Profiles of Steroid Hormones in Canine X-Linked Muscular Dystrophy via Stable Isotope Dilution LC-MS/MS.

Golden retriever muscular dystrophy (GRMD) provides the best animal model for characterizing the disease progress of the human disorder, Duchenne muscular dystrophy (DMD). The purpose of this study was to determine steroid hormone concentration profiles in healthy golden retriever dogs (control group - CtGR) versus GRMD-gene carrier (CaGR) and affected female dogs (AfCR). Therefore, a sensitive and specific analytical method was developed and validated to determine the estradiol, progesterone, cortisol, and testosterone levels in the canine serum by isotope dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To more accurately understand the dynamic nature of the serum steroid profile, the fluctuating levels of these four steroid hormones over the estrous cycle were compared across the three experimental groups using a multivariate statistical analysis. The concentration profiles of estradiol, cortisol, progesterone, and testosterone revealed a characteristic pattern for each studied group at each specific estrous phase. Additionally, several important changes in the serum concentrations of cortisol and estradiol in the CaGR and AfCR groups seem to be correlated with the status and progression of the muscular dystrophy. A comprehensive and quantitative monitoring of steroid profiles throughout the estrous cycle of normal and GRMD dogs were achieved. Significant differences in these profiles were observed between GRMD and healthy animals, most notably for estradiol. These findings contribute to a better understanding of both dog reproduction and the muscular dystrophy pathology. Our data open new venues for hormonal behavior studies in dystrophinopathies and that may affect the quality of life of DMD patients.

Plasma steroid dynamics in late- and near-term naturally and artificially conceived bovine pregnancies as elucidated by multihormone high-resolution LC-MS/MS.

The plasma levels of corticosteroids and sex steroids during pregnancy are key indicators of mammalian placental function and the onset of parturition. Steroid hormones are believed to be disturbed in pregnancies produced using assisted reproductive technologies (ARTs) due to placental dysfunction and the frequently observed lack of parturition signals. To elucidate the plasma steroid dynamics, a liquid chromatography-tandem mass spectrometry method was developed and used to determine the levels of corticosteroids (corticosterone, 11-deoxycortisol, and cortisol) and their direct precursors (progesterone and 17α-OH-progesterone) as well as sex steroids (androstenedione, estrone, estrone sulfate, testosterone, and 17ß-estradiol) in bovine plasma. The levels of these 10 steroids in recipient cows carrying naturally conceived (control), in vitro fertilized (IVF), or cloned (somatic cell nuclear transfer) conceptuses were compared during late-term pregnancy (30 days before parturition), during near-term pregnancy (1 day before parturition), and on the day of parturition (day 0). Significant differences were observed among the corticosteroid levels: higher levels of corticosterone, 11-deoxycortisol, and cortisol were detected in cloned pregnancies at day 30; lower levels of corticosterone were observed in ART-derived pregnancies at days 1 and 0; and estrone and estradiol levels were higher in IVF pregnancies throughout the final development. These results suggested an upregulation of the P450C11 and P450C21 enzymes 30 days before parturition in somatic cell nuclear transfer pregnancies and an overactivation of the aromatase enzyme in IVF pregnancies. Taken together, the monitoring of multiple steroid hormones revealed that the pregnancies obtained using ART exhibited plasma steroid concentration dynamics compatible with the dysregulation of steroidogenic tissues.

An alternative and fast method for determination of glyphosate and aminomethylphosphonic acid (AMPA) residues in soybean using liquid chromatography coupled with tandem mass spectrometry.

A simple and specific method using reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was investigated, which allowed the determination of residues of glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), in soybean samples. An aqueous extraction with liquid-liquid partition followed by protein precipitation was performed before the LC/MS/MS determination. The quantitation of glyphosate and AMPA was performed in positive and negative ESI mode, respectively, using the multiple reaction monitoring (MRM) mode with three transitions for each analyte to enhance the specificity of the method and avoid false positives. The methodology reported in this work is capable of detecting residues of glyphosate and AMPA in soybean samples with limits of quantification of 0.30 and 0.34 mg kg(-1), respectively. This alternative method has throughput advantages such as simpler sample preparation and faster chromatographic analysis.

An electrospray ionization tandem mass spectrometric study of p-tert-butylcalix[6]arene complexation with ammonium hydroxide, and ammonium and sodium ions.

The formation of complexes involving p-tert-butylcalix[6]arene with neutral and charged species has been investigated by tandem mass spectrometry combined with electrospray ionization. Complexes of p-tert-butylcalix[6]arene with NH4+ ions were observed in the ratios 1:1, 2:1, and 3:1, together with the complexes of p-tert-butylcalix[6]arene with NH4OH and Na+ ions in the ratios 1:1:1, 2:1:1, and 3:1:1. A single 1:1 complex of p-tert-butylcalix[6]arene with Na+ ions was observed. In addition, a doubly charged complex of p-tert-butylcalix[6]arene with NH4OH, Na+, and NH4+ ions in the ratio 6:1:1:1 was observed. The identity of each complex was determined by mass analysis of product ions formed by the application of a declustering potential over the range 20-220 V and by observation of product ion mass spectra wherein the collision energy was varied from 5 to 50 eV. Fragmentation of the complexes is characterized by the facile loss of the ammonia molecule, sodium and ammonium ions, loss of neutral p-tert-butylcalix[6]arene, and successive neutral losses of C4H8 from the six tert-butyl groups in each p-tert-butylcalix[6]arene molecule.