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

建立了一种高通量液相色谱-串联质谱技术检测干血点(DBS)样本中25-羟基维生素D225(OH)D2]和25-羟基维生素D325(OH)D3]的方法.以DBS为样本,以4-苯基-1,2,4-三唑啉-3,5-二酮(PTAD)为试剂进行分析物衍生化,所需样本量仅约相当于6μL全血当量的DBS样本;使用甲醇直接超声提取分析物,避开了通常情况下DBS样本前处理中的全血复溶和蛋白质沉淀等繁琐步骤;整个前处理过程使用自动化液体处理平台实现自动化操作和高检测通量;以25(OH)D2-D6和25(OH)D3-D3为同位素内标,消除基质效应的影响.前处理后的样本进行LC-MS/MS分析,使用C18柱进行分离,流动相为甲醇(含5 mmol/L甲酸铵)-水(含5 mmol/L甲酸铵)(75:25,V/V),洗脱时间为4 min,使用多反应监测模式(MRM)定量.结果表明:25(OH)D2和25(OH)D3的检出限为0.12 ng/mL(S/N=3),定量限为0.94 ng/mL(S/N=10).25(OH)D2和25(OH)D3在0.94~120.00 ng/mL范围内线性关系良好,日内相对标准偏差(RSD)分别为1.4%~8.6%和3.7%~15.5%,日间RSD分别为4.0%~5.3%和3.8%~14.9%,平均回收率分别为91.7%±7.9%~108.5%±6.5%和94.8%±6.8%~101.3%±2.9%.DBS样本在不同温度(-20℃,22℃,37℃)下储存不同时间(0,1,2,3,5,7,14天)后的稳定性实验显示样本总体RSD°15%.以25(OH)D参考物质NIST SRM 972a中的Level 3制备标准DBS样本,25(OH)D2和25(OH)D3的回收率分别为110.3%和103.0%.

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

A high-throughput method is established to determine 25-hydroxyvitamin D225(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%.