Development of a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for characterizing pomegranate extract pharmacokinetics in humans

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 233; p. 115477
• Main Authors: Wang, Yan-Hong, Mondal, Goutam, Khan, Washim, Gurley, Bill J., Yates, Charles R.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 05.09.2023
• Subjects: And human pharmacokinetics; Ellagic acid; Pomegranate; Punicalagins; UHPLC-MS/MS; Urolithin; And human pharmacokinetics; Urolithin; UHPLC-MS/MS; Pomegranate; Ellagic acid; Punicalagins
• ISSN: 0731-7085; 1873-264X; 1873-264X
• DOI: 10.1016/j.jpba.2023.115477

Pomegranate extracts standardized to punicalagins are a rich source of ellagitannins including ellagic acid (EA). Recent evidence suggests that gut microbiota-derived urolithin (Uro) metabolites of ellagitannins are pharmacologically active. Studies have evaluated the pharmacokinetics of EA, however, little is known about the disposition of urolithin metabolites (urolithin A (UA) and B (UB)). To address this gap, we developed and applied a novel ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the characterization of EA and Uro oral pharmacokinetics in humans. Subjects (10/cohort) received a single oral dose (250 or 1000 mg) of pomegranate extract (Pomella® extract) standardized to contain not less than 30 % punicalagins, < 5 % EA, and not less than 50 % polyphenols. Plasma samples, collected over 48 h, were treated with β-glucuronidase and sulfatase to permit comparison between unconjugated and conjugated forms of EA, UA and UB. EA and urolithins were separated by gradient elution (acetonitrile/water, 0.1 % formic acid) using a C18 column connected to a triple quadrupole mass spectrometer operating in the negative mode. Conjugated EA exposure was ∼5–8-fold higher than unconjugated EA for both dose groups. Conjugated UA was readily detectable beginning ∼8 h post-dosing, however, unconjugated UA was detectable in only a few subjects. Neither form of UB was detected. Together these data indicate EA is rapidly absorbed and conjugated following oral administration of Pomella® extract. Moreover, UA’s delayed appearance in the blood, primarily in the conjugated form, is consistent with gut microbiota-mediated metabolism of EA to UA, which is then rapidly converted to its conjugated form. •Quantification of pomegranate’s polyphenol and its metabolites in plasma.•Conjugated ellagic acid (EA) exposure was higher than unconjugated EA.•Eight of ten subjects showed urolithin A (UA), and none showed urolithin B (UB).•UA conjugates were detectable up to 48 h post-administration.•UA formed through the gut microbiota-mediated transformation of EA.