Normal Phase HPLC with EC Detection: 1,25-Dihydroxy Vitamin D₃ and 25-Hydroxy-16-ene-23-yne Vitamin D₃

Abstract

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 1000 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.