Tested Assay of Vitamin

General instructions are given here. All precautions (see under I) must be taken and the original literature consulted for certain details and explanations. 

We consider the suggestion of F. J. Mulder and K. H. Hanewald (private communication, N. V. Philips-Duphae, Weesp) to be valuable for safe protection of vitamin D during TLC 3,5-ditert.-butyl-4-hydroxytoluene (BHT) is added to the sample solution and to the solvent to yield 0.01% solutions sample solutions of fat-free extracts are stabilised in addition with 1% squalane. 

OUy and, to some extent, water-soluble concentrates can, for example, be directly diluted with cyclohexane or acetone, respectively. Preparations of the former type which contain little vitamin D must be largely freed from the large amount of fat-soluble ballast by dissolving in warm absolute alcohol and freezing out at 0° C. Products in powdered or pulverised form are best submitted to ammonia-cal or enzymatic digestion to liberate the vitamin D from the dry powder this is also done when added as an adsorbate or oily solution. Extraction with petrol ether is then carried out and the extract evaporated and dissolved in cyclohexane or chloroform. The most suitable procedure must be chosen and tested with every preparation. 

This solvent mixture is used if vitamin A-alcohol is present. 

In the modified colour reaction for small amounts of vitamin D (e. g., extracts with about 400 lU per 2.5 ml), the solutions for a single determination are pipetted into test tubes as described above but are treated with only 2.0 ml reagent. After briefly mixing, the contents are introduced into 1 cm cuvettes and after precisely 20 sec the extinction measured with a spectrophotometer at 500 nm against the blank solution. 

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