Equestrian chilblain: another outdoor recreational hazard.

Herein, we describe two cases and review 14 cases of equestrian chilblain or 'equestrian cold panniculitis' in the literature. The first, a 23-year-old healthy female horse trainer, presented with burning nodular swelling on her lateral thighs. The second was a 34-year-old healthy woman with recurrent nodular eruption on the lateral thighs after horseback riding in the winter. Physical examination of both patients revealed erythematous to violaceous nodules with eczema craquelé-like changes. Laboratory workup for systemic and autoimmune connective tissue disease was negative. Punch biopsies from both patients showed a superficial and deep perivascular and periadnexal lymphocytic infiltrate with focal extension into the subcutaneous fat. Parakeratosis, subtle spongiosis and increased pandermal interstitial mucin were also present. Previously reported cases generally showed a similar clinical course and similar histopathologic findings. In contrast, our cases revealed increased pandermal interstitial mucin, resembling tumid lupus erythematosus. We aim to better characterize the histopathologic findings of equestrian chilblain and discuss its relationship to other cold-induced skin injuries and autoimmune connective tissue disease, namely lupus erythematosus.

An in vivo microdialysis coupled with liquid chromatography/tandem mass spectrometry study of cortisol metabolism in monkey adipose tissue.

It is postulated that elevated tissue concentrations of cortisol may be associated with the development of metabolic syndrome, obesity, and type 2 diabetes. The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme regenerates cortisol from inactive cortisone in tissues such as liver and adipose. To better understand the pivotal role of 11beta-HSD1 in disease development, an in vivo microdialysis assay coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using stable isotope-labeled (SIL) cortisone as a substrate was developed. This assay overcomes the limitations of existing methodologies that suffer from radioactivity exposure and analytical assay sensitivity and specificity concerns. Analyte extraction efficiencies (E(d)) were evaluated by retrodialysis. The conversion of SIL-cortisone to SIL-cortisol in rhesus monkey adipose tissue was studied. Solutions containing 100, 500, and 1000 ng/mL SIL-cortisone were locally delivered through an implanted 30-mm microdialysis probe in adipose tissue. At the delivery rate of 1.0 and 0.5 microL/min, E(d) values for SIL-cortisone were between 58.7+/-5.6% (n=4) and 72.7+/-1.3% (n=4), whereas at 0.3 microL/min E(d) reached nearly 100%. The presence of 11beta-HSD1 activities in adipose tissue was demonstrated by production of SIL-cortisol during SIL-cortisone infusion. This methodology could be applied to cortisol metabolism studies in tissues of other mammalian species.

An in vitro microdialysis methodology to study 11beta-hydroxysteroid dehydrogenase type 1 enzyme activity in liver microsomes.

Microdialysis sampling coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS) was used to observe in vitro 11beta-hydroxysteroid dehydrogenase type 1 (HSD1) enzyme-catalyzed conversion of stable-isotope-labeled cortisone to cortisol in liver microsomes from dog, monkey, and human. Experimental conditions that would affect the microdialysis sampling approach including probe length, perfusion fluid flow rate, extraction efficiency (E(d)), substrate concentration, and enzyme reaction conditions were evaluated. Dialysates containing high salt concentrations (>150 mM) were directly assayed using LC/MS/MS without additional sample cleanup. The sensitivity (with lower level of quantitation at 0.1 ng/mL) and selectivity of this assay allowed detection of the enzyme reactants at physiologically relevant levels. The interconversion from M+4 cortisone to M+4 cortisol was detected in dog, human, and monkey liver microsomes. Results show species-specific reaction profiles, with a five times higher conversion rate in dog liver microsomes than in human and monkey liver microsomes. Based on M+4 cortisol production rate obtained using a microdialysis infusion of M+4 cortisone to the microsomes coincubated with a proprietary 11beta-HSD1 inhibitor of different concentrations, the degrees of enzyme inhibition were found to be 40 and 85%, consistent with values obtained by a traditional in vitro incubation method. The microdialysis sampling methodology with LC/MS/MS provided extensive information about 11beta-HSD1 activities in microsomes from different mammalian species.

Determination of M+4 stable isotope labeled cortisone and cortisol in human plasma by microElution solid-phase extraction and liquid chromatography/tandem mass spectrometry.

