Aspects of trapping efficiency and matrix effects in the development of a restricted-access-media-based trap-and-elute liquid chromatography with mass spectrometry method.

Online restricted access media with liquid chromatography and tandem mass spectrometry for the direct analysis of small molecules in biological fluids represents an interesting alternative to time-demanding traditional sample preparation techniques. In this study, important considerations concerning the development of a restricted access media with liquid chromatography and tandem mass spectrometry method for the analysis of dansylated estrogens in biological matrix are presented. Parameters influencing peak tailing and trapping efficiency were evaluated. The key factors included the ion strength of the mobile phase, a loading flow rate of the sample onto the trap column, and selection of a proper stationary phase of the trap column for a given set of analytes. These parameters have proven to be essential for minimizing any unwanted chromatographic peak tailing. The bulk derivatization of the analytes in the biological fluids and its relationship to the observed matrix effects was evaluated as well.

Bulk derivatization and direct injection of human cerebrospinal fluid for trace-level quantification of endogenous estrogens using trap-and-elute liquid chromatography with tandem mass spectrometry.

Although there are existing methods for determining estrogen in human bodily fluids including blood plasma and serum, very little information is available regarding estrogen levels in human cerebrospinal fluid (CSF), which is critical to assess in studies of neuroprotective functions and diffusion of neuroprotective estrogens across the blood-brain barrier. To address this problem, a liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of four endogenous estrogens (estrone, 17α-estradiol, 17ß-estradiol, and estriol) in human CSF was developed. An aliquot (300 µL) of human CSF was bulk derivatized using dansyl chloride in the sample and 10 µL was directly injected onto a restricted-access media trap column for protein removal. No off-line sample extraction or cleanup was needed. The limits of detection of estrone, 17α-estradiol, 17ß-estradiol, and estriol were 17, 28, 13, and 30 pg/mL, respectively, which is in the parts-per-trillion regime. The method was then applied to human CSF collected from ischemic trauma patients. Endogenous estrogens were detected and quantified, demonstrating the effectiveness of this method.

Simultaneous quantification of four native estrogen hormones at trace levels in human cerebrospinal fluid using liquid chromatography-tandem mass spectrometry.

Estrogens are known to exhibit neuroprotective effects on the brain. Their importance in this regard and in others has been emphasized in many recent studies, which increases the need to develop reliable analytical methods for the measurement of estrogen hormones. A heart-cutting two-dimensional liquid chromatography separation method coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been developed for simultaneous measurement of four estrogens, including estriol (E3), estrone (E1), 17ß-estradiol (17ß-E2), and 17α-estradiol (17α-E2), in human cerebrospinal fluid (CSF). The method was based on liquid-liquid extraction and derivatization of estrogens with dansyl chloride to enhance the sensitivity of ESI-based detection in conjunction with tandem mass spectrometry. Dansylated estriol and estrone were separated in the first dimension by an amide-C18 column, while dansylated 17ß- and 17α-estradiol were resolved on the second dimension by two C18 columns (175 mm total length) connected in series. This is the first report of a method for simultaneous quantification of all four endogenous estrogen compounds in their dansylated form. The detection limits for E1, 17α-E2, 17ß-E2, and E3 were 19, 35, 26, and 61pg/mL, respectively. Due to matrix effects, validation and calibration was carried out in charcoal-stripped CSF. The precision and accuracy were more than 86% for the two E2 compounds and 79% for E1 and E3 while the extraction recovery ranged from 91% to 104%. The method was applied to measure estrogens obtained in a clinical setting, from the CSF of ischemic trauma patients. While 17ß-estradiol was present at a significant level in the CSF of some samples, other estrogens were present at lower levels or were undetectable.

Steroids and opioid receptors.

