Tissue/fluid correlation study for the depletion of sulfadimethoxine in bovine kidney, liver, plasma, urine, and oral fluid.

Sulfonamides are among the oldest, but still effective, antimicrobial veterinary medicines. In steers and dairy cows, the sulfonamides are effective in the treatment of respiratory disease and general infections. Sulfadimethoxine (SDM) has been approved by US Food and Drug Administration (FDA) for use in steers and dairy cows with a tolerance of 100 ng/g (ppb) in edible tissues and 10 ppb in milk. The detection of SDM residue above tolerance in the animal slaughtered for food process will result in the whole carcass being discarded. This report describes a comprehensive depletion study of SDM (and its main metabolite) in plasma, urine, oral fluid, kidney, and liver. In this study, nine steers were injected intravenously with the approved dose of SDM; the loading dose was 55 mg/kg, followed by 27.5 mg/kg dose at 24 h and again at 48 h. Fluids (blood, urine, and saliva) and tissue (liver and kidney) samples were collected at intervals after the last dose of SMD. The combination of laparoscopic serial sampling technique with the liquid chromatography/mass spectrometry method provided the data to establish the tissue/fluid correlation in the depletion of SMD. A strong correlation and linearity of the log-scale concentration over time in the depletion stage has been confirmed for kidney, liver, and plasma.

A novel chemiluminescence quenching method for determination of sulfonamides in pharmaceutical and biological fluid based on luminol-Ag(III) complex reaction in alkaline solution.

A novel chemiluminescence (CL) quenching method for the determination of sulfonamides is proposed. The CL reaction between Ag(III) complex [Ag(HIO6)2]5⁻ and luminol in alkaline solution was investigated. The quenching effect of sulfonamides on CL emission of [Ag(HIO6)2]5⁻-luminol system was found. Quenching degree of CL emission was proportional to sulfonamide concentration. The effects of the reaction conditions on CL emission and quenching were examined. Under optimal conditions, the detection limits (s/n = 3) were 7.2, 17 and 8.3 ng/mL for sulfadiazine, sulfameter, and sulfadimethoxine, respectively. The recoveries of the three drugs were in the range of 91.3-110% with RSDs of 1.9-2.7% for urine samples, and 106-112% with RSDs of 1.6-2.8% for serum samples. The proposed method was used for the determination of sulfadiazine at clinically relevant concentrations in real urine and serum samples with satisfactory results.

Determination of sulfadimethoxine and 4N-acetylsulfadimethoxine in bovine plasma, urine, oral fluid, and kidney and liver biopsy samples obtained surgically from standing animals by LC/MS/MS.

A quantitative method was developed and validated to measure the concentration of sulfadimethoxine (SDM) and its major metabolite, (4)N-acetylsulfadimethoxine (AcSDM), in bovine tissues and body fluids. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) gave quantitative results for these two analytes in extracts from bovine plasma, urine, oral fluid, kidney, and liver, using SDM-d(4) as internal standard (I.S.). The lower limit of quantitation (LLOQ) for both analytes in these matrices was validated at 2, 100, and 5 ng/mL in plasma, urine, and oral fluid respectively, and 10 ng/g in both kidney (cortex) and liver. The overall accuracy (average of 4 levels) is, for plasma, 104% (SDM) and 95% (AcSDM), with standard deviation of 9% (SDM) and 15% (AcSDM); for urine, 100% (SDM) and 106% (AcSDM), with standard deviation of 5% (SDM) and 6% (AcSDM); for oral fluid, 103% (SDM) and 103% (AcSDM), with standard deviation of 4% (SDM) and 4% (AcSDM); for kidney, 101% (SDM) and 111% (AcSDM), with standard deviation of 7% (SDM) and 6% (AcSDM); and for liver, 99% (SDM) and 115% (AcSDM), with standard deviation of 11% (SDM) and 9% (AcSDM). C18 SPE cartridges were used to clean-up these matrices, except for urine which was diluted directly with buffer before analysis by LC/MS/MS.

Disposition of sulfadimethoxine in male llamas (Llama glama) after single intravenous and oral administrations.

