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The page below is a sample from the LabCE course Vitamin D. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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HPLC

High-performance liquid chromatography (HPLC) is another method which can quantitate 25-hydroxy vitamin D2 and D3. These HPLC methods are available in kit form from several companies in an effort to standardize test quality and to make the assays more cost effective and less labor intensive. One of the methods (Hitachi) uses a reverse phase column and diode array detection, which allows for highly sensitive simultaneous analysis at optimal wavelengths. This method can be used to analyze food as well as biological samples.
Newer chromatography methods have been developed to improve sensitivity, to simplify steps, and to measure all forms of vitamin D. An example is a method that was developed to analyze all forms and metabolites of vitamin D (D2, D3, and 25-hydroxy) in serum at the same time. The procedure uses an ionization detector technique known as atmospheric pressure photo ionization (APPI) to provide additional sensitivity for analysis. The method is less difficult compared with other LC methods because it doesn’t require pre-concentration steps.