Determination of Total Biotin by Liquid Chromatography Coupled with Immunoaffinity Column Cleanup Extraction: Multilaboratory Testing, Final Action 2016.02.

Single- and multilaboratory testing data have provided systematic scientific evidence that a simple, selective, accurate, and precise method can be used as a potential candidate reference method for dispute resolution in determining total biotin in all forms of infant, adult, and/or pediatric formula. Using LC coupled with immunoaffinity column cleanup extraction, the method fully meets the intended purpose and applicability statement in AOAC Standard Method Performance Requirement 2014.005. The method was applied to a cross-section of infant formula and adult nutritional matrixes, and acceptable precision and accuracy were established. The analytical platform is inexpensive, and the method can be used in almost any laboratory worldwide with basic facilities. The immunoaffinity column cleanup extraction is the key step to successful analysis.

Determination of biotin and folate in infant formula and milk by optical biosensor-based immunoassay.

Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2-70 ng/mL, recoveries of 86-102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.

Determination of vitamin K in milk and infant formulas by liquid chromatography: collaborative study.

A simple procedure for determination of vitamin K1 was developed for routine compliance monitoring of supplemented infant formula and measurement of endogenous levels in milk and milk powders. Samples are digested with lipase and extracted into hexane; and aliquot is evaporated, reconstituted in methanol, and analyzed by reversed-phase LC. Post-column zinc reduction of phylloquinone facilitates detection by fluorescence. The procedure was subjected to an AOAC collaborative study involving 8 materials, each in blind duplicate, across the range of 5-120 micrograms/100 g solids and including NIST 1846 reference material. Thirty-three laboratories returned valid data which were then statistically analyzed for outliers and precision parameters. Mean RSDR (%) was 6.53 (4.33-10.94), with a mean HORRAT value of 0.33 (0.23-0.43) and RSDr:RSDR ratio of 0.74. K1 isomers (cis and trans) were aggregated with conventional C18 columns, but may be selectively estimated with use of the C30 column.

Determination of choline in milk and infant formulas by enzymatic analysis: collaborative study.

A collaborative study was conducted on a coupled enzymatic-spectrophotometric method for the determination of choline in infant formula and milk powders. Twenty-nine laboratories participated in the analysis of 8 blind duplicates over the range of 45-175 micrograms/100 g sample. After the combined acid hydrolysis-phospholipase release of choline, incubation with choline oxidase in the presence of peroxidase and phenol generates a quinoneimine chromophore with 4-aminoantipyrine. Following raw data screening, overall mean RSDR was estimated at 5.14% (range, 4.26-6.34%) with a HORRAT value of 0.91 (range, 0.76-1.00) and an overall mean RSDr:RSDR value of 0.53 (range, 0.42-0.74). The method was also compared with alternative independent analytical techniques for several of the collaborative study samples.

Taurine analysis in milk and infant formulae by liquid chromatography: collaborative study.

A collaborative study was conducted on a liquid chromatographic (LC) method for determination of taurine in infant formula and milk powders. Twenty laboratories participated in the analysis of 8 blind duplicates over the range of approximately 3-60 mg/100 g sample. The method involved protein removal, conversion to the dansyl-derivative, and isocratic LC separation with UV and/or fluorescence detection. Following outlier treatment, overall mean RSDR has been estimated at 7.00% for supplemented products with a HORRAT value of 1.1. The poorer precision at endogenous levels establishes a lower limit of determination of about 5 mg/100 g. An overall mean RSDr:RSDR value of 0.7 for all products demonstrated acceptable performance.

Vitamin K in milk and infant formulas: determination and distribution of phylloquinone and menaquinone-4.

