Determination of 25-Hydroxyvitamin D in Human Plasma Using High-Performance Liquid Chromatography−Tandem Mass Spectrometry

• Published in: Analytical chemistry (Washington) 2005-05, Vol.77 (9), p.3001-3007
• Main Authors: Tsugawa, Naoko, Suhara, Yoshitomo, Kamao, Maya, Okano, Toshio
• Format: Article
• Language: English
• Subjects: 25-Hydroxyvitamin D 2 - blood; Analytical chemistry; Calcifediol - blood; Chemical compounds; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, High Pressure Liquid - methods; Exact sciences and technology; Female; Humans; Mass spectrometry; Middle Aged; Other chromatographic methods; Plasma; Reproducibility of Results; Sensitivity and Specificity; Spectrometric and optical methods; Tandem Mass Spectrometry - methods; Vitamin D; Vitamin D - analogs & derivatives; Vitamin D - blood
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac048249c

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.