Quantification of Serum Sulfamethoxazole and Trimethoprim by Ultra-fast Solid-Phase Extraction-Tandem Mass Spectrometry.

BACKGROUND: The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) is used to treat a number of bacterial infections. TMP/SMX concentrations in serum are conventionally monitored using high-performance liquid chromatography (HPLC) or liquid chromatography tandem mass spectrometry. These methods require laborious manual extraction techniques and relatively long sample analysis times, necessitating the development of a simple, high-throughput method. A simple, high-throughput method to measure TMP/SMX using ultra-fast solid-phase extraction (SPE)-tandem mass spectrometry has been developed. METHODS: Calibration standards, quality control materials, and patient samples were precipitated with acetonitrile containing isotopically labeled internal standards. Samples were vortexed, centrifuged for 5 minutes at 2053g, and the resulting supernatant was diluted in aqueous mobile phase and injected onto the C18 SPE cartridge. MS/MS analysis was performed by electrospray ionization in positive ion mode at a rate of <20 seconds per sample. A 5-point linear 1/x calibration curve was used to calculate sample concentrations. RESULTS: The intra-assay precision coefficients of variation were <6% and <7% for SMX and TMP, respectively, and <10% for both interassay precision coefficients of variation. Comparison studies using 50 patient and spiked serum samples showed r values of 0.9890 and 0.9853 and y-intercept values of -1.918 and -1.357, respectively compared with the HPLC reference method. All data points were <±15% of the mean. Linearity [r = 0.9952 (SMX) and 0.9954 (TMP)] was established from 12 to 400 mcg/mL with a detection limit of 0.47 mcg/mL, and 1.2-40 mcg/mL with a detection limit of 0.06 mcg/mL, for SMX and TMP, respectively. For either drug, no significant carryover was observed after samples at the upper limit of quantification. No interference was observed from any of the 77 drugs and respective metabolites tested. CONCLUSIONS: A high-throughput SPE-tandem mass spectrometry method for TMP/SMX quantification was developed. The <20 seconds analysis time is a significant improvement compared with traditional HPLC and liquid chromatography tandem mass spectrometry methods, without sacrificing analytical performance.

The drug interaction potential of daprodustat when coadministered with pioglitazone, rosuvastatin, or trimethoprim in healthy subjects.

This study was conducted to evaluate the likelihood of daprodustat to act as a perpetrator in drug-drug interactions (DDI) with the CYP2C8 enzyme and OATP1B1 transporter using the probe substrates pioglitazone and rosuvastatin as potential victims, respectively. Additionally, this study assessed the effect of a weak CYP2C8 inhibitor, trimethoprim, as a perpetrator of a DDI with daprodustat. This was a two-part study: Part A assessed the effect of coadministration of daprodustat on the pharmacokinetics of pioglitazone and rosuvastatin in 20 subjects; Part B assessed the coadministration of trimethoprim on the pharmacokinetics of daprodustat in 20 subjects. Coadministration of 100 mg of daprodustat with pioglitazone or rosuvastatin had no effect on the plasma exposures of either probe substrate. When trimethoprim was coadministered with 25-mg daprodustat plasma daprodustat AUC and Cmax increased by 48% and 28%, respectively. Additionally, AUC and Cmax for the metabolite GSK2531401 were decreased by 32% and 40%, respectively. Cmax for the other metabolites was slightly decreased (~8-15%) but no changes in AUC were observed. As 100-mg daprodustat exceeds the planned top therapeutic dose, interaction potential of daprodustat as a perpetrator with substrates of the CYP2C8 enzyme and OATP1B1 transporters is very low. Conversely, daprodustat exposure (AUC and Cmax) is likely to increase moderately with coadministration of weak CYP2C8 inhibitors.

Quantitative analysis of elevation of serum creatinine via renal transporter inhibition by trimethoprim in healthy subjects using physiologically-based pharmacokinetic model.

