尿中内因性ペプチドの電気膜抽出と液体クロマトグラフィー-タンデム質量分析法を組み合わせたハイスループット測定

To develop a precise method for analyzing urinary peptides based on electromembrane extraction (EME) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to evaluate its potential applicability in tumor biomarker screening.

A total of 15 disease-associated peptides were selected as the target analytes. A supported liquid membrane (SLM) composed of n-octanol containing 5% di (2-ethylhexyl) phosphate was employed, with the donor phase being a 1∶1 mixture of urine and 100 mmol/L formic acid and urine, and the acceptor phase being 20 mmol/L formic acid containing 50% dimethyl sulfoxide (DMSO). After EME at 40 V for 15 min, the acceptor phase solution was analyzed by LC-MS/MS. Subsequently, the method, EME combined with LC-MS/MS (EME-LC-MS/MS), was preliminarily validated utilizing urine samples from 12 healthy controls and 7 patients with urinary system tumors.

All 15 peptides exhibited excellent linearity in the range of 0.1-100.0 ng/mL (r ≥ 0.995), with the limits of detection (LODs) being 0.01-0.50 ng/mL and the limits of quantification (LOQs) being 0.03-1.50 ng/mL. The spiked recoveries ranged from 21.0% to 71.2%, with relative standard deviations (RSDs) of 0.8%-20.0% (n = 3). Small-sample analysis of clinical specimens revealed that the concentration of bradykinin 1-5 in the urine were significantly higher in tumor patients (median: 0.65 ng/mL) than that in healthy controls (median: 0.37 ng/mL) (P < 0.05), suggesting its potential as a specific biomarker for urinary system tumors.

The EME-LC-MS/MS method established in the study features simplicity, high efficiency, and high sensitivity, enabling precise determination of trace-level peptides in urine samples. Moreover, this approach provides a reliable methodological basis for disease biomarker screening and promotes the clinical application of electromembrane extraction.