Validation and Application of a Derivatization-Free RP-HPLC-DAD Method for the Determination of Low Molecular Weight Salivary Metabolites
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Study Subjects
2.3. Saliva Collection and Processing
2.4. Method Validation and Statistical Analysis
2.5. Analysis of Metabolites by RP-HPLC with UV Detection
3. Results and Discussion
3.1. Method Validation in Saliva
3.2. Metabolites Quantification in Saliva Samples
3.3. Metabolite Monitoring during Rifaximin Antibiotic Treatment
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Saliva Sample | Sex | yrs | Code | Annotations |
---|---|---|---|---|
1 | F | 51 | E_030419 | Single saliva sample |
2 | F | 51 | E_230319 | Single saliva sample |
3 | F | 51 | E_310119 | Single saliva sample |
4 | M | 28 | RN | 3 saliva samples collected in 3 days consecutively |
4pool | M | 28 | RN | Pool of 3 saliva samples collected in 3 days consecutively |
5 | M | 48 | FZ | 3 saliva samples collected in 3 days consecutively |
6 | F | 34 | LP | 3 saliva samples collected in 3 days consecutively |
7 | F | 26 | BC | Single saliva sample |
8 | F | 60 | CM | Single saliva sample |
9 | F | 51 | EB_030319 | Single saliva sample |
10 | F | 50 | EB_060119 | Single saliva sample |
11 | F | 51 | EB_090719 | Single saliva sample |
12 | F | 51 | EB_290819 | Single saliva sample |
13 | F | 51 | EB_030519 | Single saliva sample |
R1-R16 | F | 50 | Rifaximin study | 16 saliva sample collected in 16 days consecutively |
Metabolite | TR (min) | Slope (mM−1) | Intercept | R2 | Concfound in Saliva (mM) | Recovery (%) |
---|---|---|---|---|---|---|
Creatinine | 3.603 | 8633 | 7.9 | 0.9999 | 0.005 ± 0.002 | 101.7 |
Formic acid | 3.736 | 63 | 2 | 0.9974 | 0.169 ± 0.020 | 99.8 |
Malic acid | 4.015 | 173 | 528.7 | 0.9999 | 16.4 ± 0.020 | 106.3 |
Pyruvic acid | 4.240 | 1164 | 0 | 0.9999 | <LOD | 121.3 |
VAL | 4.362 | 64 | 0 | 0.9899 | <LOD | 97.0 |
Lactic acid | 4.648–4.802 | 111 | 3.8 | 0.9985 | 0.190 ± 0.020 | 100.6 |
Acetic acid | 4.954 | 39 | 15.4 | 0.9673 | 2.270 ± 0.100 | 85.5 |
Uric acid | 5.341 | 13,320 | 810 | 0.987 | 0.322 ± 0.020 | 104.3 |
Citric acid | 5.510 | 412 | 0 | 0.9999 | <LOD | 119.3 |
GSH | 5.950 | 1445 | 0 | 0.9999 | <LOD | 69.7 |
Fumaric acid | 6.170 | 23,793 | 5.25 | 0.9999 | 0.0011 ± 0.001 | 113.6 |
Succinic acid | 6.756 | 96 | 7.82 | 0.9981 | 0.4247 ± 0.030 | 108.8 |
Acetoacetic acid | 7.040 | 70 | 0 | 0.9999 | < LOD | 99.4 |
