Determination of 25-Dydroxyvitamin D2 and 25-Hydroxyvitamin D3 in Dried Blood Spots Using Liquid Chromatography- Tandem Mass Spectrometry
10.11895/j.issn.0253-3820.160745
- VernacularTitle:液相色谱-串联质谱法检测干血点样本中25-羟基维生素D2和25-羟基维生素D3
- Keywords:
Liquid chromatography-tandem mass spectrometry;
25-Hydroxyvitamin D2;
25-Hydroxyvitamin D3;
Automated liquid handling platform;
Dried blood spots
- From:
Chinese Journal of Analytical Chemistry
2017;45(3):448-454
- CountryChina
- Language:Chinese
-
Abstract:
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 SRM972 a Level 3 and the recovery is found to be 110 . 3% and 103 . 0%.
