Simultaneous determination of 17α-hydroxypregnenolone and 17α-hydroxyprogesterone in dried blood spots from low birth weight infants using LC-MS/MS
Simultaneous determination of 17α-hydroxypregnenolone and 17α-hydroxyprogesterone in dried blood spots from low birth weight infants using LC-MS/MS
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DOI：
10.1016/j.jpba.2008.05.010
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发表时间：
2008-09-10
影响因子：
3.4
通讯作者：
Maeda, Masako
中科院分区：
文献类型：
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作者：
Higashi, Tatsuya;Nishio, Tadashi;Maeda, Masako
迄今为止，17 α-羟孕烯醇酮（170 HPreg）被认为是基于免疫测定的新生儿先天性肾上腺增生（CAH）筛查中17 α-羟孕酮（170 HP）值假性升高的主要原因。为了验证这一点，我们开发了一种液相色谱-串联质谱（LC-MS/MS）方法，该方法能够同时定量干燥血液滤纸中的170 HPreg和170 HP，并测量婴儿，特别是低出生体重婴儿的血液水平。用甲醇从滤纸中提取类固醇，使用Strata-X柱纯化，用2-肼基吡啶衍生化，并进行LC-MS/MS。验证试验证明，该方法具有特异性和重现性：内源性类固醇不干扰定量，试验内和试验间的变异系数低于5.2%。当使用3个滤纸盘（直径3 mm）（对应于8 μ L全血）时，170 HPreg和170 HP的定量限分别为1.0和0.5 ng/mL。极低出生体重儿（1000-1500 g）和极低出生体重儿（< 1000 g）血170 HPreg水平高于正常出生体重儿（> 2500 g）（P < 0.05）。但是，170 HPreg浓度不足以在基于酶免疫测定的筛选中引起假阳性结果，并且认为假阳性结果来自其他内源性组分而不是170 HPreg。(c)2008 Elsevier B. V.保留所有权利。
17 alpha-Hydroxypregnenolone (170HPreg) has heretofore been considered to be the major cause of the false elevated 17 alpha-hydroxyprogesterone (170HP) value in the immunoassay-based newborn screening for congenital adrenal hyperplasia (CAH). To verify this point, we developed a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method that enables the simultaneous quantification of 170HPreg and 170HP in the dried blood filter papers and measured their blood levels in infants, especially in infants with low birth weights. Steroids were extracted from the filter papers with methanol, purified using a Strata-X cartridge, derivatized with 2-hydrazinopyridine and subjected to LC-MS/MS. Validation tests proved that this method was specific and reproducible: endogenous steroids did not interfere with the quantifications, and the intra- and inter-assay coefficients of variation were below 5.2%. The limits of quantitation were 1.0 and 0.5 ng/mL for 170HPreg and 170HP, respectively, when 3 disks (3 mm in diameter) of the filter papers (corresponding to 8 mu L of whole blood) were used. The blood 170HPreg level was elevated in the very low birth weight (1000-1500 g) infants and extremely low birth weight (< 1000 g) infants, compared to those in the normal birth weight (> 2500 g) infants (P < 0.05). However, the 170HPreg concentration was not high enough to cause the false positive results in the enzyme immunoassay-based screening, and it was considered that the false positive results come from other endogenous components rather than 170HPreg. (c) 2008 Elsevier B.V. All rights reserved.