串联质谱检测正常儿童干血滤纸片中游离肉碱和酰基肉碱含量
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摘要
目的
建立一种简便、快捷的检测干血滤纸片中肉碱类化合物的方法。通过对0～13岁正常儿童干血滤纸片中游离肉碱和酰基肉碱含量的检测,对我国南方地区儿童体内游离肉碱及酰基肉碱水平进行了统计分析,为脂肪酸代谢障碍性疾病和有机酸血症诊断提供参考范围。
方法
取直径5 mm的干血滤纸片,加入含有游离肉碱和酰基肉碱的同位素内标,用甲醇萃取,萃取物过滤后直接上机分析,采用ESI+-MS/MS进行检测,数据采集采用多反应监测模式(MRM)对13种肉碱类化合物进行了检测。以美国CDC提供的质控样品作方法学验证。应用该法对277例0～13岁正常儿童的外周血干血滤纸片酰基肉碱进行检测。分6个年龄段组1(新生儿组):年龄～28天,孕周≥37周;组2:年龄～6月;组3:年龄～12月;组4:年龄～3岁;组5:年龄～6岁;组6:年龄～13岁。应用SPSS 17.0分析软件对数据进行统计分析。
结果
本实验建立的方法重复性好,各物质在低、中、高三个浓度级别的批内变异系数和批间变异系数都小于15%。各物质实测浓度与加入浓度之间的相对偏差在0.1%-27.2%。线性范围满足临床检测需要。通过比较各个年龄段儿童干血滤纸片法酰基肉碱类化合物含量发现:1.3-羟基异戊酰肉碱(C5OH)随着年龄的增大,含量下降。2.～28天组(组1);辛酰肉碱(C8)较其它年龄组的低;乙酰肉碱(C2)、十二酰肉碱(C12)、十四酰肉碱(C14)较其它年龄组的高。3.～6月组(组2):游离肉碱(C0)比～28天组(组1)和其它较大年龄组高;乙酰肉碱(C2)比其他较大年龄组高。4.～12月(组3)乙酰肉碱(C2)比其他较大年龄组高。5.～6岁年龄(组5)十四酰肉碱(C14)比其它小年龄组的低。6.～13岁组(组6)癸酰肉碱(C10)比其它较小年龄组高。7.丙酰肉碱(C3)、丁酰肉碱(C4)、异戊酰肉碱(C5)、己酰肉碱(C6)这些中、短酰基肉碱受年龄因素影响不大。用本方法诊断脂肪酸β-氧化代谢障碍1例。
结论
同位素稀释-非衍生化串联质谱法对干血滤纸片中游离肉碱和酰基肉碱进行分析,能够达到满意的精确度和准确度,每个样品前处理加上机分析只需要3小时,是一项简便、快捷检测方法,适合应用于临床检测。本研究提供的我国南方地区0～13岁正常儿童干血滤纸片酰基肉碱含量参考值范围,可以为脂肪酸代谢障碍和有机酸血症疾病的诊断、鉴别诊断、治疗提供有意义的参考。
Objective
To establish a method for carnitine analysis in dry blood filter paper by tandem mass spectrometry for 0～13 years old children. In this study, we evaluated the variations in free carnitine and acylcarnitines concentrations with age, supplied normal reference range for clinic diagnosis.
Methods
5 mm diameter of dry blood spots were punched and extracted by methanol solution with stable isotope labeled internal standards , then filtered and analyzed by tandem mass spectrometry.Sensitivity and specificity were evaluated by the quality control samples supplied by CDC of United States.
Filter-paper blood spots were collected from 277 healthy individuals over a period of 3 months, divided into six age groups to analysis, including:1–28 days(group 1), 1–6 months(group 2),7–12 months(group 3), 13 months–3 years(group 4), 4–6 years(group 5)and 7–13 years(group 6). The concentration of free carnitine and acylcarnitines were made statistic to get normal reference range .
Results
The intraassay CVs of this method were 1% -13% ,The interassay CVs were1% -16%. The linearity of free carnitine and acylcarnitines were enough to the requitement of clinic diagnosis .Long chain carnitines was significantly higher in newborns than in older children (P <0.05), but the concentrations of several short chain acylcarnitines(C3、C4、C5、C6) tended to be no significantly difference. By this method,we identified one patient withβ-oxidation disorders deficiency which proved the method is specificity and sensitivity.
Conclusions
The precision and linearity of the method are satisfied for free carnitine and acylcarnitines analysis. The age-related reference values are useful in the diagnosis and management of inherited errors of metabolism.
