A Sensitive LC-MS Method for Anthocyanins and Comparison of Byproducts and Equivalent Wine Content
Abstract
:1. Introduction
2. Materials and Methods
2.1. Instrumentation
2.2. Reagents and Solutions
2.3. Extraction Procedure
3. Results and Discussion
3.1. Optimization of the Extraction Procedure
3.1.1. Extraction Solution Selection
3.1.2. Dried Matter–to–Solvent Volume Ratio Optimization
3.1.3. Optimization of Water Bath Extraction Duration
3.1.4. Consecutive Extraction Rounds Optimization
3.2. Mobile Phase Selection
3.3. Figures of Merit of the Proposed Method
3.4. Identification and Quantification of Anthocyanins in Wine Variety (Syrah) and Its Byproducts
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
Mv | Malvidin |
LC | liquid chromatography |
PDA | photodiode array |
MS | mass spectrometry |
References
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Name | Abrev. | R1 | R2 | R3 |
---|---|---|---|---|
Delphinidin | Dp | OH | OH | H |
Delphinidin-3-O-glucoside | Dp-3-O-glu | OH | OH | Glc |
Petunidin | Pt | OH | OCH3 | H |
Petunidin-3-O-glucoside | Pt-3-O-glu | OH | OCH3 | Glc |
Malvidin | Mv | OCH3 | OCH3 | H |
Malvidin-3-O-glucoside | Mv-3-O-glu | OCH3 | OCH3 | Glc |
Cyanidin | Cn | OH | H | H |
Cyanidin-3-O-glucoside | Cn-3-O-glu | OH | H | Glc |
Peonidin | Pn | OCH3 | H | H |
Peonidin-3-O-glucoside | Pn-3-O-glu | OCH3 | H | Glc |
Pelargonidin | Pg | H | H | H |
Pelargonidin-3-O-glucoside | Pg-3-O-glu | H | H | Glc |
Parameter | Value |
---|---|
Ionization process | ESI |
Ionization mode | Positive |
Secondary electron multiplier detector voltage | 1.8 kV |
Capillary voltage | 4.0 kV |
Mass scanning range | 200 m/z–1200 m/z |
Solvent A (volume/volume) | Water/Formic Acid (99/1) |
Solvent B (volume/volume) | Acetonitrile/Formic Acid (99/1) |
Mobile phase flow-rate | 0.5 mL/min |
Injection volume | 20 μL |
Chromatographic Column | Dionex, RP-C18, 150 mm × 4.6 mm × 5 μm |
Elution program type | Gradient |
Dry Matter Mass (g) | Solvent Volume (mL) | Dry Matter Mass (g)/Solvent Volume (mL) | mg TAC */100 g sample ** |
---|---|---|---|
0.5 | 5.0 | 1/10 | 99.9 ± 10.1 |
0.5 | 10.0 | 1/20 | 117 ± 10.9 |
0.5 | 15.0 | 1/30 | 116 ± 10.8 |
0.5 | 20.0 | 1/40 | 119 ± 11.3 |
Duration Time (h) in Water Bath | mg TAC */100 g Sample ** |
---|---|
1 | 93.4 ± 9.41 |
2 | 129 ± 11.2 |
3 | 128 ± 11.1 |
4 | 130 ± 11.6 |
Successive Extraction Number | mg TAC */100 g Sample ** |
---|---|
1 | 130 ± 14 |
2 | 27.6 ± 3.3 |
3 | 2.92 ± 0.33 |
4 | 0.08 ± 0.02 |
Slope, S (Peak Area/(mg·L−1)) | Correlation Coefficient, r | Instrumental RSD * | LOD (ng·mL−1) | Dynamic Range (mg·L−1) |
---|---|---|---|---|
1.34 × 105 | 0.9996 | 5.8% | 9.0 | 0.03–10.0 |
Samples | Concentration in Wine Byproducts (mg 1000 g−1 of DW) * | Concentration in Wine (Syrah) (mg·L−1 of Wine) * | Ratio Between Samples | |
---|---|---|---|---|
Anthocyanins Group | ||||
Simple glucosides | 4367 | 457.7 | 9.54 | |
