Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Supplies
2.2. Preparation of Buffer Solutions and Reagents
2.3. Standard and Solutions
2.4. Patients and Serum Samples
2.5. Derivatization Procedure
2.6. Detection of H2S Species in Serum Samples
2.7. Instrumentation and Analytical Methods
2.8. Method Validation and Statistical Analysis
3. Results
3.1. RP-HPLC-FLD Separation of Derivatization Product
3.2. Sulfide Levels Quantification, Method Development
3.2.1. Derivatization Method Optimization
MBB Concentration
Temperature
Serum Handling (Storage Conditions, Aging, Dilution)
Speciation Protocol Tubes
3.2.2. Calibration Curve: Optimization and Validation
3.3. Efficacy of the Method on Human Serum Samples
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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SDB Peak Area (LU * min) | RT | 50 °C |
---|---|---|
Na2S 12.5 μM | 270 | 800 |
Serum | 63 | 207 |
SBF | 2 | 230 |
Na2S 5 μM/ Serum+ Na2S 5 μM | Na2S 12.5 μM/ Serum+ Na2S 12.5 μM | Na2S 25 μM/ Serum+ Na2S 25 μM | |
---|---|---|---|
SDB peak area (LU * min) | 25/22 | 180/100 | 400/200 |
Parameter | Value |
---|---|
a (AU) | −13.2 |
sa (AU) | 6.61 |
b (AU µM−1) | 5.87 |
sb (AU µM−1) | 0.930 |
c (AU µM−2) | 0.358 |
sc (AU µM−2) | 0.0181 |
R2 | 0.998 |
RMSE (AU) | 18.16 |
model p-value | <2.20 × 10−16 |
CV | Calibration | |
---|---|---|
Intercept | − 0.108 | 0.429 |
sIntercept | 0.685 | 0.520 |
Slope | 0.999 | 0.998 |
sSlope | 0.0122 | 0.00969 |
R2 | 0.997 | 0.998 |
RMSE | 22.3 | 17.8 |
model p-value | <2.20 × 10−16 | <2.20 × 10−16 |
Parameter | Value |
---|---|
Intercept (AU) | − 0.581 |
sIntercept (AU) | 0.260 |
Slope (AU µM−1) | 3.51 |
sSlope (AU µM−1) | 0.0749 |
R2 | 0.995 |
RMSE (AU) | 0.514 |
model p-value | 4.77 × 10−13 |
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Roda, B.; Zhang, N.; Gambari, L.; Grigolo, B.; Eller-Vainicher, C.; Gennari, L.; Zappi, A.; Giordani, S.; Marassi, V.; Zattoni, A.; et al. Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment. Antioxidants 2022, 11, 939. https://doi.org/10.3390/antiox11050939
Roda B, Zhang N, Gambari L, Grigolo B, Eller-Vainicher C, Gennari L, Zappi A, Giordani S, Marassi V, Zattoni A, et al. Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment. Antioxidants. 2022; 11(5):939. https://doi.org/10.3390/antiox11050939
Chicago/Turabian StyleRoda, Barbara, Nan Zhang, Laura Gambari, Brunella Grigolo, Cristina Eller-Vainicher, Luigi Gennari, Alessandro Zappi, Stefano Giordani, Valentina Marassi, Andrea Zattoni, and et al. 2022. "Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment" Antioxidants 11, no. 5: 939. https://doi.org/10.3390/antiox11050939
APA StyleRoda, B., Zhang, N., Gambari, L., Grigolo, B., Eller-Vainicher, C., Gennari, L., Zappi, A., Giordani, S., Marassi, V., Zattoni, A., Reschiglian, P., & Grassi, F. (2022). Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment. Antioxidants, 11(5), 939. https://doi.org/10.3390/antiox11050939