Leukotriene E4 (LTE4) is quantified in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Deuterium-labeled(d5)-LTE4 internal standard and acetonitrile are added to all standards, controls, and specimens which are then positive pressure filtered to remove salts, particulates, and sediment from the urine. Additional sample clean-up is achieved via a Cyclone MAX mixed mode anion exchange, while a Waters Xbridge C8 analytical column is employed to focus and elute the LTE4 analyte. This eluent is transferred to an API 5000 MS/MS for instrumental analysis.
The specific transitions monitored are 438.3/333.1 m/z and 438.3/235.05 m/z for LTE4, while 443.4/338.25 and 443.4/240.15 m/z are monitored for the d5-LTE4 internal standard. The ratios of the integrated peak areas of LTE4 and its respective deuterium- labeled internal standard are used to calculate the concentration of the analyte. All LTE4 concentrations are normalized to urine creatinine levels measured using a Roche Cobas enzymatic method.(Package insert: Creatinine plus ver.2 (CREP2), Roche Diagnostics. Indianapolis, IN. 2013-07)