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.

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.

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).

Influence of chronic obstructive lung disease on the disposition of an acidic drug (sulfamethazine).

The influence of chronic respiratory failure (CRF) on the pharmacokinetics of an acidic drug has been studied in 11 patients and in eight normal volunteers who received 10 mg/kg of oral sulfamethazine. Blood and urine samples were collected for 24 and 48 hours, respectively. No differences were observed in the rate of sulfamethazine absorption, but bioavailability was decreased when compared with control subjects. Sulfamethazine volume of distribution (Vd) was larger in patients than in control subjects. These differences in Vd may be secondary to an increase in sulfamethazine unbound fraction. No differences were observed in sulfamethazine elimination. It is concluded that in patients with CRF sulfamethazine bioavailability decreases, and Vd increases secondary to a decrease in binding. Despite the fact that plasma concentrations of the test drug will be decreased, the administration of higher doses may not be advisable.

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.

N-acetylation phenotyping with sulfamethazine in an Iranian population.

The acetylation polymorphism is one of the most common inherited variations in the biotransformation of drugs and chemicals. Its association with drug toxicity and increased risk of developing certain diseases and certain types of chemically induced cancers has made it one of the oldest and best studied examples of pharmacogenetic conditions. N-Acetylation of sulfamethazine was studied in 74 unrelated healthy Iranian volunteers. The frequency of slow acetylators, determined by using free and total plasma sulfamethazine concentrations, was 78.4%. The mean acetylation ratio was 19.48% for slow acetylators, and the frequency of the recessive allele controlling slow acetylation was found to be 0.88. This percentage is similar to that observed in various Arab countries and higher than that observed in Europeans.

Mechanisms of nonlinear disposition kinetics of sulfamethazine.

Five healthy male subjects received oral doses of 10 and 40 mg/kg of sulfamethazine (SMZ) approximately 14 days apart in a nonrandomized crossover study. Blood and urine samples were collected for at least 24 and 72 hr, respectively. All samples were assayed by the Bratton-Marshall procedure for SMZ and apparent N-acetylsulfamethazine (NSMZ). Recovery of total drug (SMZ + NSMZ) in urine was 88.9% following the low and 79.5% following the high dose. The low and high dose plasma concentration time curves were not readily superimposable (i.e., nonlinear kinetic behavior was observed). The data suggest that several mechanisms contribute to the nonlinearity. Specifically, a dose-dependent decrease in absorption rate displaced the plasma concentration-time curve to the right in some subjects, whereas apparent metabolic clearance (Clm) decreased with increasing dose (estimated assuming dose = amount of SMZ + NSMZ in urine to 72 hr) in all subjects (0.35 ml/min/kg for the low and 0.23 for the high dose). Still greater dose-dependent effects were found when apparent Clm of unbound drug was determined, since free fraction rose from 0.11 to 0.30 over the observed plasma concentration range. Renal clearance (ClR) of Smz appeared to be a complex function of time. In the low dose study it ranged from an average of 0.071 ml/min/kg at 2 hr to 0.146 ml/min/kg at 6 hr after drug. After the high dose comparable values were 0.083 and 0.128. Interindividual variability and pronounced nonlinear kinetics of SMZ after 40 mg/kg suggest that this dose is probably a poor choice for the determination of acetylator phenotype.

Influence of double genetic polymorphism on response to sulfamethazine.

Combined effects of a double genetic polymorphism on sulfamethazine-induced changes in erythrocyte-reduced glutathione (GSH) concentration were investigated in 16 healthy Ghanaian men. The subjects were divided into four subgroups on the basis of their glucose-6-phosphate dehydrogenase (G-6-PD) activity levels and acetylator phenotypes. Each received a single oral dose of sulfamethazine (1.5 gm). Erythrocyte concentration of GSH and plasma concentrations of drug were determined in samples taken at specific times after dosing. Sulfamethazine treatment induced a decrease in erythrocyte GSH concentration in all subjects. The degree of exposure to sulfamethazine was greater in slow acetylators, as evidenced by higher plasma concentrations, longer half-lifes, and greater area under the plasma concentration-time curves. The greatest decrease in GSH concentration occurred in subjects who where both G-6-PD deficient and phenotypically slow acetylators. In these subjects the effect of the double genetic defect was approximately additive. Drug-induced hemolysis is associated with a low erythrocyte GSH concentration. Our data suggest that slow acetylator status is likely to increase susceptibility to hemolysis in G-6-PD-deficient individuals exposed to potentially hemolytic arylamines.

Kinetic discrimination of three sulfamethazine acetylation phenotypes.

