6. AOACSPIFANMethods-2018Awards

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1382 Bidlack et al. : J ournal of AOAC I nternational V ol. 98, N o. 5, 2015 INFANT FORMULA AND ADULT NUTRITIONALS

Determination of Vitamin K 1 in Infant, Pediatric, and Adult Nutritionals by HPLC with Fluorescence Detection: Single- Laboratory Validation, First Action 2015.09 Mary Bidlack, Linda D. Butler Thompson, Wesley A. Jacobs, and Karen J. Schimpf 1 Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219

methyl-substituted naphthoquinone nucleus attached to a side chain of three saturated and one unsaturated isoprene units and is a yellow viscous oil. Although vitamin K 1 occurs naturally in the trans form, during synthesis of vitamin K 1 both the cis and trans isomers are formed with the trans isomer being the major product. Vitamin K 1 is insoluble in water and sparingly soluble in methanol and ethanol. It is soluble in vegetable oils and organic solvents such as pentane, hexane, iso-octane, and 2-propanol. Vitamin K 1 has five ultraviolet absorption maxima which are at 242, 248, 260, 269, and 325 nm and can be reduced to a fluorescent hydroquinone. Vitamin K 1 is stable to air, heat, oxidizing agents, and moisture, but its activity is destroyed by light (especially UV radiation), reducing agents, and alkalies (1). Good sources of vitamin K 1 are alfalfa, cabbage, cauliflower, green vegetables, tomatoes, cheese, dairy products, meat, egg yolks, and canola and soy oil. Vitamin K 1 is also found in bacteria and is synthesized in the intestinal tract by microorganisms. Trans vitamin K 1 is biologically active, while the cis form has little if any activity (1). At the September 2013 AOAC Annual Meeting, an AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) working group developed Standard Method Performance Requirements (SMPR ® ; 2) for trans vitamin K 1 and required separation of the cis and trans isomers since cis vitamin K 1 has little if any biological activity. SPIFAN approved AOAC SMPR 2014.001 at the March 2014 AOAC Mid-Year Meeting. Subsequently, AOAC issued a call for methods. In response to AOAC’s call for methods, a new vitamin K 1 method that combined the strengths of the two current AOAC Official Vitamin K 1 methods, 992.27 and 999.15 , was developed and validated. AOAC 992.27 uses liquid–liquid extraction in separatory funnels, open column cleanup, normal phase (NP) chromatography, and UV absorbance to extract, separate, and quantitate trans vitamin K 1  (3). Although the AOAC 992.27 sample preparation procedure provides better recovery of vitamin K 1 in more complex infant, pediatric, and adult nutritional matrixes than 999.15 and the sample preparation solvents are compatible with the NP chromatography, UV detection is not very specific and the sample preparation procedure is labor-intensive. AOAC 999.15 uses an enzyme digestion and liquid–liquid extraction in glass tubes, reversed-phase chromatography, and fluorescence detection after postcolumn reduction with zinc to extract, separate, and quantitate trans or total vitamin K 1 (4). Although AOAC 999.15 uses a more specific detection system and a simpler sample preparation procedure, it will not separate cis and trans vitamin K 1 if a C 18 column is used; sample extracts must be dried down and the residue dissolved in a solvent compatible with

Received May 20, 2015. Accepted by SG July 8, 2015. This method was approved by the Expert Review Panel for Infant Formula and Adult Nutritionals as First Action. The Expert Review Panel for Infant Formula and Adult Nutritionals invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. 1 Corresponding author’s e-mail: karen.schimpf@abbott.com DOI: 10.5740/jaoacint.15-130 and average calibration errors of <1%. LOQ and LOD in ready-to-feed nutritionals were estimated to be 0.03 and 0.09 µg/100 g, respectively. The method met AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Standard Method Performance Requirements ® and was approved as a first action method at the 2015 AOAC Mid-Year Meeting. V itamin K 1 is an antihemorrhagic vitamin first isolated in 1939 after it was discovered that chicks fed diets previously extracted with nonpolar solvents developed subdural or muscular hemorrhages. Vitamin K 1 , which is also known as phylloquinone and phytonadione, consists of a This normal-phase HPLC method with postcolumn reduction and fluorescence detection allows for the quantitative determination of trans vitamin K 1 in infant, pediatric, and adult nutritionals. Vitamin K 1 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 K 1 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 K 1 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 K 1 with r 2 averaging 0.99995