Table 3.
HPLC Methodology of Analysis for Selected Anthocyanins
| Plant Sample | Anthocyanins | Column | Column Size (mm) |
Particle Size (um) |
Solvent A | Solvent B | Gradient (Changing % B) | Detector | Reference |
|---|---|---|---|---|---|---|---|---|---|
| Apples (Malus domestica) | cy glycosides | Waters Xterra RP-18 | 150×2.1 | 3.5 | water-formic acid (95:5) | methanol-formic acid (95:5) | 10% to 30% over 10 min, 40% at 17 min, 51.2% at 21 min, 64% at 26 min, 90% at 30 min | DAD, ESI-MS | [131] |
| Artichoke (Cynara scolymus) | cy mono & diglycosides | Phenomenex C18 Hydro Synergi | 150×3.0 | 4 | water-formic acid-acn (87:10:3) | water-formic acid-acn (40:10:50) | 5% for 5 min, 12% at 12 min, 18% at 20 min, 25% at 25 min, 40% at 35 min, 100% at 37 min, 100% at 42 min, 5% at 45 min, 5:% at 50 min | DAD, ESI-MS, MS-MS | [132] |
| Bilberry (Vaccinium myrtilus) | cy, dp, pt, pn, mv glycosides | Waters Xterra Phenyl | 150×4.6 | 5 | water-formic acid (90:10) | acn | 1% to 3% over 5 min, 10% at 8 min, 10% at 12 min, 30% at 13 min, 30% at 15 min | UV | [133] |
| Bilberry (V. myrtilus) | cy, dp, pt, pn, mv glycosides | Waters Xterra Phenyl | 150×2.1 | 3.5 | water-formic acid (90:10) | acn | 1% to 3% over 5 min, 10% at 8 min, 10% at 12 min, 10% at 17 min, 30% at 18 min, 30% at 20 min | MS-MS | [133] |
| Bilberry (V. myrtilus. & rabbiteye blueberry (V. ashei) | dp, cy, pt, pn, mv mono & diglycosides | Capcell Pak C18 UG120 | 150×4.6 | 5 | 0.1%TFA in water | acn-water (50:50) with 0.1% TFA | 15% to 30% over 60 min | DAD, ESI-MS | [134] |
| Black & red raspberry (Rubus occidentalis & idaeus) | cy, pg glycosides | Waters Symmetry C18 | 75×4.6 | 3.5 | water-formic acid (90:10) | acn | 0% to 10% over 20 min, 0% at 25 min | DAD, ESI-MS, MS-MS | [135] |
| Black currant (Ribes nigrum) | dp & cy glycosides | Hypersil ODS | 60×4.6 | 3 | water-formic acid (95:5) | acn | 5% to 18% over 8 min, 95% at 13 min, 95% at 14 min. 5% at 19 min, 5 % at 24 min | DAD | [136] |
| Black currant (R. nigrum), chokeberry (Aronia melanocarpa), & elderberry (Sambucus nigra) | dp, cy, pt mono & diglycosides | Capcell Pak C18 UG120 | 150×4.6 | 5 | 0.1%TFA in water | acn-water (50:50) with 0.1% TFA | 15% to 30% over 60 min | DAD, ESI-MS | [134] |
| Black rice (Oryza sativa) | cy & pn glycosides | Waters Xterra RP-18 | 50×2.1 | 2.5 | water | methanol (solvent C: 5% formic acid | A-B-C (70:10:20) to (40:40:20) over 5 min, (70:10:20) for 3 min | DAD, ESI-MS | [137] |
| Blueberry (Vaccinium corymbosum) | dp, cy, pt, pn, mv glycosides | Waters Symmetry C18 | 75×4.6 | 3.5 | water-formic acid (90: 10) | acn | 0% to 30% over 20 min, 0% at 25 min | DAD, ESI-MS, MS-MS | [135] |
| Boysenberry extract (R. loganbaccus × bailyanus) | cy mono & diglycosides | LiChroCart Superspher 100RP-18 | 250×2.0 | 5 | water-formic acid (95:5) | methanol | 25% to 60% over 40 min, 100% at 45 min, 100% at 50 min | DAD, ESI-MS | [138, 139] |
| Camu-camu (Myrciaria dubia) | dp, cy glycosides | C18 Shim-pack CLC-ODS | 250×4.6 | 5 | water-phosphoric acid (96:4) | acn | 85% to 20% over 25 min | DAD | [140] |
