NORMAL-PHASE HPLC WITH EC DETECTION - 1,25-DIHYDROXY VITAMIN-D(3) AND25-HYDROXY-16-ENE-23-YNE VITAMIN-D(3)

An analytical method for the separation and detection of 1,25-dihydrox y vitamin D3 and 25-hydroxy-16-ene-23-yne vitamin D3 by normal phase H PLC with electrochemical detector is reported. A post column electroly te technique was adopted in order to apply electrochemical detection i n a normal phase chromatographic system. Warfarin was used as an inter nal standard. The response and concentration was linear over the 10(-3 ) Mg/ml-10(-5) Mg/ml concentration range for both compounds. The sensi tivity is improved 1 000 fold by the use of amperometric detection ins tead of UV detection. The detection limit was approximately 3 pg/ml. T he amperometric detector can detect some of the degradation products o f 25-hydroxy-16-ene-23-yne vitamin D3 which can not be detected by UV.