Development of a sensitive and selective LC–MS/MS method for the determination of urea in human epithelial lining fluid

A sensitive, selective, and quantitative method for the determination of urea has been developed and validated in human epithelial lining fluid (ELF; the supernatant from bronchoalveolar lavage). The method employs a simple derivatization of urea with camphanic chloride to improve the chromatographic retention and separation. The derivatization was performed after drying an aliquot of ELF (20 μL) without prior sample clean-up. Ultra High Performance Liquid Chromatography (UHPLC) on a HSS-T3 stationary phase column with 1.8 μm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration range of 8.78–103.78 μg/mL, however the dynamic range can be further lowered if needed. The results from assay validation show that the method is rugged, precise, accurate, and well-suited to support analysis of urea in ELF samples. In addition, the relatively small sample volume (20 μL) and a run time of 1.5 min facilitate automation and allow for high-throughput analysis. This derivatization method was compared to a commercially available colorimetric assay kit, and it was used in a preclinical non-GLP mouse study where urea measurements were used as marker of bronchoalveolar lavage fluid dilution.

► Derivatization with improved chromatographic retention and separation.
► The derivatization was performed without prior sample clean-up.
► The method was validated over the concentration range of 8.78–103.78 μg/mL.
► This method was compared to a commercially available colorimetric assay kit.
► The method can also be adapted for determination of urea in plasma samples.