Serum creatinine (SCr) levels rise during trimethoprim therapy for infectious diseases. This study aimed to investigate whether the elevation of SCr can be quantitatively explained using a physiologically-based pharmacokinetic (PBPK) model incorporating inhibition by trimethoprim on tubular secretion of creatinine via renal transporters such as organic cation transporter 2 (OCT2), OCT3, multidrug and toxin extrusion protein 1 (MATE1), and MATE2-K. Firstly, pharmacokinetic parameters in the PBPK model of trimethoprim were determined to reproduce the blood concentration profile after a single intravenous and oral administration of trimethoprim in healthy subjects. The model was verified with datasets of both cumulative urinary excretions after a single administration and the blood concentration profile after repeated oral administration. The pharmacokinetic model of creatinine consisted of the creatinine synthesis rate, distribution volume, and creatinine clearance (CLcre), including tubular secretion via each transporter. When combining the models for trimethoprim and creatinine, the predicted increments in SCr from baseline were 29.0%, 39.5%, and 25.8% at trimethoprim dosages of 5 mg/kg (b.i.d.), 5 mg/kg (q.i.d.), and 200 mg (b.i.d.), respectively, which were comparable with the observed values. The present model analysis enabled us to quantitatively explain increments in SCr during trimethoprim treatment by its inhibition of renal transporters.

Electromembrane extraction of polar basic drugs from plasma with pure bis(2-ethylhexyl) phosphite as supported liquid membrane.

Electromembrane extraction (EME) of polar basic drugs from human plasma was investigated for the first time using pure bis(2-ethylhexyl) phosphite (DEHPi) as the supported liquid membrane (SLM). The polar basic drugs metaraminol, benzamidine, sotalol, phenylpropanolamine, ephedrine, and trimethoprim were selected as model analytes, and were extracted from 300 µL of human plasma, through 10 µL of DEHPi as SLM, and into 100 µL of 10 mM formic acid as acceptor solution. The extraction potential across the SLM was 100 V, and extractions were performed for 20 min. After EME, the acceptor solutions were analyzed by high-performance liquid chromatography-ultraviolet detection (HPLC-UV). In contrast to other SLMs reported for polar basic drugs in the literature, the SLM of DEHPi was highly stable in contact with plasma, and the system-current across the SLM was easily kept below 50 µA. Thus, electrolysis in the sample and acceptor solution was kept at an acceptable level with no detrimental consequences. For the polar model analytes, representing a log P range from -0.40 to 1.32, recoveries in the range 25-91% were obtained from human plasma. Strong hydrogen bonding and dipole interactions were probably responsible for efficient transfer of the model analytes into the SLM, and this is the first report on efficient EME of highly polar analytes without using any ionic carrier in the SLM.

Pharmacokinetics of mefloquine and its effect on sulfamethoxazole and trimethoprim steady-state blood levels in intermittent preventive treatment (IPTp) of pregnant HIV-infected women in Kenya.

BACKGROUND: Intermittent preventive treatment in pregnancy with sulfadoxine/pyrimethamine is contra-indicated in HIV-positive pregnant women receiving sulfamethoxazole/trimethoprim prophylaxis. Since mefloquine is being considered as a replacement for sulfadoxine/pyrimethamine in this vulnerable population, an investigation on the pharmacokinetic interactions of mefloquine, sulfamethoxazole and trimethoprim in pregnant, HIV-infected women was performed. METHODS: A double-blinded, placebo-controlled study was conducted with 124 HIV-infected, pregnant women on a standard regimen of sulfamethoxazole/trimethoprim prophylaxis. Seventy-two subjects received three doses of mefloquine (15 mg/kg) at monthly intervals. Dried blood spots were collected from both placebo and mefloquine arms four to 672 h post-administration and on day 7 following a second monthly dose of mefloquine. A novel high-performance liquid chromatographic method was developed to simultaneously measure mefloquine, sulfamethoxazole and trimethoprim from each blood spot. Non-compartmental methods using a naïve-pooled data approach were used to determine mefloquine pharmacokinetic parameters. RESULTS: Sulfamethoxazole/trimethoprim prophylaxis did not noticeably influence mefloquine pharmacokinetics relative to reported values. The mefloquine half-life, observed clearance (CL/f), and area-under-the-curve (AUC0→∞) were 12.0 days, 0.035 l/h/kg and 431 µg-h/ml, respectively. Although trimethoprim steady-state levels were not significantly different between arms, sulfamethoxazole levels showed a significant 53% decrease after mefloquine administration relative to the placebo group and returning to pre-dose levels at 28 days. CONCLUSIONS: Although a transient decrease in sulfamethoxazole levels was observed, there was no change in hospital admissions due to secondary bacterial infections, implying that mefloquine may have provided antimicrobial protection.

