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BACKGROUND: This project entails the development and validation of a method for quantification of the aminoglycoside antibiotic arbekacin in serum using liquid chromatography tandem mass spectrometry (LC-MS/MS) for therapeutic drug monitoring in future clinical trials. METHODS: Following a protein precipitation with 0.3 mol/l perchloric acid containing internal standard dibekacin at a concentration of 0.6 µg/ml, human serum samples containing arbekacin were analyzed using a Hypersil Gold PFP column and a liquid chromatography system. Elution occurred with a gradient of water and acetonitrile, each containing 0.05% (v/v) trifluoroacetic acid and 0.1% (v/v) formic acid. Analytes were detected over a 3.25 minute run time using a tandem mass spectrometer with a heated electrospray-ionization (HESI) source in positive ionization mode with selected reaction monitoring (SRM). Matrix effects, carryover, linearity, recovery, precision, and limit of quantification were carefully evaluated. RESULTS: The limit of quantification for arbekacin was 0.1 µg/ml. All simple and total precision CV's were less than 6.2%. The method was linear from 0.1 µg/ml to 45.9 µg/ml (slope of 0.973). The mean recovery ranged from 94.7 to 103.8%. No matrix effects were detected. CONCLUSIONS: This developed and validated LC-MS/MS method allows for the quantification of arbekacin in serum following protein precipitation.

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OBJECTIVE: Sulfur mustard (SM) causes blisters on the human skin. These blisters delay healing of the skin and make the victims more susceptible to infection. In vitro models have been used for protection studies against SM injury, but study on wound healing after SM exposure has not been explored. The purpose of this study was to test whether the addition of exogenous growth factors could improve the rate of SM wound healing. METHODS: The model consisted of normal human epidermal keratinocytes seeded into 6-well plates, exposed to SM, and wounded (disruption of the cell monolayer) with a sterile wounding instrument. Cells were then stained and images were captured to measure percentage wound fill. Epidermal growth factor (EGF) and keratinocyte growth factor (KGF) were tested in this model. RESULTS: EGF (1 ng/mL) significantly increased wound fill on all of the days tested (days 6, 9, and 12). KGF did not significantly improve wound healing. CONCLUSIONS: EGF showed promise as a potential therapy for SM-induced wounds. This in vitro model was a valuable tool for screening therapeutics before animal testing. These results will be used to develop a dressing that can slowly release EGF on to a debrided wound bed to help speed the healing process.

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Bis-(2-chloroethyl) sulfide (sulfur mustard; SM) is a potent alkylating agent. Three treatment compounds have been shown to limit SM damage in the mouse ear vesicant model: dimercaprol, octyl homovanillamide, and indomethacin. Microarrays were used to determine gene expression profiles of biopsies taken from mouse ears after exposure to SM in the presence or absence of treatment compounds. Mouse ears were topically exposed to SM alone or were pretreated for 15 min with a treatment compound and then exposed to SM. Ear tissue was harvested 24 h after exposure for ear weight determination, the endpoint used to evaluate treatment compound efficacy. RNA extracted from the tissues was used to generate microarray probes for gene expression profiling of therapeutic responses. Principal component analysis of the gene expression data revealed partitioning of the samples based on treatment compound and SM exposure. Patterns of gene responses to the treatment compounds were indicative of exposure condition and were phenotypically anchored to ear weight. Pretreatment with indomethacin, the least effective treatment compound, produced ear weights close to those treated with SM alone. Ear weights from animals pretreated with dimercaprol or octyl homovanillamide were more closely associated with exposure to vehicle alone. Correlation coefficients between gene expression level and ear weight revealed genes involved in mediating responses to both SM exposure and treatment compounds. These data provide a basis for elucidating the mechanisms of response to SM and drug treatment and also provide a basis for developing strategies to accelerate development of effective SM medical countermeasures.

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