Phenolic compounds are plants’ bioactive metabolites that have been studied for their ability to confer extensive benefits to human health. As currently there is an increased interest in natural compounds identification and characterization, new analytical methods based on advanced technologies have been developed.
|Plant Material||Extraction||HPLC Condition||Characteristic Fingerprint Peaks||Chemometric Analysis Approach||Ref.|
|Cranberry (Vaccinium macrocarpon)-based products (fresh and dried fruits, juice)||Lyophilized samples were extracted with an acetone:water:hydrochloric acid (70:29.9:0.1 v/v/v) solution by UAE for 10 min||Kinetex C18 (100 4.6 mm i.d., 2.6 µm particle size) column; mobile phase: 0.1% formic acid in water (v/v) and MeOH; flow rate of 1 mL/min;
monitoring wavelength range: 190–550 nm
|gallic acid, homogentisic acid, protocatechuic acid, protocatechualdehyde, (+) catechin hydrate, gentilic acid, p-salicilic acid, chlorogenic acid, vanillic acid,
(−) epicatechin, syringic acid, syringaldehyde, ethyl gallate, p-coumaric acid, ferulic acid, resveratrol and quercitrin
|Partial least square regression and PCA|||
|27 Salvia L. Species, leaf and root||Maceration in MeOH (2 × 10 mL for 24 h) followed by solvents removal on rotary evaporator under vacuum at 40 °C to dryness||RP C18 Eurospher-100 column, (5 µm particle, 125 mm × 4 mm); mobile phase: 0.2% (v/v) glacial acetic acid in water and ACN; flow rate of 1 mL/min;
monitoring wavelength: 280 nm
carnosic acid, caffeic acid, salvianolic acids A and B
|Aurantii Fructus, dried mature and imature fruits of Citrus aurantium L. (medicinal herbs in TCM);||UAE (200 W) with MEOH for
|Symmetry C18 column (250 × 4.6 mm, 5 μm); mobile phase: ACN and 0.1% aqueous phosphoric acid; flow rate of 1 mL/min;
monitoring wavelength range: 285–324 nm
|eriocitrin, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, meranzin, poncirin, naringenin, nobiletin, tangeretin and auraptene||Quantitative analysis of multiple components by single marker (QAMS); similarity analysis; standard method difference; HCA|||
|Licorice root (Liquiritiae radix) -Glycyrrhiza glabra L.||UAE with 80% MeOH-water, 120 W, 40 KHZ, 20 min||Cosmosil column (5C18-MS-II, 5 µm, 4.6 × 250 mm), at 35 °C; mobile phase: 5 mmol/L sodium heptane sulfonate solution phosphoric acid (499:1, v/v) and ACN-MeOH (9:1, v/v); flow rate of 1 mL/min;
monitoring wavelengths: 203 nm, 220 nm, 250 nm, 280 nm and 344 nm
|glycyrrhizic acid, liquiritigenin, isoliquiritigenin, isoliquiritin, liquiritin apioside, isoliquiritin apioside and glabridin||Geometric linear quantified fingerprint method (GLQFM) and PCA|||
|Flos Carthami, the dried flower of Carthamus tinctorius L.; (medicinal herbs in TCM)||UAE with 50% MeOH, 270 W,
|C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: 0.1% formic acid in water and ACN||guanosine, hydroxysafflor yellow A,
anhydrosafflor yellow B, kaempferol 3-O-β-rutinoside, rutin, quercetin, kaempferol
|Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004 A)|||
|Chamomile—Matricaria chamomilla L., commercial samples||UAE with MeOH:water mixture (80:20, v/v) at 35 °C for 30 min, three times repeated||Hypersil Gold C18 column (250 × 4.6 mm; 5 μm) at 35 °C; mobile phase: 0.05% TFA in ACN and 0.05% TFA in water; flow rate of 1 mL/min;
monitoring wavelength: 254 nm
|gallic acid, caffeic acid, syringic acid,
p-coumaric acid, ferulic acid, rutin, myricetin, quercetin and kaempferol
|ANOVA, PCA, hierarchical cluster analysis (HCA)|||
