Improvements in the high performance liquid chromatographic method for the determination of 25-hydroxycholecalciferol in cow plasma

✍ Scribed by K.T. Koshy; A.L. VanDerSlik

• Publisher: Elsevier Science
• Year: 1978
• Tongue: English
• Weight: 183 KB
• Volume: 85
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(78)90301-9

✦ Synopsis

In a previous paper [(1976) Anal. Biochem. 74, 2821 we reported a procedure for the analysis of 25hydroxycholecalciferol (25OH-Da) in cow plasma or serum by high performance liquid chromatography. Since then, we obtained synthetic 25hydroxyergocalciferol (25OH-D& and found that it had the same retention time as 25-OH-4 and interfered with the 25-OH-Ds analysis. We also encountered a relatively small number of samples that did not give sufficiently clean chromatograms by our procedure. This communication details an improvement we have made in the old procedure to overcome these problems.

EXPERIMENTAL

The sample was extracted and processed as described (1) and subjected to the following additional partition column chromatographic step prior to high performance liquid chromatography (HPLC). The column was a 5-ml disposable serological pipet.' A glass tube of the same dimension was attached to it by a Teflon adapter to hold about 8-10 ml of the mobile phase.

Celite 5452 was used as the solid support. Methanol:water, 80:20, containing 0.05% of sodium ascorbate was the stationary phase and n-pentane was the mobile phase. The solvents were saturated with each other. These solutions are stable for at least 1 week.

Two grams of the Celite was transferred to a 20-ml test tube and mixed well with 1.6 ml of 80:20 methanol:water using a spatula. A small wad of cotton was placed in the outlet of the column. The wide end was inserted ' Kimble Products,