Acetyl glucosides | 5224 | 421.3 | 12.40 | |
Cinnamoyl glucosides | 2779 | 152.7 | 18.20 | |
Pyroanthocyanins | 73.3 | 12.6 | 5.82 | |
Total anthocyanins | 12,443.3 | 1044.3 | 11.92 |
Peak No. | Identity | tr (min) | m/z (M+) | Concentration (mg 1000 g−1 of DW) * | Concentration (mg·L−1 of Wine) * |
---|---|---|---|---|---|
Simple glucosides | |||||
1 | Delphinidin-3-O-glucoside | 7.14 | 465 | 4.0 ± 0.2 | 9.6 ± 0.7 |
2 | Cyanidin-3-O-glucoside | 7.95 | 449 | - | 2.9 ± 0.1 |
3 | Petunidin-3-O-glucoside | 8.25 | 479 | 89.0 ± 5.7 | 76.8 ± 5.0 |
4 | Peonidin-3-O-glucoside | 9.06 | 463 | 51.7 ± 3.7 | 26.4 ± 1.7 |
5 | Malvidin-3-O-glucoside | 9.27 | 493 | 4222 ± 178 | 342.0 ± 14.0 |
Acetyl glucosides | |||||
6 | Dp-gls-pyruvate derivative | 7.53 | 533 | - | 6.0 ± 0.5 |
7 | Pt-gls-pyruvate derivative | 8.67 | 547 | 1.1 ± 0.1 | 2.4 ± 0.1 |
8 | Dp-3-(6″-acetylglucoside) | 9.72 | 507 | 24.9 ± 1.5 | 7.6 ± 0.5 |
9 | Mv-3-gls-pyruvate (Vitisin A) | 9.84 | 561 | 54.0 ± 3.2 | 10.7 ± 0.9 |
10 | Vitisin B (Mv derivative) | 10.20 | 517 | 1.0 ± 0.1 | 8.5 ± 0.7 |
11 | Cn-3-(6″-acetylglucoside) | 10.47 | 491 | 332.1 ± 12.5 | 3.3 ± 0.2 |
12 | Pt-3-(6″-acetylglucoside) | 10.92 | 521 | 423.3 ± 27.2 | 79.2 ± 5.0 |
13 | Pn-3-(6″-acetylglucoside) | 12.48 | 505 | 6.4 ± 0.5 | 29.9 ± 1.9 |
14 | Mv-3-(6″-acetylglucoside) | 12.57 | 535 | 4381 ± 306 | 273.7 ± 16.1 |
Cinnamoyl glucosides | |||||
15 | Dp-3-(6″-p-coumaroylglucoside) | 12.93 | 611 | 7.8 ± 0.8 | 5.7 ± 0.4 |
16 | Mv-3-(6″-caffeoylglucoside) | 14.07 | 655 | 95.3 ± 6.2 | 2.2 ± 0.2 |
17 | Cn-3-(6″-p-coumaroylglucoside) | 14.67 | 595 | 1.3 ± 0.1 | 3.4 ± 0.3 |
18 | Pt-3-(6″-p-coumaroylglucoside) | 14.91 | 625 | 55.7 ± 3.1 | 15.7 ± 0.9 |
19 | Pn-3-(6″-p-coumaroylglucoside) | 17.01 | 609 | 84.7 ± 6.1 | 27.5 ± 1.7 |
20 | Mv-3-(6″-p-coumaroylglucoside) | 17.16 | 639 | 2534 ± 142 | 98.3 ± 6.9 |
Pyroanthocyanins | |||||
21 | Mv-3-(6″-acetylglucoside) pyruvate | 10.68 | 603 | 10.0 ± 0.8 | 7.6 ± 0.5 |
22 | Mv-3-glucoside-ethyl-catechin | 11.19 | 809 | 8.5 ± 0.6 | 2.1 ± 0.2 |
23 | Mv-3-(6″-acetylglucoside)-4-vinylphenol | 21.66 | 651 | 54.9 ± 4.0 | 2.9 ± 0.2 |
Total (g/kg) | 12.44 | 1.04 |
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Trikas, E.D.; Papi, R.M.; Kyriakidis, D.A.; Zachariadis, G.A. A Sensitive LC-MS Method for Anthocyanins and Comparison of Byproducts and Equivalent Wine Content. Separations 2016, 3, 18. https://doi.org/10.3390/separations3020018
Trikas ED, Papi RM, Kyriakidis DA, Zachariadis GA. A Sensitive LC-MS Method for Anthocyanins and Comparison of Byproducts and Equivalent Wine Content. Separations. 2016; 3(2):18. https://doi.org/10.3390/separations3020018
Chicago/Turabian StyleTrikas, Evangelos D., Rigini M. Papi, Dimitrios A. Kyriakidis, and George A. Zachariadis. 2016. "A Sensitive LC-MS Method for Anthocyanins and Comparison of Byproducts and Equivalent Wine Content" Separations 3, no. 2: 18. https://doi.org/10.3390/separations3020018