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 (20μL) without prior sample clean-up. Ultra High Performance Liquid Chromatography (UHPLC) on a HSS-T3 stationary phase column with 1.8μm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration range of 8.78-103.78μg/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 (20μL) and a run time of 1.5min 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.
Bioanalytical Science and Toxicokinetics, DMPK, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA. chester.l.bowen@gsk.com
Chester L
CL
Bowen
Hermes
H
Licea-Perez
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003124:Colorimetry; D006801:Humans; D016014:Linear Models; D051379:Mice; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014508:Urea / Q000032:analysis* / Q000737:chemistry
D014508:Urea