Rapid Detection of the Antibiotic Sulfamethazine in Pig Body Fluids by Paper Spray Mass Spectrometry.

We report herein a practical method for nonlethal detection of the antibiotic sulfamethazine in pig body fluids via the combination of simple extraction and paper spray mass spectrometry (PS-MS). This method requires minimal sample preparation while still providing high sensitivities and accuracies in complex matrices including pig whole blood (LOD = 7.9 µg/L; recovery = 95.4-103.7%), pig serum (LOD = 11.5 µg/L; recovery = 103.2-106.2%), and synthetic urine (LOD = 11.2 µg/L; recovery = 99.1-103.2%). Given a known correlation between the level of sulfamethazine in body fluids and edible tissues, this method shows great promise as a practical and nonlethal solution for rapid testing of the drug, which can substantially aid managerial decision in the livestock industry.

Development of a Paper Spray Mass Spectrometry Cartridge with Integrated Solid Phase Extraction for Bioanalysis.

A novel paper spray cartridge with an integrated solid phase extraction (SPE) column is described. The cartridge performs extraction and pre-concentration, as well as sample ionization by paper spray, from complex samples such as plasma. The cartridge allows for selective enrichment of target molecules from larger sample volumes and removal of the matrix, which significantly improved the signal intensity of target compounds in plasma samples by paper spray ionization. Detection limits, quantitative performance, recovery, ionization suppression, and the effects of sample volume were evaluated for five drugs: carbamazepine, atenolol, sulfamethazine, diazepam, and alprazolam. Compared with direct paper spray analysis of dried plasma spots, paper spray analysis using the integrated solid phase extraction improved the detection limits significantly by a factor of 14-70, depending on the drug. The improvement in detection limits was, in large part, due to the capability of analyzing larger sample volumes. In addition, ionization suppression was found to be lower and recovery was higher for paper spray with integrated SPE, as compared to direct paper spray analysis. By spiking an isotopically labeled internal standard into the plasma sample, a linear calibration curve for the drugs was obtained from the limit of detection (LOD) to 1 µg/mL, indicating that this method can be used for quantitative analysis. The paper spray cartridge with integrated SPE could prove valuable for analytes that ionize poorly, in applications where lower detection limits are required, or on portable mass spectrometers. The improved performance comes at the cost of requiring a more complex paper spray cartridge and requiring larger sample volumes than those used in typical direct paper spray ionization.

The effect of breed and sex on sulfamethazine, enrofloxacin, fenbendazole and flunixin meglumine pharmacokinetic parameters in swine.

Drug use in livestock has received increased attention due to welfare concerns and food safety. Characterizing heterogeneity in the way swine populations respond to drugs could allow for group-specific dose or drug recommendations. Our objective was to determine whether drug clearance differs across genetic backgrounds and sex for sulfamethazine, enrofloxacin, fenbendazole and flunixin meglumine. Two sires from each of four breeds were mated to a common sow population. The nursery pigs generated (n = 114) were utilized in a random crossover design. Drugs were administered intravenously and blood collected a minimum of 10 times over 48 h. A non-compartmental analysis of drug and metabolite plasma concentration vs. time profiles was performed. Within-drug and metabolite analysis of pharmacokinetic parameters included fixed effects of drug administration date, sex and breed of sire. Breed differences existed for flunixin meglumine (P-value<0.05; Cl, Vdss ) and oxfendazole (P-value<0.05, AUC0→∞ ). Sex differences existed for oxfendazole (P-value < 0.05; Tmax ) and sulfamethazine (P-value < 0.05, Cl). Differences in drug clearance were seen, and future work will determine the degree of additive genetic variation utilizing a larger population.

A physiologically based pharmacokinetic model linking plasma protein binding interactions with drug disposition.

