Development and Validation of a Novel HPLC Method to Analyse Metabolic Reaction Products Catalysed by the CYP3A2 Isoform: In Vitro Inhibition of CYP3A2 Enzyme Activity by Aspirin (Drugs Often Used Together in COVID-19 Treatment)
Abstract
:1. Introduction
2. Results and Discussion
2.1. HPLC Method Development
2.2. HPLC Method Validation
2.2.1. Linearity and Range
2.2.2. Specificity and Selectivity
2.2.3. Limit of Detection (LOD) and Limit of Quantification (LOQ)
2.2.4. Precision
Intraday Precision of Dexamethasone
Interday Precision of Dexamethasone
Intraday Precision of 6β-Hydroxydexamethasone
Interday Precision of 6β-Hydroxydexamethasone
2.2.5. Stability Study
Solution Stability of Substrate (Dexamethasone)
Solution Stability of 6β-Hydroxydexamethasone (Metabolite)
2.2.6. Robustness of the Method
2.3. Optimisation of Incubation Time for Incubation System In Vitro
2.4. Inhibitory Effects of Aspirin on CYP3A2 Activity in Rat Liver Microsomes
3. Materials and Methods
3.1. Chemicals
3.2. Instruments
3.3. Cytochrome P450 Assay
3.3.1. Dexamethasone 6β-Hydroxylation Assay for CYP3A2
3.3.2. Microsomal Incubations Procedure
3.4. Preparation of Standard Substrate and Metabolite Solutions
3.5. Optimisation of Incubation Time In Vitro
3.6. Data Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Standards | Dexamethasone | 6β-Hydroxydexamethasone |
---|---|---|
Regression equation | y = 0.2505x + 0.0945 | y = 1.6775x + 0.0385 |
r2 | 0.999 | 0.998 |
Linear range | 25–200 µM | 0.2–1 µM |
Standards | Dexamethasone | 6β-Hydroxydexamethasone |
---|---|---|
LOD | 5.60 µM | 0.06 µM |
LOQ | 16.98 µM | 0.19 µM |
Dexamethasone Standards | Mean (µM) | Recovery a (%) | RSD (%) |
---|---|---|---|
Low concentration (40 µM) | 39.28 ± 0.90 | 98.21 | 2.30 |
Medium concentration (110 µM) | 99.06 ± 3.06 | 90.05 | 3.09 |
High concentration (185 µM) | 151.11 ± 0.76 | 81.68 | 0.50 |
Dexamethasone Standards | Mean (µM) | Recovery a (%) | RSD (%) |
---|---|---|---|
Low concentration (40 µM) | 45.01 ± 2.09 | 112.52 | 4.65 |
Medium concentration (110 µM) | 110.01 ± 2.17 | 100.01 | 1.98 |
High concentration (185 µM) | 178.92 ± 3.13 | 96.72 | 1.75 |
6β-Hydroxydexamethasone Standards | Mean (µM) | Recovery a (%) | RSD (%) |
---|---|---|---|
Low concentration (0.3 µM) | 0.33 ± 0.01 | 108.60 | 2.21 |
Medium concentration (0.5 µM) | 0.50 ± 0.02 | 100.01 | 3.64 |
High concentration (0.85 µM) | 1.02 ± 0.09 | 119.38 | 8.82 |
6β-Hydroxydexamethasone Standards | Mean (µM) | Recovery a (%) | RSD (%) |
---|---|---|---|
Low concentration (0.3 µM) | 0.32 ± 0.02 | 107.61 | 4.67 |
Medium concentration (0.5 µM) | 0.50 ± 0.03 | 99.28 | 5.90 |
High concentration (0.85 µM) | 0.79 ± 0.03 | 93.13 | 4.13 |
Analytical Parameters | Actual Concentration (µM) | |||
---|---|---|---|---|
Intraday | 40 | 110 | 185 | |
Calculated Concentration (µM) | 0 h | 35.40 | 88.64 | 176.98 |
5 h | 32.20 | 92.43 | 202.56 | |
10 h | 32.32 | 92.87 | 203.56 | |
% Recovery | 0 h | 88.51 | 80.58 | 95.66 |
5 h | 80.50 | 84.03 | 109.49 | |
10 h | 80.80 | 84.43 | 110.03 | |
% Accuracya | 0 h | 111.49 | 119.42 | 104.34 |
5 h | 119.51 | 115.97 | 90.51 | |
10 h | 119.20 | 115.57 | 89.97 | |
Calculated Concentration (µM) | Interday | 40 | 110 | 185 |
Interday 1 | 32.02 | 94.39 | 181.79 | |
Interday 2 | 34.99 | 100.43 | 193.84 | |
Interday 3 | 34.12 | 90.60 | 187.22 | |
% Recovery | Interday 1 | 80.04 | 85.81 | 98.26 |
Interday 2 | 87.31 | 91.30 | 104.78 | |
Interday 3 | 85.29 | 82.36 | 101.20 | |
% Accuracy a | Interday 1 | 119.96 | 114.19 | 101.74 |
Interday 2 | 112.70 | 108.70 | 95.22 | |
Interday 3 | 114.71 | 117.64 | 98.80 |
Analytical Parameters | Actual Concentration (µM) | |||
---|---|---|---|---|
Intraday | 0.3 | 0.5 | 0.85 | |
Calculated Concentration (µM) | 0 h | 0.30 | 0.47 | 0.84 |
5 h | 0.29 | 0.44 | 0.77 | |
10 h | 0.27 | 0.44 | 0.79 | |
% Recovery | 0 h | 98.18 | 94.82 | 99.34 |
5 h | 97.22 | 87.58 | 90.45 | |