|Seven species of Lavandulae flos||UAE with xylen (1:30) under reflux for 4 h followed by solvents removal on a rotary evaporator under vacuum to dryness||Kinetex RP18 column (5 μm, 150 × 4.6 mm); mobile phase: MeOHwater–0.1% formic acid (gradient 5–100% (v/v) of MEOH) at 30 °C; flow rate of 1 mL/min; monitoring wavelength: 280 nm||apigenin, myricetin, luteolin, luteolin 7-glucoside, chlorogenic acid, caffeic acid, ferulic acid||Cluster analysis using SpecAlign program (Pearson correlation coefficient, r, and Euclidean) and PCA|||
|Pomegranate (Punica granatum L.), dried peel||UAE of the dried samples with 60% ethanol, 26 min||Zorbax SB-C18 column (5 μm, 4.6 mm × 250 mm); mobile phase: glacial acetic acid (99:1, v/v; pH 3.0) and MeOH flow rate of 1 mL/min;
monitoring wavelength: 280 nm
|gallic acid, punicalagin, catechin, chlorogenic acid, caffeic acid, epicatechin, rutin, and ellagic acid||Similarity Evaluation System for Chromatographic; Fingerprint of Traditional Chinese Medicine (Version 2004A) software|||
|Sedi linearis Herba, dried whole herb||UAE of the dried samples with 70% methanol, 60 min||BDS Hypersil C18 column (4.6 m × 250 mm, 5 μm), 30 °C; Mobile phase: ACN and 0.1% acetic acid solution; flow rate of 1 mL/min;
monitoring wavelength: 265 nm
isoquercetin and astragalin
|Black tea samples||Enzymatic extraction using
immobilized polyphenol oxidase followed by fractionating using a Mitsubishi SP-207 resin chromatography with an elution gradient of 20%, 30%, 40%, 50% and 70% aqueous ethanol
|C18 column; mobile phase: water: ACN: glacial acetic acid (73.5/26/0.5, v/v/v); flow rate of 5 mL/min||caffeine, (−) epigallocatechin gallate, (−) epigallocatechin, (−) epicatechin gallate, (−) epicatechin, (+) gallocatechin gallate, (+) gallocatechin, (+) catechin gallate, (+) catechin, theaflavin, theaflavin 3-monogallate, theaflavin 3′-monogallate and theaflavin 3,3′-digallate||-|||
|Raw elderberry (Sambucus nigra L.)||UAE of the dried samples with 80% methanol, 45 min||C18 column, 35 °C, mobile phase: MeOH and acetic acid in water (1.0%, v/v); flow rate of 1 mL/min;
monitoring wavelength: 285 nm
|gallic acid, chlorogenic acid, caffeic acid, syringic acid, pcoumaric acid, ferulic acid, rutin, myricetin, quercetin kaempferol and quercetin 3-glucoside||HCA and PCA|||
|Phyllanthus emblica, dried fruits||Fruit powder was extracted in 70% ethanol (1:8) at 50 °C using a magnetic stirrer||DiKMA C18 column (250 mm × 4.6 mm × 5 μm); mobile phase: 0.2% formic acid in water and methanol; flow rate of 1 mL/min;
Monitoring wavelength: 273 nm
|gallic acid, corilagin, ellagic acid, quercetin||Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (Version, 2004A) and HCA|||
|Medicago spp. in different phenologic stages: vegetative elongation, late bud and late flower; dried leaves||MSPD extraction using C18 column. Elution with methanol: H2O (9:1, v/v)||Luna 5 U C18 column (5 μm, 150 × 4.60 mm) at 40 °C; mobile phase: acetonitrile and acetic acid in water (1.0%, v/v); flow rate of 1 mL/min; monitoring wavelength: 254 nm||puerarin, daidzin, genistin, daidzein, glycitein, genistein, pratensein,
formononetin, irilone, prunetin and biochanin A
|generalized linear model (GLM) and linear discriminant analysis (LDA)|||
|Tithonia diversifolia, dried leaves||Maceration in 80% acetone
for 72 h; evaporate to dryness at 40 °C
|C18 column (5 µm, 4.6 mm × 250 mm); mobile phase: water/acetic acid, 98:2% v/v and methanol/water/acetic acid, 70:28:2% v/v; flow rate 0.6 mL/min;
monitoring wavelengths: 254, 327, 366
|gallic acid, chlorogenic acid, caffeic acid and p-coumaric acid, and apigenin||analysis of variance model and Tukey’s test|||
|lyophilized leaves of ten mango varieties||UAE with 70% ethanol, 320 W for 30 min; evaporate to dryness by vacuum rotary evaporator at 30 °C||C18 column (250 mm × 4.6 mm, 5 μm);