Combination drug therapy increases the chance for an adverse drug reactions due to drug-drug interactions. Altered disposition for sulfamethazine (SMZ) when concurrently administered with flunixin meglumine (FLU) in swine could lead to increased tissue residues. There is a need for a pharmacokinetic modeling technique that can predict the consequences of possible drug interactions. A physiologically based pharmacokinetic model was developed that links plasma protein binding interactions to drug disposition for SMZ and FLU in swine. The model predicted a sustained decrease in total drug and a temporary increase in free drug concentration. An in vivo study confirmed the presence of a drug interaction. Neither the model nor the in vivo study revealed clinically significant changes that alter tissue disposition. This novel linkage approach has use in the prediction of the clinical impact of plasma protein binding interactions. Ultimately it could be used in the design of dosing regimens and in the protection of the food supply through prediction and minimization of tissue residues.

Effects of age on the pharmacokinetics of single dose sulfamethazine after intravenous administration in cattle.

Sulphonamides are still being used widely, influenced by the low cost and the efficacy against many common bacterial infections, since they present a broad spectrum of activity. The aim of this study was to determine the effect of age on the pharmacokinetic/pharmacodynamics (PK/PD) integration of intravenous sulfamethazine (60 mg/kgbw) in cattle, and the possible therapeutic outcomes. Six healthy female calves, at the age of one, three, seven and fifteen weeks were used. Normality analysis was assessed with the Shapiro-Wilk test. Non-parametric tests for paired data were used. Plasma concentrations were quantified using HPLC/uv. Differences were found between one-three-weeks-old calves and seven-fifteen-weeks-old calves, in pharmacokinetic parameters (clearance, area under the concentration-time curve and elimination half-life) and in the PK/PD integration. The ratios obtained in PK/PD integration (T>MIC, WAUC) confirm that it is necessary to apply twice the dose of sulfamethazine in > or = 7 weeks-old cattle to reach a satisfactory dosage regimen (MIC > or = 32 microg/mL).

Comparison of bovine in vivo bioavailability of two sulfamethazine oral boluses exhibiting different in vitro dissolution profiles.

The bolus (or oblet) is a dosage form that can be used for the oral administration of pharmaceutical compounds to ruminating species. Unlike traditional tablets, oral boluses may contain quantities of drug on the order of grams rather than milligrams. Due to its size, it is only recently that USP-like in vitro dissolution methods have been developed for this dosage form. However, whether or not these dissolution tests can predict product in vivo performance has yet to be determined. The importance of this issue is apparent when the U.S. Food and Drug Administration Center for Veterinary Medicine is faced with the decision of whether to require additional in vivo bioequivalence study data to support the approval of changes in product chemistry or manufacturing method. The current study was undertaken to determine whether an in vivo/in vitro correlation can be established for bovine sulfamethazine oral boluses and to acquire insight into the magnitude of changes in in vitro product performance that can occur before corresponding changes are seen in in vivo blood level profiles. Based upon the results of this investigation, it is concluded that marked changes in in vitro sulfamethazine bolus performance can be tolerated before resulting in altered in vivo blood level profiles. However, the data also suggest that rumenal absorption may occur for some compounds. Therefore the degree to which variation in product in vitro dissolution profiles can be tolerated may be compound specific.

Development of a physiologic-based pharmacokinetic model for estimating sulfamethazine concentrations in swine and application to prediction of violative residues in edible tissues.