This study determined the disposition of sulfadimethoxine in six, healthy, adult, gelded male llamas (Llama glama) by using a nonrandomized crossover design with i.v. dosing (58.8 +/- 3.0 mg/kg based on metabolic scaling) followed by oral dosing (59.3 mg/kg +/- 8.3). Blood samples were collected intermittently for a 72-hr period, and serum sulfadimethoxine concentrations were quantified using high-performance liquid chromatography. Serum sulfadimethoxine concentrations across time were subjected to standard pharmacokinetic analysis based on linear regression. Mean maximum serum concentration after oral dosing was 23.6 +/- 14.9 microg/ml, and extrapolated peak concentration after i.v. administration was 246.6 +/- 15.8 microg/ml. Total clearance of sulfadimethoxine was 45.4 +/- 13.9 L/kg. Half-lives after i.v. and oral administration were 541 +/- 111 min and 642.4 +/- 204.8 min, respectively. Oral bioavailability was 52.6 +/- 15%. These data suggest that the oral dose administered to llamas in this study, based on metabolic scaling from cattle, may be inadequate when compared with the reported minimum inhibitory concentration (512 microg/ml) breakpoint for sulfadimethoxine.

Sulfadimethoxine and rhodamine B as oral biomarkers for European badgers (Meles meles).

A field study was carried out on Little Island (County Waterford, Ireland) in June 2000 to evaluate the potential of a bait-marking system for use in European badgers (Meles meles). Two oral biomarkers, sulfadimethoxine (SDM) and rhodamine B, were incorporated into fishmeal baits and distributed by hand at main sets in five test territories for 3 consecutive days. In parallel, non-biomarked baits were distributed at a single control territory. The objectives of the study were to: (1) assess the effects of SDM and rhodamine B on palatability and thus bait acceptance, and (2) investigate the marking capacity of SDM and rhodamine B in serum and hair samples taken from badgers. Trapping was carried out in each territory for 5 consecutive days immediately after bait distribution. Analysis of data revealed that 90-100% of baits were removed in four of the test territories and from the control territory. In the fifth test territory, 61% of baits were removed. Of the badgers (n = 26) trapped in the test territories, 18 (69%) were positive when tested for both biomarkers. In contrast, the remaining eight animals and those captured in the control territory (n = 6 badgers) were negative. In the marked animals, the highest levels of SDM were recorded in serum samples taken soon after bait distribution. Thereafter, the levels declined in each badger over the course of the study. In contrast, rhodamine B was readily detectable by fluorescence microscopy of hair samples throughout the period of study. The results indicate that SDM and rhodamine B act as systemic markers in badgers and have potential future applications for monitoring of oral vaccine uptake.

Pharmacokinetics of sulfadimethoxine and sulfamethoxazole in combination with trimethoprim after oral single- and multiple-dose administration to healthy pigs.

The pharmacokinetics were studied of sulfadimethoxine (SDM) or sulfamethoxazole (SMX) in combination with trimethoprim (TMP) administered as a single oral dose (25 mg + 5 mg per kg body weight) to two groups of 6 healthy pigs. The elimination half-lives of SMX and TMP were quite similar (2-3 h); SDM had a relatively long half-life of 13 h. Both sulfonamides (S) were exclusively metabolized to N4-acetyl derivatives but to different extents. The main metabolic pathway for TMP was O-demethylation and subsequent conjugation. In addition, the plasma concentrations of these drugs and their main metabolites after medication with different in-feed concentrations were determined. The drug (S:TMP) concentrations in the feed were 250:50, 500:100, and 1000:200 mg per kg. Steady-state concentrations were achieved within 48 h of feed medication, twice daily (SDM+TMP) or three times a day (SMX+TMP). Protein binding of SDM and its metabolite was high (>93%), whereas SMX, TMP and their metabolites showed moderate binding (48-75%). Feed medication with 500 ppm sulfonamide combined with 100 ppm TMP provided minimum steady-state plasma concentrations (C(ss,min)) higher than the concentration required for inhibition of the growth of 90% of Actinobacillus pleuropneumoniae strains (n = 20).