A method is described for the determination of phylloquinone and menaquinones following enzymatic digestion, extraction and a single-stage HPLC technique utilizing post-column reduction with zinc and fluorescence detection. The technique is applicable to both routine compliance control of phylloquinone supplemented infant formula powders (30-150 micrograms per 100 g) and fundamental studies of the K vitamins at endogenous levels in fluid milks (0-5.0 micrograms per 100 g). Analytical figures of merit include a detection limit of 30 micrograms on-column, recoveries greater than 98% for both K1 and MK4, an RSDR of 2.35% (K1) and 2.32% (MK4) and a regression correlation of 0.9932 for a wide range of infant formulas when compared against an alternative HPLC-UV technique. MK4 and 2',3'-dihydrophylloquinone, both with undefined bioactivity, were detected at measurable levels in a range of infant formulas. Although the higher menaquinones were found to be essentially absent in the milk of several species, the significant presence of MK4 relative to K1 has been confirmed in all milks examined, with both dominant forms correlated during early lactation in the cow. These observations suggest an as yet unrecognized physiological function for MK4 in infant nutrition.

Liquid chromatographic determination of vitamin K1 in infant formulas and milk.

Vitamin K1 in infant formulas and milk products is determined by reversed-phase liquid chromatography (LC) with UV detection. The sample is hydrolyzed enzymatically, and the vitamin is extracted with hexane. Fractionation by normal-phase semi-preparative LC is followed by analytical LC, with quantitation by the internal standard technique. Recovery of the analyte was 97.4 +/- 2.8%. Linearity was established between 0.05 and 4.0 micrograms/mL. The limit of quantitation is 0.5 microgram/100 g for milk powder, which allows the method to quantitate endogenous levels of vitamin K1.

Enzymatic determination of free carnitine in milk and infant formulas.

A simple spectrophotometric assay for free carnitine in milk and supplemented infant formulas has been developed. After acid extraction, carnitine is measured enzymatically through its reaction with carnitine acetyltransferase coupled with acetyl coenzyme A and dithiobenzoate (DTNB). The manually performed method is rapid, accurate, and convenient for routine quality-control analysis of infant formulas. The chemistry is also suitable for automation, for greatly enhanced throughput.

Determination of free myo-inositol in milk and infant formula by high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for the determination of supplemental myo-inositol in infant formulas based on cows' milk. The technique incorporates pre-column derivatization with phenylisocyanate followed by reversed-phase gradient chromatography and ultraviolet detection. The protocol was also applied to a survey of free inositol at endogenous levels in the milk of cows, goats and humans. Nutritional preparations containing inositol at pharmacological levels are also amenable to a simplified isocratic analysis.

Simultaneous liquid chromatographic determination of cholesterol, phytosterols and tocopherols in foods.

A method is described for the simultaneous determination of cholesterol, phytosterols and tocopherols in dairy and non-dairy high-performance liquid chromatography. A facile saponification and extraction scheme is completed rapidly within a single reaction tube. Clean-up is not required, with analysis completed by one of two alternative reversed-phase approaches. Sterols are monitored by short-wave ultraviolet spectrophotometry, and tocopherols by means of a series fluorescence detector. Cholesterol is resolved from the principal plant phytosterols, while the tocopherol congeners are well separated, thereby offering complementary information regarding source-lipid composition in foods under regulatory quality-control conditions. Squalene, an essential metabolic sterol precursor, can also be determined concurrently at the same viewing wavelength.

Antioxidant analysis in edible oils and fats by normal-phase high-performance liquid chromatography.

Following a simple dilution in the appropriate phase, the sample is injected directly onto either of two normal-phase high-performance liquid chromatography systems (3,5-di-tert.-butyl-4-hydroxytoluene or 3-tert.-butyl-4-hydroxyanisole-tert.-butylhydroquinone) with UV detection at 280 nm. An isocratic ternary mobile phase, incorporating acetonitrile as the polar modifier, has been found to facilitate such an approach, thereby avoiding the discriminatory and recovery problems inherent in other techniques requiring prior sample manipulations. The three most commonly used antioxidants may be estimated at levels down to 3 ppm (3,5-di-tert.-butyl-4-hydroxytoluene or 3-tert.-butyl-4-hydroxyanisole) and 10 ppm (tert.-butylhydroquinone) within 30 min.