Flavonoid Detection Panel — Organized by Subclass

Each flavonoid subclass requires different sample preparation, chromatography, and ionization conditions. Mixing them in one generic method compromises data quality across all subclasses. Our panel runs subclass-specific methods and reports results in a single unified data table. Each subclass links to its dedicated analysis page with full compound lists and method details.

Subclass	Analytes	Key Compounds Covered
Flavones	10+	Apigenin, apigenin, luteolin, chrysin, baicalein, baicalin, diosmetin, acacetin. Major aglycones in herbs, vegetables, and propolis. Baicalein/baicalin are the signature flavones of Scutellaria species.
Flavonols	10+	Quercetin, kaempferol, myricetin, isorhamnetin, rutin (quercetin-3-O-rutinoside). Most widely distributed flavonoid subclass — quercetin glycosides are the dominant flavonoids in onions, apples, tea, and red wine.
Flavanones	10+	Naringenin, hesperetin, eriodictyol, homoeriodictyol. Dominant flavonoids in citrus — naringenin in grapefruit, hesperetin in oranges. Dihydroflavanones are the biosynthetic precursors.
Isoflavones	8+	Genistein, daidzein, glycitein, formononetin, biochanin A. Phytoestrogens concentrated in soy and legumes. Require enzymatic hydrolysis to release aglycones from glycoside and malonyl-glycoside conjugates — without this step, genistein and daidzein are underestimated by 30-60%.
Anthocyanins	15+	Cyanidin, delphinidin, pelargonidin, peonidin, petunidin, malvidin glycosides. pH-dependent structural interconversion — extraction and chromatography must be performed under acidic conditions (pH < 2) to stabilize the flavylium cation. Quantified as individual glycosides and as aglycone equivalents after acid hydrolysis.
Catechins (Flavan-3-ols)	8+	Catechin, epicatechin, epigallocatechin (EGC), epigallocatechin gallate (EGCG), gallocatechin. Dominant polyphenols in tea (Camellia sinensis). EGCG is the most abundant catechin in green tea and the most studied for bioactivity.
Proanthocyanidins	5+	Procyanidins B1, B2, B3, C1; prodelphinidins. Oligomeric and polymeric flavan-3-ols in grape seeds, cocoa, cranberry, and pine bark. Quantified by acid-catalyzed depolymerization to anthocyanidins (DMAC or butanol-HCl assay) with individual dimer/trimer profiling by LC-MS/MS.
O-Glycosylated Flavonoids	15+	Quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, apigenin-7-O-glucoside. O-glycosides are the predominant storage form of flavonoids in plants. Enzymatic hydrolysis (beta-glucosidase or hesperidinase) releases the aglycone for quantification by MRM.
C-Glycosylated Flavonoids	8+	Vitexin, isovitexin, orientin, isoorientin. C-glycosides resist enzymatic and acid hydrolysis — cannot be quantified as aglycones. Require direct quantification of intact glycosides against C-glycoside authentic standards. Common in cereal grains and passionflower.
Neoflavonoids	5+	Daidzein-related 4-phenylcoumarins and dalbergiones. Distinct from isoflavones in biosynthetic origin. Found in Dalbergia and Machaerium species.
Polymethoxylated Flavones (PMFs)	5+	Nobiletin, tangeretin, sinensetin, heptamethoxyflavone. Highly methoxylated flavones in citrus peel with enhanced bioavailability compared to hydroxylated flavones. GC-MS compatible due to reduced polarity from methoxy substitution.

Why Flavonoid Hydrolysis Must Be Subclass-Specific

Most flavonoids exist as glycosides — an aglycone core conjugated to one or more sugar molecules. Quantifying intact glycosides directly is impractical: hundreds of possible sugar combinations per aglycone, authentic standards available for only a handful, and MS response factors that shift with the sugar moiety.

The standard fix — enzymatic or acid hydrolysis to release the aglycone — works for O-glycosides. It fails for C-glycosides (the carbon-carbon bond resists hydrolysis). It underestimates isoflavones (malonyl-glycosides need esterase treatment). It overestimates anthocyanins if pH is not controlled. Each subclass needs its own protocol:

O-Glycosides — Flavonols, flavones, flavanones: enzymatic hydrolysis with beta-glucosidase or hesperidinase (37 degree C, 2 h) releases aglycones for MRM quantification. Aglycone concentration reported as aglycone equivalents.
Isoflavone Glycosides — Additional esterase treatment to hydrolyze malonyl- and acetyl-glycoside conjugates. Without this step, genistein and daidzein are underestimated by 30-60% in soy products.

