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.

Influence of a five-day vegetarian diet on urinary levels of antibiotics and phthalate metabolites: a pilot study with "Temple Stay" participants.

Diet is purported to be means of exposure to many environmental contaminants. The purpose of this study is to understand the influence of dietary change on the levels of exposure to several environmental chemicals - in particular, antibiotics and phthalates. For this purpose, we examined the extent to which short-term changes in diet influenced the inadvertent exposure levels to these chemicals in an adult population. We recruited participants (n=25) of a five-day 'Temple Stay' program in Korea and collected urine samples before and after the program. We also conducted a questionnaire survey on participants' dietary patterns prior to their participation. During the program, participants followed the daily routines of Buddhist monks and maintained a vegetarian diet. Urinary levels of three antibiotics and their major metabolites, metabolites of four major phthalates, and malondialdehyde (MDA) as an oxidative stress biomarker were analyzed. The frequency and levels of detection for antibiotics and phthalates noticeably decreased during the program. Urinary MDA levels were significantly lower than before program participation (0.16 versus 0.27mg/g creatinine). Although the exposure to target compounds might be influenced by other behavioral patterns, these results suggest that even short-term changes in dietary behavior may significantly decrease inadvertent exposure to antibiotics and phthalates and hence may reduce oxidative stress levels.

Development of immunoassays for the detection of sulfamethazine in swine urine.

The use of sulfonamides, such as sulfamethazine (SM2), in pig production is recognized as a public health risk as it inevitably results in sulfamethazine residues in pork. This study is aimed at establishing rapid, simple, reliable methods, with both sensitivity and specificity, for detecting sulfamethazine residues. For this purpose, monoclonal antibodies against sulfamethazine were prepared and characterized. No cross-reaction of the monoclonal antibodies was identified with other sulfonamides or analytes. Based on the competitive immunoassay principle, an indirect competitive ELISA kit (SM2 kit) and a rapid detection strip for detecting sulfamethazine residues were developed using monoclonal antibodies and the colloidal gold technique. The indirect competitive ELISA kit and the strip assay could be performed within 2 h and 5-10 min, respectively. The results showed that the detection limits were 1 ng ml(-1) for the indirect competitive ELISA kit and 8 ng ml(-1) with the unaided eye and 1 ng ml(-1) with the strip reader for the rapid strip assay. Comparing the HPLC method with the SM2-kit or the strip in pig urine spiked with standards of SM2, the difference was <4.6% for SM2-kit and 4.3% for the strip. The two methods are suitable for the rapid screening of sulfamethazine residues in swine urine. Experimental data revealed that the two methods, especially the strip, proved to be sensitive, specific, rapid and easy to use for the quantitative, semi-quantitative or qualitative detection of SM2 residues in swine urine.

Preliminary evaluation of a lateral flow immunoassay device for screening urine samples for the presence of sulphamethazine.

A lateral flow immunoassay (LFIA) device was developed and applied to testing urine samples for residues of the antimicrobial sulphamethazine (SMZ). This report describes the preparation of a rat monoclonal antibody to SMZ and its characterisation in an ELISA format. Apart from SMZ, the antibody showed high (> or =50%) cross-reactivity to N4-acetyl-sulphamethazine (55%), sulphamerazine (59%) and sulphisoxazole (50%) and lower cross-reactivity of 18% to sulphachlorpyridazine and sulphadiazine. The LFIA device consisted of a nitrocellulose membrane spotted with SMZ-ovalbumin and goat anti-mouse antibody as capture line and control line, respectively. Mouse anti-rat IgG F(ab')2 fragment specific antibody, adsorbed to colloidal carbon, was used as the detection ligand in the LFIA. The LFIA device had a cut-off value of 6.3 ng/ml in diluted (1/10) urine. Urine samples from SMZ-treated pigs, and bovine and porcine urine samples fortified with SMZ were used for a blind, four-laboratory evaluation of the performance of the LFIA device. Concentrations of SMZ in the test samples (n=29), as determined by LC-MS/MS, ranged from 0 (<3) to 1174 ng/ml. The evaluation of the LFIA device showed an overall sensitivity of 100%, a specificity of 71%, and positive and negative prediction values of 73% and 100%, respectively. The LFIA device has been fabricated as a test kit for determining SMZ residues in animals produced for slaughter.

