Determination of trans and Total Vitamin K1 in Infant, Pediatric, and Adult Nutritionals by HPLC with Post Column Reduction and Fluorescence Detection: Multilaboratory Testing Study, AOAC Final Action 2015.09.

A multilaboratory study was completed with AOAC INTERNATIONAL First Action Official MethodSM 2015.09, "Determination of trans and Total (cis+trans) Vitamin K1 in Infant, Pediatric, and Adult Nutritionals by HPLC with Post Column Reduction and Fluorescence Detection." Eight laboratories from six countries participated in the multilaboratory study. Each laboratory analyzed 19 infant, pediatric, and adult nutritionals in duplicate. The product matrixes analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders; milk-based infant formula ready-to-feed liquids; pediatric powders; adult low- and high-fat powders; and high-protein ready-to-drink nutritionals. Vitamin K1 was extracted from product matrixes with isooctane after precipitation of proteins and the release of lipids with methanol. Prepared samples were injected onto a silica HPLC column in which cis and trans vitamin K1 were separated with an isooctane-isopropanol mobile phase. The column eluent was mixed with a dilute ethanolic solution of zinc chloride, sodium acetate, and acetic acid, and cis and trans vitamin K1 were reduced to fluorescent derivatives in a zinc reactor column. Overall, trans vitamin K1 repeatability averaged 3.06% relative standard deviation (RSD) with a range of 0.99-7.16% RSD and reproducibility averaged 6.36% RSD with a range of 3.15-16.1% RSD. Repeatability Standard Method Performance Requirements (SMPR®) were met for all 19 matrixes, and reproducibility SMPRs were met for 18 of the 19 product matrixes analyzed. Repeatability and reproducibility for total (cis + trans) vitamin K1 averaged 3.15% RSD with a range of 1.06-6.87% RSD and 6.11% RSD with a range of 2.94-16.7% RSD, respectively.

Determination of Carotenoids in Infant, Pediatric, and Adult Nutritionals by HPLC with UV-Visible Detection: Single-Laboratory Validation, First Action 2017.04.

This reversed-phase HPLC method uses C30 chromatography and UV-Vis spectroscopy to determine cis and trans isomers of lutein, ß-carotene, and lycopene in infant, pediatric, and adult nutritionals. Samples are saponified with a mixture of potassium hydroxide, tetrahydrofuran, and methanol, and carotenoids are extracted from saponified samples with 75 + 25 hexane-methyl tertiary butyl ether (MtBE). After extraction, a portion of the organic layer is evaporated to dryness, and the residue is dissolved in 75 + 25 10% butylated hydroxytoluene in methanol-MtBE. Prepared samples are injected into a C30 HPLC column where cis and trans isomers of lutein, ß-carotene, and lycopene are separated with a methanol-MtBE gradient and detected with UV-Vis spectroscopy at 445 nm. Total carotenoid concentrations are calculated by comparison of sample peak areas with the areas of trans carotenoid standards of known concentration. During a single-laboratory validation of this method, total lutein repeatability and intermediate precision ranged from 1.89 to 14.9 and 1.93 to 14.0%, respectively, in infant and adult nutritional matrixes with concentrations >1 µg/100 g. Total ß-carotene repeatability and intermediate precision ranged from 1.81 to 6.77 and 3.07 to 16.2%, respectively, in infant and adult nutritional matrixes with concentrations >1 µg/100 g, and total lycopene repeatability and intermediate precision ranged from 3.01 to 6.37 and 4.29 to 10.3%, respectively, in infant and adult nutritional matrixes with concentrations >1 µg/100 g. Mean overspike recoveries ranged from 90.3 to 95.3, 89.3 to 108, and 97.3 to 109% for lutein, ß-carotene, and lycopene, respectively. The method also demonstrated good linearity. For lutein, r averaged 0.99991 over a standard range of approximately 10-250 µg/L trans-lutein. with average calibration errors of <1%. For ß-carotene and lycopene, r averaged 0.99993 and 0.9998 over standard ranges of approximately 25-500 and 5-100 µg/L with calibration errors of <1 and <1.5%, respectively. Lutein, ß-carotene, and lycopene LOQs in ready-to-feed nutritionals were estimated to be 0.4, 0.1, and 0.3 µg/100 g, respectively. This method met AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Standard Method Performance Requirements and was approved as a First Action Official Method at the AOAC INTERNATIONAL 2017 midyear meeting.

