Citation: HAN Ji-Chun, WANG Xiao-Chen, LI Guang-Lin, ZHAO Ya-Jun, LUO Y. Ruben. Determination of 25-Dydroxyvitamin D₂ and 25-Hydroxyvitamin D₃ in Dried Blood Spots Using Liquid Chromatography-Tandem Mass Spectrometry[J]. Chinese Journal of Analytical Chemistry, ;2017, 45(3): 448-454. doi: 10.11895/j.issn.0253-3820.160745 shu

Determination of 25-Dydroxyvitamin D₂ and 25-Hydroxyvitamin D₃ in Dried Blood Spots Using Liquid Chromatography-Tandem Mass Spectrometry

Quantum Health Medical Laboratory, Hangzhou 310052, China

Received Date: 8 October 2016
Revised Date: 5 January 2017

A high-throughput method is established to determine 25-hydroxyvitamin D₂[25(OH)D₂] and 25-hydroxyvitamin D₃[25(OH)D₃] in dried blood spots (DBS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which only needs a DBS sample prepared from about 6 μL of whole blood. The DBS sample processing includes ultrasonic extraction of analytes, addition of 25(OH)D₂-D6 and 25(OH)D₃-D3 as internal standard, application of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) as derivatization reagent. The procedure is carried out in a 96-well plate format in an automated liquid handling platform to facilitate high-throughput analysis. The processed sample is separated in a C₁₈ column with water-methanol gradient elution, and quantitated by mass spectrometry in multiple reaction monitoring (MRM) mode. For both 25(OH)D₂ and 25(OH)D₃, the linear range of quantitation is 0.94-120 ng/mL, the limit of detection is 0.12 ng/mL (S/N=3), and the limit of quantitation is 0.94 ng/mL (S/N=10). The intra-day relative standard deviation (RSD) values of 25(OH)D₂ and 25(OH)D₃ are 1.4%-6.6%, 3.7%-7.0%, respectively. The inter-day RSD values of 25(OH)D₂ and 25(OH)D₃ are 4.0%-5.3%, 3.8-10.6%, respectively. The recovery (mean±SD) in 5 consecutive of 25(OH)D₂ is 98.7%±4.6%-108.5%±6.5%, and that of 25(OH)D₃ is 94.8%±6.8%-101.3%±2.9%. DBS sample stability is confirmed by measuring identical DBS samples stored for 0, 1, 2, 3, 5, 7 and 14 days at -20℃, 22℃, and 37℃, and an overall RSD<15% was observed under each temperature. DBS sample stability in freeze-thaw cycles is also confirmed by experimenting identical DBS samples up to 14 free-thaw cycles (each cycle consisting of 23 h freezing at -80℃ followed by 1 h thaw at room temperature), and an overall RSD<15% is observed. The accuracy of 25(OH)D₂ and 25(OH)D₃ quantitation is validated by measuring a DBS sample prepared from NIST reference material SRM972a Level 3 and the recovery is found to be 110.3% and 103.0%.
- Liquid chromatography-tandem mass spectrometry,
- 25-Hydroxyvitamin D₂,
- 25-Hydroxyvitamin D₃,
- Automated liquid handling platform,
- Dried blood spots
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通讯作者: 陈斌, bchen63@163.com

Determination of 25-Dydroxyvitamin D₂ and 25-Hydroxyvitamin D₃ in Dried Blood Spots Using Liquid Chromatography-Tandem Mass Spectrometry