The relationship between sulfamethazine disposition kinetics and acetylator phenotype was studied in 19 healthy subjects. Various kinetic parameters for sulfamethazine and its N4-acetylated metabolite were determined after a dose of a rapidly absorbed oral solution. When plotted on a frequency distribution histogram, the results exhibited a well-defined trimodal pattern for acetylation clearance values and overall elimination or metabolic rate constants. These data were consistent with the well-recognized acetylation polymorphism for sulfamethazine, except that they clearly subdivided the previously acknowledged "fast" acetylator mode into intermediate and rapid acetylator groups. The apparent distribution volume and renal clearance for sulfamethazine and acetylsulfamethazine did not differ significantly among the 3 phenotypes. Of special interest was the observation that rapid acetylators initially produce much greater amounts of acetyl metabolite than intermediate acetylators. The potential clinical implications of identifying rapid and intermediate acetylators are discussed in view of evidence showing that acetyl metabolites may be pharmacologically active or function as intermediates in toxic metabolic pathways.

Caffeine as a metabolic probe: validation of its use for acetylator phenotyping.

The use of two caffeine metabolite ratios for acetylator phenotyping was validated by demonstrating concordance with two sulfamethazine tests in 178 unrelated healthy subjects. The caffeine metabolites used for this purpose were 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U). The ratio AAMU/(AAMU + 1X + 1U), referred to as molar ratio or N-acetyltransferase, was compared with the ratio AAMU/1X. The results indicated that, for screening purposes, the acetylator phenotype can be determined by analysis of a 6-hour urine sample after a cup of coffee or strong tea or a can of caffeine-containing soft drink. The ratio AAMU/1X is the ratio of choice for the study of subjects in whom variability of xanthine oxidase can be neglected; use of the ratio AAMU/(AAMU + 1X + 1U) appears appropriate for special purposes. Gender, ethnic origin, habitual or moderate consumption of coffee, tea, soft drinks, or ethanol, or cigarette smoking have little if any effect on the caffeine tests for acetylator phenotyping.

Inhibition and induction of cytochrome P4502E1-catalyzed oxidation by isoniazid in humans.

We studied the effect of isoniazid administration on the cytochrome P4502E1-catalyzed elimination of chlorzoxazone and acetaminophen. Isoniazid, 300 mg daily, was administered for 7 days to a group of 10 volunteer slow acetylators. Acetaminophen, 500 mg, and chlorzoxazone, 750 mg, were administered on separate occasions before isoniazid, during the period of isoniazid administration, and after the discontinuation of isoniazid. Isoniazid inhibited the clearance of chlorzoxazone by 58%, as assessed from plasma data, and inhibited the formation of acetaminophen thioether metabolites (a measure of the formation of the hepatotoxin N-acetyl-p-benzoquinone imine and catechol oxidative metabolites of acetaminophen, as determined from their recovery in urine, by 63% and 49%, respectively. Two days after the discontinuation of isoniazid, the clearance of chlorzoxazone was increased over the value before isoniazid by 56%. Acetaminophen thioether but not catechol metabolites were increased by 56% 1 day after the discontinuation of isoniazid and had returned to the pre-isoniazid value 3 days after the discontinuation of isoniazid. We conclude that the time course of the interaction with regard to chlorzoxazone elimination and formation is compatible with an inhibition-induction effect of isoniazid on cytochrome P4502E1. The mechanism of this biphasic effect is probably induction by protein stabilization, which results in inhibition of catalytic activity while isoniazid is present.

Evidence for the carriage of silver by sulphadimidine: inhibition of proteolytic enzymes.

1 Trypsin in free solution and trypsin-sepharose were shown to be inhibited by Ag+ in the absence of Cl-. 2 In the presence of Cl- and absence of a suitable carrier, Ag+ has no inhibitory action on trypsin or chymotrypsin. 3 Sulphadimidine bound Ag+ in the presence of Cl-, and carried the Ag+ to both trypsin and chymotrypsin in free solution as well as to trypsin-sepharose leading to the inhibition of all these enzyme systems. 4 The neutral protease of tumour cell surfaces was inhibited by Ag+ transported by sulphadimidine in the presence of Cl-. 5 Kinetic data demonstrated the requirements for both Ag+ and carrier to effect inhibition, the degree of inhibition being directly related to the molarity of each of these reagents. 6 The known inhibition of trypsin by Ag+ binding to histidine in the active site has been defined in mechanistic terms employing the sulphonamide drug, sulphadimidine, to illustrate this exchange mechanism.

The regulation of oxidative drug metabolism by growth hormone in the dwarf goat: differences from and similarities to the mechanisms in rats.

