A sensitive and reliable HPLC method for complete separation and quantitation of vitamins D2and D3in complicated biological mixtures such as feedingstuffs has been developed and described. The method has been applied to the quantitative determination of D2and D3in feedingstuffs and related matrices at both high premix levels and low feed levels ranging down to a detection limit near 100 IU/1b or 0.22 IU/g sample. The procedures consist of the initial step of sample preparation by extraction; four sample cleanup stages: Sep/Pak/Silica Cartridge Cleanup, Millipore-Teflon Cleanup, Gel Permeation/Sephadex LH-20 Column Cleanup, and HPLC/Partisil-PAC Column Cleanup; and the final step of Reverse-Phase HPLC Separation, Identification, and Quantitation. The analytical Column used was Rainin Accupak 20 cm-3 um C-18 & Guard Columns. The Waters Associates Model 440 Fixed Wavelength UV Detector at 254 nm was used for all measurements. All separations and quantitations were carried out isocratically at room temperature and under subdued lighting. By these procedures, the sensitivity for both D2and D3is about the same (20 ng), and the resolution is excellent. Normally, the D2peak eluted at 30–31 min. and the D3peak followed at 2–3 min. later. By using the standard calibration and standard addition methods, the percent recovery ranges from 90.0%–104.8% with the mean value of 97.4%, whereas the accuracy is from 85.3% to 108.9% with the average of 97.8%. The standard deviation is ±5.2% and the coefficient of variation is 5.3%.
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