Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane
Abstract
:1. Introduction
2. Results and Discussion
2.1. Sample Preparation
2.1.1. Disulfide Bonds Cleavage
2.1.2. Derivatization via the –SH Group
2.2. Chromatography
2.3. Method Validation
2.3.1. Linearity
2.3.2. Limits of Detection and Quantification
2.3.3. Precision and Accuracy
2.3.4. Matrix Effect
2.4. Application to Real Plasma Samples
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. HPLC Instrumentation and Chromatographic Conditions
3.3. Preparation of Stock and Buffer Solutions
3.4. Human Plasma Sample Collection and Storage
3.5. Analytical Method Validation
3.5.1. Preparation of Calibration Standards
3.5.2. Calibration Curves
3.5.3. Limits of Detection and Quantification
3.5.4. Precision and Accuracy
3.5.5. Matrix Effect Evaluation
3.6. Application to Real Samples
3.7. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
Appendix A
References
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Analyte | Regression Equation | R2 | Linear Range (nmol mL−1) | Precision (%) | Recovery (%) | LOD (nmol mL−1) | LOQ (nmol mL−1) | ||
---|---|---|---|---|---|---|---|---|---|
Min. | Max. | Min. | Max. | ||||||
HSA | y = 150,925x + 10,000,000 | 0.9953 | 1.76–30.0 a | 0.40 | 5.80 | 92.94 | 113.67 | 0.59 a | 1.76 a |
α-LA | y = 3487.6x + 4187.2 | 0.9977 | 0.29–5.0 | 0.26 | 6.48 | 83.38 | 106.88 | 0.10 | 0.29 |
GSH | y = 6153x + 18,335 | 0.9991 | 1.16–35.0 | 0.24 | 10.92 | 84.71 | 99.15 | 0.39 | 1.16 |
Cys | y = 6765.9x + 806,715 | 0.9994 | 9.83–450.0 | 0.89 | 6.24 | 87.27 | 110.74 | 3.28 | 9.83 |
Hcy | y = 6621.9x + 34,488 | 0.9994 | 0.55–40.0 | 0.90 | 11.93 | 83.89 | 115.77 | 0.18 | 0.55 |
NAC | y = 34,376x + 379.58 | 0.9999 | 0.34–50.0 | 0.17 | 1.26 | 81.87 | 106.07 | 0.11 | 0.34 |
Cys-Gly | y = 3370.3x + 54,775 | 0.9998 | 1.45–45.0 | 0.90 | 5.33 | 96.34 | 114.56 | 0.48 | 1.45 |
Analyte | Concentration (nmol mL−1) | Precision (%) | Accuracy (%) | ||
---|---|---|---|---|---|
Intra-Day | Inter-Day | Intra-Day | Inter-Day | ||
HSA a | 1.76 | 0.35 | 1.49 | 119.48 | 99.31 |
5.0 | 0.10 | 4.83 | 96.77 | 109.77 | |
15.0 | 0.35 | 1.99 | 97.49 | 94.78 | |
30.0 | 0.37 | 2.47 | 100.65 | 101.05 | |
α-LA | 0.29 | 0.55 | 0.18 | 97.16 | 84.30 |
0.9 | 0.34 | 0.03 | 104.90 | 95.83 | |
1.75 | 0.60 | 7.34 | 97.31 | 107.24 | |
4.0 | 0.20 | 9.18 | 100.28 | 98.91 | |
GSH | 1.16 | 1.50 | 2.87 | 89.10 | 116.57 |
4.0 | 0.71 | 1.25 | 100.67 | 100.87 | |
15.0 | 0.05 | 0.28 | 101.31 | 97.14 | |
30.0 | 0.13 | 0.22 | 99.68 | 100.68 | |
Cys | 9.83 | 2.23 | 4.25 | 85.56 | 97.18 |