mobile phase: 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile; flow rate: 0.8 mL/min;
all wavelengths scanning detection from 200 to 600 nm
|neomangiferin, galic acid, 5-caffeoylquinic acid, 3-chlorogenic acid, mangiferin, 4-hydroxybenzoic acid, sinpic acid, isoquercitrin, quercetin||similarity analysis, PCA, HCA, discriminant analysis|||
|Plant Material||Extraction||Hyphenated Technique Used||Instrumental Methodology (HPLC Condition, Ionization, Acquisition Mode)||Selected Polyphenols||Ref.|
|Flowers, leaves, and stalks of alfalfa (Medicago sativa L.) and goldenrod (Solidago virgaurea L.); flowers, leaves, stalks, and roots of phacelia (Phacelia tanacetifolia Benth.); buckwheat (Fagopyrum esculentum); licorice root (Glycyrrhiza glabra); and lavender flower (Lavandula spica L.)||solid–liquid extraction
(SLE) consecutively using H2O; mixture H2O/EtOH (1:1; v/v), mixture H2O/MeOH (1:1; v/v), and finally NeOH shaking for 5 h; SPE purification using a C18 column
|UHPLC-MS/MS||Zorbax Eclipse XDB-C18 column (50 × 2.1 mm, 1.8 μm); mobile phase: 0.1% v/v formic acid in water and ACN;
ESI ionization source in negative mode,
acquisition in selected reactions monitoring mode, SRM
|3-(4-hydroxyphenyl) propionic acid,
4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid, quercetin, rutin, glabridin, and naringenin
(Nelumbo nucifera) rhizome knot
|Enzymatic hydrolysis with cellulose and pectinase, at 62 °C, pH 4, 90 min followed by
|HPLC–QqQ-MS/MS||ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm; mobile phase: aqueous 0.4% acetic acid, and acetonitrile;
ESI in negative ion mode
Acquisition in MRM mode
|chlorogenic acid, B-type procyanidin
dimer, (+)-catechin, B-type procyanidin dimer, (−)-epicatechin, propyl
gallate, caffeic acid, (−)-epicatechin-3-gallate, and rutin
|Trifolium pratense L. (Red Clover), dried leaves||MAE, 300 W, with MeOH at 70 °C||HPLC-ESI-MS/MS||Zorbax Eclipse XDB-C18 50 mm × 4.6 mm × 1.8 μm column; mobile phase: 0.05% aqueous formic acid and MeOH;
ESI ionisation source in negative mode, acquisition in SRM
|43 phenolic including: genistein, daidzein, p-hydroxy-benzoic acid, caffeic acids, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, hyperoside|||
|Heartwood samples of Dalbergia odorifera (medicinal herbs in TCM);||UAE with 70% methanol (v/v) for 45 min||UHPLC–QqQ-MS/MS
|Acquity HSS T3 column (100 mm × 2.1 mm, 1.8 µm); mobile phase: ACNand water containing 0.05% formic acid
ESI negative ion mode.
Acquisition in MRM mode for QqQ analysis.
|17 flavonoids including:
daidzein, dalbergin, 30-hydroxydaidein, liquiritigenin, isoliquiritigenin, alpinetin, butein, naringenin, butin, prunetin, eriodictyol, tectorigenin, pinocembrin, formononetin, genistein, sativanone
|Green, black and oolong tea (Camellia sinensis)||Extraction at high temperature (80 °C) for 3 min with mild stirring||HPLC–QqQ-MS/MS||Capcell Pak C18 MGIII (2.0 mm × 100 mm, 3 m) column;
ESI in positive and negative ion mode.
Acquisition in MRM mode.
|gallocatechin, epigallocatechin, catechin, epigallocatechin gallate, epicatechin, gallocatechin gallate, epicatechin gallate, catechin gallate, theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate; theaflavin-3,3′-digallate.|||
|Sour Guava (Psidium friedrichsthalianum Nied) lyophilized whole fruits||UAE of lyophilized fruits with acetone:water (7:3). The extract was submitted to successive partitions with ethyl ether, ethyl acetate, and N-butanol.||UPLC-ESI/QqQ-MS/MS||UPLC BEH C18-column (2.1 × 100 mm, 1.7 µm); mobile phase water/formic acid (99.9:0.1, v/v), and ACN/formic acid (99.9:0.1, v/v)
ESI ionization source in negative mode.