Rapid and sensitive screening of sulfamethazine in porcine urine with an enzyme-linked immunosorbent assay and a field-portable immunofiltration assay.

An enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunofiltration assay (ELIFA) were developed for the screening of sulfamethazine (SMZ) in porcine urine. Incurred urine samples were measured by ELISA with a working range of 0 to 10 ng of SMZ per ml. The assay showed good accuracy and precision, with recoveries above 99.8% and intra- and interassay coefficients of variation (CVs) ranging from 2.6 to 5.6% and from 5.9 to 12.7%, respectively. Good agreement was observed when the results of the immunoassay were compared with those of liquid chromatography/tandem mass spectrometry analysis. For the ELIFA, a nylon membrane is placed on top of an absorbent material and covalently coated with rabbit anti-rat immunoglobulins. Free binding sites are blocked, and monoclonal anti-SMZ antibodies, SMZ standard or urine, and SMZ-horse radish peroxidase conjugate are subsequently dropped onto the membrane. During the assay, the reactants are drawn through the membrane because of its close contact with the absorbent pad. Finally, a substrate solution is added for blue color development. The blue spot produced can be visually evaluated or instrumentally measured (numeric deltaE*ab value), and the intensity of its color is inversely proportional to the analyte concentration. When a blue dot appears on the membrane, even if its color is less intense than that of the negative control, the sample is considered "negative," i.e., it is thought to contain a concentration of SMZ that is below the visual detection limit. If no color appears on the test membrane, the sample is considered "positive," i.e., it is thought to contain a concentration of SMZ that is equal to or above the visual detection limit. Validation of the assay showed good inter- and intra-assay precision (CV < 10%). Because samples can be analyzed after a simple dilution in <30 min with this assay format, it has strong potential for application in the field.

[Diagnosis of acetylation phenotype in the protection of production personnel]. / Diagnostika fenotipa atsetilirovaniia pri obespechenii bezopasnosti proizvodstvennogo personala.

The authors elaborated noninvasive method to diagnose acetylation phenotype through photocolorimetric studying kinetics of sulfadimesine urinary excretion. Suggestion is approach to people protection through selection of inductors which purposefully increase individual activity of N-acetyltransferase.

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.

Liquid chromatography tandem mass spectrometric quantitation of sulfamethazine and its metabolites: direct analysis of swine urine by triple quadrupole and by ion trap mass spectrometry.

This work describes a new method for the quantitation of trace amounts of sulfamethazine (SMZ) and its main metabolite, N4-acetylsulfamethazine (Ac-SMZ), in swine urine, using high-performance liquid chromatography (HPLC) tandem mass spectrometric analysis of crude urine after addition of internal standard and simple dilution with water. The aim was to determine whether residues of this sulfamidic drug, normally administered to swine in order to prevent infectious diseases, were present in urine at levels lower than those permitted by regulatory authorities before human consumption (EU Project SMT, contract number CT 96-2092). A 10 microL volume of diluted urine was injected into a very short, narrow-bore chromatographic column (Zorbax SB-C18 2.1 i. d. x30 mm length, 3.5 microm pore size). Elution of the analytes of interest was achieved in less than seven minutes using a rapid gradient (from 20 to 80% methanol in 3 minutes). Either a PE Sciex API 365 triple quadrupole (QqQ), operated in the selected reaction monitoring (SRM) mode, or a Finnigan LCQ ion trap (IT) mass spectrometer, operated in narrow-range product ion scan, was used as the final detector. Electrospray (ESI) was used as the ionization technique. A comparison of the two tandem mass spectrometers was performed by analyzing the same set of test samples, at three concentration levels, on three different days. Linearity of responses of the calibration standards, intra- and inter-assay precision of the samples, specificity and limits of detection were evaluated for both systems. Both the QqQ and the IT instrument was suitable for rapid, sensitive and specific determination of the analytes, although the overall performance of the QqQ was slightly superior in terms of linearity, precision and sensitivity.

[Clinical significance of oxidation and acetylation genetic polymorphism in patients with Parkinson's disease]. / Kliniczne znaczenie polimorfizmu oksydacji sparteiny i acetylacji sulfadimidyny u chorych na chorobe Parkinsona.

