HPLC-DAD Determination of Nitrite and Nitrate in Human Saliva Utilizing a Phosphatidylcholine Column
Abstract
:1. Introduction
2. Results and Discussion
2.1. Optimization of Nitrate/Nitrite Separation
2.2. Quantification of Nitrate/Nitrite by Oxidation Using Potassium Permanganate
2.3. The Kinetic Study of the Permanganate–Nitrite Reaction in Acidic Media
2.4. Linearity and Sensitivity
2.5. Recovery Study
2.6. Stability Tests
2.7. Application of the Assay
3. Materials and Methods
3.1. Materials and Sample
3.2. HPLC Conditions
3.3. Method Validation
3.3.1. Linearity, Recovery, and Precision
3.3.2. Recovery and Precision
3.4. Saliva Sample Preparation
3.5. Data Analysis
4. Conclusion
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Not available. |
Linear Regression Parameters | Nitrates NO3− | Nitrites NO2− | |
---|---|---|---|
LOD * [ng mL−1] | 4.56 | 4.21 | |
LOQ * [ng mL−1] | 15.21 | 14.03 | |
Linear range ** [µg mL−1] | 0.50–35.00 | 0.50–15.00 | |
Regression equation ***(y = ax + b) **** | a ± sa | 7809.31 ± 97.92 | 8966.61 ± 194.40 |
b ± sb | 1739.12 ± 5114.79 | −1743.60 ± 9500.16 | |
R2seF | 0.9992 6360.71 8703.09 | 0.9972 2127.55 18575.24 |
Parameters | Nitrate | ||
Fortification level (µg/mL) | 3.00 | 7.50 | 15.00 |
Average recovery (n = 6) (%) | 98.12 | 98.65 | 93.33 |
Intra-day precision, (RSDr,%) | 3.21 | 2.65 | 3.41 |
Inter-day precision, (RSDwR,%) | 4.73 | 4.71 | 4.20 |
Parameters | Nitrite | ||
Fortification level (ng/mL) | 3.00 | 7.50 | 15.00 |
Average recovery (n = 6) (%) | 86.90 | 87.74 | 104.23 |
Intra-day precision, (RSDr,%) | 3.51 | 3.83 | 3.34 |
Inter-day precision, (RSDwR,%) | 5.90 | 4.19 | 4.76 |
Water System | 3 µg mL−1 | 7.5 µg mL−1 | 15 µg mL−1 | ||
Without oxidation | tr | 4.54 (±0.03) | 4.58 (±0.05) | 4.52 (±0.01) | |
H | 23604 (±342) | 58198 (±989) | 115198 (±1727) | ||
As | 1.15 (±0.02) | 1.29 (±0.03) | 1.28 (±0.02) | ||
N | 5591 (±331) | 5771 (±295) | 5859 (±307) | ||
P | 0.9996 (±0.0003) | 0.9996 (±0.0001) | 1.0000 (±0.0000) | ||
R2 | 0.9907 (±0.0014) | 0.9907 (±0.0009) | 0.9910 (±0.0003) | ||
After oxidation | tr | 4.57 (±0.05) | 4.53 (±0.03) | 4.53 (±0.02) | |
H | 59305 (±771) | 136869 (±1916) | 262669 (±3415) | ||
As | 1.26 (±0.03) | 1.34 (±0.04) | 1.33 (±0.04) | ||
N | 6122 (±262) | 5960 (±311) | 5944 (±270) | ||
P | 1.0000 (±0.0000) | 0.9996 (±0.0002) | 0.9999 (±0.0001) | ||
R2 | 0.9908 (±0.0011) | 0.9910 (±0.0007) | 0.9908 (±0.0009) | ||
Samples of Saliva | Without oxidation | tr | 4.53 (±0.03) | 4.55 (±0.04) | 4.58 (±0.06) |
H | 72097 (±901) | 108175 (±1293) | 167554 (±1508) | ||
As | 1.36 (±0.03) | 1.28 (±0.02) | 1.25 (±0.04) | ||
N | 5446 (±369) | 5365 (±291) | 5850 (±283) | ||
P | 0.9614 (±0.0097) | 0.9731 (±0.0054) | 0.9982 (±0.0016) | ||
R2 | 0.9909 (±0.0012) | 0.9908 (±0.0008) | 0.9907 (±0.0008) | ||
After oxidation | tr | 4.51 (±0.02) | 4.53 (±0.01) | 4.55 (±0.04) | |
H | 123968 (±1759) | 197560 (±2374) | 340325 (±3544) | ||
As | 1.37 (±0.05) | 1.22 (±0.02) | 1.35 (±0.03) | ||
N | 6499 (±321) | 6036 (± 279) | 6450 (±332) | ||
P | 0.9997 (±0.0002) | 0.9998 (±0.0002) | 1.0000 (±0.0000) | ||
R2 | 0.9906 (±0.0005) | 0.9904 (±0.0012) | 0.9904 (±0.0007) |
Nitrate | Nitrite | ||
---|---|---|---|
Concentration Range [µg mL−1] | Mean ± SD [µg mL−1] | Concentration Range [µg mL−1] | Mean ± SD [µg mL−1] |
8.98–18.52 | 13.76 ± 3.20 | 3.50–5.34 | 4.24 ± 0.65 |
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Tatarczak-Michalewska, M.; Flieger, J.; Kawka, J.; Płaziński, W.; Flieger, W.; Blicharska, E.; Majerek, D. HPLC-DAD Determination of Nitrite and Nitrate in Human Saliva Utilizing a Phosphatidylcholine Column. Molecules 2019, 24, 1754. https://doi.org/10.3390/molecules24091754
Tatarczak-Michalewska M, Flieger J, Kawka J, Płaziński W, Flieger W, Blicharska E, Majerek D. HPLC-DAD Determination of Nitrite and Nitrate in Human Saliva Utilizing a Phosphatidylcholine Column. Molecules. 2019; 24(9):1754. https://doi.org/10.3390/molecules24091754
Chicago/Turabian StyleTatarczak-Michalewska, Małgorzata, Jolanta Flieger, Justyna Kawka, Wojciech Płaziński, Wojciech Flieger, Eliza Blicharska, and Dariusz Majerek. 2019. "HPLC-DAD Determination of Nitrite and Nitrate in Human Saliva Utilizing a Phosphatidylcholine Column" Molecules 24, no. 9: 1754. https://doi.org/10.3390/molecules24091754