Richard A. Sams

Single dose pharmacokinetic study of florfenicol in Atlantic salmon (Salmo salar) in seawater at 11°C

Abstract The pharmacokinetics of intravenously and orally administered florfenicol were determined in Atlantic salmon (Salmo salar) weighing 194±40 g (mean±s.d.). The study was performed at 10.8±1.5°C. A dose of 10 mg florfenicol/kg body weight was administered either intravenously or orally to groups of 85 fish each. At seven time points, from 3 h to 120 h after administration, blood was sampled from 10 individual fish in each group. The plasma was assayed for florfenicol using an HPLC method. The pharmacokinetic modelling of the results was performed using the computer program PCNONLIN. Following intravenous administration, the plasma concentration-time data of florfenicol were best described by a two-compartment open model. The volume of distribution at steady state, Vd(ss), and the total body clearance, ClT, were 1.122 l/kg and 0.086 l/h·kg, respectively. The elimination halflife, t 1 2 β , was estimated as 12.2 h. Following oral administration, the plasma concentration-time data of florfenicol were best described by a one-compartment open model with first order absorption and elimination. Peak plasma concentration, Cmax, was estimated at 4.0 μg/ml and was estimated to occur at 10.3 h (Tmax) following dosing. The bioavailability, F, was estimated at 96.5%. Based on the median minimum inhibitory concentrations (MICs) of 0.8 μg/ml reported for Aeromonas salmonicida, Vibrio anguillarum and Vibrio salmonicida, plasma concentrations should remain above the MIC for 36–40 h following a single oral dose of 10 mg florfenicol/kg.

Hysteresis and calcium set-point for the calcium parathyroid hormone relationship in healthy horses.

Abnormalities in calcium (Ca(2+)) homeostasis are reported in horses with several pathological conditions; however, there is little information on Ca(2+) regulation in horses. The objectives of the present study were to determine the Ca(2+) set-point in healthy horses, to determine whether the Ca(2+)/parathyroid hormone (PTH) response curves were characterized by hysteresis, and to determine if the order of experimentally induced hypocalcemia or hypercalcemia had an effect on PTH secretion. The Ca(2+) set-point and hysteresis were determined in 12 healthy horses by infusing Na(2)EDTA and calcium gluconate. The Ca(2+) set-point was 1.37 +/- 0.05 mmol/L, which is higher than values reported for humans and dogs (1.0-1.2 mmol/L). Hysteresis was present during hypocalcemia and hypercalcemia. Horses in which hypocalcemia was followed by hypercalcemia secreted more PTH (7440 +/- 740 pmol min/L) than horses in which hypercalcemia was followed by hypocalcemia (5990 +/- 570 pmol min/L). This study has demonstrated that the Ca(2+) set-point in the horse is higher than in other domestic animals and man. We have shown that the Ca(2+)/PTH relationship in horses is sigmoidal and displays hysteresis during both hypocalcemia and hypercalcemia, and that extracellular Ca(2+) concentrations may affect the response of the parathyroid gland to hypocalcemia.

Determination of naloxone and naltrexone as perfluoroalkyl ester derivatives by electron-capture gas-liquid chromatography

An electron-capture gas chromatographic method is described for the determination of naloxone and naltrexone as the perfluoroalkyl esters. Each compound serves as internal standard for determination of the other. The method permits quantitation of 2-100 ng of either compound. Conditions for derivatization with heptafluorobutyric anhydride (HFBA), pentafluoropropionic anhydride (PFPA), and trifluoroacetic anhydride (TFAA) have been investigated. When catalyzed with pyridine, derivatization with HFBA and PFPA at 70 degrees gives naloxone and naltrexone triesters. Evidence for triester formation was obtained from gas chromatography-methane chemical ionization mass spectrometry and infrared spectral analysis. It was found that both the HFB and PFP triesters are suitable for quantitation of the narcotic antagonists, the HFB derivatives having greater stability than the PFP derivatives. The TFA derivatives are substantially less stable.

