GC-MS衍生化法检测先天性畸形羊水中的非极性氨基酸

目的利用气相色谱质谱(GC-MS)衍生化法对孕先天性畸形儿孕妇羊水中的非极性氨基酸,包括丙氨酸(Ala)、亮氨酸(Leu)、异亮氨酸(Ile)、脯氨酸(Pro)和苯丙氨酸(Phe)进行定量检测;探讨疾病组与正常组孕妇羊水中非极性氨基酸的差异及其意义。方法取100μL羊水上清液,加入甲醇去蛋白后提取上层清液吹干,进行MSTFA衍生化,利用GC-MS检测并分析检测结果。结果该方法具有良好的灵敏度、精密度、准确度以及回收率。在最优化的检测条件下,被检测氨基酸最低检出限为0.12~0.38 mg/L,线性范围为0.5~10 mg/L,添加回收率为91.12%~104.41%,日内及日间相对标准偏差(RSD)为0.84%~9.33%。用建立的方法对17例疾病组和13名正常组孕妇的羊水样本进行测定,统计分析结果表明,疾病组孕妇羊水中亮氨酸和异亮氨酸含量显著降低(P0.01),其余3种氨基酸水平无明显差异。结论本方法经方法学验证,可作为羊水中氨基酸含量的测定方法,具有良好的应用前景;亮氨酸的减少可能参与了先天性畸形的发生。

Quantification of the nonpolar amino acids in amniotic fluid of congenital malformation by gas chromatography mass spectrometry (GC-MS) derivatization method

Objective To develop a gas chromatography-mass spectrometry (GC-MS) detection method for quantification of the nonpolar amino acids in amniotic fluid of congenital malformation including alanine (Ala),leu cine (Leu),isoleucine (Ile),proline (Pro) and phenylalanine (Phe).To compare the different amino acids in amniotic fluid between pregnant women with congenital malformation and normal control and analyze the corresponding pathogenesis.Methods After precipitated protein of the 100 μL supernatant of amniotic fluid by methyl alcohol,the supernatant was dried by nitrogen.The extractions were treated with MSTFA for derivatization.Then gas chromatography-mass spectrometry was used to detect and analyze the amino acids.Results This method was proved to have good sensitivity,precision,accuracy and recoveries.Under the optimum testing conditions,the limit of detection (LOD) was 0.12-0.38 mg/L.The calibration curves showed good linearity over the investigated concentration range between 0.5 and 10 mg/L.The recoveries were 91.12％ to 104.41％.The relative standard deviation of intra and inter-day was 0.84％ to 9.33％.The developed method was applied to the quantification of 5 nonpolar amino acids in amniotic fluid of 17 pregnant women with congenital malformation and 13 normal control pregnant woman.The contents of leucine and isoleucine decreased in disease group compared to controls.The difference of the other three amino acids between the two groups had no statistical significance.Conclusions The validation results showed that the method was suitable for detection of the amino acids in amniotic fluid and having broad prospect of clinical application.Leucine may participate in the pathogenesis of congenital malformation.