Development of Analyses by High-Performance Liquid Chromatography and Liquid Chromatography/Tandem Mass Spectrometry of Bilberry (Vaccinium myrtilus) Anthocyanins in Human Plasma and Urine

Abstract

Anthocyanins are potent antioxidants that may possess chronic disease preventive properties. Here, rapid, reliable, and reproducible solid-phase extraction, high-performance liquid chromatography (HPLC), and mass spectrometry techniques are described for the isolation, separation, and identification of anthocyanins in human plasma and urine. Recoveries of cyanidin-3-glucoside (C3G) were 91% from water, 71% from plasma, and 81% from urine. Intra- and interday variations for C3G extraction were 9 and 9.1% in plasma and 7.1 and 9.1% in urine and were less than 15% for all anthocyanins from a standardized bilberry extract (mirtoselect). Analysis of mirtoselect by HPLC with UV detection produced spectra with 15 peaks compatible with anthocyanin components found in mirtoselect within a total run time of 15 min. Chromatographic analysis of human urine obtained after an oral dose of mirtoselect yielded 19 anthocyanin peaks. Mass spectrometric analysis employing multiple reaction monitoring suggests the presence of unchanged anthocyanins and anthocyanidin glucuronide metabolites.

Antibacterial and antioxidant activities of bilberry (Vaccinium myrtillus L.) in vitro

“…Recent studies confirm these findings also for phenolic composition and antioxidant capacity of bilberry leaves (Martz et al, 2010). Literature data indicate that the V. myrtillus is a medicinal plant in traditional medicine and it is applied as a source of active substances (Taruscio et al, 2004;Faria et al, 2005;Cooke et al, 2006). Leaves of V. myrtillus can prevent urinary tract infections.…”

Development and validation of an LC‐MS/MS analysis for simultaneous determination of delphinidin‐3‐glucoside, cyanidin‐3‐glucoside and cyanidin‐3‐(6‐malonylglucoside) in human plasma and urine after blood orange juice administration

“…Despite the high sensitivity and selectivity that could be achieved by LC-MS/MS techniques in quantitative analysis, the most recent reports described quantitative analysis of anthocyanins in plasma and urine samples based on HPLC-DAD, using mass spectrometric techniques only for identification purposes and not for quantitation [14 -18]. Furthermore, except for the work of Cooke et al [18], in which a validated SPE technique for the isolation of cyanidin-3-glucoside (C3G) from human plasma and urine was described, no validation of these methods for quantification of anthocyanins in plasma and urine has been performed. For these reasons, to better characterise the precise pharmacokinetic properties of anthocyanins, it was important to develop a highly sensitive and accurate analytical method for their quantification in biological samples.…”

Pilot Study of Oral Anthocyanins for Colorectal Cancer Chemoprevention

“…Anthocyanin species in portal blood were found by spectroscopic detection in patients for whom the delay between dosing and sampling was 5.7 ± 1 hours (n = 3), but they were below the detection limit (∼0.5 ng/mL, 1 nmol/L; ref. 20) in patients in whom this delay was 8.1 ± 2.1 hours (n = 9). Mass spectrometric analysis and comparison of chromatograms with information contained in the Indena datasheet allows allocation of at least 14 and 17 discrete molecular species to individual peaks in colorectal tissue and urine, respectively ( Table 2).…”

“…The supernatant was dried (nitrogen), and the residue was reconstituted in water/formic acid. Solid phase eluate (for plasma, urine, colorectal tissue) and liver extract were analyzed by HPLC with detection by VIS (510 nm) or tandem mass spectrometry (MS/MS), the latter with selected reaction monitoring (SRM), operated in the positive ion mode, as described and validated previously (20). Anthocyanin concentrations were estimated semiquantitatively using a standard curve for authentic cyanidin-3-glucoside, based on the simplifying assumption that all detected anthocyanins possess similar molar absorption coefficients.…”