N(1)-methylnicotinamide as an endogenous probe for drug interactions by renal cation transporters: studies on the metformin-trimethoprim interaction.

PURPOSE: N(1)-methylnicotinamide (NMN) was proposed as an in vivo probe for drug interactions involving renal cation transporters, which, for example, transport the oral antidiabetic drug metformin, based on a study with the inhibitor pyrimethamine. The role of NMN for predicting other interactions with involvement of renal cation transporters (organic cation transporter 2, OCT2; multidrug and toxin extrusion proteins 1 and 2-K, MATE1 and MATE2-K) is unclear. METHODS: We determined inhibition of metformin or NMN transport by trimethoprim using cell lines expressing OCT2, MATE1, or MATE2-K. Moreover, a randomized, open-label, two-phase crossover study was performed in 12 healthy volunteers. In each phase, 850 mg metformin hydrochloride was administered p.o. in the evening of day 4 and in the morning of day 5. In phase B, 200 mg trimethoprim was administered additionally p.o. twice daily for 5 days. Metformin pharmacokinetics and effects (measured by OGTT) and NMN pharmacokinetics were determined. RESULTS: Trimethoprim inhibited metformin transport with K i values of 27.2, 6.3, and 28.9 µM and NMN transport with IC50 values of 133.9, 29.1, and 0.61 µM for OCT2, MATE1, and MATE2-K, respectively. In the clinical study, trimethoprim increased metformin area under the plasma concentration-time curve (AUC) by 29.5 % and decreased metformin and NMN renal clearances by 26.4 and 19.9 %, respectively (p ≤ 0.01). Moreover, decreases of NMN and metformin renal clearances due to trimethoprim correlated significantly (r S=0.727, p=0.010). CONCLUSIONS: These data on the metformin-trimethoprim interaction support the potential utility of N(1)-methylnicotinamide as an endogenous probe for renal drug-drug interactions with involvement of renal cation transporters.

Pharmacokinetic interaction of enrofloxacin/trimethoprim combination following single-dose intraperitoneal and oral administration in rats.

The pharmacokinetic interaction of enrofloxacin and trimethoprim was evaluated after single-dose intraperitoneal or oral co-administration in rats. Plasma concentrations of the two drugs were determined by high-performance liquid chromatography. Following intraperitoneal combination, a significant (P < 0.05) increase in mean values of plasma half-life (t 1/2) and maximum plasma concentration (C max) was observed for enrofloxacin and trimethoprim, respectively. There was a significant (P < 0.05) increase in mean values of area under the plasma drug concentration versus time from time zero to infinity (AUC0-∞) and C max between combined oral doses (10, 30 and 100 mg/kg) of both antibacterial drugs. Also, after oral conjugation a significant difference in mean values of MRT0-∞ was observed between lower (10 mg/kg) and higher (100 mg/kg) doses of both drugs. A significant increase in pharmacokinetic parameters of both drugs in combined intraperitoneal and oral doses indicated pharmacokinetic interaction of enrofloxacin and trimethoprim. Further study is recommended in other species of animals.

No pharmacokinetic interaction between paliperidone extended-release tablets and trimethoprim in healthy subjects.

OBJECTIVE: The effect of trimethoprim, a potent organic cation transport inhibitor, on the pharmacokinetics (PK) of paliperidone extended-release tablets (paliperidone ER), an organic cation mainly eliminated via renal excretion, was assessed. METHODS: Open-label, two-period, randomized, crossover study in 30 healthy males. Single dose of paliperidone ER 6 mg was administered either alone on day 1 or day 5 during an 8-day treatment period of trimethoprim 200 mg twice daily. Serial blood and urine samples were collected for PK and plasma protein binding of paliperidone and its enantiomers. The 90% confidence interval (CI) of ratios with/without trimethoprim for PK parameters of paliperidone and its enantiomers calculated. RESULTS: Creatinine clearance decreased from 119 to 102 mL min(-1) with trimethoprim. Addition of trimethoprim increased unbound fraction of paliperidone by 16%, renal clearance by 13%, AUC(infinity) by 9%, and t((1/2)) by 19%. The 90% CIs for ratios with/without trimethoprim were within the 80-125% range for C(max), AUC(last), and renal clearance. For AUC(infinity), 90% CI was 79.37-101.51, marginally below the lower bound of the acceptance range. Paliperidone did not affect steady-state plasma concentrations of trimethoprim. CONCLUSIONS: No clinically important drug interactions are expected when paliperidone ER is administered with organic cation transport inhibitors.

