[Measurement of 25-hydroxyvitamin D3 in human serum by liquid chromatography-tandem mass spectrometry and the control for analytical conditions].
OBJECTIVE
Serum 25-hydroxyvitamin D is very important in evaluating the true vitamin D status of humans. This study is to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantitative determination of 25-(OH)D3 in human serum.


METHODS
Protein in serum sample (400 microl) was precipitated by methanol and the supernatant was extracted with n-hexane. 25-(OH) D3 was detected by using multiple reaction monitoring (MRM) at atmospheric pressure chemical ionization (APCI) in the positive ion mode. Column: Waters ACQUITY UPLC BEH C18 (50 mm x 2.1 mm, 1.7 microm). Mobile phase: 100% methanol. Flow rate: 0.2 ml/min. The results of serum 25-hydroxyvitamin D detected by LC-MS/MS assay were compared with radioimmunoassay (RIA, Diasorin).


RESULTS
The limit of quantitation of LC-MS/MS assay was 1.0 ng/ml for 25-(OH) D3 with a linearity in the range of 6-120 ng/ml (r = 0.999). The recovery was 99.5% (range: 97.1% - 103.4%), and RSD was 4.7%. The average value of 25-(OH) D3 of 53 serum samples was 23.69 ng/ml and 20.13 ng/ml for LC-MS/MS and RIA, respectively (P = 0.083). The correlation of the two methods was: RIA = LC-MS/MS-3.57, r = 0.883.


CONCLUSIONS
LC Tandem MS assay is accurate, convenient and precise at physiologically relevant 25-(OH) D3 concentrations, and the results are comparable well with RIA. This new method can be used for the measurement of 25-(OH)D3 in human serum.
Institute of Nutrition Research, Qingdao University Medical College, Qingdao 266021, China. zhengxiaoyan522@sina.com
Xiaoyan
X
Zheng
Aiguo
A
Ma
Huizhen
H
Zhang
Zhi
Z
Wang
Qiuzhen
Q
Wang
Hui
H
Liang
D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol / Q000097:blood*; D002853:Chromatography, Liquid; D006586:Hexanes; D006801:Humans; D011863:Radioimmunoassay; D053719:Tandem Mass Spectrometry; D014807:Vitamin D / Q000031:analogs & derivatives
D006586:Hexanes; D014807:Vitamin D; D015652:25-Hydroxyvitamin D 2; C026385:n-hexane; C104450:25-hydroxyvitamin D; D002112:Calcifediol