OBJECTIVE: To develop a flow-limited, physiologic-based pharmacokinetic model for use in estimating concentrations of sulfamethazine after IV administration to swine. SAMPLE POPULATION: 4 published studies provided physiologic values for organ weights, blood flows, clearance, and tissue-to-blood partition coefficients, and 3 published studies provided data on plasma and other tissue compartments for model validation. PROCEDURE: For the parent compound, the model included compartments for blood, adipose, muscle, liver, and kidney tissue with an extra compartment representing the remaining carcass. Compartments for the N-acetyl metabolite included the liver and the remaining body. The model was created and optimized by use of computer software. Sensitivity analysis was completed to evaluate the importance of each constant on the whole model. The model was validated and used to estimate a withhold interval after an IV injection at a dose of 50 mg/kg. The withhold interval was compared to the interval estimated by the Food Animal Residue Avoidance Databank (FARAD). RESULTS: Specific tissue correlations for plasma, adipose, muscle, kidney, and liver tissue compartments were 0.93, 0.86, 0.99, 0.94, and 0.98, respectively. The model typically overpredicted concentrations at early time points but had excellent accuracy at later time points. The withhold interval estimated by use of the model was 120 hours, compared with 100 hours estimated by FARAD. CONCLUSIONS AND CLINICAL RELEVANCE: Use of this model enabled accurate prediction of sulfamethazine pharmacokinetics in swine and has applications for food safety and prediction of drug residues in edible tissues.

Pharmacokinetics and urinary excretion of sulphadimidine in buffalo calves.

Pharmacokinetics and urinary excretion of sulphadimidine (SDI) were determined in buffalo calves following single oral administration (150 mg/kg). The plasma levels of free sulphadimidine were above minimum effective therapeutic concentration (> 40 micrograms/ml) between 4 and 12 h and the N4-acetylated form of the drug was in the range of 7.2-19.3%. Kinetic evaluation of plasma levels was performed using a two-compartment open model. The absorption and elimination half-lives of SDI were 3.01 and 11.94 h, respectively. Based on this study, an optimal dosage regimen of sulphadimidine in buffalo calves would be 100 mg/kg, followed by 50 mg/kg at 12 h intervals. Sulphadimidine was mainly excreted in the urine as free amine. The percentage of N4-acetyl sulphadimidine in urine was comparatively higher than in plasma.

Pharmacokinetic interactions between flunixin and sulphadimidine in horses.

The pharmacokinetic aspects of sulphadimidine were studied in clinically healthy (control) and Flunixin-medicated horses after a single intravenous and oral administration of 100 mg/kg body weight. Plasma sulphadimidine concentration were determined by high-performance liquid chromatography (HPLC). Following the intravenous injection, all plasma sulphadimidine data were best approximated by a two-compartment open model using sequential, weight non-linear regression. Flunixin induced a 67% increase in the rate of sulphadimidine return to the central compartment from peripheral tissues (K21) and there were a trend to a 30% increase in K12. The sulphadimidine elimination half-life was decreased 21%, the Vdss was reduced by 18% and MRT was decreased by 20%. Following the oral administration, sulphadimidine was rapidly absorbed in control and Flunixin-medicated horses with absorption half-lives (t1/2 ab) of 0.5 and 0.43 hours respectively. The peak plasma concentration (Cmax) were 93.7 and 109 micrograms/ml attained at (tmax) 2.36 and 1.9 hours respectively. The elimination half-life after oral administration (t1/2 ab) was shorter in flunixin pre-medicated horses than in control ones. The systemic bioavalability percentages (F%) of sulphadimidine after oral administration of 100 mg/kg body weight was 79.3 and 71.2% in control and flunixin medicated horses, respectively. Therefore care should be exercised in the use of sulphadimidine in equine patients concurrently treated with flunixin.

Pharmacokinetics, bioavailability and dosage regimen of sulphadimidine in camels (Camelus dromedarius) under hot, arid environmental conditions.

A two-way crossover study was conducted in young Bikaneri camels (aged between 12 and 18 months) during the hot summer season to determine the bioavailability, pharmacokinetics and dosage regimens of sulphadimidine (SDM). A dose of 100 mg.kg-1 of SDM was used to study both the intravenous and oral pharmacokinetics of the drug. Analysis of the intravenous data according to a two-compartment pharmacokinetic model revealed that SDM was well distributed in the body (Vd(area):0.862 L.kg-1), had an overall body clearance of 0.035 +/- 0.019 L.h-1.kg-1 and the elimination of half-lives was in the range of 14.2 to 20.6 h. The mean maximum plasma SDM concentration following oral administration was 63.23 +/- 2.33 micrograms.mL-1, which was achieved 24 h after the oral administration. The mean bioavailability of SDM following oral administration was approximately 100%. To achieve and maintain the therapeutically satisfactory plasma sulphadimidine levels of > or = 50 micrograms.mL-1, the optimum dosage regimen for camels following either intravenous or oral administration would be 110 mg.kg-1 as the priming dose and 69 mg.kg-1 as the maintenance dose, to be repeated at 24 h intervals.