Cardiorespiratory, gastrointestinal, and analgesic effects of morphine sulfate in conscious healthy horses

OBJECTIVE To compare the cardiorespiratory, gastrointestinal, analgesic, and behavioral effects between IV and IM administration of morphine in conscious horses with no signs of pain. ANIMALS 6 healthy adult horses. PROCEDURES Horses received saline (0.9% NaCl) solution (IM or IV) or morphine sulfate (0.05 and 0.1 mg/kg, IM or IV) in a randomized, masked crossover study design. The following variables were measured before and for 360 minutes after drug administration: heart and respiratory rates; systolic, diastolic, and mean arterial blood pressures; rectal temperature; arterial pH and blood gas variables; intestinal motility; and response to thermal and electrical noxious stimuli. Adverse effects and horse behavior were also recorded. Plasma concentrations of morphine, morphine-3-glucuronide, and morphine-6-glucuronide were measured via liquid chromatography-mass spectrometry. RESULTS No significant differences in any variable were evident after saline solution administration. Intravenous and IM administration of morphine resulted in minimal and short-term cardiorespiratory, intestinal motility, and behavioral changes. A decrease in gastrointestinal motility was detected 1 to 2 hours after IM administration of morphine at doses of 0.05 and 0.1 mg/kg and after IV administration of morphine at a dose of 0.1 mg/kg. Morphine administration yielded no change in any horses response to noxious stimuli. Both morphine-3-glucuronide and morphine-6-glucuronide were detected in plasma after IV and IM administration of morphine. CONCLUSIONS AND CLINICAL RELEVANCE Clinically relevant doses of morphine sulfate yielded minimal and short-term behavioral and intestinal motility effects in healthy horses with no signs of pain. Neither dose of morphine affected their response to a noxious stimulus.

Specific and sensitive determination of digoxin and metabolites in human serum by high performance liquid chromatography with cyclodextrin solid-phase extraction and precolumn fluorescence derivatization

A precolumn fluorescence derivatization high performance liquid chromatographic method has been developed for the simultaneous determination of digoxin and its metabolites digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside digoxigenin, and dihydrodigoxin (20-R and 20-S epimers) in human serum. Digoxin and its metabolites were extracted from serum samples (containing digitoxin as internal standard) with a cyclodextrin solid-phase extraction (SPE) column. Fluorescent derivatives were formed by reaction of the analytes with 1-naphthoyl chloride in the presence of 4-dimethylaminopyridine under a nitrogen atmosphere in a glove box with controlled relative humidity (26% r.h. or less). The derivatives were isolated using cyclodextrin and C1 SPE columns sequentially, and determined by HPLC using silica column separation and fluorescence detection. Calibration curves were linear over the concentration range from 0.25 to 4.0 ng ml-1. Recoveries of digoxin and its metabolites from serum ranged from 62 to 86%, and coefficients of variation from repetitive analyses ranged from 6.9 to 20.9% and from 5.8 to 12.2% at 0.5 ng ml-1 and 2.0 ng ml-1, respectively. This method has been shown capable of specifically determining digoxin and its major metabolites in serum, and has been successfully used in the determination of digoxin and its metabolites in serum samples collected from patients undergoing digoxin therapy. This method thus permits the investigation of digoxin metabolism and pharmacokinetics after the administration of commercial dosage forms.

Inhibitory effects of intravenous chloramphenicol sodium succinate on the disposition of phenylbutazone in horses

The effects of i.v. chloramphenicol sodium succinate on the pharmacokinetics of i.v. phenylbutazone in six healthy adult horses were investigated. Administration of chloramphenicol sodium succinate to mares reduced mean (±SD) phenylbutazone clearance from 0.600±0.222 to 0.339±0.123 ml/min per kg and increased mean (±SD) half life from 244 ± 59.8 to 371 ± 80.8 min and mean residence time from 333 ± 86.2 to 533 ± 124 min. The mean steady-state volume of distribution of phenylbutazone was unchanged, with mean (±SD) values of 187 ± 28.9 ml/kg in control animals and 170 ± 32.4 ml/kg after chloramphenicol sodium succinate.

