Research and Development Center , University of Health Sciences , Ankara 06011 , Turkey.
Department of Medical Biochemistry, Faculty of Medicine , Gazi University , Ankara 06011 , Turkey.
J Am Soc Mass Spectrom. 2020 Feb 5;31(2):379-385. doi: 10.1021/jasms.9b00007. Epub 2020 Jan 9.
Tryptophan is an essential amino acid that plays an important role in cell metabolism, and kynurenine is its main metabolic pathway. By using ultra-high-performance liquid chromatography coupled to electrospray ionization triple-quadrupole mass spectrometry, tryptophan and kynurenine were determined using amlodipine as an internal standard. The analysis was carried out on an ACE-C18 (4.6 mm × 50 mm, 5 μm) reversed-phase analytical column using the gradient elution mode. For quantitative determination, amlodipine was used as an internal standard. Detection was performed using multiple reaction monitoring in electrospray ionization mode at / 205.1 → 117.7 and 187.9 for tryptophan, / 209.1 → 146 and 93.9 for kynurenine, and / 409.2 → 294.1 for the internal standard. Good linearity of the analyte to internal standard peak area ratios was seen in the concentration range 1.25-4000 ng/mL for tryptophan and 0.5-1600 ng/mL for kynurenine. The method showed excellent linearity with regression coefficients of 0.99 for kynurenine and 0.996 for tryptophan. The limits of quantification were 0.55 ng/mL for tryptophan and 0.47 ng/mL for kynurenine. The % RSD for all analytes ranged from 0.3 to 3.4% for intraday and 0.4 to 8.9% for interday experiments. A simple LC-MS/MS method has been developed and validated for measuring Kyn and Trp by using an affordable and more easily available internal standard, which is amlodipine.
色氨酸是一种必需氨基酸，在细胞代谢中发挥重要作用，而犬尿氨酸是其主要代谢途径。本研究采用超高效液相色谱-电喷雾串联三重四极杆质谱法，以氨氯地平为内标，测定色氨酸和犬尿氨酸。分析采用 ACE-C18（4.6mm×50mm，5μm）反相分析柱，梯度洗脱模式。定量测定时，以氨氯地平为内标。采用电喷雾电离模式下的多反应监测进行检测，色氨酸为 / 205.1→117.7 和 187.9，犬尿氨酸为 / 209.1→146 和 93.9，内标为 / 409.2→294.1。色氨酸在 1.25-4000ng/mL 浓度范围内，内标峰面积比与分析物呈良好线性关系；犬尿氨酸在 0.5-1600ng/mL 浓度范围内，内标峰面积比与分析物呈良好线性关系。犬尿氨酸和色氨酸的回归系数分别为 0.99 和 0.996。犬尿氨酸和色氨酸的定量下限分别为 0.55ng/mL 和 0.47ng/mL。日内和日间实验的所有分析物的%RSD 范围为 0.3-3.4%和 0.4-8.9%。本研究建立并验证了一种采用价格合理、更易获得的内标物（氨氯地平）测定犬尿氨酸和色氨酸的 LC-MS/MS 方法。