Pharmacokinetics and bioavailability of sulfadiazine and trimethoprim following intravenous, intramuscular and oral administration in ostriches (Struthio camelus).

A pharmacokinetic and bioavailability study of sulfadiazine combined with trimethoprim (sulfadiazine/trimethoprim) was carried out in fifteen healthy young ostriches after intravenous (i.v.), intramuscular (i.m.) and oral administration at a total dose of 30 mg/kg body weight (bw) (25 and 5 mg/kg bw of sulfadiazine and trimethoprim, respectively). The study followed a single dose, three periods, cross-over randomized design. The sulfadiazine/trimethoprim combination was administered to ostriches after an overnight fasting on three treatment days, each separated by a 2-week washout period. Blood samples were collected at 0 (pretreatment), 0.08, 0.25, 0.50, 1, 2, 4, 6, 8, 12, 24 and 48 h after drug administration. Following i.v. administration, the elimination half-life (t(1/2beta)), the mean residence time (MRT), volume of distribution at steady-state (V(d(ss))), volume of distribution based on terminal phase (V(d(z))), and the total body clearance (Cl(B)) were (13.23 +/- 2.24 and 1.95 +/- 0.19 h), (10.06 +/- 0.33 and 2.17 +/- 0.20 h), (0.60 +/- 0.08, and 2.35 +/- 0.14 L/kg), (0.79 +/- 0.12 and 2.49 +/- 0.14 L/kg) and (0.69 +/- 0.03 and 16.12 +/- 1.38 mL/min/kg), for sulfadiazine and trimethoprim, respectively. No significant difference in C(max) (35.47 +/- 2.52 and 37.50 +/- 3.39 microg/mL), t(max) (2.47 +/- 0.31 and 2.47 +/- 0.36 h), t((1/2)beta) (11.79 +/- 0.79 and 10.96 +/- 0.56 h), V(d(z))/F (0.77 +/- 0.06 and 0.89 +/- 0.07 L/kg), Cl(B)/F (0.76 +/- 0.04 and 0.89 +/- 0.07) and MRT (12.39 +/- 0.40 and 12.08 +/- 0.36 h) were found in sulfadiazine after i.m. and oral dosing, respectively. There were also no differences in C(max) (0.71 +/- 0.06 and 0.78 +/- 0.10 microg/mL), t(max) (2.07 +/- 0.28 and 3.27 +/- 0.28 h), t((1/2)beta) (3.30 +/- 0.25 and 3.83 +/- 0.33 h), V(d(z))/F (6.2 +/- 0.56 and 6.27 +/- 0.77 L/kg), Cl(B)/F (21.9 +/- 1.46 and 18.83 +/- 1.72) and MRT (3.68 +/- 0.19 and 4.34 +/- 0.14 h) for trimethoprim after i.m. and oral dosing, respectively. The absolute bioavailability (F) was 95.41% and 86.20% for sulfadiazine and 70.02% and 79.58% for trimethoprim after i.m. and oral administration, respectively.

Simultaneous determination and validation of antimicrobials in plasma and tissue of actinomycetoma by high-performance liquid chromatography with diode array and fluorescence detection.

