High Performance Liquid Chromatographic Determination of Vitamin A in Margarine, Milk, Partially Skimmed Milk, and Skimmed Milk

Abstract

Saponification and subsequent evaporation of extracts can cause losses of vitamin A during analysis and thus cause erratic results. These steps were therefore avoided by measuring retinyl palmitate and β-carotene directly in hexane extracts of milk and margarine. Extracts were purified before high performance liquid chromatography(HPLC) by washing with aqueous alcohol. Retinyl palmitate was measured at 325 nm after chromatography on LiChrosorb Si60, 5 μm, using ethyl ether–hexane (2+98) as the solvent system. Milk (2 mL) was shaken with absolute ethanol (5 mL) and hexane (5 mL). Water (3 mL) was added and, after mixing and centrifugation, 100 μL hexane layer was injected. Margarine (5 g) was dissolved in hexane (100 mL). After 5 mL of the hexane solution was washed with 60% ethanol and centrifuged, a 50 μL aliquot was injected. The results of the retinyl palmitate estimation in milk agreed with those obtained by a fluorometric method; the results in margarine agreed with those obtained by saponification, extraction, and HPLC. The coefficients of variation on analysis of 10 replicate samples of milk and margarine were 3 and 4.5%, respectively. The analysis of margarine for vitamin A was completed by measuring β-carotene with a detector set at 453 nm; the coefficient of variation of this measurement was 3% (10 replicates).

Isocratic nonaqueous reversed-phase liquid chromatography of carotenoids

“…Although modern liquid chromatography (HPLC) is gradually emerging as a method of choice for carotenoid separation, no systematic study of its merits and shortcomings has yet been conducted. Rather, a diversity of HPLC systems has been devised for specific applications, often of limited scope, such as the determination of /3-carotene and/or some selected derivatives in pizza (4), oil, margarine, and milk (5)(6)(7)(8)(9), orange and citrus juice (10)(11)(12)(13)(14), tomatoes (15), and tobacco leaves (16,17). HPLC has proven particularly useful for photosynthetic pigment profiling in algae and higher plants (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

Vitamin Bullets. Microencapsulated Feeds to Fortify Shellfish and Tackle Human Nutrient Deficiencies

“…Vitamin A was determined as the sum contribution of retinol and carotenes, and the limit of detection (LOD) was 10 μg 100 g −1 . Measurement of retinol followed UKAS protocol C-TM-021; retinol was saponified with alcoholic KOH and extracted into hexane, then the cis and trans isomers the determined using High-performance Liquid Chromatography (HPLC) with UV detection at 325 nm ( 35 ). Measurement of carotenes followed UKAS protocol C-TM-087; samples were saponified with alcoholic KOH and carotenes extracted into hexane, then the alpha- and ß-carotenes were determined using reverse-phase HPLC with visible detection ( 36 ).…”

Isomerization of retinyl palmitate in fortified skim milk under retail fluorescent lighting

“…Skim milk was extracted for the RP isomers according to Thompson et al (1980) by using a hexane/ethanol mixture. All samples were filtered through a 0.45-µ Alpha-450 (Fisher) before chromatography.…”