Rapid determination of urinary oxalic acid concentration aids in the screening and monitoring of kidney diseases: An LC-ESI-MS/MS method that does not require complex derivatization, acidification treatment, or large-volume sample consumption

The balance of oxalic acid in the body is intricately regulated. Importantly, elevated levels of oxalic acid in urine or blood have been linked to the progression of various diseases. There is strong interest in developing efficient methods to quantify oxalic acid in various bio-matrices like urine. However, due to its small molecular weight, high polarity, acidic nature, and endogenous properties, establishing an analysis method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for oxalic acid presents significant challenges, and such analysis methods that meet clinical needs are not widely available. Faced with the aforementioned challenging issues, in this study, we have discovered innovative strategies to improve the ionization, chromatographic separation, and enrichment of oxalic acid and its internal standard, leading to the successful LC-MS/MS method development and clinical application of the analysis method. These include: (1) simultaneous use of two low concentrations of electrolytes (NH 3 ·H 2 O and NH 4 Ac) in the mobile phase; (2) alkalinization of urine; (3) addition of 0.3 % NH 3 ·H 2 O to the internal standard oxalic acid-1,2- 13 C solution; (4) selection of ultrapure water (UPW) as an alternative matrix; (5) urine sample processing using an easy-to-use protein precipitation method. These measures have created a relatively homogeneous alkaline environment, addressing the issues in the ionization, chromatographic separation, and enrichment of oxalic acid and its internal standard. Of note, chromatographic separation of oxalic acid and oxalic acid-1,2- 13 C was achieved on the Waters Symmetry C18 column (4.6 mm × 50 mm, 3.5 μm) with an isocratic elution at a flow rate of 0.40 mL/min. The target analyte was completely separated by elution with 30 % acetonitrile containing 0.01 % NH 3 ·H 2 O and 1 mM NH 4 Ac for 3 min, without any endogenous interference. The method showed good linearity over the 0.020–50.0 μg/mL concentration range with a correlation coefficient r 2  = 0.9998. The intra-assay and inter-run precision and accuracy, recovery, and stability of the method were all within the acceptable parameters. Collectively, this study developed and validated a simple, fast, and sensitive LC-ESI-MS/MS method that did not require complex derivatization, acidification, large-volume sample consumption. Moreover, this method was successfully applied for routine urinary oxalic acid detection in children with kidney disease, providing valuable information for clinicians.