George A. Maylin

Determination of drugs in biological samples by thin-layer chromatography-tandem mass spectrometry.

In this work thin-layer chromatography-tandem mass spectrometry (TLC-MS-MS) allowed detection and confirmation of caffeine and nicotine in human urine and of butorphanol, betamethasone, and clenbuterol in equine urine. In most cases of trace analysis of labile compounds the drugs could not be identified unless they were developed on a TLC plate, scraped from the plate and the TLC scrape eluted with a suitable organic solvent prior to MS-MS. Usually a sample prepared in this way still had several components in it, but was sufficiently cleaned up to allow collision-induced dissociation (CID) experiments to unequivocally identify the drug. In contrast, trace levels of labile drugs could not be identified by CID experiments either directly from the raw urine extracts or by thermally desorbing them from the TLC scrape.

The Effects of Phenylbutazone on the Morphology and Prostaglandin Concentrations of the Pyloric Mucosa of the Equine Stomach

Phenylbutazone, a nonsteroidal anti-inflammatory drug known to produce gastric ulcers, was administered intravenously (13.46 mg/kg body weight) daily to 12 horses. Horses were euthanatized daily after 24,48,72, and 96 hours following the initial injection. Eight untreated horses served as controls. Small multifocal pyloric erosions were seen after 24 hours and then progressed in severity over time. The erosions were characterized by sloughing of the surface epithelium, subepithelial bleb formation, necrosis of the lamina propria, degeneration of the walls of subsurface capillaries, and microthrombosis of the capillaries of the pyloric mucosa. Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Eroded pyloric mucosa and adjacent macroscopically intact mucosa were examined ultrastructurally. In both the mac-roscopically eroded mucosa and multifocally in the adjacent macroscopically uneroded mucosa, there was cellular swelling of the endothelium, pericytes, and smooth muscle cells of arterioles. In capillaries and post-capillary venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and edema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular injury that is associated with the formation of pyloric erosions in horses. The pyloric mucosa of six horses was assayed for prostacyclin and prostaglandin E2 at 48 and 96 hours following the initial injection. There was no statistically significant difference between prostaglandin concentrations in the mucosa of control and treated horses. It was concluded that there was little correlation between pyloric mucosal prostaglandin concentrations and pyloric erosions after 48 hours.

Dexamethasone metabolism in the horse.

Abstract Dexamethasone and a metabolite, 9-fluoro-16α-methyl-6β, 11β, 16β-trihydroxy-1, 4-androstadiene-3, 17-dione, were detected in the urine of horses injected parenterally with the parent drug. The structure of the metabolite was elucidated by thin-layer chromatography, infrared spectroscopy, mass spectroscopy and nuclear magnetic resonance spectroscopy.

Thermospray liquid chromatography—mass spectrometry of corticosteroids

A high-performance liquid chromatographic method was developed for thermospray mass spectrometric analysis of steroidal hormones. Using a Nova-Pak C18 reversed-phase column and isocratic elution with a solvent comprised of 25 mM ammonium formate in 30% acetonitrile, corticosteroids were separated within 10 min. This solvent also permitted ultraviolet absorbance detection down to 220 nm with low-nanogram sensitivity. The use of acetonitrile was favourable for thermospray mass spectrometric analysis because mass spectra were obtained with a pseudomolecular ion as the base peak. A combination of liquid chromatography, ultraviolet absorbance detection and thermospray mass spectrometry provided a sensitive and reliable method for unequivocal confirmation of the presence of steroidal drugs in equine urine.

