Service de Biochimie, Hôpital A. Trousseau, APHP, 75012 Paris, France.
Laboratoire de métabolomique clinique Hôpital Saint Antoine, 75012 Paris, France.
J Chromatogr A. 2020 Jul 5;1622:461135. doi: 10.1016/j.chroma.2020.461135. Epub 2020 Apr 17.
Here we describe a new HPLC-MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds. Validation studies evaluated accuracy, linearity, within- and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism.
在这里，我们描述了一种新的 HPLC-MS/MS 方法，该方法使用混合模式固定相和二元洗脱梯度，无需衍生化或添加离子对试剂，即可快速分离和定量人血浆中的氨基酸。样品制备程序包括在磺基水杨酸蛋白沉淀后进行单一稀释步骤。该方法允许在 18 分钟的色谱运行中对 52 种氨基酸和相关化合物进行明确的鉴定和分析，包括异构体和同位素的分离，包括条件和平衡时间。氨基酸通过选择性反应监测进行检测。内部校准用于定量 37 种氨基酸，包括 25 种使用相应同位素标记的内标。外部校准（无内标）用于另外 5 种分析物。对其余化合物进行定性检测。验证研究评估了 37 种常见质控样品中存在的氨基酸的准确性、线性、精密度（日内和日间）、检测限和定量限。对于所有化合物，日内精密度 CV 平均为 3.8%（n=30）。对于所有化合物，日间精密度 CV 平均为 8.6%（n=20）。还对 32 个血浆样品进行了与常见的标准离子交换色谱-柱后衍生化方法的相关性研究。虽然该方法的灵敏度至少高 50 倍，但数据显示与斜率等于或高于 0.9 的相关性良好，相关系数大多高于 0.90。该方法成功应用于检测氨基酸代谢遗传障碍的血浆样品分析。