Acquisition in MRM
|22 phenolic compounds including several hydroxybenzoic, phenylacetic, and hydroxycinnamic acid derivatives|||
|Barks of Connarus var. angustifolius, and leaves of Cecropia obtusa, Cecropia palmata and Mansoa alliacea; dried samples||UAE in 70% hydroethanolic, butanol/ethyl acetate, 4 h
Butanol and ethyl acetate were evaporated at 40 °C
|HPLC-ESI/QqQ-MS/MS||SB-C18 Rapid Resolution HD column (2.1 × 50 mm, 1.8 μm; mobile phase 0.1% acetic acid in water and ACN;
ESI ionization source in negative mode
Acquisition in MRM
|gallic acid, catechin, caffeic acid, rutin, ferulic acid, quercitrin and resveratrol|||
|Instrumental Methodology (LC Condition, Ionization, Acquisition Mode)||Data Processing Approach Used for Tentatively Identification||Ref.|
|Ground dried culinary herbs and spices: dill (Anethum graveolens), marjoram (Origanum majorana), turmeric (Curcuma longa), caraway (Carum carvi), and nutmeg (Myristica fragans)||UAE with 5 mL of 50% ethanol in 0.1% formic acid in ultrapure water; 5 min||UHPLC- LTQ Orbitrap MS||Atlantis T3 C18 (100 × 2.1 mm, 3 µm) column; mobile phase; 1% formic acid in water and 0.1% formic acid in ACN.
Full scan MS mode at 60,000 FWHM and MS/MS mode with the Orbitrap at 30,000 FWHM
Comparison with HR-MS data found in literature
(traditional Chinese medicine prescription)
|Ultrasonic extraction with MeOH, 30 min||UHPLC-Q/Orbitrap –HRMS/MS||Acquity T3 (2.1 × 100 mm, 1.8 μm) column; mobile phase: 0.1% formic acid in water (A) and 0.1% formic acid in MeOH.
HESI II ionization source in positive and negative mode; collision energy: 25–60 V.
Data-dependent acquisition (DDA).
Searching in Chemspider; Pubchem.
|Aster tataricus rhizoma||UAE with MeOH for 30 min||UHPLC-Q-TOF-MS||Poroshell 120 EC-C18 column (100 mm × 2.1 mm, 2.6 µm); mobile phase: water + 0.1% formic acid and ACN + 0.1% formic acid.
ESI source in both positive and negative ion mode.
DDA and DIA acquisition methods.
Multiple mass defect filter (MMDF) and dynamic background subtraction (DBS) by AB Sciex software
|Searching for reported metabolites; in-house database.
Searching in Chemspider database using MasterViewTM1.0.
Product ions strategy (KPIs).
Clog P (calculated by Chemdraw Ultra 12.0 software)—used for distinguishing isomers.
|Ophryosporus triangularis Meyen, dried aerial parts (leaves and stems) and flowers||UAE with MeOH, 30 min||UHPLC-Q-
|Acclaim UHPLC C18 column, (150 mm × 4.6 mm ID, 2.5 µm); mobile phases: 1% formic aqueous solution and ACN;
HESI negative ionization mode;
full scan at 70,000 FWHM followed by targeted MS/MS at 17,500 FWHM;
collision energy (HCD cell)–30 kv
comparison with HR-MS data found in literature
|Dried leaves of green perilla (Perilla frutescens)||Extraction with MeOH by shaking for 8 h at ambient temperature||LC- TOF-MS/MS||Poroshell 120 EC-C18, 150 × 2.1 mm, I.D., 4 mm; mobile phase: 0.1% formic acid in water and MeOH;
ESI source in negative ion mode; resolution of 2700; collision energy 10 eV.
|The analyst TF software (version 1.7);
identification of the compounds by comparison with previous studies.
Tentatively characterized by fragmentation pathway identification.
|Goji berries (Lycium barbarum L.)||Extraction in 1% formic acid in 80:20 methanol/water solution by centrifugation at
25,000 rpm/3 min
|UHPLC-ESI-QTOF-MS||Zorbax eclipse plus C18 column (50 × 2.1 mm, 1.8 μm); mobile phase: water and MeOH
ESI ionisation source in positive;
full scan MS2 (30,000 FWHM);
mass accuracy ≤ 5
|Profinder B.07 software.
Phenol-Explorer 3.6 database
|Stellera chamaejasme extracts||reflux with MeOH for 30 min at 50 °C.||UHPLC–LTQ-Orbitrap MSn||Agilent Zorbax Ecipse Plus C18 column (100 × 3.0 mm, 1.8 µm) at 50 °C; mobile phase water with 0.5% FA and ACN; HESI in positive and negative mode
DDA, MSn scan (four ions for MS2 and one ion for MS3); resolution of MS2–60,000 and MS3–30,000;
collision energy: MS2–30 V and MS3–35 V.
