Determination of 25-hydroxyvitamin D in human plasma using high-performance liquid chromatography-tandem mass spectrometry

We report here the development of a precise and sensitive method to determine 25-hydroxyvitamin D (25-OH-D₂/ -D₃) in human plasma using high-perfomance liquid chromatography-tandem mass-mass spectrometry with atomospheric pressure chemical ionization (LC-APCI-MS/ MS). The method involves the use of deuterated 25-OH-D₃ as an internal standard compound for 25-OH-D₂/-D₃, which was synthesized in our laboratory, and the selection of a precursor and product ion with a MS/MS multiple reaction monitoring method. The average intraassay and interassay variation values (relative standard deviation) were 5.7 and 2.5%, respectively, for 25-OH-D₃ and 4.5 and 5.1%, respectively, for 25-OH-D₂. The average spiked recoveries from authentic compounds added to normal human plasma samples for 25-OH-D₃ and 25-OH-D₂ were 103.8 and 98.8%, respectively. Mean plasma concentrations of 25-OH-D₃ and 25-OH-D₂ in healthy subjects were 20.5 and 0.4 ng/mL, respectively. We conclude that this novel LC-APCI-MS/MS method would be useful for the evaluation of the vitamin D status in postmenopausal women and elderly subjects and provide useful information in the diagnosis of vitamin D insufficiency/ deficiency, as well as for the treatment and prevention of osteoporosis with vitamin D.