Application of High-Performance Liquid Chromatography with Diode Array Detection to Simultaneous Analysis of Reference Antioxidants and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) in Free Radical Scavenging Test
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. HPLC-DAD Conditions
2.3. DPPH Free Radical Scavenging Activity Assay
2.4. Statistical Analysis
3. Results and Discussion
3.1. Stability of DPPH-R in Gradient Elution Mode with Acetonitrile–Water Mobile Phase
3.2. Influence of Analysis Time and UV Radiation Emitted by a Detector on DPPH Stability
3.3. Influence of DPPH-R Concentration on the DPPH-R/DPPH-H Ratio
3.4. Stability of DPPH Free Radical under Acidic Conditions
3.5. Antioxidant Properties of AO Standards Measured by DPPH-HPLC-DAD
3.6. AGREE—Analytical GREEnness Metric Approach
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gradient Profile | LOD [µmol L−1] | LOQ [µmol L−1] | Linearity Equation: (y = ax + b) | ||||
---|---|---|---|---|---|---|---|
a ± SD | b ± SD | R2 | F | se | |||
λ = 517 nm | |||||||
5% ACN | 107.31 | 325.18 | 28,435,512 ± 935,113 | −8,015,561 ± 1,444,263 | 0.9957 | 1,611,206 | 924.68 |
10% ACN | 55.23 | 167.35 | 28,440,890 ± 1,303,644 | −8,499,618 ± 2,013,452 | 0.9917 | 2,246,187 | 475.96 |
20% ACN | 45.10 | 136.65 | 27,462,657 ± 1,417,624 | −7,311,386 ± 2,189,491 | 0.9895 | 2,442,574 | 375.29 |
30% ACN | 31.64 | 95.86 | 27,090,228 ± 1,681,039 | −6,070,836 ± 2,596,330 | 0.9848 | 2,896,440 | 259.70 |
λ = 330 nm | |||||||
5% ACN | 1677.05 | 5081.96 | 3,343,277 ± 81,109 | −744,633 ± 125,272 | 0.9977 | 139,752 | 1699.04 |
10% ACN | 642.58 | 1947.20 | 3,421,269 ± 132,552 | −902,170 ± 204,725 | 0.9940 | 228,389 | 666.19 |
20% ACN | 200.13 | 606.45 | 3,661,783 ± 245,725 | −1,342,050 ± 379,518 | 0.9823 | 423,386 | 222.07 |
30% ACN | 189.41 | 573.97 | 3,246,670 ± 237,833 | −566,917 ± 367,329 | 0.9790 | 409,788 | 186.35 |
ACN [% (v/v)] | AC (0) 1 | AA (t) 2 | Peak Inhibition | ||
---|---|---|---|---|---|
[%] ± SD | Mean [%] | Relative Error [%] | |||
quercetin | |||||
5% | 56,744,541 | 6,236,679 | 89.01 ± 0.64 | 89.96 | 2.45 |
10% | 56,920,403 | 5,940,782 | 89.56 ± 0.61 | ||
20% | 57,213,505 | 5,701,898 | 90.03 ± 0.50 | ||
30% | 58,620,394 | 5,129,109 | 91.25 ± 0.35 | ||
resveratrol | |||||
5% | 56,550,363 | 13,284,246 | 76.51 ± 1.49 | 77.35 | 2.06 |
10% | 56,725,622 | 12,896,570 | 77.27 ± 1.41 | ||
20% | 57,017,721 | 12,821,575 | 77.51 ± 0.96 | ||
30% | 58,419,796 | 12,782,251 | 78.12 ± 0.74 | ||
Trolox | |||||
5% | 56,576,945 | 13,941,691 | 75.36 ± 1.87 | 76.33 | 2.33 |
10% | 56,752,287 | 13,618,846 | 76.00 ± 1.19 | ||
20% | 57,044,523 | 13,246,879 | 76.78 ± 0.10 | ||
30% | 58,447,257 | 13,349,353 | 77.16 ± 0.05 | ||
chlorogenic acid | |||||
5% | 56,608,329 | 14,141,327 | 75.02 ± 0.05 | 75.75 | 1.92 |
10% | 56,783,768 | 13,921,676 | 75.48 ± 1.43 | ||
20% | 57,076,167 | 13,691,431 | 76.01 ± 0.97 | ||
30% | 58,479,679 | 13,748,572 | 76.49 ± 0.11 | ||
hesperetin | |||||
5% | 56,768,577 | 39,634,117 | 30.18 ± 4.95 | 30.71 | 3.92 |
10% | 56,944,512 | 39,710,256 | 30.27 ± 0.22 | ||
20% | 57,237,739 | 39,506,060 | 30.98 ± 2.92 | ||
