la Marca Giancarlo, Malvagia Sabrina, Pasquini Elisabetta, Innocenti Marzia, Donati Maria Alice, Zammarchi Enrico
Metabolic Unit, Department of Paediatrics, Meyer Children's Hospital and Pharmaceuticals Department, University of Florence, Florence, Italy.
Clin Chem. 2007 Jul;53(7):1364-9. doi: 10.1373/clinchem.2007.087775. Epub 2007 May 17.
The expansion of newborn screening programs has increased the number of newborns diagnosed with inborn errors of metabolism in the presymptomatic phase, but it has also increased the number of costly, stress-producing false-positive results. Because propionylcarnitine (C3) is one of the analytes most frequently responsible for false-positive results, we aimed to develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to identify free methylmalonic (MMA) and 3-OH propionic (3OH-PA) acids in blood spots.
We studied newborn screening spots from 250 healthy controls; 124 from infants with abnormal C3, of whom only 5 (4%) were truly affected; 124 from infants with altered isolated methylmalonylcarnitine; and 4 from clinically diagnosed patients. Whole blood was eluted from a 3.2-mm dried blood spot by a CH(3)CN/H(2)O 7:3 and 5 mL/L formic. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray without derivatization. Total analysis time was 5 min per sample.
The assays were linear up to 3300 nmol/L for both metabolites. Intra- and interassay imprecision data were 3.6%-8% and 3.1%-6%, respectively, for MMA and 5.2%-20% and 3.6%-17% for 3OH-PA. Limit of detection and limit of quantitation were 1.95 and 4.2 micromol/L, respectively, for MMA and 8 and 10 micromol/L for 3OH-PA. The recoveries were 92.9%-106.1%. No deterioration was noted on the columns after 500 chromatographic runs. If the new method had been used as a 2nd-tier test for the 124 samples, only the 5 true positives would have been recalled for additional samples, and the positive predictive value would have been 100%.
This method has the potential to markedly reduce false-positive results and the associated costs and anxiety. It may also be suitable for diagnosing and routinely monitoring blood spots for methylmalonic aciduria and propionic acidemia.
新生儿筛查项目的扩展增加了在症状前阶段被诊断出患有先天性代谢缺陷的新生儿数量，但同时也增加了代价高昂且令人产生压力的假阳性结果数量。由于丙酰肉碱（C3）是最常导致假阳性结果的分析物之一，我们旨在开发一种快速液相色谱 - 串联质谱（LC-MS/MS）方法，用于鉴定血斑中的游离甲基丙二酸（MMA）和3-羟基丙酸（3OH-PA）。
我们研究了250名健康对照的新生儿筛查血斑；124名C3异常婴儿的血斑，其中只有5名（4%）是真正患病的；124名孤立的甲基丙二酰肉碱改变婴儿的血斑；以及4名临床诊断患者的血斑。用7:3的CH(3)CN/H(2)O和5 mL/L甲酸从3.2毫米的干血斑中洗脱全血。将该提取物注入配备气动辅助电喷雾且无需衍生化的LC-MS/MS中。每个样品的总分析时间为5分钟。
两种代谢物的测定在高达3300 nmol/L时呈线性。MMA的批内和批间不精密度数据分别为3.6% - 8%和3.1% - 6%，3OH-PA的批内和批间不精密度数据分别为5.2% - 20%和3.6% - 17%。MMA的检测限和定量限分别为1.95和4.2 μmol/L，3OH-PA的检测限和定量限分别为8和10 μmol/L。回收率为92.9% - 106.1%。在进行500次色谱运行后，柱子未出现劣化。如果将新方法用作对124个样品的二线检测，那么只需召回5个真正的阳性样品进行额外检测，阳性预测值将为100%。
该方法有可能显著减少假阳性结果以及相关的成本和焦虑。它也可能适用于诊断和常规监测血斑中的甲基丙二酸血症和丙酸血症。