Tyrosine | 7.504 | 18,408 | 40.7 | 0.9996 | 0.0115 ± 0.010 | 110.1 |
GSSG | 8.167 | 7214 | 0 | 0.9999 | <LOD | 133.5 |
Propionic acid | 10.25 | 49 | 1.7 | 0.9975 | 0.1979 ± 0.020 | 95.2 |
Phenylalanine | 17.547 | 3642 | 6.24 | 0.9999 | 0.0089 ± 0.001 | 109.4 |
Butyric acid | 22.838 | 67 ± 7 | 4.9 ± 2.7 | 0.981 | 0.370 ± 0.040 | 101 |
Tryptophane | 23.392 | 70,892 | 34.6 | 0.9999 | 0.0025 ± 0.002 | 110.8 |
N. Missing Values | Min | Max | Mean | SD (n−1) | Median | IQR | Literature Value (μM) | |
---|---|---|---|---|---|---|---|---|
Creatinine | 0 | 2.3 | 70.9 | 22.7 | 22.6 | 10.0 | 23.4 | 2- > 10 [74] 5 ± 3 [55] 6.5 ± 2 [17,75] 18–37 [76] |
Formic acid | 0 | 137.3 | 4687.8 | 1466.4 | 1442.8 | 1066.1 | 1420.6 | 7–244 [77] 600 ± 750 [51] |
Malic acid | 0 | 7290.1 | 57,645.1 | 24,038.7 | 13,731.4 | 23,367.3 | 19,916.7 | 6 ± 3 [55] 20 ± 11 [75] 20.45 ± 10.87 [17] |
VAL | 2 | 9.8 | 8303.0 | 2327.9 | 2452.0 | 2118.7 | 3326.6 | 16.2 ± 12.3 [78] 48 ± 34 [55] 4 ± 2 [75] |
Lactic acid | 0 | 190.0 | 8040.9 | 2467.7 | 2470.2 | 1637.9 | 2462.1 | 527 ± 690 [55] 511 ± 612 [75] 73–208 [76] |
Acetic acid | 0 | 14.1 | 22,898.6 | 4265.0 | 5968.6 | 2163.0 | 2279.3 | 6815 ± 4311 [55] 1000–1500 [52] 1200–3261 [76] |
α-ketoglutaric | 9 | 4.1 | 65.9 | 23.5 | 24.8 | 11.9 | 19.1 | 5.27 ± 3.61 [75] |
uric acid | 0 | 95.6 | 358.9 | 209.9 | 82.5 | 216.5 | 122.0 | 184 ± 22 [79] 179 ± 84 [80] |
Citric acid | 11 | 139.4 | 289.9 | 214.6 | 75.2 | 214.6 | 75.2 | 29 ± 10 [55] 18 (1–338) [75] |
GSH | 10 | 32.8 | 44.1 | 37.0 | 5.1 | 34.0 | 5.7 | 7 ± 6 [55] |
Fumaric acid | 0 | 1.1 | 85.7 | 18.2 | 26.8 | 7.4 | 10.2 | 2 ± 0.7 [55] 1 ± 0.5 [75] |
cis-aconitic acid | 9 | 0.7 | 25.9 | 7.4 | 10.7 | 1.5 | 6.5 | 3 ± 1 [17,75] |
Succinic acid | 3 | 51.1 | 4755.7 | 946.9 | 1341.0 | 345.9 | 928.5 | 125 ± 181 [55] 2260 (60–4460) [75] 8–21 [76] |
Acetoacetic acid | 10 | 164.3 | 823.8 | 593.7 | 303.9 | 792.9 | 329.8 | 10.7 ± 4.33 [55] |
TYR | 0 | 3.6 | 81.0 | 26.1 | 23.9 | 11.5 | 32.9 | 40 ± 25 [55] 36 ± 15 [75] |
GSSG | 5 | 6.6 | 56.2 | 18.4 | 16.3 | 9.4 | 14.1 | 0.9 ± 0.4 [17] |
Propionic acid | 0 | 12.7 | 6990.2 | 1018.5 | 1797.4 | 333.3 | 1078.5 | 1412 ± 1090 [55] 1180 ± 1270 [75] 6.5–338 [76] |
PHE | 0 | 0.2 | 233.0 | 41.8 | 59.9 | 14.3 | 32.6 | 44 ± 23 [55] 18 (1–112) [75] |
Butyric acid | 0 | 9.8 | 13,621.2 | 2916.8 | 3635.7 | 2189.4 | 4171.7 | 277 ± 199 [55] 1470 (0–2940) [75] |
TRP | 0 | 0.5 | 16.2 | 6.2 | 5.5 | 2.7 | 9.1 | 4.8 ± 3 [55] 0.5 ± 0.5 [75] |