Comparative pharmacokinetics of sulfamethazine in plasma and parotid saliva of sheep.

Salivary output in sheep is large enough to be considered a physiologic body fluid compartment. The hypothesis for this work was that pharmacokinetics of sulfamethazine in saliva was similar to that in plasma. A reliable technique was developed to measure parotid salivary output. Mean output of saliva was 3.18 +/- 1.04 L from a single parotid gland per day with a mean flow of 2.21 +/- 0.43 mL/min. Using concentrations of sulfamethazine in parotid saliva made it possible to calculate the total passage of sulfamethazine to parotid saliva, which was calculated to be 3.5% of the total dose. Pharmacokinetic variables obtained for sulfamethazine in plasma and in saliva were closely related (AUC 1408 micrograms.h/mL and AUC 1484 micrograms.h/mL; Vdarea 0.434 L/kg and Vdarea 0.374 L/kg; t 1/2 beta 4.30 h and 3.46 h, respectively) and no substantial differences were observed. The convenience of using salivary concentrations of sulfamethazine for drug monitoring is discussed.

[Biotransformation of phenazone and sulfadimidine as markers of liver metabolism of drug and xenobiotic oxidation and acetylation in women with breast cancer]. / Biotransformacja fenazou i sulfadimidyny jako wskazników watrobowego metabolizmu leków i ksenobiotyków droga utleniania i acetylacji u kobiet chorych na raka sutka.

The aim of our study was to determine phenazone pharmacokinetics as an index of hepatic microsomal enzymes activity and acetylation phenotype in women with breast cancer. Phenazone test was made in 41 women with breast cancer and in 25 healthy women as a control group. Acetylation phenotype was measured in 40 women with breast cancer and in 25 healthy women as a control group. The plasma concentration of phenazone was estimated by the spectrophotometric method of Brodie and associates. Acetylation phenotype was determined by the Bratton-Marshall method in Varley's modification. The mean phenazone half-life time (t0,5), shortened significantly and the mean elimination rate constant (K) increased in women compared with a group of healthy persons. Mean clearance rate was increased in women with breast cancer too, compared with a group of healthy women. Acceleration of phenazone elimination may suggest that in patients with breast cancer elimination of the other drugs metabolized by the pathway similar to phenazone also may be changed. This should be considered in selection of their dosage. We have observed the predominance of rapid acetylators in women with breast cancer comparing with healthy persons. This difference was not statistically significant. Our results of acetylation phenotype in women with breast cancer suggests that rapid acetylation may be a factor of susceptibility to breast cancer.

Application of an enzyme immunoassay for the determination of sulphamethazine (sulphadimidine) residues in swine urine and plasma and their use as predictors of the level in edible tissue.

The potential of an enzyme immunoassay (EIA) with high cross-reactivity towards the major metabolite (N4-acetyl-sulphamethazine) of sulphamethazine was tested for screening fluids and tissues. Healthy pigs were given 20 mg sulphamethazine per kg body weight per day in their drinking water for 2 days. Groups of four pigs were slaughtered after 3, 4 and 7 days withdrawal. The results were compared with liquid chromatographic analysis for urine, plasma, kidney, liver, gluteal muscle and diaphragm. In general, concentrations found by the EIA were higher than those found by liquid chromatography (LC) because sulphamethazine metabolites were detected by the EIA and not by LC. Using the EIA for the detection of sulphamethazine and the major metabolite in urine and plasma, predictive relationships (tissue-fluid ratios) for the concentration of the parent drug in tissue, determined by LC, were calculated. The tissue-plasma ratios for muscle, liver and kidney were 0.1, 0.2 and 0.1, respectively. The tissue-urine ratios for muscle, liver and kidney were 0.02, 0.03 and 0.03, respectively. Owing to the higher concentration of the parent drug in both fluids, the presence of the major metabolite in urine and the sensitivity of the EIA, tissue can be screened for low concentrations of sulphamethazine.

