Simultaneous determination of urinary quinolinate, gentisate, 4-hydroxybenzoate, and α-ketoglutarate by high-performance liquid chromatography-tandem mass spectrometry

Abstract

Renal cell carcinoma (RCC) remains a difficult-to-detect cancer for which new detection methodologies are urgently needed to improve screening efficacy. Urinary metabolomic profiling has identified several potential RCC biomarkers, including quinolinic acid, 4-hydroxybenzoic acid, gentisic acid and aketoglutaric acid. These four endogenous metabolites have unique pathophysiological mechanisms associated with RCC, suggesting that they may be promising biomarkers for earlier RCC detection.However, the lack of targeted analytical methods for their simultaneous determination in urine has hindered efforts to evaluate their clinical applicability. In this study, a robust high-performance liquid chromatography -tandem mass spectrometry (HPLC-MS/MS) technique using multiple reaction monitoring was developed to simultaneously monitor these four urinary biomarkers. The method was validated by multiple figures of merit including method detection limits (0.05 ng mL À1 to 1 ng mL À1 ), spiked recovery accuracy (88.6-114.8%) and reproducibility (0.4-9.3% RSD). The applicability of the method was further demonstrated in clinically relevant 100-fold diluted urine specimens. All four potential biomarkers were successfully quantified with urinary concentrations that were in agreement with previous reports. In conclusion, an accurate and sensitive method has been described for the simultaneous determination of quinolinic acid, 4-hydroxybenzoic acid, gentisic acid and a-ketoglutaric acid. We anticipate this method to significantly benefit clinical translational research aiming to evaluate the clinical applicability of these four potential biomarkers.

Multivalued Logic Assay of the Disease Marker of α-Ketoglutaric Acid by a Luminescent MOF-Based Biosensor

“…Therefore, it is of vital importance to trace the abnormal expression of α-KA for monitoring the disease progression. Despite the biological importance of α-KA, it still remains a challenge to construct a simple, fast, and accurate method for α-KA analysis because some traditional methods such as chromatography either require laborious pretreatment or involve time-consuming test. − In contrast, fluorescence materials provide a facile detection method for various species with several advantages of simple operation/procedure, cost-saving apparatus, rapid analysis time, and even naked-eye visibility. Nevertheless, most of the reported fluorescence platforms for α-KA suffer from a series of drawbacks including working with the assistance of extra certain surfactant (CTAB) , or in organic media, requiring long reaction time , or complicated material fabrication process, , and relying on an enzymatic transamination .…”

Development and validation of high performance liquid chromatographic method for determination of gentisic acid and related Renal Cell Carcinoma biomarkers in urine

“…Clearly, there is a need for an accurate, fast, and inexpensive analytical method to quantify quinolinic acid, gentisic acid and 4-hydroxybenzoic acid in urine. The only reported procedure in the literature to quantify these three bio-markers simultaneously was developed by Ma [9] which utilizes liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, the cost of analysis per sample using LC/MS/MS is high due to the expense of the instrument and its maintenance cost.…”

“…These three bio-markers are present in relatively high concentration in urine. In the LC/MS/MS method previously reported by Ma, each urine sample was diluted one-hundred fold prior to analysis [9]. Furthermore, Ma normalized the chromatograms using specific gravity to take into account renal dilution.…”