柱前衍生-稳定同位素标记-超高效液相色谱-四极杆静电场轨道阱高分辨质谱法测定血清中25-羟基维生素D

Determination of 25-hydroxyvitamin D in serum by pre-column derivatization-stable isotope labeling-ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry

目的建立快速、准确检测血清中25-羟基维生素D_(3)[25-hydroxyvitamin D_(3),25(OH)D_(3)]、25-羟基维生素D_(2)[25-hydroxyvitamin D_(2),25(OH)D_(2)]含量的柱前衍生-稳定同位素标记-超高效液相色谱-四极杆静电场轨道阱高分辨质谱的方法,并用此方法诊断维生素D缺乏症,评估人群维生素D营养状况。方法加入同位素内标的血清样品采用乙酸乙酯-正己烷溶液(2∶1,V/V)提取,离心后氮气吹干,以4-苯基-1,2,4-三唑啉-3,5-二酮(4-phenyl-1,2,4-triazoline-3,5-dione,PTAD)作为衍生化试剂与目标化合物进行衍生化反应。反应产物采用BEH C_(18)色谱柱(2.1 mm×50 mm,1.7μm)进行分离,以0.1%甲酸水溶液-乙腈梯度洗脱。质谱采用电喷雾正离子模式和平行反应监测对目标物进行检测,内标法定量。结果25(OH)D_(3)和25(OH)D_(2)的检出限为0.01 ng/mL,定量限为0.03 ng/mL。25(OH)D_(2)浓度的线性范围为0.5~40.0 ng/mL,25(OH)D_(3)浓度的线性范围为2.5~200.0 ng/mL,二者在浓度范围内线性良好,相关系数(r^(2))大于0.995。25(OH)D_(3)和25(OH)D_(2)的回收率分别为95.7%~101.3%和98.7%~108.6%,相对标准偏差分别为0.5%~4.9%和2.2%~3.4%(n=5)。测定参考物质NIST SRM 972a的Level 2血清中25(OH)D_(3)和25(OH)D_(2)的准确度分别为104.8%和94.9%。利用该方法检测北京市116名孕妇孕早期血清中25(OH)D_(3)和25(OH)D_(2)水平[M(P25,P75)]分别为25.7(20.8,32.6)ng/mL和0.8(0.4,1.1)ng/mL。结论本方法灵敏度高、定性准确,适用于人群维生素D营养状况的评价和监测。

OBJECTIVE To establish a rapid and accurate method for the determination of 25-hydroxyvitamin D_(3)(25(OH)D_(3))and 25-hydroxyvitamin D_(2)(25(OH)D_(2))in serum by pre-column derivatization with stable isotope labeling and ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry,which could be used to diagnose vitamin deficiency and to assess the nutritional status of vitamin D in the population.METHODS The serum samples with isotopic internal standard were extracted by mixed extraction solvent(ethyl acetate∶hexane=2∶1,V/V),centrifuged and dried by the nitrogen blowing,and derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD).The reaction products were separated on a BEH C_(18)column(2.1 mm×50 mm,1.7μm),and eluted with a 0.1%formic acid/water solution-acetonitrile gradient.The mass spectrometry was performed in positive electrospray ionization(ESI+)and parallel reaction monitoring(PRM)for the detection of the targets,and quantified by isotope internal standard.RESULTS The limits of detection both for 25(OH)D_(3)and 25(OH)D_(2)were 0.01 ng/mL and the limits of quantification were 0.03 ng/mL.The concentration series of 25(OH)D_(2)ranged from 0.5 to 40.0 ng/mL and the concentration series of 25(OH)D_(3)ranged from 2.5 to 200.0 ng/mL.The recoveries(n=5)were 95.7%-101.3%for 25(OH)D_(3),and 98.7%-108.6%for 25(OH)D_(2),with the relative standard deviations(RSDs)of 0.5%-4.9%and 2.2%-3.4%,respectively.The accuracy of the determination of 25(OH)D_(3)and 25(OH)D_(2)in Level 2 serum of the 25(OH)D standard reference material NIST SRM 972a was 104.8%and 94.9%,respectively.The M(P25,P75)serum levels of 25(OH)D_(3)and 25(OH)D_(2)for 116 pregnant women at the first trimester from Beijing were 25.7(20.8,32.6)ng/mL and 0.8(0.4,1.1)ng/mL,respectively.CONCLUSION This method is highly sensitive,qualitatively accurate and suitable for evaluation and monitoring the nutritional status of vitamin D in the population.