Evaluation of Matrix Effects from Urine for Estrogen Metabolites using HILIC Coupled with ESI-MS

摘要

Estrogens and estrogen metabolites are important in the scientific community due to their many associated positive and negative heath effects. In this work, we investigate the use of HILIC-ESI-MS for quantification of estrogen glucuronide metabolites, focusing specifically on obtaining a better understanding of matrix effects. Urine was processed using reversed phase solid phase extraction SPE prior to addition of metabolite standards to measure matrix effects on calibration sensitivity. The matrix was prepared using two different SPE procedures. A preliminary study in the absence of matrix showed that when comparing RPLC, HILIC, and modified RPLC separations, HILIC provided better sensitivity. Yet, in HILIC mode, the propensity for retaining potentially interfering salts from urine is a potential problem for ESI-MS detection. In the columns tested, effect of matrix was different for each SPE preparation, and between different HILIC columns. Using an amide-HILIC stationary phase, significant suppression effects were observed relative to calibration in pure standards. In contrast, and interestingly, using a diol-HILIC stationary phase, the presence of matrix components appeared to enhance signal. These differences in effects can be attributed to the presence of different interferences co-eluting with the compounds of interest, which influence the ionization efficiency of analytes during ESI-MS detection. Significant care should always be taken when developing quantitative methods for biomonitoring compounds from biological fluids. This work shows that very significant effects can arise, and should be carefully considered when ESI-MS detection is used in conjunction with HILIC separations.