| Camu-camu (M. dubia) | dp, cy glycosides | Synergi MaxRO RP-12 | 250×4.6 | 5 | water-acn-formic acid (87:3:10) | water-acn-formic acid (40:50: 10) | 6% to 20% over 20 min, 40% at 35 min, 60% at 40 min, 90% at 45 min | UV, ESI-MS, MS-MS | [140] |
| Crabapple (Malus domestica) | cy glycosides | Waters Xterra RP-18 | 250×4.6 | 5 | water-phosphoric acid (96:4) | acn | isocratic at 10% | DAD | [141] |
| Crabapple (Malus domestica) | cy glycosides | Waters Xterra RP-18 | 250×4.6 | 5 | 0.1% TFA in water | water-acn-acetic acid-TFA (50.4:48.5:1:.1) | 20% for 26 min, 60% at 30 min, 20% at 35 min | ESI-MS | [141] |
| Cranberry (Vaccinium macrocarpon | cy & pn glycosides | Waters Nova-Pak C18 | 150×3.9 | 5 | water-phosphoric acid (96:4) | acn | 5% to 25% over 60 min | DAD | [142] |
| Cranberry (V. macrocarpon) | cy & pn glycosides | Waters Symmetry C18 | 100×2.1 | 3.5 | water-formic acid (98:2) | methanol-formic acid (98:2) | 1% to 20% over 30 min, 40% at 45 min, 95% at 60 min | DAD, ESI-MS | [142] |
| Bee pollen (Echium plantagineum) | dp, cy, pt, pn, mv glycosides & acylglycosides | Phenomenex AQUA RP-C18 | 150×4.6 | 5 | 0.1% TFA in water | acn | 10% for 5 min, 13% at 20 min, 15% at 35 min, 35% at 55 min, 10% at 65 min | DAD, ESI-MS | [143] |
| Flower petal (Clitoria ternatea) | dp acylglycosides | Develosil C30-UG-3 | 150×1.5 | 3 | water-acn (95:5) with 0.05% TFA | water-acn (60:40) with 0.05% TFA | 14% to 86% over 30 min | DAD | [144] |
| Flower petal (C. ternatea) | dp acylglycosides | Develosil C30-UG-3 | 150×1.5 | 3 | water-acn (95:5) with 0.05% TFA | water-acn (60:40) with 0.05% TFA | 0% to 60% over 45 min | DAD | [144] |
| Flower petal (C. ternatea) | dp acylglycosides | Develosil Ph-UG-5 | 150×1.5 | 5 | water-acn (95:5) with 0.1% TFA | acn-water (80:20) with 0.1% TFA | 5% to 25% over 45 min | DAD | [144] |
| Flower petal (C. ternatea) | dp glycosides & acylglycosides | Develosil CC30-UG-5 | 250×1.5 | 5 | water-acn (95:5) with 0.1% TFA | water-acn (60:40) with 0.1% TFA | 14% to 86% over 45 min | DAD, MS-MS | [145] |
| Fruit (Coriaria myrtifolia) | dp, cy, pt, pn, mv glycosides | Nova Pak C18 | 250×4.5 | 5 | water-formic acid (90:10) | acn | 5% to 9% over 5 min, 11% at 15 min, 15% at 40 min, 20% at 50 min, 30% at 65 min, 40% at 70 min | DAD | [146] |
| Fruit-derived foods | mv glycosides | Lichrospher 100-RP18 | 250×4.0 | 5 | water-formic acid (98:2) | acn-water-formic acid (80:18:2) | 5% to 30% over 40 min, 40% at 50 min, 100% at 55 min | DAD | [147] |
| Fruit-derived foods | mv glycosides | Lichrospher 100-RP18 | 250×4.0 | 5 | water-formic acid (98:2) | acn-water-formic acid (80:18:2) | 10% for 4 min, 15% at 15 min, 50% at 40 min, 100% at 45 min | ESI-MS | [147] |
| Grapes (Vitis vinifera) | cy, dp, pt, pn, mv glycosides & acylglycosides | Agilent Zorbax SB-C18 | 250×3.6 | 5 | water-formic acid (95:5) | methanol-acn-water (33:60:70) | 10% to 11% over 6 min, 12% at 17 min, 14% at 21 min, 23% at 27 min, 47% at 37 min, 85% at 39 min, 100% at 40 min, 100% at 48 min | DAD | [148] |