Field evaluation of a dog owner, participation-based, bait delivery system for the oral immunization of dogs against rabies in Tunisia.

We evaluated a dog owner, participation-based, bait delivery system for the oral immunization of dogs against rabies. In a field study in a semirural area of northern Tunisia, dog owners were asked to come to temporary bait delivery sites. A total of 314 baits were given to 178 dog owners in four sites. The experimental baits used consisted of a freeze-dried core unit containing sulfadimethoxine (SDM) as a biological marker and an aromatized paraffin envelope. No vaccine was used. Preliminary tests had shown that by using a rapid commercial card test, positive SDM serum levels were detected in more than 95% of dogs up to two days after bait ingestion. During the two days following bait delivery, we visited more than 95% of all households in the study area and took blood samples from as many owned dogs as possible. Unconsumed baits were recovered and human contacts with the bait matrix were recorded. The campaign required 7.6 person-min per bait and 13.5 person-min per dog owner for providing baits, gloves, and instructions. The estimated average cost effectiveness ratio per dog accepting a bait was 1.7 US dollars. From the indications given by the dog owners and the results of the SDM test, it was concluded that 85-90% of the owned dogs in the study area had consumed a bait at least partially. Of 314 baits delivered, 78.7% were fully consumed by dogs and 4.1% were recovered during the household survey. The remaining baits (17.2%) that were not recovered were either not consumed or only partially consumed by the target dogs (3.7 baits per 100 inhabitants). These baits probably remained within the highly populated areas and were potentially accessible to other domestic animals and other nontarget species, including humans. Twenty-five unprotected human contacts with baits were recorded (1.7% of all inhabitants). Our study has demonstrated the potential of dog owner based bait delivery. This technique is simple and efficient, particularly if the human population is accustomed to mass immunization in defined centers. Before applying this method on a large scale with live vaccine loaded baits, further studies should focus on minimizing the number of human contacts with the vaccine bait, systematizing contact identification and establishing structures in ensuring proper treatment if exposure to vaccine should occur.

Plasma/muscle ratios of sulfadimethoxine residues in channel catfish (Ictalurus punctatus).

Channel catfish (n = 84) maintained at a water temperature of 27 degrees C were used in a feeding study to determine the plasma to muscle concentration ratios of sulfadimethoxine (SDM) and 4-N-acetylsulfadimethoxine residues. Sulfadimethoxine medicated feed was provided free choice at 42 mg SDM/kg body weight once daily for 5 days and the plasma and muscle concentrations of SDM were determined at selected withdrawal times (6, 12, 24, 48, 72, and 96 hours) following the last dose. Considerable variation in total SDM tissue concentration among fish within a sampling period was observed. For fish (n = 12) at six hours post-dose, total SDM concentrations ranged from 1.4-24.8 micrograms/mL and 0.6-12.6 micrograms/g, with mean total SDM concentrations of 9.1 micrograms/mL and 5.3 micrograms/g for plasma and muscle, respectively. However, a mean plasma:muscle concentration ratio of 1.8:1 +/- 0.3:1 was obtained over all concentrations and sampling periods. The plasma:muscle 95% t distribution interval for individual fish was 1.2:1 to 2.4:1. A correlation coefficient of 0.967 was obtained for the relationship between plasma and muscle total SDM concentration among individual fish (n = 25). Results of this study indicate that plasma total SDM concentration may be used to identify samples containing violative SDM muscle residue. No fish contained total SDM muscle residues greater than the FDA tolerance (0.1 microgram/g) by 48 hours following the final dose.

Transfer of dietary sulphamonomethoxine and sulphadimethoxine into various tissues of laying hens.

1. Sulphamonomethoxine (SMM) or sulphadimethoxine (SDM) were fed to laying hens at a dietary concentration of 400 mg/kg. Concentrations (mg/kg) of SMM and SDM in the blood, kidney, liver, ovary, muscle and adipose tissue, collected at 4, 8, 16 and 24 h after the start of feeding, were determined by HPLC. 2. The relationships between the sulphonamide concentrations (mg/kg) in the tissues and times (h) after the start of the feeding were analysed statistically. 3. Dietary SMM and SDM were transferred throughout the whole body, and concentrations in all tissues became constant 8 h after the start of feeding. 4. Among the 6 tissues examined the constant values (mg/kg) of both SMM and SDM were highest in the kidney and were lowest in adipose tissue. 5. With the exception of adipose tissue, the values of SDM in the tissues were statistically greater than those of SMM.