The relationship between genetically determined polymorphic oxidation and acetylation and susceptibility to some disease has aroused much interest. The aim of our study was to evaluate whether patients with Parkinson's disease differ from healthy persons in their ability to oxidize sparteine and acetylate sulfadimidine as model drugs. Oxidation and acetylation phenotypes were estimated in 50 patients with Parkinson's disease. The control group consisted of 160 healthy volunteers for comparison of oxidation phenotype and 60 healthy volunteers for comparison of acetylation phenotype. The phenotyping of oxidation revealed two distinct populations among 50 patients with Parkinson's disease: 47 persons (94%) were extensive metabolizers of sparteine and 3 persons (6%) were poor metabolizers. In 160 healthy persons, 146 persons (91.2%) were extensive metabolizers of sparteine and 14 persons (8.8%) were poor metabolizers. The difference between frequency distribution of PMs and EMs in healthy persons and in patients with Parkinson's disease was not statistically significant. The phenotyping of acetylation showed among 50 patients with Parkinson's disease 38 persons (76%) slow acetylators and 12 persons (24%) rapid acetylators. In 60 healthy volunteers the phenotype of slow acetylation was observed in 29 persons (48.3%) and rapid acetylation in 31 persons (51.7%). The prevalence of slow acetylators among patients with Parkinson's disease in comparison to healthy volunteers was statistically significant (chi 2 = 8.7677/p < 0.003). The results of our study may suggest that the slow acetylation phenotype is associated with increased risk of the development of Parkinson's disease.

Development of a one step strip test for the detection of sulfadimidine residues.

The one step strip test described is a competitive immunoassay in which the detector reagent consists of colloidal gold particles coated with affinity purified polyclonal anti-sulfadimidine (SDD) antibodies. The capture reagent in the assay is an SDD-ovalbumin conjugate which is immobilised on the lateral flow membrane of the test device. In the test procedure, 150 microliters (four drops) of a liquid sample (buffer, urine or milk) are brought into the sample well of the test device and allowed to migrate over the membrane. The more analyte present in the sample, the more effectively it will compete with the SDD immobilised on the membrane for binding to the limited amount of antibodies of the detector reagent. A sufficient amount of SDD in the sample will therefore prevent the binding of the detector reagent to the SDD immobilised on the membrane. Therefore, a positive sample will not show a test line in the read-out zone. With spiked buffer or calf urine this was obtained at a level of > 10 ng ml-1 of SDD and with spiked (diluted) fresh cow milk at a level > 20 ng ml-1 of SDD. At these levels, the test is applicable only as a qualitative assay. The presence or absence of a test line indicates lower or higher levels of SDD, respectively. The major advantages of the one step strip test are that results can be obtained within 10 min and that all reagents are included in the test device.

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.

[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.

The production of pig tissue sulphadimidine reference material.

The production of stable homogeneous reference materials containing the antimicrobial agent sulphadimidine in pig tissue is described. These were commissioned by the Community Bureau of Reference (BCR), established by the Commission of the European Communities, to promote improvements in analytical accuracy and to ensure uniformity of results determined by member states. Sulphadimidine-containing tissue powders (400 vials each of muscle, liver and kidney) were prepared by orally dosing pigs with drug, producing lyophilized tissue powders and blending these with negative tissues from unmedicated animals to achieve target concentrations. Details of the production process, the stabilizing procedure developed and the analytical assessments of homogeneity and stability are given.

[Drug residues in untreated swine]. / Arzneimittelrückstände bei unbehandelten Schweinen.

The concentration of sulfadimidine was measured in the urine of pigs which were housed (over five days) in boxes where other pigs have been treated orally with sulfadimidine before. Sulfadimidine was measured in the urine of the unmedicated pigs in a concentration of up to 4 micrograms/ml. Considering these urine concentrations, violative sulfadimidine tissue residues would be expectable in the carcass after slaughter. The practice of fixing withdrawal times must be considered again to ensure that drug residues in tissues are below the MRL before slaughter also in unmedicated animals.

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.

Sulfamethazine as a model compound to assess sex hormone-dependent cytochrome P-450 activity in rats.