Quantitation of interference in digoxin immunoassay in renal, hepatic, and diabetic disease

A comparison of the results of a newly developed fluorescence‐derivatization/HPLC method and a commercial immunoassay method (ACA, Dupont) for the measurement of serum digoxin concentrations in patients indicates that (1) the results from the ACA method agree well with those from the HPLC method in patients with cardiovascular disease but without renal, diabetic, and liver disease, (2) serum digoxin concentrations determined by the ACA method are overestimated in patients with renal, diabetic, or liver disease, and (3) the steady‐state serum concentrations of hydrolyzed and reduced metabolites are relatively insignificant in patients receiving digoxin therapy, including patients with renal failure. The excellent reproducibility of the HPLC and immunoassay methods (coefficient of variation < 9.0%), together with the demonstrated specificity of the HPLC method with respect to potential interference from digoxin metabolites, endogenous digoxin‐like immunoactive substances, and coadministered drugs and their metabolites, allows quantitation of the degree of interference in digoxin immunoassays under actual therapeutic drug monitoring conditions. Clinically significant interferences (0.3 to 1.1 ng/ml) with immunoassay determination were found in the majority of patients in all three diseases studied.

Chemical assay of avermectins by high performance liquid chromatography with fluorescence detection

Chemical methods for determining the concentrations of the avermectins in feeds, dosage forms, and biological samples are reviewed. The earliest methods for measuring the avermectins made use of the intense ultraviolet absorption due to the conjugated diene (olefinic bonds between carbons 8 and 9 and carbons 9 and 10) that has an ultraviolet absorption maximum at 245 nm and a molar absorptivity of over 30,000 l mol-cm-1. High performance liquid chromatographic (HPLC) separation of the avermectins and photometric measurement of their absorption at 245 nm has permitted the determination of plasma or serum concentrations to approximately 10 ng ml-1. Increased sensitivity of detection has been achieved by dehydration of the dihydroxycyclohexene ring of the avermectins in the presence of various catalysts that produces an intensely fluorescent derivative with an absorption maximum at 365 nm and emission maximum at 475 nm. These derivatives have been separated by HPLC and detected by fluorescence detectors at concentrations less than 1.0 ng ml-1 of plasma or serum. Recent improvements in the use of more efficient catalysts to effect the dehydration have reduced analysis time and decreased the formation of by-products, thereby improving the performance of these methods. A method based on HPLC with fluorescence detection of the dehydrated derivative was developed to determine ivermectin in horse dung after oral ivermectin doses of 200 micrograms kg-1 of body weight. Ivermectin concentrations greater than 0.05 microgram g-1 of wet feces were detectable for 1-2 days after dosing.

DIGOXIN AND METABOLITES IN URINE AND FECES: A FLUORESCENCE DERIVATIZATION-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC TECHNIQUE

A high-performance liquid chromatography method is described for the determination of digoxin and its metabolites digoxigenin, digoxigenin monodigitoxoside, digoxigenin bis-digitoxoside and dihydrodigoxin (20S and 20R) excreted in urine and feces. The urine sample or fecal supernatant is extracted with methylene chloride in the presence of digitoxigenin or digitoxin as internal standard. Pre-column derivatization is achieved using 1-naphthoyl chloride with subsequent separation of the derivatized compounds on either a normal- or reversed-phase system with fluorescence detection. Recoveries for digoxin and all metabolites from fecal samples were in the range 60-74%, which is comparable to that previously determined for urine samples. Standard curve data revealed linearity over a wide range of concentrations. Coefficients of variation for the analysis were less than 10% for all compounds over a range of 5-125 ng per ml urine and 10-250 ng per 200 mg feces. Peaks for digoxin and metabolites in urine and feces were obtained when human excreta were analyzed.

Determination of Reserpine in Plasma Using High-Performance Liquid Chromatography with Fluorescence Detection

Abstract A high-performance liquid chromatographic method for determining reserpine in plasma has been developed. The procedure involves extraction of reserpine from buffered plasma into benzene, oxidation of reserpine to a fluorophor by treatment with vanadium pentoxide in phosphoric acid, and chromatographic separation of the reserpine fluorophor on an octadecylsilane column by ion-pairing with heptanesulfonate ions. Fluorescence monitoring of the column effluent provides high sensitivity of detection and increases the specificity of the procedure. A detection limit of approximately 100 pg of reserpine per ml of plasma was obtained following analysis of 2 ml samples. Analysis of a number of samples demonstrated the applicability of this method in confirming the presence of reserpine in equine plasma specimens collected at various horse shows and in evaluating the pharmacokinetic behavior of reserpine following intramuscular administration to horses.