Min | Max | Mean | SD (n−1) | CV% (n−1) | Median | IQR | |
---|---|---|---|---|---|---|---|
Creatinine (basal) | 2.9 | 7.1 | 4.7 | 2.1 | 46.2 | 3.9 | 2.1 |
During rifaximin | 3.7 | 6.6 | 4.7 | 1.1 | 23.3 | 4.6 | 0.6 |
After rifaximin | 4.0 | 5.9 | 4.8 | 0.7 | 14.2 | 4.7 | 1.1 |
Formic acid (basal) | 206.3 | 317.5 | 254.0 | 57.2 | 22.5 | 238.1 | 55.6 |
During rifaximin | 39.7 | 214.3 | 131.7 | 73.9 | 56.1 | 150.8 | 111.1 |
After rifaximin | 35.7 | 198.4 | 112.5 | 62.7 | 55.7 | 107.1 | 85.5 |
Malic acid (basal) | 24736.1 | 26234.6 | 25426.5 | 756.1 | 3.0 | 25308.6 | 749.2 |
During rifaximin | 13765.4 | 42098.8 | 23364.2 | 10861.2 | 46.5 | 20648.1 | 1234.6 |
After rifaximin | 13688.3 | 29876.5 | 18776.2 | 5416.4 | 28.8 | 16354.9 | 6159.7 |
VAL (basal) | 833.3 | 1136.4 | 1,007.6 | 156.5 | 15.5 | 1053.0 | 151.5 |
During rifaximin | 818.2 | 5,401.5 | 2,195.5 | 1941.1 | 88.4 | 1166.7 | 1742.4 |
After rifaximin | 818.2 | 3,712.1 | 1,747.2 | 993.2 | 56.8 | 1284.1 | 1075.8 |
Lactic acid (basal) | 2500.0 | 3272.7 | 2856.1 | 389.9 | 13.7 | 2795.5 | 386.4 |
During rifaximin | 790.9 | 6363.6 | 3367.3 | 2239.5 | 66.5 | 2422.7 | 2613.6 |
After rifaximin | 650.0 | 3695.5 | 1969.3 | 905.2 | 46.0 | 1825.0 | 719.3 |
Acetic acid (basal) | 1847.8 | 2717.4 | 2235.5 | 442.4 | 19.8 | 2141.3 | 434.8 |
During rifaximin | 847.8 | 3815.2 | 1997.8 | 1103.1 | 55.2 | 1728.3 | 423.9 |
After rifaximin | 1130.4 | 2260.9 | 1773.1 | 380.7 | 21.5 | 1722.8 | 418.5 |
α-ketoglutaric acid (basal) | 103.6 | 144.3 | 125.5 | 20.5 | 16.4 | 128.4 | 20.4 |
During rifaximin | 39.6 | 215.4 | 113.0 | 64.7 | 57.3 | 105.1 | 34.8 |
After rifaximin | 12.2 | 103.6 | 66.4 | 27.0 | 40.7 | 63.8 | 18.4 |
Uric acid (basal) | 289.7 | 298.3 | 294.5 | 4.4 | 1.5 | 295.5 | 4.3 |
During rifaximin | 13.7 | 295.5 | 181.4 | 136.5 | 75.2 | 263.8 | 228.8 |
After rifaximin | 12.0 | 334.9 | 166.5 | 117.7 | 70.6 | 194.2 | 183.2 |
GSH (basal) | 12.3 | 14.5 | 13.7 | 1.2 | 8.7 | 14.2 | 1.1 |
During rifaximin | 13.0 | 19.5 | 17.3 | 2.6 | 15.0 | 18.1 | 2.2 |
After rifaximin | 0.7 | 21.2 | 11.8 | 6.8 | 58.1 | 13.1 | 5.4 |
Fumaric acid (basal) | 17.9 | 20.3 | 19.0 | 1.3 | 6.6 | 18.6 | 1.2 |
During rifaximin | 5.8 | 29.8 | 16.2 | 8.7 | 53.4 | 15.5 | 3.1 |
After rifaximin | 3.1 | 14.5 | 9.5 | 3.3 | 35.1 | 9.8 | 2.3 |
Succinic acid (basal) | 1022.7 | 1096.6 | 1056.8 | 37.3 | 3.5 | 1051.1 | 36.9 |
During rifaximin | 221.6 | 1340.9 | 750.0 | 455.3 | 60.7 | 642.0 | 613.6 |
