Determination of Vitamin K1 in Infant, Pediatric, and Adult Nutritionals by HPLC with Fluorescence Detection: Single-Laboratory Validation, First Action 2015.09

• Published in: Journal of AOAC International 2015-09, Vol.98 (5), p.1382-1389
• Main Authors: Bidlack, Mary, Butler Thompson, Linda D, Jacobs, Wesley A, Schimpf, Karen J
• Format: Article
• Language: English
• Subjects: 2-Propanol - chemistry; Acetic Acid - chemistry; Adult; Child; Chlorides - chemistry; Chromatography, High Pressure Liquid - standards; Fluorescence; Food, Formulated - analysis; Humans; Infant; Limit of Detection; Octanes - chemistry; Reproducibility of Results; Sodium Acetate - chemistry; Solid Phase Extraction - methods; Vitamin K 1 - isolation & purification; Zinc Compounds - chemistry
• ISSN: 1060-3271; 1944-7922
• DOI: 10.5740/jaoacint.15-130

This normal-phase HPLC method with postcolumn reduction and fluorescence detection allows for the quantitative determination of trans vitamin K1 in infant, pediatric, and adult nutritionals. Vitamin K1 is extracted from products with iso-octane after precipitation of proteins and release of lipids with methanol. Prepared samples are injected onto a silica HPLC column where cis and trans vitamin K1 are separated with an iso-octane-isopropanol mobile phase. The column eluent is mixed with a dilute ethanolic solution of zinc chloride, sodium acetate, and acetic acid, and vitamin K1 is reduced to a fluorescent derivative in a zinc reactor column. The resulting hydroquinone is then detected by fluorescence at an excitation wavelength of 245 nm and an emission wavelength of 440 nm. During a single-laboratory validation of this method, repeatability and intermediate precision ranged from 0.6 to 3.5% RSD and 1.1 to 6.0% RSD, respectively. Mean overspike recoveries ranged from 91.9 to 106%. The method demonstrated good linearity over a standard range of approximately 2-90 μg/L trans vitamin K1 with r2 averaging 0.99995 and average calibration errors of