建立一种简便、快捷的检测干血滤纸片中肉碱类化合物的方法。通过对0～13岁正常儿童干血滤纸片中游离肉碱和酰基肉碱含量的检测,对我国南方地区儿童体内游离肉碱及酰基肉碱水平进行了统计分析,为脂肪酸代谢障碍性疾病和有机酸血症诊断提供参考范围。
方法
取直径5 mm的干血滤纸片,加入含有游离肉碱和酰基肉碱的同位素内标,用甲醇萃取,萃取物过滤后直接上机分析,采用ESI+-MS/MS进行检测,数据采集采用多反应监测模式(MRM)对13种肉碱类化合物进行了检测。以美国CDC提供的质控样品作方法学验证。应用该法对277例0～13岁正常儿童的外周血干血滤纸片酰基肉碱进行检测。分6个年龄段组1(新生儿组):年龄～28天,孕周≥37周;组2:年龄～6月;组3:年龄～12月;组4:年龄～3岁;组5:年龄～6岁;组6:年龄～13岁。应用SPSS 17.0分析软件对数据进行统计分析。
结果
本实验建立的方法重复性好,各物质在低、中、高三个浓度级别的批内变异系数和批间变异系数都小于15%。各物质实测浓度与加入浓度之间的相对偏差在0.1%-27.2%。线性范围满足临床检测需要。通过比较各个年龄段儿童干血滤纸片法酰基肉碱类化合物含量发现:1.3-羟基异戊酰肉碱(C5OH)随着年龄的增大,含量下降。2.～28天组(组1);辛酰肉碱(C8)较其它年龄组的低;乙酰肉碱(C2)、十二酰肉碱(C12)、十四酰肉碱(C14)较其它年龄组的高。3.～6月组(组2):游离肉碱(C0)比～28天组(组1)和其它较大年龄组高;乙酰肉碱(C2)比其他较大年龄组高。4.～12月(组3)乙酰肉碱(C2)比其他较大年龄组高。5.～6岁年龄(组5)十四酰肉碱(C14)比其它小年龄组的低。6.～13岁组(组6)癸酰肉碱(C10)比其它较小年龄组高。7.丙酰肉碱(C3)、丁酰肉碱(C4)、异戊酰肉碱(C5)、己酰肉碱(C6)这些中、短酰基肉碱受年龄因素影响不大。用本方法诊断脂肪酸β-氧化代谢障碍1例。
结论
同位素稀释-非衍生化串联质谱法对干血滤纸片中游离肉碱和酰基肉碱进行分析,能够达到满意的精确度和准确度,每个样品前处理加上机分析只需要3小时,是一项简便、快捷检测方法,适合应用于临床检测。本研究提供的我国南方地区0～13岁正常儿童干血滤纸片酰基肉碱含量参考值范围,可以为脂肪酸代谢障碍和有机酸血症疾病的诊断、鉴别诊断、治疗提供有意义的参考。
Objective
To establish a method for carnitine analysis in dry blood filter paper by tandem mass spectrometry for 0～13 years old children. In this study, we evaluated the variations in free carnitine and acylcarnitines concentrations with age, supplied normal reference range for clinic diagnosis.
Methods
5 mm diameter of dry blood spots were punched and extracted by methanol solution with stable isotope labeled internal standards , then filtered and analyzed by tandem mass spectrometry.Sensitivity and specificity were evaluated by the quality control samples supplied by CDC of United States.
Filter-paper blood spots were collected from 277 healthy individuals over a period of 3 months, divided into six age groups to analysis, including:1–28 days(group 1), 1–6 months(group 2),7–12 months(group 3), 13 months–3 years(group 4), 4–6 years(group 5)and 7–13 years(group 6). The concentration of free carnitine and acylcarnitines were made statistic to get normal reference range .
Results
The intraassay CVs of this method were 1% -13% ,The interassay CVs were1% -16%. The linearity of free carnitine and acylcarnitines were enough to the requitement of clinic diagnosis .Long chain carnitines was significantly higher in newborns than in older children (P <0.05), but the concentrations of several short chain acylcarnitines(C3、C4、C5、C6) tended to be no significantly difference. By this method,we identified one patient withβ-oxidation disorders deficiency which proved the method is specificity and sensitivity.
Conclusions
The precision and linearity of the method are satisfied for free carnitine and acylcarnitines analysis. The age-related reference values are useful in the diagnosis and management of inherited errors of metabolism.
引文
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