title: An LC/MS/MS method for stable isotope dilution studies of β-carotene bioavailability, bioconversion, and vitamin A status in humans.
abstract: Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as β-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [(13)C10]β-carotene and 1 mg [(13)C10]retinyl acetate in human subjects over a 2 week period. A reverse phase C18 column and binary mobile phase solvent system separated β-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [(12)C]β-carotene and [(12)C] retinoids; m/z 547→330 and m/z 274→98 for [(13)C10]β-carotene and [(13)C5] cleavage products; and m/z 279→100 for metabolites of [(13)C10]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [(13)C10]retinyl acetate with [(13)C10]β-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual's vitamin A status.
authors.affiliation: Human Nutrition Research Centre, Newcastle University, Newcastle Upon Tyne, UK.
authors.forename: Anthony
authors.initials: A
authors.lastname: Oxley
authors.forename: Philip
authors.initials: P
authors.lastname: Berry
authors.forename: Gordon A
authors.initials: GA
authors.lastname: Taylor
authors.forename: Joseph
authors.initials: J
authors.lastname: Cowell
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hall
authors.forename: John
authors.initials: J
authors.lastname: Hesketh
authors.forename: Georg
authors.initials: G
authors.lastname: Lietz
authors.forename: Alan V
authors.initials: AV
authors.lastname: Boddy
mesh_terms: D000293:Adolescent; D000328:Adult; D001682:Biological Availability; D001711:Biotransformation; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D007554:Isotopes; D008297:Male; D008875:Middle Aged; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors; D014801:Vitamin A / Q000097:blood / Q000378:metabolism*; D055815:Young Adult; D019207:beta Carotene / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics*
chemical_list: D007554:Isotopes; D019207:beta Carotene; D014801:Vitamin A
keywords: carotenoid metabolism; retinol metabolism; retinyl esters; tandem mass spectrometry; β-carotene 15,15′-monooxygenase