Micro LC/MS in Drug Analysis and Metabolism Studies

LC/MS has become a routine research tool in some laboratories. Although no single approach to LC/MS presently is without limitations, each approach offers encouraging results and prompts continued improvements. The remaining hurdles appear to be the introduction of total LC effluent into the MS, increased sensitivity, and the ability to analyze higher molecular weight, polar molecules. The micro LC/MS results discussed in this paper offer an opportunity for significantly increased sensitivity by allowing total micro LC effluent introduction into the MS. It remains to be seen whether the development of micro LC column and equipment technology will allow practical micro LC/MS. It may someday provide improved separation of drugs, metabolites, and their conjugates from high levels of endogenous materials in reasonable time periods. Currently, the impressive efficiencies demonstrated for micro LC columns involve mixtures containing low molecular weight solutes that are perhaps more amenable to GC analysis. The analyst routinely involved with problem solving must deal with complex mixtures composed of components with large concentration differences. The future of micro LC, and hence micro LC/MS, will depend upon how well the technique helps solve problems. We believe the future is bright for micro LC/MS. The techniques may require some fine tuning of operating procedures, just as with capillary GC/MS, but certainly the potential for increased efficiency and sensitivity is worth the effort. Currently we are testing a new micro LC/MS DLI probe wherein the exit of the micro column is within 1 cm of the MS ion source [82]. The micro LC column is contained within the DLI probe and should offer the lowest dead volume and the least extracolumn effects yet achieved by LC/MS. Initial testing of this new probe is under way, and experimental results will be reported subsequently. The analytical potential of micro LC/MS is receiving considerable interest. Practical micro LC performance can provide increased capabilities to all types of LC/MS reported to date, and perhaps offer new insight into alternative methods of LC/MS not yet reported. We look forward to learning of these breakthroughs as they become available.

Milk production of cows exposed to industrial fluoride pollution

Milk sales records for a dairy herd showed that milk production did not decrease during the first 4 yr of exposure of cows to fluoride pollution from an aluminum plant. During the next 3 yr milk production decreased, but not significantly. From yr 8 of exposure there was a significant decrease, which persisted through yr 19, when the dairy operation was terminated. From yr 15 through yr 19, the milk yield averaged less than 60% of the expected value.

A water-extractable toxic compound in vinyl upholstery fabric.

SummaryA compound, highly toxic to goldfish, was found to be released from a vinyl (polyvinyl chloride) automotive upholstery fabric when the material was immersed in their water. The compound, a flame retardant used in such material, was identified by specific detector gas chromatography and mass spectrometry as triphenyl phosphate. Fish exposed to the immersed fabric or pure triphenyl phosphate showed neurologic intoxication and extensive histopathologic lesions.

Toxicity to fish of flame retardant fabrics immersed in their water. Part I

SummaryA number of commercial and candidate flame retardants were studied with regard to their toxicity to fish when released from fabrics immersed in their water. Immersion of laundered or unlaundered flame retardant 100% polyester or polyester blend fabrics used in childrens sleepwear in water containing goldfish resulted in release of the anticholin-esterase flame retardant TDBPP (tris(2,3-dibromopropyl) phosphate) and death of all fish within 24 hours. TDBPP undergoes loss of HBr in water and production of a metabolite.

Excretion of triclopyr herbicide in the bovine

Triclopyr, (3,5,6-trichloro-2-pyridinyloxyacetic acid) is a herbicide for control of woody plants and broad leaf weeds on rights-of-way, forests, industrial sites and turfs. A number of reports in the news media and legal literature (Lehoczky 1986) have associated application of Triclopyr and certain other herbicides near farm land with toxicity and death of cattle. Triclopyr has similarities in chemical structure and herbicide properties to Picloram (4-amino-3,5,6-trichloropicolinic acid) and sometimes the two herbicides are applied as a mixture. In an earlier study, it was shown that when Picloram was fed to a lactating dairy cow, 97.7% was excreted intact in the urine (Fisher et el. 1965). It was of interest to determine the mode of excretion of Triclopyr herbicide in the bovine. In the work reported here, Triclopyr was fed to a catherized, lactating dairy cow and milk, urine and feces were collected and analyzed for residues of the herbicide.

The extraction and isolation of dexamethasone-related compounds

Radiolabeled dexamethasone (9-fluoro-16 alpha-methyl-11 beta, 17, 21-trihydroxy-1,4-pregnadiene-3,20-dione) was utilized to develop a practical and relatively expedient method of detection for dexamethasone and its major metabolite in horse urine. The effects of solvent, pH, salt saturation, and back extraction on the extraction efficiency of dexamethasone-related compounds and the presence of endogenous background were evaluated. Final isolations of dexamethasone and its major urinary metabolite were procured by successive thin-layer chromatography (TLC) in three different developing systems.