A simple, precise, and reliable chromatographic method was developed for the simultaneous determination in plasma and infected tissue of five antimicrobials proposed for the treatment of actinomycotic mycetoma: amoxicillin, trimethoprim, linezolid, sulfamethoxazole and garenoxacin. Separation of the analytes was achieved on an Atlantis dC18 column (150 mm x 4.6 mm, ID 5 microm) with a mobile phase composed of acetonitrile and trifluoroacetic acid (ATF) 0.1% (v/v) using a gradient program. The detection was carried out using a diode array detector at 254 nm and in a fluorescence detector at wavelengths of excitation and emission of 292 nm and 392 nm for linezolid and sulfamethoxazole, and 292 nm and 408 nm for garenoxacin, respectively. The intraday precision was in the range of 0.7-15% of relative standard deviations (%R.S.D.) for plasma and 1-18% for tissue. Linearity range was from 2.4 to 20 microg/ml for amoxicillin, 0.3 to 20 microg/ml for trimethoprim, sulfamethoxazole and linezolid, and 0.3 to 10 microg/ml for garenoxacin. Acetonitrile was used to precipitate proteins from plasma. Recoveries in plasma ranged from 71% to 118% and in infected tissue from 78% to 122%. Limits of detection (LODs) were 1.2 and 0.5 microg/ml for amoxicillin in plasma and tissue, respectively and 0.15 and 1.2 microg/ml in plasma and tissue, respectively for the other antimicrobials. The method can be applied for individual or simultaneous determination of the antimicrobials in plasma and tissue of mouse infected with actinomycetoma.

Liquid chromatographic method for the simultaneous determination of cefalexin and trimethoprim in dog plasma and application to the pharmacokinetic studies of a coformulated preparation.

A liquid chromatographic method is described for the simultaneous determination of cefalexin and trimethoprim in dog plasma. A simple protein precipitation procedure was adopted for the sample preparation with satisfactory extraction recoveries for both analytes. Chromatographic separation of the analytes was achieved on a C(18) column using a mixture of 2 mol/l formate buffer (pH 3.5), methanol and acetonitrile (22:7:7, v/v/v) containing a 0.002 mol/l sodium dodecyl sulfate as mobile phase and detection was performed at 240 nm. The linearity was obtained over the concentration ranges of 1.0-100.0 microg/ml for cefalexin and 0.5-50.0 microg/ml for trimethoprim. For each level of QC samples including the lower limit of quantification, both inter- and intra-day precisions (R.S.D.) were < or =14.0% for cefalexin and < or =11.4% for trimethoprim, and accuracy (RE) was -1.4% for cefalexin and -3.0% for trimethoprim. The present LC method was successfully applied to the pharmacokinetic studies of coformulated cefalexin dispersible tablets after oral administration to beagle dogs.

Micellar electrokinetic chromatography method for the determination of sulfamethoxazole, trimethoprim and their main metabolites in human serum.

A complete analytical procedure, including sample clean-up and a micellar electrokinetic chromatographic method, is presented for the determination of sulfamethoxazole, trimethoprim, and their main metabolites by using 20 mmol L(-1) borate buffer (pH 9.3), 25 mmol L(-1) sodium dodecylsulfate, and 5% v/v acetonitrile as electrolyte. The separation was carried out at 30 kV and 20 degrees C in a fused silica capillary (60.2 cm x 75 microm inner diameter) fitted with a window in the capillary cartridge of 100 x 800 microm. The detector response was linear from the limit of quantification to 3 mg L(-1) for the individual components. The limits of quantification ranged from 0.13 up to 0.24 mg L(-1). The method was applied to human serum, previously spiked at different concentrations of all the analytes, and recoveries between 95% and 108% were obtained.

Sustained availability of trimethoprim in drinking water to achieve higher plasma sulphonamide-trimethoprim antibacterial activity in broilers.

(1) In order to make trimethoprim (TMP) available to broilers throughout the day, a sustained release formulation (SRF) of the drug in the form of granules was added to the water tank that supplies drinking water. (2) Broilers were initially dosed with sulphachloropiridazine-TMP (SCP-TMP 5:1) and then further medicated throughout the day, achieving in the end a dose of 30 mg/kg each of SCP and TMP (group A). Group B received a preparation with the same dose of SCP and TMP (1:1) as group A, but administered as a single dose without the SRF of TMP. Group C received the customary SCP-TMP 5:1 preparation (30 and 6 mg/kg, respectively). Water tanks were completely consumed in 3 to 4 h. (3) Broilers were bled at different times and concentration of antibacterial activity in serum determined by correlating the composite antibacterial activity of SCP and TMP with actual concentrations of these drugs by means of a microbiological agar diffusion assay. (4) Time vs serum concentrations of activity were higher in group B; the increments in the maximum serum concentration for group B over groups A and C being 39 and 67%, respectively. (5) However, the sustained concentration of activity over time, measured as the area under the cu)rve, was highest in group A. Group B had higher values for area under the curve than group C. (6) An additional dose of TMP to achieve 30 mg/kg of both SCP and TMP improves the serum concentration of this combination over the customary 5:1 proportion. The best values for sustaining antibacterial activity were obtained using a 1:1 ratio as in group A. The use of a SRF as in group A may translate into better clinical results.

