Development of a multi-matrix LC–MS/MS method for urea quantitation and its application in human respiratory disease studies

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 133; pp. 96 - 104
• Main Authors: Wang, Jianshuang, Gao, Yang, Dorshorst, Drew W., Cai, Fang, Bremer, Meire, Milanowski, Dennis, Staton, Tracy L., Cape, Stephanie S., Dean, Brian, Ding, Xiao
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 30.01.2017
• Subjects: Bioanalysis; Buffers; Carbon Radioisotopes - analysis; Carbon Radioisotopes - blood; Chromatography, High Pressure Liquid - methods; Culture Media - chemistry; Humans; LC–MS/MS; Nitrogen Isotopes - analysis; Nitrogen Isotopes - blood; Reproducibility of Results; Respiratory disease; Respiratory Tract Diseases - blood; Tandem Mass Spectrometry - methods; Urea; Urea - analysis; Urea - blood; Volume normalization; Urea; Bioanalysis; Respiratory disease; LC–MS/MS; Volume normalization
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2016.11.001

[Display omitted] •Sample volume normalization by urea concentration ratio in support of human respiratory disease studies.•A simple and reliable multi-matrix LC–MS/MS method for absolute quantitation of urea.•Salting-out assisted liquid-liquid extraction of extremely polar small molecule. In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states. Here, we report an easy and reliable LC–MS/MS method for cross-matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal standard, respectively. This approach provides the best measurement consistency across different matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM, SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only dramatically reduces the matrix interferences but also improves the assay recovery. The use of an HILIC column largely increases the analyte retention. The typical run time is 3.6min which allows for high throughput analysis.