| Grapes (V. vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | C18 Krornasil 100. | 250×4.0 | 4 | water-formic acid (90:10) | acn-water-formic acid (45:45:10) | 25% to 35% over 15 min, 50% at 20 min, 55% at 25 min, 65% at 40 min, 25% at 45 min | DAD | [149] |
| Grapes (V. vinifera) | dp, cy, pt, pn, pg, mv glycosides & acylglycosides | Waters Symmetry C18 | 75×4.6 | 3.5 | water-formic acid (90: 10) | acn | 0% to 20% over 13 min, 30% at 20 min, 0% at 25 min | DAD, ESI-MS, MS-MS | [135] |
| Grapes (V. vinifera) | dp, pt, mv glycosides | Synergi Hydro-RP 80 A | 150×2.1 | 4 | water-formic acid (95:5) | acn-water-formic acid (80:15:5) | 10% to 35% over 35 min, 60% at 60 min, 60% at 61 min | DAD, ESI-MS | [150] |
| Grapes (V. vinifera) | pn, cy, mv, pt, dp glycosides and acylglycosides | LiChroCart Superspher 100RP-18 | 250×4.0 | 5 | water-formic acid (95:5) | methanol | 2% to 32% over 30 min, 40% at 40 min, 95% at 50 min, 95% at 55 min | DAD, ESI-MS, MS-MS | [151,152] |
| Huckleberry (Vaccinium membranaceum & V. ovatum) | dp, cy, pt, pn glycosides | Phenomenex Prodigy ODS (3) | 250×4.6 | 5 | water-acetic acid-phosphoric acid (89:10:1) | acn | 2% to 20% over 25 min, 40% at 30 min | DAD | [153] |
| Huckleberry (V. membranaceum & V ovatum) | dp, cy, pt, pn glycosides | Synergi Hydro-RP 80 A | 250×2 | 4 | water-formic acid (95:5) | acn-water-formic acid (80:15:5) | 10% to 30% over 30 min, | DAD, ESI-MS | [153] |
| Isla oca tubers (Oxalis tuberosa) | dp, pt, pn, mv mono & diglycosides | Phenomenex AQUA RP-C18 | 150×4.6 | 5 | 0.1%TFA in water | acn | 10% for 5 min, 15 % at 20 min, 15% at 25 min, 18% at 30 min, 35% at 50 min | DAD, ESI-MS | [154] |
| Kidney bean (Phaseolus vulgaris) | cy, dp, pt, pg mono & diglycosides | Supelco TSK gel ODS-120T | 150×4.6 | 5 | water-formic acid (95:5) | acn-formic acid (95:5) | 1-% to 18% over 10 min, 28% at 18 min, 40% at 19 min, 40% at 21 min, 10% at 23 min, 10% at 25 min | DAD, ESI-MS | [155] |
| Mashua tubers (Tropaeolum tuberosum) | dp, cy, pg mono & diglycosides | Waters Atlantis C18 | 150×4.6 | 5 | water-phosphoric acid (100:.85) | water-acn-acetic acid-phosphoric acid (50.3:49.1:.4:.2) | 20% to 38% over 13 min, 40% at 14 min, 50% at 25 min | DAD | [43] |
| Mashua tubers (T. tuberosum) | dp, cy, pg mono & diglycosides | Waters Symmetry C18 | 75×4.6 | 3.5 | water-formic acid (90:10) | acn | 5% to 15% over 20 min | DAD, ESI-MS, MS-MS | [43] |
| Myrtle liqueur berry (Myrtus communis) | dp, cy, pt, pn, mv glycosides | Waters u-Bondapack C18 | 300×7.8 | 10 | 0.1%TFA in water | 0.1% TFA in acn | 20% to 33% over 18 min, 40% at 30 min, 40% at 40 min | DAD, ESI-MS, MS-MS | [156] |
| Pinta boca tubers (Solanum stenotomum) | pt, pn, mv, dp acylglycosides | Phenomenex AQUA RP-C18 | 150×4.6 | 5 | 0.1%TFA in water | acn | 10% for 5 min, 15% at 20 min, 15% at 25 min, 18% at 30 min, 35% at 50 min | DAD, ESI-MS | [157] |
| Port wine (Vitis vinifera) | mv glycosides | Merck RP-C18 | 250×4.6 | 5 | water-formic acid (95:5) | acn | 10 to 65% for 50 min | DAD | [158, 159] |
| Port wine (V. vinifera) | mv glycosides | Phenomenex AQUA RP-C18 | 150×4.6 | 5 | 0.1%TFA in water | acn | 10% for 5 min, 15% at 20 min, 15% at 25 min, 18% at 30 min, 35% at 50 min | ESI-MS, MS-MS | [158, 159] |
