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Identification of conjugation positions of urinary glucuronidated vitamin D 3 metabolites by LC/ESI–MS/MS after conversion to MS/MS‐fragmentable derivatives
Author(s) -
Yoshimura Yusuke,
Hibi Ryuichiro,
Nakata Akiho,
Togashi Moeka,
Ogawa Shoujiro,
Ishige Takayuki,
Satoh Mamoru,
Nomura Fumio,
Higashi Tatsuya
Publication year - 2019
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.4538
Subject(s) - chemistry , glucuronidation , derivatization , chromatography , glucuronide , mass spectrometry , electrospray ionization , tandem mass spectrometry , urine , glucuronates , liquid chromatography–mass spectrometry , glucuronic acid , microsome , organic chemistry , biochemistry , polysaccharide , enzyme
A liquid chromatography/electrospray ionization–tandem mass spectrometry‐based method was developed for the identification of the conjugation positions of the monoglucuronides of 25‐hydroxyvitamin D 3 [25(OH)D 3 ] and 24,25‐dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] in human urine. The method employed derivatization with 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D 3 ‐3‐ and ‐25‐glucuronides, or 24,25(OH) 2 D 3 ‐3‐, ‐24‐ and ‐25‐glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D 3 and 24,25(OH) 2 D 3 , respectively. The 3‐glucuronide was not present for 24,25(OH) 2 D 3 , unlike 25(OH)D 3 , thus we found that selective glucuronidation occurs at the C24‐hydroxy group for 24,25(OH) 2 D 3 .