The effects of bovine GH (BST), administered in different dose patterns, on in-vivo oxidative drug metabolism, were studied in female dwarf goats. Animals received recombinantly derived methionyl BST at a dose of 500 micrograms/kg body weight per 24 h for 6 days. It was administered to one group of goats as one s.c. injection per day, another group received a similar 24-h dose divided into three s.c. injections given at 8-h intervals, and the third group received 50 micrograms BST/kg body weight every 2.5 h by a pulsative i.v. infusion. Oxidative metabolic capacity was assessed by determining plasma sulphadimidine (SDD) elimination and urinary metabolite excretion. SDD shows a marked sex-dependent plasma elimination in dwarf goats, with male goats having a lower plasma clearance than female goats. When BST was given by daily injection, no clear effects on SDD plasma clearance or urinary metabolite excretion were observed. However, when the total dose was divided into three injections given at 8-h intervals, the plasma SDD elimination rate decreased. This was associated with a decrease in urinary excretion of the two main hydroxy SDD metabolites. When BST was given by discontinuous i.v. infusion, simulating the male endogenous plasma GH pattern, a marked decrease in SDD plasma clearance was observed. In addition, the excretion of the two urinary hydroxy metabolites was considerably reduced. These results suggest that GH can affect drug oxidation in dwarf goats via mechanisms similar to those suggested for rats. However, in the dwarf goat, the sex differences in drug metabolism are opposite to those in rats.

Polymorphic acetylation of the antibacterials, sulfamethazine and dapsone, in South Indian subjects.

A group of South Indian subjects was studied for their capacities to acetylate sulfamethazine (SMZ) and dapsone (DDS) and to clear DDS from the circulation. An apparent trimodal distribution of acetylator phenotypes was found in 49 subjects (51% slow, 12% intermediate, and 37% rapid acetylators) from measurements of the percentage acetylation of SMZ in 6-hour plasma samples after administration of 10 mg SMZ/kg. The intermediate phenotype was not discernible from either the percentage acetylation of SMZ in urine (collected concurrently with the plasma after SMZ) or that of DDS in plasma after the ingestion of 50 mg DDS by the same subjects. The latter two measurements yielded a bimodal distribution of 59% slow and 41% rapid acetylators, nearly identical to earlier reported distributions of isoniazid inactivator phenotypes in larger numbers of South Indian tuberculosis patients. In the current group, acetylation of DDS and SMZ was positively correlated. The half-time of disappearance (T 1/2) of DDS, an expression of the rate of clearance from the plasma, ranged from 13 to 40 hours. No correlation was found between the subject's capacity to acetylate DDS and the T 1/2 value for DDS. These results were generally consistent with earlier observations made during similar studies of American and Filipino subjects.

Simple high-pressure liquid chromatographic determination of trisulfapyrimidines in human serum.

A simple and rapid high-pressure liquid chromatographic method was developed for the determination of sulfadiazine, sulfamerazine, and sulfamethazine in human serum. After the trichloroacetic acid precipitation of the serum proteins, an aliquot of the supernate is injected into a high-pressure liquid chromatograph equipped with a reversed-phase microparticulate column and a fixed wavelength UV detector. For each of the three components of trisulfapyrimidines, a linear calibration curve was observed in the 1-30-microgram/ml range, with the precision of the assay estimated to be +/- 2% (RSD). Preliminary pharmacokinetic data are also presented.

Bioavailability and dissolution behavior of trisulfapyrimidine suspensions.

The bioavailability of seven commercial trisulfapyrimidine suspensions was studied in 14 adult male volunteers. Fifteen blood samples were collected over a 48-hr period following administration of a 1-g dose of each suspension. Serum was assayed for each component (sulfadiazine, sulfamerazine, and sulfamethazine) by high-pressure liquid chromatography. Analysis of variance indicated several significant differences among the seven commercial preparations with respect to Cmax Tmax, and AUC for sulfadiazine, sulfamerazine, and sulfamethazine, The in vitro behavior of each suspension was then studied by the paddle method of the Food and Drug Administration. A 0.5-ml sample was introduced into 900 ml of hydrochloric acid (2.2 x 10(-4) M) at 37 degree and dissolved using a paddle speed of 25 rpm. Samples withdrawn at 15 and 30 min were analyzed by high-pressure liquid chromatography, and the percent of sulfadiazine, sulfamerazine, and sulfamethazine was calculated. Significant correlation was obtained between an in vivo parameter (Cmax for sulfadiazine) and an in vitro parameter (percent sulfadiazine dissolved in 30 min). Results indicate that this method is suitable for the in vitro screening of trisulapyrimidine suspensions.

Fish and antibiotics: pharmacokinetics of sulphadimidine in carp (Cyprinus carpio).

Pharmacokinetics, metabolism and clearance of sulphadimidine (SDM) were studied after a single intraperitoneal injection of SDM in carp at 20 degrees C. SDM was acetylated and hydroxylated to a small extent. The main metabolite was N4-acetyl derivative amounting only 2% of the total drug dose excreted; hydroxylation was less important (0.41% of the dose). The elimination half-life for SDM in carp was 17.5 h. The clearance values for SDM and its metabolites were equivalent. The importance of pharmacokinetic studies in different fish species is discussed.

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.