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Ultra‐Performance Liquid Chromatographic (UPLC) Method with Photodiode Array (PDA) Ultraviolet Detection for Simultaneous Determination of 25‐Hydroxyvitamin D3 and D2, Retinol, and Tocopherols in Human Plasma
Author(s) -
Cui Hongjie,
Schulze Kerry J,
Charurat Manhattan,
Christian Parul
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1019.5
Subject(s) - chromatography , analyte , detection limit , high performance liquid chromatography , chemistry , extraction (chemistry) , reproducibility , vitamin , vitamin d and neurology , analytical chemistry (journal) , biochemistry , medicine , endocrinology
Challenges in simultaneous, accurate measurement of vitamins A, D, and E in human plasma limit our ability to comprehensively evaluate vitamin status. For vitamin D in particular, methods must be sufficiently sensitive to determine 25‐hydroxyvitamin D, 25(OH)D, as 25(OH)D 3 and 25(OH)D 2 to identify vitamin D deficiency and monitor the effect of supplemental vitamin D 2 . We developed a method to determine 25(OH)D 3 and 25(OH)D 2 , retinol, and α‐ and γ‐tocopherol in a single run using a Waters Acquity Hclass system with a PDA detector. Extraction of fat‐soluble vitamin forms was achieved using liquid‐liquid extraction with hexanes. Chromatographic separation was done with an Acquity UPLC carbon‐18 column using a gradient mobile phase. The method yields low intra‐ and inter‐assay variability (<8.5% for all analytes) and good recovery for 25(OH) D 3 (97%) and D 2 (93%) with a limit of quantitation of 9.93 nmol/L and 3.19 nmol/L for 25(OH)D 3 and D 2 , respectively, using 100 uL of plasma. All analytes elute within 11 minutes and peaks are assessed in 4 channels. The accuracy of the method was validated using NIST Standard Reference Material 972 and 968d, with results in the range of 94–106% of expected for each analyte. The sensitivity, reproducibility, low sample volume requirement, and speed of the method make it suitable for routine clinical and research use. Support: NIAID.