[Demonstration of two trimethoprim/sulfonamide combinations in bronchoalveolar lavage fluid of horses and determination of blood levels]. / Nachweis zweier Trimethoprim/Sulfonamid-Kombinationen im Bronchialsekret von Pferden und Bestimmung der Blutspiegel.

Five healthy horses were given a sulfadoxine/trimethoprim combination (Borgal, Hoechst AG) i.v. on day 1. The next ten days the horses got once a day a sulfadimethoxine/trimethoprim combination orally (Trafigal, Hoechst AG). The doses were given as recommended. One horse received no medicaments for control. On each horse six bronchoalveolar lavages were performed. Blood samples were taken to calculate blood levels and elimination half lives. To determine the amount of substances in lavage fluid and plasma the high performance liquid chromatography (HPLC) was used. Regularly low quantities of sulfonamides and trimethoprim were detected in lavage-samples. The mean plasma concentration (n = 4) of sulfadoxine and trimethoprim 30 min after i.v. administration was 71.6 and 1.13 micrograms/g respectively. 24 h after injection the sulfadoxine blood level was 3.0 micrograms/g, while trimethoprim was no longer detectable. The average elimination half lives of sulfadoxine and trimethoprim were 7.94 h and 1.35 h respectively. 8 h after oral application (n = 5) the highest mean sulfadimethoxine blood levels of 53.8 micrograms/g were measured. The elimination half life of sulfadimethoxine was 9.77 h. Two hours after feeding the drug the first blood samples were taken. They already contained the highest mean trimethoprim concentration of 0.32 microgram/g plasma.

Distribution and elimination of sulphadimethoxine and its metabolites in treated chicken.

Sulphadimethoxine (SDM), and its metabolites, N4-acetyl SDM, N1-(2-methyl-6-hydroxy-4-pyrimidinyl) sulphanilamide (6-OH-SDM), N1-(6-methyl-2-hydroxy-4-pyrimidinyl) sulphanilamide (2-OH-SDM), N1-(2,6-dihydroxy-4-pyrimidinyl) sulphanilamide (2,6-diOH-SDM) and SDM N1-glucuronide in chicken tissues were extracted, partially purified by Bond Elute SCX cartridges, and assayed and identified by HPLC/LC-MS after administration of SDM to chickens. During the administration and 24 h after withdrawal, SDM and 6-OH-SDM were observed in almost all tissues and excreta. N4-Acetyl SDM and 2,6-diOH-SDM were observed in some tissues, but 2-OH-SDM and SDM N1-glucuronide were observed in a few limited tissues. Twenty four hours after withdrawal, SDM and its metabolites, except 6-OH-SDM, decreased. SDM and its metabolites were eliminated from all tissues within 48 h of withdrawal.

Extraction and liquid chromatographic analysis of sulfadimethoxine and 4-N-acetylsulfadimethoxine residues in channel catfish (Ictalurus punctatus) muscle and plasma.

A simple and rapid method was developed for the simultaneous extraction and liquid chromatographic (LC) determination of sulfadimethoxine (SDM) and 4-N-acetylsulfadimethoxine (N-acetyl SDM) in channel catfish muscle and plasma. Tissues fortified at 0, 50, 100, 200, 400, and 1000 ppb were examined. Matrix solid phase dispersion (MSPD) was used for muscle extraction. Plasma was extracted with a modified MSPD procedure in which 100 microL plasma and 400 mg C18 were blended by Vortex mixing in a disposable chromatographic column. Recovery of SDM based on radioactivity was 79% for muscle and 67% for plasma. Standard curves based on extracted fortified samples were used for quantitation of N-acetyl SDM. LC run times of 12 min were obtained using a microbore analytical column and an isocratic mobile phase of aqueous 0.017M phosphoric acid-acetonitrile at ratios of 71:29 for muscle and 73:27 for plasma extracts. Method detection limits were 26 ng SDM and 26 ng N-acetyl SDM/g muscle, and 33 ng SDM and 11 ng N-acetyl SDM/mL plasma. Intra-assay variation was < 10% for both compounds at all concentrations examined. Inter-assay variation for SDM was 13% for muscle and 14% for plasma, and for N-acetyl SDM was 11% for muscle and 10% for plasma.