Sprayed medicated feed with sulfadimidine for piglets.

The bioavailability of two different forms of medicated feed containing 2000 mg sulfadimidine (SDM) per kg was determined in three groups of eight piglets. In the first group, pharmacokinetic parameters of SDM were determined after a single intravenous dose of 10 mg/kg body weight and after single oral doses of 45 mg/kg body weight ingested either as an oleus solution sprayed directly onto the feed pellets ready for use (SPR) or as a commercially available premix incorporated into the feed before pelletising (PMX). After the single intravenous administration, the mean +/- SD of the volume of distribution was 0.34 +/- 0.05 l/kg, the total body clearance 0.37 +/- 0.07 ml/min.kg, the mean residence time 15.5 +/- 2.5 h, and the elimination half-life 11.1 +/- 2.0 h. Although no statistical significance existed, a single meal with PMX was associated with slightly higher mean values for the maximum serum concentration (Cmax), the time to reach Cmax, and the bioavailability (52.98 +/- 6.60 micrograms/ml, 6.8 +/- 1.1 h, 59.7 +/- 12.1%, respectively, vs. 40.04 +/- 13.19 micrograms/ml, 6.0 +/- 1.4 h, 49.0 +/- 18.6 for SPR). The remaining two groups of piglets received medicated feed with either SPR or PMX during a 3-day period both with restrictive (twice-daily) or ad libitum feeding according to a cross-over design. In all four cases, potentially efficacious plasma SDM concentrations between 50 and 150 micrograms/ml were obtained within 24 h after initiation of the treatment. With PMX, plasma concentrations tended to be higher than with SPR with both feeding regimens. Ad libitum feeding was associated with a significantly higher food intake and hence a higher SDM intake resulting in higher plasma concentrations. Additionally, plasma concentrations were more constant over time with ad libitum feeding whereas they declined considerably between meals in restrictively fed animals. In vitro dissolution tests of the two types of medicated feed revealed that SDM was rapidly released from SPR (58% within 15 min) and that SDM release from PMX was markedly slower (3% within 15 min). Despite the relatively slow rate of in vitro dissolution, in vivo absorption of SDM was satisfactory. It is concluded that both forms of SDM medicated feed may be considered bioequivalent and potentially efficacious in piglets.

Effect of antibacterial growth promoters on the immune system of broiler chicks.

Administration of either 0.001 g/kg ampicillin (A), 0.05 g/kg oxytetracycline (O) or 0.05 g/kg sulphadimidine (S) in feed to broiler chicks for 50 days caused an increased serum concentration of the drug compared to the control birds that were given no drugs. O and S but not A resulted in a significant decrease of the total number of leukocytes, lymphocytes and the size of bursa of Fabricius and thymus but not spleen or body weight. The antibacterials significantly reduced the macrophage phagocytic activity compared to controls. It is suggested that the prolonged administration of O and S to chickens may induce an immunosuppressant effect.

Relative bioavailability of microgranulated sulfadimidine in veal calves.

The kinetics of free and microgranulated sulfadimidine were compared in milk-fed calves dosed orally (180 mg/kg) in a crossover study. Microgranulation results in delayed absorption of sulfadimidine and poor bioavailability, with the area under the plasma concentration-time curve (AUC(0-infinity)) reduced from 7400 to 3781 micrograms.h/mL, and maximum plasma concentration (Cmax) reduced from 188.1 +/- 39.0 to 84.41 +/- 22.6 micrograms/mL. It is concluded that sulfadimidine microgranulated with long chain fatty acids is not suitable for use in milk-fed calves; the gastrointestinal transit time is too rapid to allow full release of the drug, markedly limiting its bioavailability. In adult animals, or in the young of other animal species in which digesta transit time is slower than in calves, the bioavailability of microgranulated sulfadimidine may be much greater.