Serum activity/concentration profiles of a sustained-released formulation of sulphathiazole-trimethoprim (2.5:1) in calves.

Thirty-six two-week-old healthy Holstein-Friesian calves weighing between 52 and 58 kg were divided at random into three groups of 12; group A calves were given a single oral bolus containing 2.5 g sulphathiazole and 1 g trimethoprim in a sustained-release formulation; group B received the same doses of the drugs but the trimethoprim was not in a sustained-release formulation; group C received a bolus containing 2.5 g sulphathiazole and 0.5 g conventional trimethoprim. Blood samples were collected at intervals for two days, the serum was separated and the composite antibacterial activity profiles of the mixture were analysed by an agar-diffusion microbiological method. The mean maximum activities in the serum of the three groups were 23.4 microg/ml in group A, 9.25 microg/ml in group B and 8.01 microg/ml in group C. The mean areas under the curves of the serum activity time curves were 838 microg/ml/hour in group A, 216 microg/ml/hour in group B and 182 microg/ml/hour in group C.

Effects of trimethoprim-sulfadiazine on thyroid function of horses.

Trimethoprim-sulfadiazine was administered to horses in a randomized, placebo controlled study to determine the effects of potentiated sulfonamides on thyroid function in normal horses. The treatment group included eight horses that received trimethoprim-sulfadiazine mixed with molasses orally at 30 mg/kg once daily for eight weeks. The control group included 8 horses that received an oral placebo (flour mixed with molasses) once daily for the same period. Thyroid function was evaluated prior to initiation of treatment and after 8 weeks of treatment. Serum concentrations of total and free triiodothyronine (T3), total and free thyroxine (T4), and thyroid stimulating hormone (TSH) were determined at rest and after a thyrotropin-releasing hormone (TRH) stimulation test. There was no detectable difference between treatment and control groups.

Determination of trimethoprim in low-volume human plasma by liquid chromatography.

Trimethoprim is an anti-infective agent used in the treatment of urinary and respiratory tract infections and mild to moderate pneumocystis carinii pneumonia. Trimethoprim is also a selective in vitro inhibitor of cytochrome P450 2C8 and may have utility as an in vivo inhibitor of this enzyme. A simplified high performance liquid chromatography (HPLC) method was developed to determine trimethoprim in human plasma. Samples are processed by protein precipitation with perchloric acid and chromatographic separation is achieved on a Synergi Polar-RP column (4 micron, 150 mm x 4.6 mm) using a mobile phase consisting of 50 mM ammonium formate-acetonitrile-methanol (pH=3.0; 90:6:4 (v/v/v)). Detection is monitored at 280 nm. Intra- and inter-day precision ranged from 1.1 to 1.9 and 0.9 to 4.1%, respectively. The assay is simple, economical, precise, and is directly applicable to human studies involving steady state trimethoprim pharmacokinetics.

Simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by capillary zone electrophoresis.

A capillary electrophoretic method for the simultaneous determination of sulfamethoxazole and trimethoprim in plasma was developed. Sulfamethoxazole and trimethoprim extracted from human plasma with ethyl acetate were analyzed at 20 kV and 25 degrees C using 15 mm phosphate buffer (pH 6.2) as the electrolyte. The detection was by UV at 220 nm. The run time was 8.0 min and the limit of quantification was 10.00 microg/mL for sulfamethoxazole and 2.00 microg/mL for trimethoprim. The recovery was >99% for both compounds. This method enabled the detection of sulfamethoxazole and trimethoprim in plasma of patients after oral ingestion of their combined formulation. The present simple and rapid method is applicable to drug monitoring in immunocompromised patients who are taking the combined formulation of these compounds for the treatment or prophylaxis of Pneumocystis carinii pneumonia.

Pharmacokinetics and oral bioavailability of sulfadiazine and trimethoprim in broiler chickens.