C-Glycosides — Resistant to enzymatic and acid hydrolysis. Quantified directly as intact glycosides against C-glycoside authentic standards (vitexin, isovitexin, orientin). Reported as individual C-glycoside concentrations.
Anthocyanins — Acidified extraction (1% HCl in methanol) stabilizes the flavylium cation. Quantified as individual anthocyanin glycosides by MRM. Acid hydrolysis (1.2 M HCl, 90 degree C, 2 h) converts to anthocyanidin aglycones for aglycone-equivalent reporting.

HILIC + RP LC-MS/MS Platform for Flavonoid Quantification

LC-MS/MS Platform

SCIEX QTRAP 6500+ with scheduled MRM. Reversed-phase C18 for aglycone separation (flavonoid core structure determines retention). HILIC for intact glycoside profiling (sugar moiety determines retention — complementary to RP). AB SCIEX TripleTOF 5600 for UHPLC-qTOF untargeted flavonoid profiling and glycoside identification via MS/MS fragmentation (sugar neutral loss patterns identify glycosylation type and position).

Complementary: HPLC-DAD — Agilent 1260 Infinity II with diode array. Flavonoids have characteristic UV spectra (Band I: 300-380 nm, Band II: 240-280 nm) that distinguish subclasses: flavones/flavonols absorb at 350-365 nm, flavanones at 280-290 nm with a shoulder at 330 nm, isoflavones at 260 nm. DAD provides subclass confirmation orthogonal to MS.

Method Performance

Parameter	Specification
LOD	0.01-0.5 ng/mL (analyte-dependent); quercetin: 0.05 ng/mL, EGCG: 0.1 ng/mL
Linear Range	3-4 orders of magnitude; R2 above or equal to 0.995 per analyte
Quantification	Absolute — 6-point authentic standard calibration, subclass-specific IS
Precision (CV)	Intra-batch: below 5% (aglycones), below 10% (glycosides). Inter-batch: below 15%
Hydrolysis Efficiency	Validated per subclass — spike recovery of glycoside standard converted to aglycone: 85-115%

Flavonoid Analysis Workflow

Subclass-Specific Sample Preparation

Plant tissue freeze-dried, ground, extracted with methanol:water (80:20) + 0.1% formic acid
Anthocyanin aliquot: acidified methanol (1% HCl) stabilizes flavylium cation
Isoflavone aliquot: saponification cleaves ester-linked malonyl-glycosides
C-glycoside aliquot: direct extraction — no hydrolysis possible
Authentic standard cocktail and subclass-specific IS spiked at homogenization

Enzymatic Hydrolysis & Glycoside Profiling

O-glycoside aliquots: beta-glucosidase + hesperidinase (37 degree C, 2 h) → aglycones extracted with ethyl acetate
C-glycoside aliquots: direct injection — the C-C bond between sugar and aglycone resists enzymatic and acid hydrolysis
Intact glycoside profiling by UHPLC-qTOF on TripleTOF 5600 with MS/MS neutral loss annotation: 162 Da (hexose), 146 Da (rhamnose), 132 Da (pentose)

RP + HILIC LC-MS/MS Acquisition

Aglycones: RP C18, scheduled MRM on SCIEX QTRAP 6500+, 2-3 transitions per analyte. Intact glycosides: HILIC, SWATH DIA on TripleTOF 5600. Anthocyanins: RP C18 with acidified mobile phase (0.1% TFA). Sequence: blank, 6 calibrators, matrix-matched QC, randomized samples, QC every 8-10 injections.

Quantification & Report Delivery

Aglycones: 6-point authentic standard calibration, 1/x2 weighted regression. Aglycone equivalents reported for hydrolyzed O-glycosides. Intact C-glycosides reported as individual compounds. QC report with hydrolysis efficiency validation, calibration curves, IS recovery. Methods documentation included.

