■氨基酸是许多先天代谢错误的关键生物标志物,但是由于这些小分子固有的化学性质范围,氨基酸分析具有挑战性。技术可用于氨基酸分析,但是他们可能会遭受长时间的折磨,费力的衍生化,和/或同量异位化合物的分辨率差。
■开发和验证使用混合模式色谱法和串联质谱法对血浆和尿液样品中未衍生的游离氨基酸谱进行定量的方法。
■优化色谱条件以分离亮氨酸,异亮氨酸,和异亮氨酸,并保持分析运行时间小于15分钟。样品制备包括快速蛋白沉淀,随后进行LC-MS/MS分析。矩阵效应,干扰,线性度结转,可接受的稀释极限,精度,准确度,并在血浆和尿液标本类型中评估稳定性。
■用这种方法成功地定量了总共38种氨基酸和相关化合物。此外,对精氨酸琥珀酸进行了定性分析。进行了完整的临床验证,包括与血浆和尿液参考实验室的方法比较,Deming回归斜率范围为0.38至1.26。
■该方法代表了基于衍生化的方法的替代方法,尤其是在尿液样本中,代谢物和药物的干扰很普遍。
UNASSIGNED: Amino acids are critical biomarkers for many inborn errors of metabolism, but amino acid analysis is challenging due to the range of chemical properties inherent in these small molecules. Techniques are available for amino acid analysis, but they can suffer from long run times, laborious derivatization, and/or poor resolution of isobaric compounds.
UNASSIGNED: To develop and validate a method for the quantitation of a non-derivatized free amino acid profile in both plasma and urine samples using mixed-mode chromatography and tandem mass spectrometry.
UNASSIGNED: Chromatographic conditions were optimized to separate leucine, isoleucine, and allo-isoleucine and maintain analytical runtime at less than 15 min. Sample preparation included a quick protein precipitation followed by LC-MS/MS analysis. Matrix effects, interferences, linearity, carryover, acceptable dilution limits, precision, accuracy, and stability were evaluated in both plasma and urine specimen types.
UNASSIGNED: A total of 38 amino acids and related compounds were successfully quantitated with this method. In addition, argininosuccinic acid was qualitatively analyzed. A full clinical validation was performed that included method comparison to a reference laboratory for plasma and urine with Deming regression slopes ranging from 0.38 to 1.26.
UNASSIGNED: This method represents an alternative to derivatization-based methods, especially in urine samples where interference from metabolites and medications is prevalent.