Division of Pharmacology, Drug Research and Development Focus Area, School of Pharmacy, North West University, Potchefstroom, South Africa.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Jun 1;898:121-9. doi: 10.1016/j.jchromb.2012.04.030. Epub 2012 May 4.
It is highly beneficial to monitor the activity of the kynurenine pathway in a large series of samples with high accuracy and reliability in a single experimental protocol. We have developed a rapid specific solid-phase extraction (SPE)-liquid chromatography-electrospray ionization tandem mass spectrometry method for assaying tryptophan, kynurenine, kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid and quinolinic acid (QA) in rat plasma. We also evaluated picolinic acid (PA) in this method, but it presented with unacceptable validation parameters. The assay involves pre-purification by SPE followed by chromatographic separation by C18 reversed phase chromatography. Mass spectrometric detection was performed using a mass spectrometer in positive and negative electrospray ionization; with a flow rate of 0.2 mL/min and an injection volume of 10 μL. Total run time including sample clean-up was 12 min. The assay method was found to be linear (R² > 0.95) and all the validation parameters were within acceptance range. The developed technique also demonstrated a significant elevation in plasma tryptophan, kynurenine, anthranilic acid and QA, and a significant decrease in KYNA, in rats subjected to post-weaning social isolation rearing, a putative animal model of relevance for depression and schizophrenia. This method can therefore be applied to measure metabolites of the kynurenine pathway in plasma accurately and precisely by LC-MS/MS, thereby helping to realize new opportunities in pharmacological and diagnostic research.
在单个实验方案中，以高精度和高可靠性监测犬尿氨酸途径的活性，对大量样本进行监测是非常有益的。我们开发了一种快速、特异的固相萃取（SPE）-液相色谱-电喷雾串联质谱法，用于测定大鼠血浆中的色氨酸、犬尿氨酸、犬尿喹啉酸（KYNA）、3-羟基犬尿氨酸（3OHAA）、邻氨基苯甲酸和喹啉酸（QA）。我们还在该方法中评估了吡啶酸（PA），但它的验证参数不可接受。该测定法包括 SPE 预纯化，然后通过 C18 反相色谱进行色谱分离。质谱检测采用正、负离子电喷雾；流速为 0.2 mL/min，进样量为 10 μL。包括样品净化在内的总运行时间为 12 分钟。该测定方法呈线性（R²>0.95），所有验证参数均在可接受范围内。该技术还表明，在经历了断奶后社交隔离饲养的大鼠中，血浆色氨酸、犬尿氨酸、邻氨基苯甲酸和 QA 显著升高，KYNA 显著降低，这是一种与抑郁症和精神分裂症相关的动物模型。因此，该方法可以通过 LC-MS/MS 准确、精确地测量犬尿氨酸途径的代谢产物，从而为药理学和诊断研究提供新的机会。