Flavonoid Analysis Workflow — Four-Step Pipeline from Subclass-Specific Sample Preparation to Quantification

What You Receive from Flavonoid Analysis

Quantitative Concentration Table — Absolute concentrations (mg/g dry weight, ug/mL, or mg/100 g) for each flavonoid per sample. Aglycone equivalents reported for hydrolyzed O-glycosides. Intact C-glycosides reported individually. Hydrolysis efficiency per subclass documented.
QC Report — Calibration curves (6-point, 1/x2 weighted, R2 and back-calculated accuracy per analyte). Pooled QC RSD. IS recovery. Glycoside-to-aglycone conversion efficiency. Blank carryover.
Chromatograms & Spectral Data — MRM traces for each aglycone. UHPLC-qTOF MS/MS spectra for glycoside identification (sugar neutral loss annotation). HPLC-DAD UV spectra confirming subclass assignment.
Methods Documentation — Extraction protocol per subclass, hydrolysis conditions and enzyme specifications, LC-MS/MS parameters. Formatted for manuscript methods section.

Flavonoid Analysis — Chromatograms, Spectra & Quantification

Flavonoid HPLC-DAD Chromatogram — Subclass Identification by UV Spectra and RP C18 Separation

HPLC-DAD chromatogram (280/350 nm) showing subclass-specific UV absorption patterns: flavones and flavonols at 350 nm, flavanones at 280 nm, and isoflavones at 260 nm.

Flavonoid MRM Chromatogram — Quercetin Kaempferol Naringenin and Genistein Aglycone Separation

RP C18 MRM chromatogram of flavonoid aglycones after enzymatic hydrolysis, showing baseline separation of quercetin, kaempferol, naringenin, hesperetin, and genistein.

Flavonoid Glycoside Profiling — UHPLC-qTOF MS/MS Sugar Neutral Loss Identification

UHPLC-qTOF MS/MS spectrum of quercetin-3-O-rutinoside (rutin) showing characteristic neutral losses of rhamnose (146 Da) and glucose (162 Da) for glycoside identification.

Flavonoid Quantification — Box Plots of Aglycone Concentrations Across Plant Treatment Groups

Flavonoid aglycone concentration box plots across plant treatment groups with FDR significance, showing quercetin, kaempferol, and naringenin response to UV-B exposure.

Case Study — Flavonoid Profiling Links Aronia Supplement to Improved Gut Function

Effects of Aronia melanocarpa juice-powder on hindgut function and performance in post-weaned pigs

Pearce, S.C., Anderson, C.L., & Kerr, B.J. | Journal of Functional Foods, 2024, 116, 106196 | IF: 3.8

DOI: 10.1016/j.jff.2024.106196

The Challenge

Post-weaning is the most stressful period in a pig's life — intestinal inflammation, reduced feed intake, and growth stasis caused by abrupt dietary and environmental changes. Aronia melanocarpa (black chokeberry) is one of the richest dietary sources of flavonoids and phenolic acids, with documented anti-inflammatory and antioxidant properties. The question was whether supplementing post-weaning pig feed with Aronia juice powder could mitigate intestinal inflammation and improve growth performance — and critically, what specific flavonoids and phenolic acids were present in the supplement at bioactive concentrations. Without comprehensive flavonoid profiling, the link between supplement composition and biological effect would remain a black box.

The Results

The Aronia juice powder was sent to a commercial laboratory for targeted flavonoid and phenolic acid quantification by LC-MS/MS. The analysis identified and quantified the full spectrum of Aronia polyphenols, including anthocyanins (cyanidin-3-galactoside, cyanidin-3-arabinoside), flavonols (quercetin glycosides), and phenolic acids (chlorogenic acid, neochlorogenic acid). Pigs receiving Aronia-supplemented feed showed improved hindgut barrier function and reduced intestinal inflammation markers compared to unsupplemented controls — effects directly attributable to the characterized flavonoid profile of the supplement.

Why It Matters

This study demonstrates the essential role of flavonoid profiling in nutritional intervention research: without quantifying which flavonoids were present and at what concentrations, the biological effect cannot be attributed to specific compounds, the dose-response relationship cannot be established, and the results cannot be replicated. Our flavonoid panel provides the same analytical framework — identify and quantify the flavonoid profile of your plant material, food product, or supplement, then connect the chemistry to the biology.