A sensitive microElution solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma. In this method, M+4 cortisone and M+4 cortisol were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well microElution SPE plate using 70 microL methanol as the elution solvent, and chromatographed on a Waters Symmetry C18 column (4.6 x 50 mm, 3.5 microm). M+9 cortisone and M+9 cortisol were used as the internal standards. A PE Sciex API 4000 tandem mass spectrometer interfaced with the liquid chromatograph via a turboionspray source was used for mass analysis and detection. The selected reaction monitoring (SRM) of precursor --> product ion transitions were monitored at m/z 365.2 [M+H](+) --> 167.0 and at m/z 367.3 [M+H](+) --> 125.1 for M+4 cortisone and M+4 cortisol, respectively. The lower limit of quantitation was 0.1 ng mL(-1) and the linear calibration range was from 0.1 to 100 ng mL(-1) for both analytes. This method demonstrated to be very reproducible and reliable.

Application of a novel ultra-low elution volume 96-well solid-phase extraction method to the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma.

A novel extraction method has been utilized in the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma. In this method, 300 microl of plasma sample was loaded onto a Waters Oasis 96-well HLB microElution plate, the stationary phase was washed using 2 x 400 microl of 5% methanol in water, and the analytes were eluted using 35 microl of 95/5 acetonitrile/H(2)O twice. The sample extracts were diluted with 40 microl of methyl ammonium acetate (1mM, pH 4.5). Chromatography was performed on a Phenomenex Synergi Max-RP column (2.0 mm x 50 mm, 4 microm). A PE Sciex API 3000 tandem mass spectrometer interfaced with a turbo ionspray source was used for mass detection. Compared to solid-phase extraction, liquid-liquid extraction and solid-supported liquid-liquid extraction methods that were developed and previously used in our laboratory, this method reduced the labor cost and was less time consuming in sample preparation, due to the fact that post-extraction solvent evaporation and reconstitution steps were avoided using this microElution solid-phase extraction plate. The method has been proved to be fast, reliable and reproducible.

A semi-automated 96-well protein precipitation method for the determination of montelukast in human plasma using high performance liquid chromatography/fluorescence detection.

A simple, semi-automated, protein precipitation assay for the determination of montelukast (SINGULAIR, MK-0476) in human plasma has been developed. Montelukast is a potent and selective antagonist of the cysteinyl leukotriene receptor used for the treatment of asthma. A Packard MultiPROBE II EX is used to transfer 300 microl of plasma from sample, standard, and QC sample tubes to a microtiter plate (96-well). After addition of the internal standard by a repeating pipettor, a Tomtec QUADRA 96 adds 400 microl of acetonitrile to all plasma sample wells, simultaneously, in the microtiter plate. The Tomtec is also used to transfer the acetonitrile supernatant from the plasma protein precipitation step, batchwise, to another microtiter plate for analysis by HPLC with fluorescence detection. This assay has been validated and implemented for a clinical study of over 1300 plasma samples and is comparable to manual assays in the LLOQ (lower limit of quantitation, 3 ng/ml) and in stability. This is the first semi-automated protein precipitation assay published for the analysis of montelukast in human plasma and it results in significant time savings over the manual methods, both in sample preparation and in HPLC run time.

Effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of analytes in liquid chromatographic/ionspray tandem mass spectrometric determination.

The effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of drug molecules in liquid chromatographic/ionspray tandem mass spectrometric (LC/MS/MS) determination were evaluated for simvastatin (SV) and its hydroxy acid (SVA). The objective was to improve further the sensitivity for SV by overcoming the unfavorable condition caused by the formation of multiple major adduct ions and multiple major fragment ions when using ammonium as LC mobile phase buffer. Mobile phases (70:30 acetonitrile-buffer, 2 mM, pH 4.5) with buffers made from ammonium, hydrazine or alkyl (methyl, ethyl, dimethyl or trimethyl)-substituted ammonium acetate were evaluated. Q1 scan and product ion scan spectra were obtained for SV in each of the mobile phases under optimized conditions. The results showed that, with the alkylammonium buffers, the alkylammonium-adducted SV was observed as the only major molecular ion, while the formation of other adduct ions ([M + H](+), [M + Na](+) and [M + K](+)) was successfully suppressed. On the other hand, product ion spectra with a single major fragment ion were not observed for any of the alkylammonium-adducted SVs. The affinity of the alkylammoniums to SV and the basicity of the alkylamines are believed to be factors influencing the formation and abundance of molecular and fragment ions, respectively. Methylammonium acetate provided the most favorable condition among all the buffers evaluated and improved the sensitivity several-fold for SV in LC/MS/MS quantitation compared with that obtained using ammonium acetate buffer. Better precision for SV in both Q1 and SRM scans was observed when using methylammonium buffer compared with those using ammonium buffer. The mobile phase buffer contents did not seem to affect the ionization, fragmentation and chromatography of SVA. The results of this evaluation can be applied to similar situations with other organic molecules in ionspray LC/MS/MS determination.