Determination of Vitamin B12 in Infant, Adult, and Pediatric Formulas by HPLC-UV and Column Switching: Collaborative Study, Final Action 2011.10.

AOAC First Action Method 2011.10, Vitamin B12 in Infant and Pediatric Formulas and Adult Nutritionals, was collaboratively studied. This method uses a pH 4.5 sodium acetate buffer and potassium cyanide at 105°C to extract and convert all biologically active forms of vitamin B12 present to cyanocobalamin; octylsilyl (C8) or C18 SPE cartridges to purify and concentrate cyanocobalamin; a combination of size-exclusion and RPLC to isolate cyanocobalamin; and visible absorbance at 550 nm to detect and quantitate cyanocobalamin in infant, pediatric, and adult nutritionals with vitamin B12 concentrations greater than 0.025 µg/100 g ready-to-feed (RTF) liquid. During this collaborative study, nine to 11 laboratories from eight different countries analyzed blind duplicates of 12 infant, pediatric, and adult nutritional formulas. Per the AOAC Expert Review Panel (ERP) on Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Nutrient Methods the method demonstrated acceptable repeatability and reproducibility and met SPIFAN Standard Method Performance Requirements (SMPRs®) for the majority of product matrixes analyzed. Vitamin B12 SPIFAN SMPRs for repeatability were ≤15% RSD at vitamin B12 concentrations of 0.01 µg/100 g RTF liquid and ≤7% RSD at vitamin B12 concentrations of 0.2-5.0 µg/100 g RTF liquid. Vitamin B12 SPIFAN SMPRs for reproducibility were ≤11% RSD in products with vitamin B12 concentrations ranging from 0.3 to 5.0 µg/100 g RTF liquid. During this collaborative study, the RSDr ranged from 2.98 to 9.77%, and the RSDR ranged from 3.54 to 19.5%. During previous single-laboratory validation studies, the method LOQ was estimated to be 0.025 µg/100 g RTF liquid.

Determination of Myo-Inositol in Infant, Pediatric, and Adult Formulas by Liquid Chromatography-Pulsed Amperometric Detection with Column Switching: Collaborative Study, Final Action 2011.18.

AOAC First Action Method 2011.18, Myo-Inositol (Free and Bound as Phosphatidylinositol) in Infant and Pediatric Formulas and Adult Nutritionals, was collaboratively studied. With this method free myo-inositol and phosphatidylinositol bound myo-inositol are extracted using two different sample preparation procedures, separated by ion chromatography using a combination of Dionex Carbo Pac PA1 and MA1 columns with column switching, and detected with pulsed amperometry using a gold electrode. Free myo-inositol is extracted from samples with dilute hydrochloric acid and water. Phosphatidylinositol is extracted from samples with chloroform and separated from other fats with silica SPE cartridges. Myo-inositol is then released from the glycerol backbone with concentrated acetic and hydrochloric acids at 120°C. During this collaborative study, nine laboratories from five different countries analyzed blind duplicates of nine infant and pediatric nutritional formulas for both free and phosphatidylinositol bound myo-inositol, and one additional laboratory only completed the free myo-inositol analyses. The method demonstrated acceptable repeatability and reproducibility and met the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Standard Method Performance Requirements (SMPRs®) for free myo-inositol plus phosphatidylinositol bound myo-inositol for all the matrixes analyzed. SMPRs for repeatability were ≤5% RSD at myo-inositol concentrations of 2-68 mg/100 g ready-to-feed (RTF) liquid. SMPRs for reproducibility were ≤8% RSD in products with myo-inositol concentrations ranging from 2 to 68 mg/100 g RTF liquid. During this collaborative study, repeatability RSDs ranged from 0.51 to 3.22%, and RSDs ranged from 2.66 to 7.55% for free myo-inositol plus phosphatidylinositol bound myo-inositol.

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

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 <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.