|SciFinder database (https://scifinder.cas.org) for chemical formula annotation;
comparison with HR-MS data found in literature
|The male flowers of date palm (Phoenix dactylifera)||Soxhlet extraction with 80% MeOH for 6 h at 50 °C, fractionation in a C18 column||HPLC-ESI-ITMS||Symmetry C18 column (5 µm, 2.1 mm × 150 mm; mobile phase: ACN containing 0.03% (v/v) formic acid and water containing 0.03% (v/v) formic acid;
ESI ion source in negative ion mode full-scan followed by MS-MS
|Density functional theory (DFT) study|||
|Mentha rotundifolia (L.) Huds, aerial parts||maceration in MeOH for 24 h at room temperature||UHPLC-ESI-Q- Orbitrap- HRMS/MS||Kromasil RP-18 column (250 mm 10 mm); mobile phase: water/ACN 75:25, 0.25% FA and ACN, 0.25% FA;
HESI ionization source in negative mode;
full MS followed by DDA scan
|In-house database, confirmation by NMR approaches|||
|Baoyuan decoction (traditional Chinese medicine formula)||reflux with water for 1.5 h||UHPLC–Q-TOF-MS/MS||Cortecs UPLC C18 column (1.6 μm, 2.1 × 100 mm; mobile phase: 0.05% aqueous formic acid and ACN containing 0.05% formic acid; ESI ionization in negative mode;
|The bark, twigs, leaves, and fruits of Alnus japonica, Alnus hirsuta and Alnus hirsuta var. sibirica||UAE with MeOH (1:10 g DW/g), 60 °C, 60 min, at 60 kHz||UHPLC–Q-TOF-MS/MS||Acquity BEH C18 (100 × 2.1 mm 1.7 μm) column; mobile phase: H2O and MeCN, both of which were acidified with 0.1% formic acid;
ESI ionization mode in negative ion;
DDA (full MS followed by MS/MS scans for the three most intense ion).
|Spectral preprocessing using MZmine; in silico annotation with network; annotation propagation
GNPS molecular networking;
integration of annotation data using MolNetEnhancer
|Medicago sativa L. and Trifolium pratense L. dried sprouts||UAE with 70% ethanol (1:10 g DW/g), 60 °C, 60 min, at 60kHz||UHPLC-Q-
|Accucore U-HPLC Column C18 (150 × 2.1 mm, 2.6 μm); mobile phase: water containing 500 μL/L−1 formic acid (pH 2.5) and MeOH;
HESI ionization in negative mode;
DIA (Full scan 70,000 FWHM, MS/MS 35,000 FWHM).
Normalized collision energy: 30, 60 and 80 NCE
NORMAN MassBank, mzCloudeTM Advanced Mass Spectral Database; in silico fragmentation with ACDLabs MS Fragmenter 2019.2.1 software
|Blackcurrant (Ribes nigrum L.) leaves||UAE with ethanol/water (1:1), 20 °C, 60 min||ESI-LTQ-Orbitrap MS||Kinetex Evo C18 5 μm column; mobile phase: water + 0.1% formic acid and ACN + 0.1% formic acid.
ESI source in negative ion mode.
|Persimmon leaves (Chinese traditional medicine)||UAE with methanol/water (80:20, v/v)||LC-ESI-LTQ- Orbitrap-MS||Atlantis T3 column 2.1 × 100 mm, 3 m; mobile phase water/0.1% formic acid and ACN;
ESI source in both positive and negative ion mode;
DDA approach-full scan at 30,000 FWHM and MS/MS at 15,000 FWHM;
Collision energy (HCD cell)–35 kv
|Identification by generating the molecular formula using accurate mass (C = 30, H = 100, O = 15), and matching with the isotopic pattern.
Searching in polyphenol database: (http://phenol-explorer.eu/).
Confirmation by comparison with HR-MS data in literature and databases
|Flowers and leaves of Chilean Mistletoe
(Quintral, Tristerix tetrandus)
|Lyophilized flowers and leaves were defatted thrice with N-hexane (1:10) and then extracted by UAE with 0.1% HCl in MeOH (1:10) for 60 min. Purification with XAD-7 column.||UHPLC–Q/Orbitrap/HRMS/MS||UHPLC C18 Column, Acclaim, 150 mm × 4.6 mm ID, 5 µm. Mobile phase: 0.1% aqueous formic acid and ACN 0.1% formic acid.
HESI II ionization source in positive and negative mode.
Full scan MS (70,000 FWHM) acquisition followed by targeted MS/MS analysis (17,500 FWHM).
Collision energy: 30 kv.
Mass accuracy ≤ 5
|Trace Finder 3.2 software|||