| Port wine (V. vinifera) | mv glycosides | Phenomenex AQUA RP-C18 | 150×4.6 | 5 | 0.1%TFA in water | acn | 10% for 5 min, 15 % at 20 min, 15 % at 25 min, 18% at 30 min, 35% at 50 min | DAD, ESI-MS | [160] |
| Potato plants (S. tuberosum cv. Desiree) | pg, pt acylglycosides | Superspher 100 RP | 250×4.0 | 5 | water-formic acid (90:10) | acn-formic acid (90:10) | 10% for 2 min, 30% at 25 min, 70% at 27 min | UV | [161] |
| Potato plants (S. tuberosum cv. Desiree) | pg, pt acylglycosides | Superspher 100 RP | 250×4.0 | 5 | water-acn-formic acid (95:4.5:.5) | acn-water-formic acid (95:4.5:35) | 0% to 100% over 15 min, 100% at 25 min | UV | [161] |
| Purple corn cob (Zea mays cv. Morado) | cy, pg, pn glycosides & acylglycosides | Phenomenex AQUA RP-C18 | 150×4.6 | 5 | 0.1%TFA in water | acn | 10% for 5 min, 15% at 20 min, 15% at 25 min, 18% at 30 min, 35% at 50 min | DAD, ESI-MS, MS-MS | [162] |
| Red onion (Allium cepa) | cy glycosides | ODS Hypersil | 250×3.0 | 5. | water-formic acid (90:10) | water-methanol-formic acid (40:50:10) | 10% to 100% over 17 min, 100% at 23 min, 10% at 24 min | DAD, ESI-MS | [163] |
| Red onion (A. cepa) | cy glycosides | ODS Hypersil | 250×4.0 | 5 | water-formic acid (95:5) | methanol-water-formic acid (50:40:10) | 10% to 100% over 23 min, 100% at 28 min, 10% at 29 min | DAD | [164] |
| Red wine (Visit vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Discovery RP-Amide C16 | 150×4.6 | 5 | 1% formic acid in water | acn | 5% to 15% over 2 min, 15% at 10 min, 20% at 25 min, 25% at 35 min, 30% at 44 min, 40% at 49 min, 50% at 54 min, 60% at 60 min | DAD, MS | [165] |
| Red wine (V. vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Superspher 100 RP | 250×4.6 | 18.5 | water-formic acid (90:10) | methanol-water-formic acid (45:45:10) | 35 to 95% in 20 min, 100% at 25 min, 100% at 30 min | UV, APCI-MS | [166] |
| Red wine (V. vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Phenomenex Luna C18 | 250×4.6 | 5 | water-formic acid (92.5:7.5) | acn-formic acid (92.5:7.5) | 3%for 1 min, 15% at 12 min, 25% at 24 min, 30% at 28 min, 30% at 32 min | DAD | [130] |
| Red wine (V. vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Phenomenex Luna C18 | 250×2.0 | 3 | water-formic acid (92.5:7.5) | acn-formic acid (92.5:7.5) | 3% for 1 min, 15% at 12 min, 25% at 24 min, 30% at 28 min, 30% at 32 min | ESI-MS | [130] |
| Red wine (V. vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Phenomenex Luna C18 | 150×2.0 | 5 | water-acn (95:5) with 0.1% TFA | water-acn (10:90) with 0.1% TFA | 2% for 2 min, 10% at 8 min, 13% at 30 min, 20% at 50 min, 30% at 75 min | DAD, ESI-MS, MS-MS | [167] |
| Red wine (V. vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Waters Symmetry C18 | 150×1.0 | 3.5 | water-formic acid (90:10) | water-formic acid-acn (40:10:50) | 12% for 2 min, 50% at 20 min, 90% at 25 min | UV, ESI-MS | [168] |
| Red wine (V. vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Supelco Discovery Biopeptide C18 | 100×.32 | 3 | water-formic acid (90: 10) | water-formic acid-acn (40:10:50) | 2% for 4. min, 12% at 6 min, 50% at 20 min, 90% at 25 min | UV, ESI-MS | [168] |
