Methods for isolating, identifying, and quantifying anthocyanin metabolites in clinical samples.

The metabolic fate of anthocyanins until recently was relatively unknown, primarily as a result of their instability at physiological pH and a lack of published methods for isolating and identifying their metabolites from biological samples. The aim of the present work was to establish methods for the extraction and quantification of anthocyanin metabolites present in urine, serum, and fecal samples. 35 commercial and 10 synthetic analytes, including both known and predicted human and microbial metabolites of anthocyanins, were obtained as reference standards. HPLC and MS/MS conditions were optimized for organic modifier, ionic modifier, mobile phase gradient, flow rate, column type, MS source, and compound dependent parameters. The impact of sorbent, solvent, acid, preservative, elution, and evaporation on solid phase extraction (SPE) efficiency was also explored. The HPLC-MS/MS method validation demonstrated acceptable linearity (R(2), 0.997 ± 0.002) and sensitivity (limits of detection (LODs): urine, 100 ± 375 nM; serum, 104 ± 358 nM; feces 138 ± 344 nM), and the final SPE methods provided recoveries of 88.3 ± 17.8% for urine, 86.5 ± 11.1% for serum, and 80.6 ± 20.9% for feces. The final methods were applied to clinical samples derived from an anthocyanin intervention study, where 36 of the 45 modeled metabolites were detected within urine, plasma, or fecal samples. The described methods provide suitable versatility for the identification and quantification of an extensive series of anthocyanin metabolites for use in future clinical studies exploring absorption, distribution, metabolism, and elimination.

Phenolic metabolites of anthocyanins following a dietary intervention study in post-menopausal women.

SCOPE: Numerous studies feeding anthocyanin-rich foods report limited bioavailability of the parent anthocyanins. The present study explores the identity and concentration of the phenolic metabolites of anthocyanins in humans. METHODS AND RESULTS: Anthocyanin metabolites were quantified in samples collected from a previously conducted 12-wk elderberry intervention study in healthy post-menopausal women. Individual 1-, 2- and 3-h post-bolus urine samples and pooled plasma samples following acute (single bolus) and chronic (12-wk supplementation) anthocyanin consumption (500 mg/day) were analysed using HPLC-ESI-MS/MS. Twenty-eight anthocyanin metabolites were identified in urine and 21 in plasma (including sulfates of vanillic, protocatechuic and benzoic acid). Phenolic metabolites reached peak concentrations of 1237 nM in plasma, while anthocyanin conjugates only reached concentrations of 34 nM. Similarly, in urine, phenolic metabolites were detected at concentrations of 33,185 ± 2549 nM/mM creatinine, while anthocyanin conjugates reached concentrations of 548 ± 219 nM/mM creatinine. There was no evidence that chronic exposure had any impact on either the profile or quantity of metabolites recovered relative to acute exposure. CONCLUSION: An extensive range of phenolic metabolites of anthocyanin was identified following elderberry consumption in humans, including 11 novel metabolites, which were identified at much higher concentrations than their parent compounds.