Sulfonamides and trimethoprim are chemotherapeutics that are extensively used in various animal species. Little information about the pharmacokinetics of these compounds in chickens exists in the literature. In this study, a new commercial formulation of sulfadiazine in combination with trimethoprim was administered both intravenously and orally, according to a crossover design, to healthy, 7-week-old broilers. The plasma concentrations of the drugs were determined by validated high-performance liquid chromatographic methods, and pharmacokinetic parameters were calculated. After intravenous or oral administration of trimethoprim (6.67 mg/kg body weight) and sulfadiazine (33.34 mg/kg body weight), both active substances were rapidly eliminated from the plasma. There was a mean half-life of 1.61 h for trimethoprim and 3.2 h for sulfadiazine. The apparent volumes of distribution (2.2 and 0.43 L/kg, respectively, indicated that the tissue distribution of trimethoprim was more extensive than that of sulfadiazine. The oral bioavailability was approximately 80% for both components.

Comparison of a liquid chromatographic method with ultraviolet and ion-trap tandem mass spectrometric detection for the simultaneous determination of sulfadiazine and trimethoprim in plasma from dogs.

A method for the simultaneous determination of sulfadiazine and trimethoprim in plasma from Beagle dogs was developed and validated. Samples were deproteinized with acetonitrile and extracted with ethyl acetate. Sulfachloropyridazine and ormethoprim were used as internal standards for the sulfadiazine and trimethoprim analysis, respectively. The chromatography was carried out both on an LC-UV (liquid chromatography-ultraviolet detection) and ion-trap LC-MS(n) (liquid chromatography-mass spectrometric detection) instrument, operating in the positive APCI mode (atmospheric pressure chemical ionization). The purpose of this work was to compare the quantification results of both methods. Both the LC-UV and LC-MS-MS methods were validated for their linearity, accuracy, precision, limit of detection and limit of quantification, according to the requirements defined by the European Community. Calibration curves using plasma fortified between 0.1 and 1 microg/ml of sulfadiazine, 0.1 and 2 microg/ml of trimethoprim, 1 and 20 microg/ml of sulfadiazine showed a good linear correlation (r> or =0.9990, goodness-of-fit< or =8.4%). The results for the accuracy and precision at 1 microg/ml of sulfadiazine and trimethoprim and at 20 microg/ml of sulfadiazine fell within the ranges specified. The limits of quantification of both methods were 0.1 microg/ml. The limits of detection were 0.019 microg/ml of sulfadiazine and 0.024 microg/ml of trimethoprim for the LC-UV method, and 0.020 microg/ml of sulfadiazine and 0.062 microg/ml of trimethoprim for the LC-MS-MS method. The methods have been successfully applied in a pharmacokinetic study to determine the drug concentrations in plasma samples from dogs. A good correlation between the results of both methods was observed (R=0.9724, slope=1.0239, intercept=-0.2080 microg/ml for sulfadiazine and R=0.9357, slope=1.0433, intercept=0.0325 microg/ml for trimethoprim). The precision of both methods was also tested on the results of the same samples using an F-test (alpha=0.05), indicating that both methods did not differ in precision.

Efficacy of trimethoprim-sulfadoxine against Escherichia coli in a tissue cage model in calves.

Tissue cages implanted subcutaneously in calves were infected with Escherichia coli. Twenty-four hours later, the calves were treated either with single doses of 2.5 + 12.5 or 5 + 25 mg/kg trimethoprim (TMP) + sulfadoxine (SDX) or with five doses of 7.5 + 37.5 mg/kg TMP + SDX at 12-h intervals. In addition, one cage in each of three calves in the highest dose group was infected 3 h after initiation of treatment. Untreated calves were kept as controls. Concentrations of TMP and SDX in plasma and tissue cage fluid (TCF) and counts of viable bacteria in TCF were determined. In the highest dose group, concentrations of TMP in TCF remained above the minimum inhibitory concentration of the test strain for 94-101 h and peak to minimum inhibitory concentration (MIC) ratio was close to 10. In spite of this, an effect of treatment was noted only in cages infected after initiation of treatment. In vitro studies and analysis of thymidine content in serum and TCF from calves suggest that levels of thymidine in TCF are high enough to antagonize the antibacterial effect of TMP. The results indicate that soft tissue infections in secluded infection sites of calves are refractory to treatment with TMP + SDX.