Study of Matrix Effect in Metabolomic Analysis of Urinary Markers of Chronic Kidney Disease by Hydrophilic Interaction Chromatography Tandem Mass Spectrometry

Metabolomic analysis of biological samples is an important direction in the development of diagnostic methods for chronic kidney disease (CKD) in children. Hydrophilic interaction chromatography tandem mass spectrometry (HILIC–MS/MS) is widely used in metabolomics; however, this method is associated with the problem of matrix effect (ME). In this study, we evaluate the ME arising from the analysis of nine low-molecular weight polar marker metabolites of CKD under HILIC conditions in samples of new model and real urine. Amino acids and their polar metabolites involved in the pathophysiologic processes of CKD development were selected as nine biomarkers to be determined. The ME on the first quadrupole was assessed by calculating the ratio of the coelution parameters of low-molecular weight clusters consisting of buffer formate anions and salt cations in urine and L-valine-¹³C₅ standard. In real urine, the signal intensity of the L-valine-¹³C₅ standard was reduced by more than 50% relative to methanol when the cluster signal was superimposed, whereas in artificial urine, the suppression effect was comparable to the real sample under all elution conditions. The addition method was also applied to evaluate the ME of isotope-labeled endogenous markers in real and artificial matrices. It was shown that a preliminary assessment of signal quenching can be studied on model urine of a new composition. The results demonstrate the importance of evaluating the optimal signal resolution of not only marker compounds but also inorganic clusters, which can significantly reduce the analysis errors under real matrix conditions. The evaluation of this effect should improve the accuracy of polar metabolite analysis in real samples in CKD metabolomics. The applied artificial urine samples showed comparable ME to the real sample, which confirms its promising potential for optimizing the HILIC–MS/MS analysis conditions.

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