NORMAL-PHASE HPLC WITH EC DETECTION - 1,25-DIHYDROXY VITAMIN-D(3) AND 25-HYDROXY-16-ENE-23-YNE VITAMIN-D(3)

An analytical method for the separation and detection of 1,25-dihydroxy vitamin D3 and 25-hydroxy-16-ene-23-yne vitamin D3 by normal phase HPLC with electrochemical detector is reported. A post column electrolyte technique was adopted in order to apply electrochemical detection in a normal phase chromatographic system. Warfarin was used as an internal standard. The response and concentration was linear over the 10(-3) Mg/ml-10(-5) Mg/ml concentration range for both compounds. The sensitivity is improved 1 000 fold by the use of amperometric detection instead of UV detection. The detection limit was approximately 3 pg/ml. The amperometric detector can detect some of the degradation products of 25-hydroxy-16-ene-23-yne vitamin D3 which can not be detected by UV.