Objective 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.Methods 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.Results 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.Conclusion 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.

A method of direct injection and liquid chromatography coupled with tandem mass spectrometric (LC-MS / MS) was developed for simultaneous determination of 5 aniline compounds including aniline, 3-nitroaniline, 4-nitroaniline, 2,6-dichloro-4-nitroaniline and hexanitrodiphenylamine in drinking water and source water. The samples were filtered using a 0. 22-μm polyethersulfone membrane prior to HPLC analysis. Five target compounds were chromatographically separated on an HSS T3 column with gradient elution. Chromatographic data were acquired by tandem mass spectrometric detection in multiple reaction monitoring (MRM) mode, and thus favorable resolutions of all target compounds were achieved within 4 min. Under the optimal analytical conditions, the peak area of each analyte and its concentration had a good correlation within the linear range (R≥0. 995). The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0. 773-1. 88 μg / L (S / N=3) and 2. 58-6. 27 μg / L (S / N=10), respectively. The intra- and inter-day relative standard deviations ( RSDs) of the mix standard solution were 0. 8% -1. 9% and 3. 3% -4. 9% , respectively. The spiked recoveries of the analytes were 84. 1% -105% and the RSDs of the spiked samples were 1. 0% -3. 1% . This proposed method was applied in the analysis of 35 samples from drinking water, source water and surface water, which indicated that the novel LC-MS / MS method could detect 5 aniline compounds in water without any complicated sample pretreatment in an accurate, sensitive and rapid way, and it also could provide technique support for evaluation of the contamination caused by aniline compounds.