| Red wine (V. vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Lichrospher 100-RP18 | 250×4.0 | 5 | 9mM aqueous o-phosphoric acid | water-acn (75:25) with 9mM o-phosphoric acid | 0% for 20 min, 100% at 120 min, 100% at 140 min | DAD | [85, 169] |
| Red wine (V. vinifera) | mv glycosides | Macherey-Nagel NucleosilC18 | 125×4.0 | 3 | water-formic acid (98:2) | acn-water-formic acid (80: 18:2) | 15% to 75% over 15 min, 100% at 20 min | DAD | [170] |
| Red wine (V. vinifera) | mv glycosides | Lichrospher 100-RP18 | 250×2.0 | 5 | water-formic acid (98:2) | acn-water-formic acid (80:18:2) | 10% for 4 min, 15% at 15 min, 50% at 40 min, 100% at 45 min | ESI-MS | [170] |
| Red wine (V. vinifera) | thiolysed mv glycosides | Superspher 100 RP | 125×2.0 | 3 | water-formic acid (98:2) | acn-water-formic acid (80:18:2) | 10% to 50% over 17 min, 60% at 20 min, 100% at 22 min | ESI-MS | [170] |
| Red wine and grape skins (V vinifera) | dp, cy, pt, pn, mv glycosides & acylglycosides | Spherisorb ODS2 | 150×4.6 | 5 | water-formic acid (90: 10) | methanol-water- formic acid (45:45:10) | 35% to 95% over 20 min, 100% at 25 min, 100% at 30 min | DAD, ESI-MS | [171] |
| Rosé and blanc de noir wine (V. vinifera) | mv, pn glycosides | Waters Nova-Pak C18 | 150×3.9 | 4 | water-formic acid (90:10) | water-methanol- formic acid (45:45:10) | 15% to 80% over 30 min, 80% at 43 min | DAD, ESI-MS | [172] |
| Rosé cider (Malus × domestica) | cy glycosides | Inertsil ODS3 | 250×4.6 | 5 | water-formic acid (95:5) | methanol | 15% for 15 min, 38% at 105 min, 50% at 110 min, 50% at 120 in | DAD, ESI-MS | [173] |
| Rose (Rosa chinensis) | cy & pg diglycosides | Nomura ODS-VP C18 | 250×2.0 | 5 | 0.1% formic acid in water | 0.1% formic acid in methanol | 5% for 5 min, 45% at 90 min, 45% at 100 min, 5% at 101 min | DAD, ESI-MS | [86] |
| Rose hip (Rosa canina) | cy glycosides | Supelcosil LC-18-DB | 250×2.1 | 5 | water-formic acid (90:10) | methanol-formic acid (90:10) | 5% for 3 min, 30% at 16 min, 100% at 26 min, 100% at 29 min, 5% at 32 min | DAD, ESI-MS | [174] |
| Strawberry (Fragaria × ananassa) | cy, pg mono, di, & acylglycosides | Phenomenex AQUA RP-C18 | 150×4.6 | 5 | 0.1%TFA in water | acn | 10% for 5 min, 15 % at 20 min, 15 % at 25 min, 18% at 30 min, 35% at 50 min | DAD, ESI-MS | [175] |
| Tart cherry (Prunus cerasus), elderberry (Sambucus nigra), and bilberry (Vaccinium myrtillus) | cy, dp, pt, pn, mv glycosides | HP ODS hypersil | 125×4.0 | 5 | 0.1%TFA in water | water-acn-acetic acid-TFA (50.4:48.5:1:.1) | 20% to 60% over 26 min, 60% at 30 min, 20% at 35 min | DAD, ESI-MS | [34] |
Abbreviations are as follows: cy, cyanidin; dp, delphinidin; pt, petunidin; pn, peonidin: pg, pelargonidin; mv, malvidin. Glycosides consist of any of the following: glucoside, arabinoside, xyloside, rutinoside, sophoroside, sambubioside, or lathyroside. Acylating groups consist of any of the following: acetyl, coumaryl, caffeoyl, malonyl, feruloyl, sinapoyl. or dioxaloyl. Solvent abbreviations are: acn, acetonitrile; TFA, trifluoroacetic acid.