10 h | 88.89 | 87.75 | 92.98 | |
% Accuracy a | 0 h | 101.82 | 105.18 | 100.66 |
5 h | 102.79 | 112.42 | 109.55 | |
10 h | 111.11 | 112.25 | 107.02 | |
Interday | 0.3 | 0.5 | 0.85 | |
Calculated Concentration (µM) | Interday 1 | 0.27 | 0.46 | 0.72 |
Interday 2 | 0.32 | 0.47 | 0.69 | |
Interday 3 | 0.32 | 0.46 | 0.68 | |
% Recovery | Interday 1 | 89.49 | 92.64 | 84.06 |
Interday 2 | 105.46 | 94.15 | 81.57 | |
Interday 3 | 106.90 | 92.10 | 80.51 | |
% Accuracy a | Interday 1 | 110.51 | 107.36 | 115.94 |
Interday 2 | 94.54 | 105.85 | 118.43 | |
Interday 3 | 93.10 | 107.90 | 119.49 |
Analytes of Interest | Average tR | Average Peak Area | Resolution |
---|---|---|---|
Normal Conditions (0.6 mL/min, 243 nm and 25 °C) | |||
Aspirin | 2.04 | 16,162.67 | All compounds were well separated, and a good resolution was achieved. |
6β-Hydroxydexamethasone | 2.64 | 127,567.67 | |
Dexamethasone | 3.08 | 58,676.00 | |
4-Hydroxyoctanophenone | 6.67 | 31,991.33 | |
A: Temperature (30 °C) | |||
Aspirin | 2.04 | 12,351.00 | All compounds were separated well, with a faster elution pattern as the temperature increased. |
6β-Hydroxydexamethasone | 2.64 | 120,462.67 | |
Dexamethasone | 3.07 | 55,890.67 | |
4-Hydroxyoctanophenone | 6.51 | 29,137.67 | |
B: Wavelength (248 nm) | |||
Aspirin | 2.08 | 10,039.33 | All four compounds were separated, but there was a decrease in intensity of metabolite peak. |
6β-Hydroxydexamethasone | 2.67 | 75,995.00 | |
Dexamethasone | 3.08 | 49,674.00 | |
4-Hydroxyoctanophenone | 6.61 | 53,310.00 | |
C: Flow rate (0.8 mL/min) | |||
Aspirin | 1.42 | 6458.00 | Peaks were separated with a 0.8 mL/min flow rate. All compounds showed a faster and narrow elution pattern. |
6β-Hydroxydexamethasone | 1.99 | 97,253.00 | |
Dexamethasone | 2.33 | 38,095.00 | |
4-Hydroxyoctanophenone | 5.02 | 22,253.33 |
Aspirin Concentration | (Inhibition Parameters) | ||||
---|---|---|---|---|---|
Km (µM) | Vmax (µM−1∙min−1) | Clint (µM−2∙min−1) | á | %Inhibition | |
0 µM Aspirin | 21.23 ± 0.51 | 0.0127 ± 1.53 × 10−4 | 0.0006 ± 1.10 × 10−5 | - | - |
50 µM Aspirin | 23.83 ± 0.31 | 0.0123 ± 1.15 × 10−4 | 0.0005 ± 4.00 × 10−6 | 1.03 ± 0.01 | 12.44 ± 1.20 |
100 µM Aspirin | 26.13 ± 0.70 | 0.0127 ± 7.94 × 10−4 | 0.0005 ± 5.00 × 10−5 | 1.02 ± 0.06 | 23.29 ± 3.30 |
200 µM Aspirin | 32.57 ± 0.35 | 0.0123 ± 1.73 × 10−4 | 0.0004 ± 1.00 × 10−5 | 1.04 ± 0.01 | 53.64 ± 1.76 |
Aspirin | 6β-Hydroxydexamethasone | Dexamethasone | Internal Standard | |||||
---|---|---|---|---|---|---|---|---|
Retention Time (tR) | 2.09 | 2.65 | 3.08 | 6.66 | ||||
Efficiency (N) | 1078 | 970 | 3363 | 6137 | ||||
Plate Height (H) | 1.39 × 10−2 | 1.55 × 10−2 | 4.46 × 10−3 | 2.44 × 10−3 | ||||
Resolution (Rs) | 1.58 | 1.33 | 11.01 | |||||
Asymmetry Factor (AsF) | 0.97 | 1.03 | 1.07 | 1.07 |
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Hussain, A.; Naughton, D.P.; Barker, J. Development and Validation of a Novel HPLC Method to Analyse Metabolic Reaction Products Catalysed by the CYP3A2 Isoform: In Vitro Inhibition of CYP3A2 Enzyme Activity by Aspirin (Drugs Often Used Together in COVID-19 Treatment). Molecules 2022, 27, 927. https://doi.org/10.3390/molecules27030927
Hussain A, Naughton DP, Barker J. Development and Validation of a Novel HPLC Method to Analyse Metabolic Reaction Products Catalysed by the CYP3A2 Isoform: In Vitro Inhibition of CYP3A2 Enzyme Activity by Aspirin (Drugs Often Used Together in COVID-19 Treatment). Molecules. 2022; 27(3):927. https://doi.org/10.3390/molecules27030927
Chicago/Turabian StyleHussain, Amira, Declan P. Naughton, and James Barker. 2022. "Development and Validation of a Novel HPLC Method to Analyse Metabolic Reaction Products Catalysed by the CYP3A2 Isoform: In Vitro Inhibition of CYP3A2 Enzyme Activity by Aspirin (Drugs Often Used Together in COVID-19 Treatment)" Molecules 27, no. 3: 927. https://doi.org/10.3390/molecules27030927