UHPLC-ESI-MS/MS Quantification of Relevant Substrates and Metabolites of the Kynurenine Pathway Present in Serum and Peritoneal Fluid from Gastric Cancer Patients—Method Development and Validation

Abstract

Metabolites and enzymes involved in the kynurenine pathway (KP) are highly promising targets for cancer treatment, including gastrointestinal tract diseases. Thus, accurate quantification of these compounds in body fluids becomes increasingly important. The aim of this study was the development and validation of the UHPLC-ESI-MS/MS methods for targeted quantification of biologically important KP substrates (tryptophan and nicotinamide) and metabolites(kynurenines) in samples of serum and peritoneal fluid from gastric cancer patients. The serum samples were simply pretreated with trichloroacetic acid to precipitate proteins. The peritoneal fluid was purified by solid-phase extraction before analysis. Validation was carried out for both matrices independently. Analysis of the samples from gastric cancer patients showed different accumulations of tryptophan and its metabolites in different biofluids of the same patient. The protocols will be used for the evaluation of tryptophan and kynurenines in blood and peritoneal fluid to determine correlation with the clinicopathological status of gastric cancer or the disease’s prognosis.

Electrochemical Determination of Kynurenine Pathway Metabolites—Challenges and Perspectives

“…Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) allows for monitoring and quantification of a whole panel of KP metabolites simultaneously with compounds from other metabolic routes during a single run, and in a relatively short analysis time [34]. The analysis can be performed in positive and negative ion scan modes; however, the negative mode offers lower LODs for 3HAA and AA [27]. Electrospray ionization (ESI) is prone to so-called matrix effects (MEs) caused, e.g., by co-eluting matrix components, and leads to ion suppression or ion enhancement [80].…”

“…Electrospray ionization (ESI) is prone to so-called matrix effects (MEs) caused, e.g., by co-eluting matrix components, and leads to ion suppression or ion enhancement [80]. MEs are considered as a "weak" point of LC-MS-based protocols, because they affect the accuracy, precision, and sensitivity of the method, and are observed during KP metabolites' determination in biological samples [27,34]. Matrix-matched calibration and using internal standards are popular strategies for compensation of the matrix-dependent effects effecting the analyte signal and correcting the loss of a target compound during a sample preparation step.…”

“…All the above suggest that new electrode materials with high electrocatalytic activity or other solutions should be exploited to allow direct electrochemical detection of QA and Kyna at low physiologically relevant levels. Addressing these limitations, QA and Kyna are currently preferably measured in biological samples by LC-MS-based methods [27,54,70]. It is noteworthy that QA is difficult to quantify by LC-MS or LC-MS/MS because of short retention time, low m/z, and poor ionization efficiency in the MS source [37].…”

“…Mounting evidence from many laboratories associates KP with tumor immune surveillance [2], neuropathology [22], autoimmune disorders [23], dermatological pathologies [24], infections [25], obesity, preeclampsia, depression and stress, suicide, schizophrenia [26], and cognitive deficits [19]. Furthermore, KP metabolites are promising diagnostic and therapeutic targets in gastric cancer [27], amyotrophic lateral sclerosis [26], major depressive disorder [28], type 2 diabetes [6], and psychiatric disorders [19]. Within the last few years, a number of comprehensive reviews have been published on the role of KP in the regulation of biological processes [5,6,10,[18][19][20][21][22][23][24][25][26]; thus, this issue is only briefly discussed here.…”

“…Meanwhile, liquid chromatography (LC) employing detection modalities like UV absorbance, fluorescence, and mass spectrometry (MS) has attracted considerable interest for studying Trp metabolism [33].In particular, expanding evidence suggests the LC-MS-based method to be the analytical gold standard for the monitoring and determination of KP metabolites in body fluids, tissues, and cultured cells. Although many LC-based protocols for KP metabolites determination have been developed [27,[33][34][35][36], the number of new analytical approaches published is constantly growing from year to year. Importantly, some KP metabolites (3HKyn, QA) have unfavorable chromatographic properties (short retention time, poor peak shape, low ionization efficiency in the MS source), making their quantitative LC-MS analysis in a rich biological matrix difficult.…”