After rifaximin | 187.5 | 858.0 | 573.2 | 227.5 | 39.7 | 579.5 | 268.5 |
TYR (basal) | 6.7 | 8.9 | 7.7 | 1.1 | 14.6 | 7.5 | 1.1 |
During rifaximin | 6.0 | 19.3 | 10.9 | 5.2 | 48.2 | 9.3 | 4.5 |
After rifaximin | 5.2 | 11.7 | 8.6 | 2.6 | 30.4 | 8.2 | 4.5 |
Propionic acid (basal) | 245.1 | 294.1 | 264.7 | 25.9 | 9.8 | 254.9 | 24.5 |
During rifaximin | 34.3 | 281.4 | 148.2 | 109.3 | 73.7 | 156.9 | 182.4 |
After rifaximin | 49.0 | 134.3 | 93.3 | 33.9 | 36.3 | 86.3 | 60.8 |
PHE (basal) | 19.2 | 22.2 | 20.6 | 1.5 | 7.4 | 20.3 | 1.5 |
During rifaximin | 8.1 | 39.7 | 21.0 | 12.2 | 57.8 | 17.9 | 11.3 |
After rifaximin | 8.6 | 19.2 | 13.6 | 4.1 | 30.0 | 13.4 | 4.8 |
Butyric acid (basal) | 1363.6 | 1515.2 | 1441.9 | 75.9 | 5.3 | 1447.0 | 75.8 |
During rifaximin | 1204.5 | 2030.3 | 1527.3 | 378.4 | 24.8 | 1325.8 | 590.9 |
After rifaximin | 424.2 | 1924.2 | 1218.8 | 550.8 | 45.2 | 1197.0 | 611.7 |
TRP (basal) | 2.0 | 2.5 | 2.2 | 0.3 | 12.3 | 2.3 | 0.3 |
During rifaximin | 1.3 | 4.2 | 2.4 | 1.2 | 49.3 | 1.8 | 1.4 |
After rifaximin | 0.4 | 2.4 | 1.5 | 0.6 | 41.0 | 1.5 | 0.5 |
R2 | p-Value (Pearson) | |
---|---|---|
VAL | 0.6339 | 0.0002 |
Acetic acid | 0.5915 | 0.0005 |
a-ketoglutaric acid | 0.6436 | 0.0002 |
Fumaric acid | 0.6601 | 0.0001 |
Succinic acid | 0.6021 | 0.0004 |
TYR | 0.4554 | 0.0041 |
PHE | 0.6099 | 0.0004 |
TRP | 0.4196 | 0.0067 |
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Campanella, B.; Lomonaco, T.; Benedetti, E.; Onor, M.; Nieri, R.; Bramanti, E. Validation and Application of a Derivatization-Free RP-HPLC-DAD Method for the Determination of Low Molecular Weight Salivary Metabolites. Int. J. Environ. Res. Public Health 2020, 17, 6158. https://doi.org/10.3390/ijerph17176158
Campanella B, Lomonaco T, Benedetti E, Onor M, Nieri R, Bramanti E. Validation and Application of a Derivatization-Free RP-HPLC-DAD Method for the Determination of Low Molecular Weight Salivary Metabolites. International Journal of Environmental Research and Public Health. 2020; 17(17):6158. https://doi.org/10.3390/ijerph17176158
Chicago/Turabian StyleCampanella, Beatrice, Tommaso Lomonaco, Edoardo Benedetti, Massimo Onor, Riccardo Nieri, and Emilia Bramanti. 2020. "Validation and Application of a Derivatization-Free RP-HPLC-DAD Method for the Determination of Low Molecular Weight Salivary Metabolites" International Journal of Environmental Research and Public Health 17, no. 17: 6158. https://doi.org/10.3390/ijerph17176158