Simultaneous determination of ormethoprim and sulphadimethoxine in plasma and muscle of Atlantic salmon (Salmo salar).

A rapid clean-up and high-performance liquid chromatographic method for the simultaneous determination of ormethoprim and sulphadimethoxine in plasma and muscle of Atlantic salmon (Salmo salar) has been developed. Sample preparation is based on protein precipitation using trichloroacetic acid or methanol for plasma and muscle, respectively. The drugs are separated using a reversed-phase C18 analytical column and phosphate buffer-acetonitrile (80:20, v/v) containing 1-heptanesodiumsulphonate and triethylamine, as mobile phase. Detection was performed at 270 nm. The average recovery of ormethoprim was 97.2% in muscle and 95.7% in plasma, whereas the average recovery of sulphadimethoxine was 86.5% in muscle and 90.2% in plasma. The limit of detection at a signal-to-noise ratio of 3 was 50 ng/g and 30 ng/ml for ormethoprim in muscle and plasma respectively and 30 ng/g and 15 ng/ml in muscle and plasma respectively for sulphadimethoxine.

Renal tubular excretion of the N4-acetyl metabolites of sulphasomidine and sulphadimethoxine in the dog.

To investigate whether dogs are able to excrete acetylated drugs by active transport, the plasma kinetics and renal excretion of the N4-acetyl metabolites of sulphasomidine and sulphadimethoxine were studied in the beagle dog after a rapid intravenous bolus injection. Two doses of N4-acetylsulphasomidine (1050 and 105 mg) and one dose of N4-acetylsulphadimethoxine (472 mg) were administered on separate occasions. The renal clearance (CLR) was as follows: N4-acetylsulphasomidine (1050 mg) 34 mL min-1; N4-acetylsulphasomidine (105 mg) 28 mL min-1; and N4-acetylsulphadimethoxine (472 mg) 24 mL min-1. CLR was higher than expected on the basis of the measured glomerular filtration rate, indicating that the N4-acetyl metabolites may be excreted by the renal tubules by active tubular transport. Saturation of the excretion process of N4-acetylsulphasomidine occurred with a transport maximum of 930 +/- 190 micrograms min-1 and a Michaelis-Menten constant of 37 +/- 10 micrograms mL-1. It may be concluded that the dog renal organic anion transport system is able to secrete acetylated sulphonamides.

Pharmacokinetics of sulphadimethoxine in channel catfish (Ictalurus punctatus).

1. Plasma clearance, bioavailability, tissue disposition and elimination of 14C-sulphadimethoxine (SDM) were studied in channel catfish (Ictalurus punctatus) after intravenous (i.v.) and oral dosing (per os; p.o.) at 40 mg/kg body weight. 2. Analysis of blood SDM concentrations over time for intravascularly administered SDM showed that disposition and elimination were best described by a two-compartment pharmacokinetic model; estimated half-lives for SDM in blood were 0.09 and 12.6 h for the distribution and elimination phases, respectively. 3. SDM was found primarily in muscle tissue immediately after oral administration; however, clearance from muscle was rapid, with a half-life of 13.1 h. 4. With time, SDM-derived radioactivity became concentrated in the bile and was eliminated slowly (t 1/2 = 115.5 h). 5. Binding of SDM in channel catfish plasma was low (18%) and was non-specific and dose-independent. 6. With the exception of the initial, rapid clearance of SDM from blood, the pharmacokinetic parameters describing SDM distribution and elimination in channel catfish were similar to values reported for other vertebrate species; the rapid distribution of SDM from blood to the tissues in the catfish may be related to species differences in the plasma binding of SDM.

