storage Conditions | PTU | PTU-GLU | |||||
---|---|---|---|---|---|---|---|
0.5 µM | 5 µM | 25 µM | 0.5 µM | 5 µM | 25 µM | ||
Benchtop (n = 6) 20 °C, 6 h | Measured a | 0.428 ± 0.011 | 5.13 ± 0.15 | 24.9 ± 2.7 | 0.558 ± 0.035 | 5.63 ± 0.54 | 25.47 ± 1.2 |
%Bias | −14.5% | 2.69% | 0.473% | 11.8% | 12.6% | 1.87% | |
%RSD | 2.58% | 2.83% | 11.0% | 6.22% | 9.62% | 2.34% | |
Auto-Sampler (n = 6) 4 °C, 72 h | Measured a | 0.496 ± 0.030 | 5.54 ± 0.16 | 24.8 ± 1.0 | 0.49 ± 0.039 | 4.47 ± 0.32 | 23.5 ± 0.9 |
%Bias | −7.67% | 10.8% | 0.885% | −12% | −10.5% | −6.06% | |
%RSD | 5.97% | 2.91% | 0.416% | 7.86% | 7.36% | 3.91% | |
Long-Term (n = 6) −80 °C, 20 d | Measured a | 0.481 ± 0.029 | 4.95 ± 0.46 | 23.8 ± 2.6 | 0.429 ± 0.45 | 5.88 ± 0.82 | 22.7 ± 3.0 |
%Bias | −3.97% | −0.893% | −4.76% | −14.4% | 7.53% | −8.78% | |
%RSD | 6.06% | 9.28% | 10.9% | 10.5% | 14.5% | 13.2% | |
Freeze–Thaw (n = 6) −80 °C, Up to 3 Cycles | Measured a | 0.455 ± 0.056 | 5.41 ± 0.68 | 26.3 ± 1.0 | 0.517 ± 0.067 | 5.20 ± 0.42 | 25.7 ± 2.6 |
%Bias | −9.04% | −8.19% | 5.09% | 3.43% | 4.01% | 2.66% | |
%RSD | 12.3% | 12.6% | 3.96% | 13.0% | 8.15% | 10.0% |
Magnolol IC50 (µM) | |
---|---|
HLMs | 1.028 |
UGT1A9 | 1.160 |
Enzyme | Km (µM) a | Vmax (nmol/min/mg) a | Clearance (mL/min/mg) b |
---|---|---|---|
HLMs | 22.76 ± 12.29 | 220.0 ± 43.35 | 9.67 |
UGT1A9 | 15.27 ± 7.73 | 352.3 ± 56.38 | 23.07 |
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Li, M.; He, Q.; Yao, L.; Wang, X.; Tang, Z.; Zhu, X.; Lin, H.-S.; Xiang, X. Simultaneous Quantification of Propylthiouracil and Its N-β-d Glucuronide by HPLC-MS/MS: Application to a Metabolic Study. Pharmaceuticals 2021, 14, 1194. https://doi.org/10.3390/ph14111194
Li M, He Q, Yao L, Wang X, Tang Z, Zhu X, Lin H-S, Xiang X. Simultaneous Quantification of Propylthiouracil and Its N-β-d Glucuronide by HPLC-MS/MS: Application to a Metabolic Study. Pharmaceuticals. 2021; 14(11):1194. https://doi.org/10.3390/ph14111194
Chicago/Turabian StyleLi, Min, Qingfeng He, Li Yao, Xiaofeng Wang, Zhijia Tang, Xiao Zhu, Hai-Shu Lin, and Xiaoqiang Xiang. 2021. "Simultaneous Quantification of Propylthiouracil and Its N-β-d Glucuronide by HPLC-MS/MS: Application to a Metabolic Study" Pharmaceuticals 14, no. 11: 1194. https://doi.org/10.3390/ph14111194
APA StyleLi, M., He, Q., Yao, L., Wang, X., Tang, Z., Zhu, X., Lin, H. -S., & Xiang, X. (2021). Simultaneous Quantification of Propylthiouracil and Its N-β-d Glucuronide by HPLC-MS/MS: Application to a Metabolic Study. Pharmaceuticals, 14(11), 1194. https://doi.org/10.3390/ph14111194