Simultaneous determination of multiple urine biomarkers for kidney injury using SPE combined with LC-MS/MS

Urinary biomarkers such as low molecular weight proteins and small molecular weight metabolites are crucial in the diagnosis of kidney injury. The objective of this study was to develop and preliminarily validate a sensitive and specific method using solid-phase extraction (SPE) in conjunction with liquid chromatography–tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of these biomarkers in human urine. This study presents the development of a solid-phase extraction method integrated with LC-MS/MS analyzing biomarkers including creatinine, urea, β2-microglobulin, α1-microglobulin, and cystatin C in human urine. An enhanced solid-phase cartridge technique was employed for peptide purification and dilution of small molecule metabolites during sample preparation. The developed LC-MS/MS method achieved satisfactory separation of the five analytes within 15 min. Accuracy levels ranged from −8.6% to 13.6%. Both intra-assay and inter-assay imprecision rates were maintained below 7.9% for all analytes. The established LC-MS/MS method effectively quantifies creatinine, urea, β2-microglobulin, α1-microglobulin and cystatin C concurrently. This offers a viable alternative for the detection of kidney injury biomarkers in human urine, demonstrating potential for clinical application in kidney injury diagnosis.