[Clinical significance of the determination of oxidation and acetylation phenotype in patients with multiple sclerosis]. / Kliniczne znaczenie badania fenotypu oksydacji i acetylacji u chorych na stwardnienie rozsiane.

Genetically determined individual differences in the ability of oxidation and acetylation of certain drugs have raised in recent years a considerable interest in view of their clinical importance. The purpose of the study was finding out of a possible difference in the ability to oxidized sparteine and to acetylate sulfamidine as model drugs between patients with multiple sclerosis and healthy control volunteers. The study was carried out in 23 patients with MS. The control group comprised 160 healthy subjects for comparison of oxidation phenotype. The results of determination of acetylation phenotype were obtained in 45 healthy controls. The study showed that in 160 controls 146 were extensive (rapid) metabolizers (91.3%) and 14 were weak (slow) metabolizers of sparteine (8.7%). In the group of MS patients 21 were extensive metabolizers (91.3%) and 2 were weak metabolizers (8.7%). The determination of acetylation phenotype in 45 controls showed 51% of rapid acetylation (23 subjects) and 49% of slow acetylation (22.

Determination of sulfamethazine residue in chicken serum and egg by high-performance liquid chromatography with chemiluminescence detection.

A sensitive method that involves high-performance liquid chromatography with chemiluminescence detection is developed for the determination of sulfamethazine residue in samples of chicken serum and egg. Sulfamethazine is extracted from the samples and derivatized with fluorescamine. The derivatized samples are eluted by reversed-phase chromatography using a mixture of 10 mM potassium phosphate and 30% acetonitrile on a Nova-Pak C18 column. The post-column reagents in the chemiluminescence system are 1 mM bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl] oxalate and 0.3M hydrogen peroxide in acetonitrile. The detection limit in the standard solution is 1 ng/mL, and the calibration curve is linear between 1 and 100 ng/mL. The recoveries of spiked samples (50 ppb) are 95.8 +/- 9.7% in chicken serum samples and 84.9 +/- 10.7% in egg samples. In actual sample analysis, the lowest detectable concentration of sulfamethazine (0.073 microgram/mL) was found at 48 h in serum and on day 9 (0.017 microgram/g) in egg after oral administration of a dose of 100 mg/kg body weight to hens.

Depletion of residues from milk and blood of cows dosed orally and intravenously with sulfamethazine.

Cows were dosed orally (n = 4) or intravenously (n = 4) with sulfamethazine [sulmet; 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide] for 5 consecutive days (220 mg/kg of body weight on day 1 and 110 mg/kg on days 2-5). The concentrations of sulmet, N4-acetylsulfamethazine (Ac-sulmet), and the N4-lactose conjugate of sulfamethazine (lac-sulmet) were measured in milk and blood collected at 24 h intervals after the last doses of sulmet were given. The method of analysis included (1) spiking of samples with known amounts of 13C6-labeled reference compounds, (2) resolution of the 3 compounds by reversed-phase chromatography, (3) hydrolysis of lacsulmet, (4) treatment with diazomethane to yield N1-methyl derivatives, and (5) gas chromatography/mass spectrometry. The ratios of intensities of selected mass spectral ions containing 12C6 and the corresponding ions containing 13C6 were used for residue quantitation. Sulmet, which was always the most abundant residue in the blood, decreased to less than 100 ppb 4 days after the last doses were given and to less than 10 ppb 7 days after the last doses. The concentrations of sulmet in milk were approximately one fifth the concentrations of sulmet in blood. The concentrations of lac-sulmet and Ac-sulmet in milk were lower than the concentrations of sulmet in milk.