Gas Chromatography–Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva
Abstract
:1. Introduction
2. Results and Discussion
2.1. Sample Preparation
2.1.1. Disulfides Reduction
2.1.2. Proteins Removal
2.1.3. Analytes Preconcentration
2.1.4. Derivatization
2.2. GC Separation and MS Detection
2.3. Validation of the Method
2.4. Application of the Method
3. Materials and Methods
3.1. Reagents and Materials
3.2. Instrumentation
3.3. Stock Solutions
3.4. Biological Samples Collection
3.5. Saliva Specimen Preparation for Met-Related Sulfur-Containing Compounds Quantification by GC-MS
3.6. GC-MS Conditions
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
CMLT | 2-chloro-1-methyllepidinium tetrafluoroborate |
Cys | cysteine |
CysGly | cysteinyl-glycine |
CV | coefficient of variation |
DTT | dithiothreitol |
EI | electron ionization |
GC | gas chromatography |
GSH | glutathione |
Hcy | homocysteine |
HPLC | high-performance liquid chromatography |
HSA | human serum albumin |
HTL | homocysteine thiolactone |
LOQ | limit of quantification |
MeCN | acetonitrile |
Met | methionine |
MS | mass spectrometry |
MSTFA | N-trimethylsilyl-N-methyl trifluoroacetamide |
PCA | perchloric acid |
R | correlation coefficient |
SD | standard deviation |
SIM | selected ion monitoring mode |
TCEP | tris(2-carboxyethyl)phosphine |
THP | tris(hydroxymethyl)phosphine |
TMCS | trimethylchlorosilane |
TMS | trimethylsilyl group |
UV | spectrophotometric detection |
2-ME | 2-mercaptoethanol |
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Analyte | HTL | Met | Cys | Hcy | |
---|---|---|---|---|---|
Retention time | (min) | 3.9 | 6.7 | 7.5 | 9.9 |
CV of retention time (n = 10) | (%) | 0.442 | 0.060 | 0.049 | 0.038 |
Identification ions | (m/z) | 128.1; 161.1 | 128.1; 176.1 | 218.1; 220.1 | 128.1; 234.1 |
Quantification ions | (m/z) | 128.1 | 176.1 | 220.1 | 234.1 |
Time programmed SIM group | 1st | 2nd | 2nd | 3rd | |
SIM group start time | (min) | 2.2 | 5.0 | 5.0 | 8.0 |
Analyte | HTL | Met | Cys | Hcy | |
---|---|---|---|---|---|
Acceptance Criteria | Value | ||||
CV of retention time | ≤1% | 0.442% | 0.060% | 0.049% | 0.038% |
Assymetry factor | 0.8–1.5 | 1.39 | 1.47 | 1.16 | 1.50 |
Number of theoretical plates | ≥2000 | 10,384 | 11,375 | 22,278 | 19,274 |
Analyte | Concentration (µmol L−1) | Precision (%) | Accuracy (%) | ||
---|---|---|---|---|---|
Intra-Assay | Intermediate | Intra-Assay | Intermediate | ||
HTL | 1 | 12.80 | 11.77 | 89.46 | 92.53 |
10 | 8.53 | 12.24 | 102.21 | 96.91 | |
20 | 9.46 | 10.98 | 96.04 | 101.51 | |
Met | 1 | 14.30 | 13.72 | 91.54 | 102.74 |
10 | 5.75 | 7.83 | 88.75 | 107.58 | |
20 | 10.69 | 9.54 | 100.41 | 101.96 | |
Cys | 1 | 9.87 | 11.23 | 112.92 | 91.37 |
10 | 3.60 | 5.89 | 103.15 | 104.15 | |
20 | 7.83 | 6.24 | 105.09 | 99.87 | |
Hcy | 1 | 10.60 | 12.36 | 99.59 | 103.67 |
10 | 6.58 | 4.87 | 96.79 | 94.53 | |
20 | 12.03 | 9.56 | 102.91 | 100.22 |
Analyte | Regression Equation | R (R2) | CV Slope (%) | Linear Range (µmol L−1) | Intra-Assay Precision (%) | Intra-Assay Accuracy (%) | LOQ (µmol L−1) | ||
---|---|---|---|---|---|---|---|---|---|
Min | Max | Min | Max | ||||||
HTL | y = 2.935x + 1.890 | 0.9970 (0.9940) | 1.9 | 1.0–20 | 8.53 | 12.80 | 89.46 | 105.17 | 0.05 |
Met | y = 26.10x + 43.51 | 0.9940 (0.9880) | 3.5 | 0.5–20 | 5.75 | 14.30 | 88.75 | 106.35 | 0.1 |
Cys | y = 70.31x + 5.908 | 0.9952 (0.9904) | 1.4 | 0.5–20 | 3.60 | 11.57 | 94.93 | 112.92 | 0.1 |
Hcy | y = 28.94x + 26.63 | 0.9969 (0.9938) | 1.2 | 0.5–20 | 1.52 | 13.54 | 94.63 | 102.91 | 0.1 |
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Piechocka, J.; Wieczorek, M.; Głowacki, R. Gas Chromatography–Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva. Int. J. Mol. Sci. 2020, 21, 9252. https://doi.org/10.3390/ijms21239252
Piechocka J, Wieczorek M, Głowacki R. Gas Chromatography–Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva. International Journal of Molecular Sciences. 2020; 21(23):9252. https://doi.org/10.3390/ijms21239252
Chicago/Turabian StylePiechocka, Justyna, Monika Wieczorek, and Rafał Głowacki. 2020. "Gas Chromatography–Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva" International Journal of Molecular Sciences 21, no. 23: 9252. https://doi.org/10.3390/ijms21239252