The genomic mode of action is believed to represent the predominant effect of a steroid hormone. Recently, however, rapidly manifesting, non-genomic effects have also been observed. These are mediated mostly by allosteric interaction of a steroid with heterologous target structures such as membrane receptors, a prototype example being the GABAA. Here we describe our studies considering two interdependent questions: (1) do steroids also interact with opioid receptors in brain? Twenty different steroids, i.e. estrogens, androgens, glucocorticoids, mineralocorticoids, gestagens and a cardiac glycoside were tested with respect to their ability to compete for in vitro binding to rat brain membranes of 3H-ligands specific for delta, mu and kappa opioid receptors, respectively. Among all classes of steroids, only the estrogens were effective, all others were 20 to 100 times less effective or ineffective. The rank order among the estrogens was diethylstilbestrol > 17 alpha-estradiol > 17 alpha-ethinyl-estradiol > estriol > estrone > 17 beta-estradiol. Next potent to estrogens (although far less) were--on average as a group--glucocorticoids, followed by mineralocorticoids, androgens, gestagens and digoxin. This global as well as within-group rank order, was, with rare exceptions, qualitatively equal irrespective of which radioligand was used, yet displayed the various radioligands different sensitivities with respect of being inhibited by steroids (irrespective of kind), i.e. in the order [3H]naloxone > or = [3H]DAGO > or = [3H]DADL > [3H]DPDP >> [3H]etorphine. The IC50 of diethylstilbestrol for displacing [3H]DAGO was approximately 30 microM and that of 17 beta-estradiol was approximately 200 microM. (2) What are the concentrations of the major steroid hormones in the brain's extracellular fluid? We have analyzed in 56 matched (i.e. simultaneously withdrawn) peripheral serum and cerebrospinal fluid (CSF) samples (from endocrinologically grossly normal patients) the concentrations of the unconjugated steroid hormones testosterone, androstenedione, dehydroepiandrosterone (DHEA), progesterone and cortisol (all being more or less lipophilic) as well as those of their hydrophilic counterparts, i.e. DHEA-sulfate, or their hydrophilic binding proteins, i.e. sex hormone binding globulin, corticosterone binding globulin, and albumin. Total (i.e. free plus protein-bound) CSF levels of all these steroids were found to be in the 0.02-2 nM range except for cortisol (approximately 20-50 nM), thus 3 to 4 orders of magnitude lower than the IC50 of estrogens for [3H]DAGO (see above). These total CSF values were quite similar to the reported and calculated free serum levels of these steroids and no difference existed between those of patients with intact or with disturbed (abnormally leaky) blood-brain barrier function.(ABSTRACT TRUNCATED AT 400 WORDS)

Endocrine studies in patients with pseudotumor cerebri. Estrogen levels in blood and cerebrospinal fluid.

In 15 patients fulfilling conventional diagnostic criteria for pseudotumor cerebri, anterior and posterior pituitary functions were examined. Eight of 12 female patients with pseudotumor were grossly overweight. Except for a subnormal response of growth hormone level to insulin-induced hypoglycemia in four patients, no major disturbances were found in pituitary, gonadal, thyroid, or adrenal functions. Vasopressin concentration in the cerebrospinal fluid was increased in patients with pseudotumor, whereas cerebrospinal fluid concentration of estrogens was below detection limit.

Cellular and humoral pathways in the neuroendocrine regulation of reproductive function.

PIP: Studies carried out in recent years, including those conducted by the author on the rhesus monkey, which have added more information on the possible cellular and humoral pathways by which the neural and endocrine functions concerned with reproductive phenomena are integrated are reviewed. The possible involvement of the circumventricular structures, especially the tanycyte ependyma and the pineal gland in the detection of the endogenous levels of gonadal hormones, and the cerebrospinal fluid (CSF) in the transmission of hormonal information to the various parts of the brain indicated further areas of research to be explored. Such exploration should be conducted in diverse, yet complimentary, disciplines so that the whole spectrum of events known to occur in the neuroendocrine regulation of reproduction can be studied.^ieng