How We Deliver the Same

Targeted LC-MS/MS quantification of anthocyanins, flavonols, flavanones, and phenolic acids with authentic standard calibration
Aglycone-equivalent reporting after enzymatic hydrolysis — the standard format for nutritional flavonoid data
Subclass-specific extraction and hydrolysis protocols — anthocyanins require acidified conditions, which our method handles separately from flavonols

Reference

Pearce, S.C., Anderson, C.L., & Kerr, B.J. Effects of Aronia melanocarpa juice-powder on hindgut function and performance in post-weaned pigs. Journal of Functional Foods 116, 106196 (2024).

Frequently Asked Questions About Flavonoid Analysis

Why are flavonoids reported as aglycone equivalents rather than intact glycosides?

There are hundreds of possible glycoside combinations for each flavonoid aglycone (different sugars, different attachment positions, different numbers of sugar units), and authentic standards exist for only a small fraction of them. Quantifying every individual glycoside is analytically impossible. The standard approach — enzymatic hydrolysis to release the aglycone — converts all O-glycosides of a given flavonoid (e.g., all quercetin glycosides → quercetin) into a single quantifiable peak. Results are reported as "mg quercetin equivalents per g" — meaning the total quercetin content from all glycoside sources. C-glycosides are an exception: the C-C bond resists hydrolysis, so these are quantified directly as intact compounds against C-glycoside standards (vitexin, orientin). Your report clearly distinguishes aglycone-equivalent values from directly quantified C-glycosides.

How do you handle the different hydrolysis requirements across flavonoid subclasses?

We split your sample into subclass-specific aliquots after homogenization. Flavonols, flavones, and flavanones: beta-glucosidase + hesperidinase (37 degree C, 2 h). Isoflavones: additional esterase step to cleave malonyl- and acetyl-glycoside conjugates — without this, genistein and daidzein are underestimated by 30-60%. Anthocyanins: acid hydrolysis (1.2 M HCl, 90 degree C, 2 h) converts to anthocyanidin aglycones. C-Glycosides: no hydrolysis — quantified directly. Hydrolysis efficiency is validated per batch by spiking a known glycoside standard (e.g., rutin → quercetin) and measuring aglycone recovery.

What flavonoid subclasses can you quantify?

11 subclasses: flavones, flavonols, flavanones, isoflavones, anthocyanins, catechins (flavan-3-ols), proanthocyanidins, O-glycosylated flavonoids, C-glycosylated flavonoids, neoflavonoids, and polymethoxylated flavones. Each subclass has its own extraction, hydrolysis (if applicable), chromatography, and MRM method. The panel covers 50+ individual flavonoids. Representative compounds per subclass are listed in the flavonoid detection panel on the Service Details tab.

What sample types do you accept for flavonoid analysis?

Plant tissue (leaves, roots, flowers, fruits — 100-500 mg freeze-dried, ground to powder). Food and beverage (tea, wine, juice, chocolate, soy products — 1-5 mL or 1-5 g). Herbal medicine (dried extracts, tinctures, decoctions — 100-500 mg). Biological samples (plasma, urine, tissue — flavonoid concentrations are typically low; 200-500 uL plasma needed). Plant samples should be freeze-dried and shipped at room temperature; biological samples shipped on dry ice. For anthocyanin-containing samples, acidified extraction must be performed immediately upon receipt — contact us before shipping.

Can you profile intact flavonoid glycosides for identification purposes?

Yes — this is a core part of the service. Before hydrolysis, an aliquot of each sample is analyzed by UHPLC-qTOF-MS/MS on the AB SCIEX TripleTOF 5600. MS/MS fragmentation spectra reveal the glycosylation pattern through characteristic neutral losses: 162 Da (hexose — glucose or galactose), 146 Da (rhamnose), 132 Da (pentose — arabinose or xylose), and 176 Da (glucuronic acid). This tells you which glycosides are present in your sample — information that is lost after hydrolysis. The intact glycoside profiling data is included in your report alongside the aglycone quantification data.

Flavonoid Analysis Service — LC-MS/MS Quantification of 50+ Flavonoids Across 11 Subclasses