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


  • Sawsan Mohammed ali Hadi nutrition research institute
  • Alaa Shalan Hussen head of nutrition research institute
  • Azhar Abdul jabbar Benone chemist/nutrition research institute






Background; Human breast milk is the milk produced by the breasts (or mammary glands) of a human female for her infant offspring. Milk is the primary source of nutrition for newborns 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.

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