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 Biotin in Infant, Pediatric, and Adult Nutritionals by High-Performance Liquid Chromatography and Fluorescence Detection: Single-Laboratory Validation, First Action 2016.11.

A reversed-phase HPLC method with postcolumn protein conjugation and fluorescence detection for the quantitative determination of biotin in infant, pediatric, and adult nutritionals was developed and evaluated in a single-laboratory validation (SLV). Sample of appropriate size is mixed with 2% metaphosphoric acid to precipitate out the protein. The filtrate is injected onto a C18 HPLC column in which biotin and riboflavin are separated with an appropriate mobile phase. The biotin, after eluting from the column, binds with the streptavidin fluorescein to become a fluorescent conjugate. The conjugate is then detected by fluorescence at λex = 495 nm and λem = 518 nm. A column switch is used in the method as an option to shorten the run time from 30 to 15 min, by eluting out riboflavin at a higher flow rate. In this SLV, a total of 19 AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals matrixes representing a range of infant, pediatric, and adult formulas were evaluated for their biotin content. The analytical range was 1.66-142 µg/100 g reconstituted final product. The repeatability and intermediate precision ranged from 0.5 to 3.0% RSDr and from 1.3 to 4.5% RSDiR, respectively. Recovery from spiked matrixes varied from 95 to 111%, and accuracy of quantification using Standard Reference Material 1849a ranged from 99 to 105%. The LOQ in reconstituted product was estimated to be 0.8 µg/100 g. The method was approved by the Expert Review Panel as First Action at the 2016 AOAC INTERNATIONAL Mid-Year Meeting.

Determination of biotin by high-performance liquid chromatography in infant formula, medical nutritional products, and vitamin premixes.

A solid-phase extraction sample preparation procedure was developed for use with a high-performance liquid chromatography (HPLC) method for biotin analysis. The HPLC method used a reversed-phase C18 column; chromatography run time was 8.5 min. After eluting from the column, biotin went through postcolumn reaction to form a conjugate with streptavidin-fluorescein isothiocyanate, which was then detected by a fluorescence detector. This method was tested with infant formula, medical nutritional products, and vitamin premix samples.