Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay

Abstract

A new analytical method suitable for high throughput measurements of LTE(4) in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500pg/ml in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE(4) from healthy adults and children are reported.

Figure 1. Evaluation of ion suppression on response of LTE4 and LTE4-d3 in urine and water

Injection Experiments: Extracted ion chromatogram (EIC) of MRM transitions for LTE4-d3 (m/z 443.2>304.3) spiked at 200 pg/ml in a human urine sample (A) and water (B), and MRM transition of endogenous LTE4 (m/z 440.2>301.3) in a human urine sample (C, hatched lines). Data demonstrate the response of internal standard is not affected by ion suppression. Data was collected in positive electrospray ionization mode. The peak area for urine and water spiked with LTE4-d3 was 115754 and 107672 respectively. Post- Column Infusion experiments: LTE4 was infused and the 440.2 > 301.2 transition was monitored during injection of urine (D) and water (E) samples. Data demonstrate the absence of ion suppression in a urine matrix.

Figure 2. Comparison of ELISA and LC/MS/MS analysis of LTE₄ in urine from asthmatics

Urine from 10 asthmatic subjects was obtained, centrifuged within 15 hours, and processed as follows: One aliquot was analyzed in-house using LC/MS/MS on the day of collection (LCMS-1) and several aliquots were frozen at -80°C immediately after collection. One frozen aliquot was analyzed by Cayman using ELISA (ELISA) according to the manufacturer's protocol and a frozen aliquot was analyzed in-house using LC/MS/MS (LCMS-2). Creatinine levels were obtained from Cayman and used to normalize all data. For LC/MS/MS samples were injected in duplicate and their average and CV were determined. CV values for ELISA were obtained from Cayman. Methods used are on the x-axis and Panel A shows normalized LTE4 values in pg/mg creatinine (y-axis) and Panel B shows CVs (y-axis).