Rapid HPLC method and sample extraction procedures for measuring 25-hydroxyvitamin D3 concentrations in human breast milk

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  • Abstract

    Background; Human breast milkis themilk produced by thebreasts (or mammary glands) of ahuman female for her infant offspring. Milk is the primary source of nutrition fornewborns before they are able to eat and digest other foods. Vitamin D describes a group of fat-soluble steroids. Vitamins D2 and D3 can be converted to the active steroid hormone in the human body. The active form of vitamin D is hydroxylated in two places. The most accurate measurement of vitamin D levels in the body is a blood test that detects the levels of circulating 25-hydroxylated vitamin D.

    Aims of the study; the purpose of the present study was to develop a protocol for the extraction of cholecalciferol from human breast milk for analysis by HPLC using retinyl acetate as internal standards.

    Methods: The HPLC proposed enables successful separation and quantitation of Vitamin D3 (cholecalciferol) and their respective internal standards (retinyl acetate) in less than 10 minutes; RP- C18 column (100 x 4.6 mm I.D.; particle size, 5 micron) at a flow-rate of 1 ml/min, the mobile phase was methanol. The eluate was monitored with a photodiode-array detector with wavelengths 265 nm.

    Results: No interference was found from other fat soluble vitamins (vitamin A) that are commonly presents with vitamin D. Reproducibility studies carried out with pooled breast milk showed a within day and between day precision of the analysis did not exceed 2.6% and 4 % respectively for cholecalciferol. The detection limits were 2.8ng/ml, the linearity of the standard was excellent (r2 > 0.999), over the concentration range of 0-100ng/ml.

    Conclusions: This method separates fat-soluble vitamins in human breast milk, including cholecalciferol using retinyl acetate as internal standards. HPLC method is a rapid determination and quantification of vitamins D in human breast milk, time-consuming steps and has been shown to be sensitive, and reliable.




Article ID: 1057
DOI: 10.14419/ijbas.v2i3.1057

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