Plasma disposition and urinary recovery of sulfamethazine (SMZ), its N4-acetylated metabolite (N4AcSMZ), and two of its hydroxylated metabolites [5-hydroxysulfamethazine (5OHSMZ) and 6-hydroxymethylsulfamethazine (6CH2OHSMZ)] were determined in male and female rats, in castrated males, and in rats pretreated with various steroid hormones. Male rats had a 2-fold higher SMZ plasma clearance than females, castrates, and males treated with flutamide (a testosterone antagonist). When castrated male rats were treated with testosterone or trenbolone, SMZ plasma clearance returned to normal values. Higher SMZ plasma clearance rates in the presence of androgens went together with higher urinary recoveries of the 6CH2OHSMZ metabolite. In hepatic microsomes of male rats lower apparent KM values, and higher Vmax values for 6CH2OHSMZ and 5OHSMZ formation were found than in microsomes of female rats. Castration or treatment of male rats with flutamide markedly reduced microsomal SMZ hydroxylation rates. Pretreatment of male or female rats with phenobarbital or triacetyl-oleandomycin had no effect on microsomal SMZ hydroxylation, whereas a continuous infusion with bovine somatotropin in male rats caused a marked decrease in SMZ hydroxylation rate. Finally, SMZ hydroxylation to 6CH2OHSMZ and 5OHSMZ in hepatic microsomes from male rats was strongly inhibited by monoclonal antibodies against P-4502C11. These results suggest that the male-specific P-4502C11 enzyme plays an important role in the hydroxylation of SMZ to 6CH2OHSMZ and 5OHSMZ in rats. SMZ seems a useful model compound to assess hormone effects on oxidative biotransformation (in vivo and in vitro) in rats.

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.

Isoniazid induced neuropathy in slow versus rapid acetylators: an electrophysiological study.

Clinical, biochemical and nerve conduction studies were performed in 100 cases of tuberculosis taking isonicotinic acid hydrozide (isoniazid) for more than 12 weeks. Electro-physiological studies were carried out in a similar number of normal age and sex matched controls. In 16 percent of cases an abnormality was documented in the motor nerve conduction velocity, amplitude and terminal latency of the common peroneal, ulnar and median nerves; of these, only two patients had objective evidence of neuritis. The occurrence of isoniazid neuropathy was found to be more in the fourth decade of life (10 of 16), in those who had taken the drug for over six months (13 of 16), and in 'slow' inactivators (10 of 16).

The effect of moderate haemodilution with Fluosol-DA or Hespan on the nonmicrosomal acetylation of sulphadimidine in the rat.

The effects of Fluosol-DA (Fluosol) and Hespan haemodilution on the nonmicrosomal acetylation of sulphadimidine were studied in male rats. Fluosol increased the acetylsulphadimidine percent excreted in urine, the metabolic formation rate constant (kf), and the formation clearance (CLF) for 72 h after haemodilution without any significant changes in the sulphadimidine apparent volume of distribution (Vd) or total body clearance (CL). Hespan haemodilution increased the acetylsulphadimidine percent excreted in urine only at 48 h while significantly decreasing the sulphadimidine clearance, urinary excretion rate constant (ku), and renal clearance (CLR) for 72 h. The enhanced N-acetyltransferase activity after Fluosol haemodilution may have therapeutic consequences for concomitantly given drugs metabolized by this enzyme.

N-acetylation and oxidative capacity in aged volunteers determined with sulfamethazine and antipyrine.

The elimination of antipyrine (AP 15 mg/kg) and sulfamethazine (SM 500 mg) was measured in healthy volunteers of rapid and slow acetylator phenotype. Nineteen males were 20-32 years of age, 11 males and 6 females between 62-86 years of age. Apparent volume of distribution of AP was reduced in advanced age independent of the acetylator status of the individuals. Total body clearance was significantly lower and half-time and mean residence time were higher only in slow but not in rapid acetylators. In the elderly of both phenotypes, the acetylation ratios of SM were significantly enhanced. Renal and metabolic clearance were decreased and AUC-values of SM and its acetylated metabolite were increased in slow but unchanged in rapid acetylators. Physiological peculiarities of distribution and renal excretion of SM and its acetylated metabolite in advanced age may have caused the contradictory results.