30.0 | 2.66 | 2.22 | 93.51 | 115.62 | |
100.0 | 0.57 | 6.94 | 104.43 | 94.38 | |
350.0 | 0.10 | 10.71 | 99.70 | 100.35 | |
Hcy | 0.55 | 3.85 | 14.57 | 97.45 | 82.17 |
1.7 | 5.34 | 2.55 | 88.30 | 90.84 | |
15.0 | 0.43 | 0.27 | 102.69 | 103.24 | |
30.0 | 0.01 | 3.69 | 99.37 | 99.22 | |
NAC | 0.34 | 0.25 | 0.11 | 85.24 | 98.98 |
1.0 | 0.48 | 2.05 | 100.83 | 115.95 | |
20.0 | 0.29 | 0.73 | 100.42 | 98.48 | |
40.0 | 0.04 | 0.10 | 99.90 | 100.37 | |
Cys-Gly | 1.45 | 1.43 | 5.09 | 108.52 | 93.58 |
4.5 | 0.19 | 1.58 | 85.16 | 82.99 | |
20.0 | 0.19 | 0.36 | 105.41 | 108.83 | |
35.0 | 0.70 | 0.19 | 98.46 | 97.41 |
No. | HSA | GSH | Cys | Hcy | Cys-Gly | |||||
---|---|---|---|---|---|---|---|---|---|---|
Mean (SD) | RSD (%) | Mean (SD) | RSD (%) | Mean (SD) | RSD (%) | Mean (SD) | RSD (%) | Mean (SD) | RSD (%) | |
1 | 42.62 (3.01) | 7.06 | 4.14 (0.25) | 6.00 | 65.54 (2.08) | 3.22 | 3.07 (0.28) | 9.05 | 30.71 (0.29) | 0.96 |
2 | 17.58 (1.51) | 8.61 | 7.93 (0.67) | 8.44 | 54.11 (3.06) | 5.66 | 2.99 (0.11) | 3.70 | 62.55 (3.40) | 5.43 |
3 | 11.73 (0.72) | 6.11 | 5.80 (0.28) | 4.85 | 74.72 (7.48) | 10.01 | 4.76 (0.22) | 4.59 | 46.49 (4.98) | 10.71 |
4 | 40.34 (2.64) | 6.53 | 6.00 (0.40) | 6.67 | 89.34 (4.60) | 5.14 | 5.46 (0.54) | 9.95 | 80.28 (3.14) | 3.91 |
5 | 10.71 (0.49) | 4.56 | 4.77 (0.22) | 4.64 | 67.38 (5.56) | 8.25 | 3.72 (0.25) | 6.67 | 55.48 (4.38) | 7.89 |
6 | 13.63 (1.25) | 9.18 | 5.22 (0.33) | 6.29 | 70.00 (3.38) | 4.83 | 3.42 (0.14) | 4.03 | 90.16 (7.00) | 7.76 |
7 | 14.92 (0.84) | 5.64 | 5.25 (0.23) | 4.32 | 36.85 (3.55) | 9.64 | 2.48 (0.07) | 2.62 | 42.90 (1.40) | 3.27 |
8 | 24.99 (1.02) | 4.08 | 5.69 (0.24) | 4.21 | 53.95 (4.28) | 7.94 | 5.47 (0.48) | 8.83 | 45.75 (3.14) | 6.86 |
9 | 38.12 (3.18) | 8.33 | 5.33 (0.41) | 7.66 | 41.59 (3.31) | 7.95 | 3.32 (0.31) | 9.23 | 19.63 (1.83) | 9.31 |
10 | 25.15 (1.31) | 5.22 | 2.73 (0.10) | 3.79 | 77.44 (5.79) | 7.48 | 4.56 (0.45) | 9.83 | 28.08 (3.22) | 11.48 |
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Kurpet, K.; Głowacki, R.; Chwatko, G. Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane. Molecules 2021, 26, 3321. https://doi.org/10.3390/molecules26113321
Kurpet K, Głowacki R, Chwatko G. Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane. Molecules. 2021; 26(11):3321. https://doi.org/10.3390/molecules26113321
Chicago/Turabian StyleKurpet, Katarzyna, Rafał Głowacki, and Grażyna Chwatko. 2021. "Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane" Molecules 26, no. 11: 3321. https://doi.org/10.3390/molecules26113321