30% | 58,645,224 | 40,224,759 | 31.41 ± 0.09 | ||
coumarin | |||||
5% | 56,338,341 | 44,578,275 | 20.87 ± 5.57 | 21.29 | 4.83 |
10% | 56,512,943 | 44,653,137 | 20.99 ± 4.43 | ||
20% | 56,803,947 | 44,675,168 | 21.35 ± 0.55 | ||
30% | 58,200,765 | 45,437,337 | 21.93 ± 2.52 |
Reference Antioxidant | n | Statistic | p-Value |
---|---|---|---|
chlorogenic acid | 12 | 118.28 | 0.0004 |
coumarin | 12 | 0.05 | 0.9830 |
hesperetin | 12 | 17.27 | 0.0130 |
quercetin | 12 | 10.13 | 0.0210 |
resveratrol | 12 | 0.85 | 0.5300 |
Trolox | 12 | 9.22 | 0.0330 |
Compound | ACN | n | Mean | Homogenic Group | Mean Group 1 | Mean Group 2 |
---|---|---|---|---|---|---|
chlorogenic acid | 5% | 3 | 75.017 | a | 75.490 | NA |
chlorogenic acid | 10% | 3 | 75.453 | ab | 75.490 | 76.493 |
chlorogenic acid | 20% | 3 | 76.000 | ab | 75.490 | 76.493 |
chlorogenic acid | 30% | 3 | 76.493 | b | NA | 76.493 |
coumarin | 5% | 3 | 20.740 | c | 21.227 | NA |
coumarin | 10% | 3 | 20.903 | c | 21.227 | NA |
coumarin | 20% | 3 | 21.360 | c | 21.227 | NA |
coumarin | 30% | 3 | 21.903 | c | 21.227 | NA |
hesperetin | 5% | 3 | 30.073 | de | 30.427 | 31.413 |
hesperetin | 10% | 3 | 30.267 | d | 30.427 | NA |
hesperetin | 20% | 3 | 30.940 | de | 30.427 | 31.413 |
hesperetin | 30% | 3 | 31.413 | e | NA | 31.413 |
quercetin | 5% | 3 | 89.000 | f | 89.530 | NA |
quercetin | 10% | 3 | 89.557 | fg | 89.530 | 91.250 |
quercetin | 20% | 3 | 90.033 | fg | 89.530 | 91.250 |
quercetin | 30% | 3 | 91.250 | g | NA | 91.250 |
resveratrol | 5% | 3 | 76.487 | h | 77.329 | NA |
resveratrol | 10% | 3 | 77.217 | h | 77.329 | NA |
resveratrol | 20% | 3 | 77.500 | h | 77.329 | NA |
resveratrol | 30% | 3 | 78.110 | h | 77.329 | NA |
trolox | 5% | 3 | 75.327 | ij | 76.030 | 77.160 |
trolox | 10% | 3 | 75.983 | ij | 76.030 | 77.160 |
trolox | 20% | 3 | 76.780 | i | 76.030 | NA |
trolox | 30% | 3 | 77.160 | j | NA | 77.160 |
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Tatarczak-Michalewska, M.; Flieger, J. Application of High-Performance Liquid Chromatography with Diode Array Detection to Simultaneous Analysis of Reference Antioxidants and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) in Free Radical Scavenging Test. Int. J. Environ. Res. Public Health 2022, 19, 8288. https://doi.org/10.3390/ijerph19148288
Tatarczak-Michalewska M, Flieger J. Application of High-Performance Liquid Chromatography with Diode Array Detection to Simultaneous Analysis of Reference Antioxidants and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) in Free Radical Scavenging Test. International Journal of Environmental Research and Public Health. 2022; 19(14):8288. https://doi.org/10.3390/ijerph19148288
Chicago/Turabian StyleTatarczak-Michalewska, Małgorzata, and Jolanta Flieger. 2022. "Application of High-Performance Liquid Chromatography with Diode Array Detection to Simultaneous Analysis of Reference Antioxidants and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) in Free Radical Scavenging Test" International Journal of Environmental Research and Public Health 19, no. 14: 8288. https://doi.org/10.3390/ijerph19148288