Estrogen analysis by liquid chromatography - mass spectrometry
Estrogens are well known as female hormones associated with both health and disease. Although they have been reported as potential carcinogens, their use as therapy in the treatment of neurodegeneration has recently received a great deal of attention. The main goal of this work was to advance estrogen analysis using liquid chromatography – mass spectrometry (LC-MS) and associated techniques in order to support pre-clinical and clinical research on the neuroprotective effects of estrogens.
Native estrogen analyses were performed in different types of biological samples, including human cerebrospinal fluid (CSF), horse serum, phosphate buffered saline – bovine serum albumin (PBS-BSA), mouse serum, and mouse brain. A derivatization step with dansyl chloride was used in sample preparation prior to the introduction of LC-MS to enhance the electrospray ionization efficiency of native estrogens, since they are poorly ionizable in aqueous liquid phases. The four dansylated estrogens were successfully analyzed in cerebrospinal fluid (CSF) using a heart-cutting two dimensional reversed-phase (2D RP) LC-tandem MS system; detection limits in the part-per-trillion concentration range were achieved. Interestingly, later elution and 2D-separation were considered to be factors that reduced interferences in the analysis of 17α- and 17β-estradiol from CSF matrices. The matrix effects were further studied in other biological samples for these two estrogens, which are of most interest for their neuroprotective effects, using two sequentially connected C18 columns coupled with tandem MS. The data revealed that matrix effects were observed in mouse brain and gelded horse serum but not in mouse serum and PBS-BSA.
In direct support of clinical trials, the stability of Premarin®Intravenous, a potential first response treatment for traumatic brain injury, was investigated under the conditions that mimic ambulance environment. The drug is a cocktail of estrogen metabolites mainly in the form of sulfates, generated from natural sources of pregnant mare's urine. The experiment was designed in a time frame of six months and Premarin®Intravenous was studied in the form of dry powder and reconstituted solutions. Although individual major components of the drug in reconstituted samples significantly varied at elevated temperatures of 100 and 120°F, they were considered to be fairly stable in the form of dry power with the variation ≤ 10%. It was noted that the variation was larger for reconstituted samples stored at elevated versus room temperature.
Based on the diversity of estrogens and their metabolites, they were used as a model to study the mechanism of hydrophilic interaction chromatography (HILIC) in the conjunction with MS. The retention behavior of estrogens and their metabolites was investigated on five polar stationary phases. Two interaction models were applied, the partition and the surface adsorption model. Retention of the compounds, especially estrogen glucuronides, on the amide- and diol-bonded stationary phases could be best fit the surface adsorption model while mixed modes were observed on most stationary phases. The advantages of electrospray (ESI) – MS detection in conjunction with HILIC were also emphasized. The combination is considered to be ideal for providing an increased sensitivity over reversed phase separation approaches in many cases. A comprehensive literature review was carried out on this topic.