The role of plasma protein binding on the metabolism and renal excretion of sulphadimethoxine and its metabolite N4-acetylsulphadimethoxine in pigs.

The effects of plasma protein binding on the elimination of sulphadimethoxine (SDM) were examined after intravenous administration of 6.25, 12.5, 25, 50, 100 and 150 mg/kg to pigs. At an early stage of the experiment, the animals were anaesthetised by inhalation of enflurane to obtain a more exact relationship between plasma concentration and the renal excretion. SDM and its acetylated conjugate, N4-acetylsulphadimethoxine (N4-SDM) were detected in plasma and urine of all animals, and the recovery of the doses was almost complete in two animals with negligible renal excretion of SDM. The percentages of plasma protein binding of SDM and N4-SDM were almost similar, and ranged from 30 to 95%, depending on the plasma concentration. The metabolic clearance of SDM by acetylation increased when the plasma protein binding decreased. These results suggested that the main elimination route of SDM in pigs is acetylation, and that the plasma protein binding can have a large effect on the elimination of SDM in pigs. The effect of plasma protein binding on the renal clearance of SDM was not so evident, because urine pH had a much greater effect on it. The deacetylation of N4-SDM was detected after 25 mg/kg intravenous administration of N4-SDM, which suggests that the metabolic clearance of SDM is part of an acetylation-deacetylation equilibrium. Saturation of the active tubular reabsorption of SDM and of the active tubular secretion of N4-SDM was also suggested after higher doses of SDM.

High-performance liquid chromatography of sulphadimethoxine and its N1-glucuronide, N4-acetyl and N4-acetyl-N1-glucuronide metabolites in human plasma and urine.

Sulphadimethoxine is metabolized in humans by N1-glucuronidation and by N4-acetylation. Sulphadimethoxine-N1-glucuronide can be measured by the direct high-performance liquid chromatographic analysis and without enzymic deglucuronidation. The N1-glucuronide can be measured by an isocratic as well as by a gradient mobile phase. The group contribution of the N1-glucuronide moiety to the capacity factor is a reduction of 0.24 in the isocratic system and 0.55 in the gradient system. N4-Acetylation increases the capacity factor by a factor 1.4 in the isocratic system and by 1.06 in the gradient system.

Nonlinear pharmacokinetics of intravenous sulphadimethoxine and its dosage regimen in pigs.

The time courses of the total (Ct) and unbound plasma (Cf) concentration after the i.v. injection of 20, 50 and 100 mg/kg of sulphadimethoxine (SDM) were examined in pigs. The area under the Ct-time curve per unit dose decreased dose-dependently. Vdarea and total body clearance of Ct increased dose-dependently. The concentration-dependent plasma protein binding of SDM was evident after 50 and 100 mg/kg. The time courses of Cf en Ct after 3 doses were analyzed by a one compartment open model with nonlinear plasma protein binding. The agreement between calculated curves of Cf and Ct and the observed values, and relative constancy of pharmacokinetic parameters were obtained over 3 doses. These results suggested that the nonlinear pharmacokinetics of SDM was caused by saturable plasma protein binding. The multiple i.v. dose of SDM was based on the dosage regimen using the nonlinear pharmacokinetic model (50 mg/kg, 24 hour interval, 4 days). The observed Cf was maintained in the intended range by the dosage regimen. Therefore, the dosage regimen based on the nonlinear pharmacokinetics may allow the unbound concentration after i.v. injection of SDM in pigs to be controlled.

[Differential effect of sodium bicarbonate on disposition of sulfadimethoxine and sulfisoxazole in rabbits].

The orally co-administered sodium bicarbonate significantly enhanced the blood concentration of sulfadimethoxine at the early stage after oral administration to rabbits, by increasing its intestinal absorption. On the other hand, the sodium bicarbonate significantly reduced the blood concentration of sulfisoxazole at the elimination phase after oral administration to rabbits, by increasing its urinary excretion. The fact that sodium bicarbonate exhibits different effects in the disposition of these two sulfonamides is an interesting example to gain a better understanding for the complexity of drug interaction.