Vitamin D is determined by high performance liquid chromatography (HPLC) in samples containing other fat-soluble vitamins. The vitamin D in the unsaponifiable residue is extracted and separated from interferences by straight phase chromatography, and the fraction corresponding to vitamin D3 is collected and quantitated using the AOAC official final action HPLC method for vitamin D3. Analysis of a synthetic mixture gave reasonable recoveries. The method measures potential vitamin D3 content in milkpowder samples containing 2IU vitamin D/g in the presence of all known vitamin D3 isomers, vitamin A, and vitamin E.
Nine laboratories determined the vitamin D3 content of 4 samples in oil containing, in addition to vitamin D3, different amounts of tachysterol3, isotachysterol3, trans-vitamin D3, lumisterol3, and 7-dehydrocholesterol, in order to assess the effect of these isomers on the assay. The analyst selected the method to be used. Three high performance liquid chromatographic (HPLC) methods were applied: 2 were direct and the third required prior isolation of the unsaponifiable material. The 6 remaining analysts used gas-liquid chromatographic (CLC) methods; all GLC techniques required saponification and silylation, and 3 required an additional pretreatment. The results from the GLC and HPLC methods were compared with the known composition of the samples and the results obtained by the official AOAC method, 43.B14–43.B24. The samples were also analyzed by the AOAC method without the maleic anhydride reaction and by the AOAC method with correction for the isotachysterol content. The results of the HPLC assays showed that this technique is promising, but improvements are still needed. The precision and accuracy of the GLC assays varied. However, satisfactory results were obtained after extensive prepurifications. The AOAC chemical method was satisfactory for those samples shown suitable by the confirmation test, i.e., in the absence of isotachysterol.
A specific liquid chromatographic method was developed for determination of nifursol in premixes and turkey feeds. Nifursol is extracted from test sample into tetrahydrofuran. Butylhydroxytoluene is added to prevent degradation of nifursol. The extract is diluted with tetrahydrofuran–water (50 + 50, v/v); an aliquot is injected onto a Zorbax ODS column. The mobile phase is water-acetonitrile (525 + 475, v/v) adjusted to an apparent pH of 3.5 with formic acid and ammonia. The wavelength of detection is 380 nm. The system can separate nifursol from dimetridazole and ronidazole. The method is specific; has linearity of more than one order of magnitude; and has a limit of quantitation in the 1–2 ppm range. Recovery averaged 98% or more, and the reproducibility had a coefficient of variation of better than 2.5% in pelleted feed.
Further study of vitamin D methodology solved the discrepancy between the AOAC chemical method, 43.068-43.078, and the HPLC assay for vitamin D3 in resin containing dry powders. The discrepancy is caused by the difference in solubility of the vitamin D3 resin in benzene and in pentane. The method has been modified accordingly, and has been adopted official first action for vitamin D3 resins and vitamin D3 resin containing powders and aqueous dispersions.
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