Development and validation of method for the simultaneous determination of sulfamethazine, trimethoprim and doxycycline in veterinary formulation using high performance liquid chromatography.

Sulfamethazine (SMZ), trimethoprim (TMP) and doxycycline (DOXY) are drugs of choice used in the treatment of intestinal and respiratory infections that affect poultry and swine. The aim of this study was develop and validate a simple, sensitive and fast method for the simultaneous determination of SMZ, TMP and DOXY in veterinary formulations by high-performance liquid chromatography. The separation was performed on a Macherey-Nagel C8 analytical column (4 × 125 mm, 5 µm), with a flow rate of 0.5 ml min-1 and detection at 268, 270 and 350 nm, for SMZ, TMP and DOXY, respectively. All measurements were performed in acetonitrile-water (45:55 v/v; pH 3.0). The analytical curves were linear (r > 0.9997) in the concentration range of 5.0-35.0 µg ml-1 for SMZ, 1.0-7.0 µg ml-1 for TMP and 7.0-13.0 µg ml-1 for DOXY. The method proved to be precise, robust, accurate and selective. In accelerated stability, the sample was analyzed for 6 months, with no major variations observed in organoleptic analysis and pH. Therefore, the developed method was proved to be suitable for routine quality control analyses for the simultaneous determination of SMZ, TMP and DOXY in pharmaceutical formulations.

Simultaneous attenuation of pharmaceuticals, organic matter, and nutrients in wastewater effluent through managed aquifer recharge: Batch and column studies.

Batch and column experiments were conducted to evaluate the removal of organic matter, nutrients, and pharmaceuticals and to identify the removal mechanisms of the target contaminants. The sands used in the experiments were obtained from the Youngsan River located in South Korea. Neutral and cationic pharmaceuticals (iopromide, estrone, and trimethoprim) were removed with efficiencies greater than 80% from different sand media during experiments, due to the effect of sorption between sand and pharmaceuticals. However, the anionic pharmaceuticals (sulfamethoxazole, ketoprofen, ibuprofen, and diclofenac) were more effectively removed by natural sand, compared to baked sand. These observations were mainly attributed to biodegradation under natural conditions of surface organic matter and ATP concentrations. The removal of organic matter and nitrogen was also found to increase under biotic conditions. Therefore, it is indicated that biodegradation plays an important role and act as major mechanisms for the removal of organic matter, nutrients, and selected pharmaceuticals during sand passage and the managed aquifer recharge, which is an effective treatment method for removing target contaminants. However, the low removal efficiencies of pharmaceuticals (e.g., carbamazepine and sulfamethoxazole) require additional processes (e.g., AOPs, NF and RO membrane), a long residence time, and long travel distance for increasing the removal efficiencies.

Mechanism considerations for photocatalytic oxidation, ozonation and photocatalytic ozonation of some pharmaceutical compounds in water.

Aqueous solutions of four pharmaceutical compounds, belonging to the group of emergent contaminants of water: atenolol (ATL), hydrochlorothiazide (HCT), ofloxacin (OFX) and trimethoprim (TMP), have been treated with different oxidation systems, mainly, photocatalytic oxidation, ozonation and photocatalytic ozonation. TiO2 has been used as semiconductor for photocatalytic reactions both in the presence of air, oxygen or ozone-oxygen gas mixtures. Black light lamps mainly emitting at 365 nm were the source of radiation. In all cases, the influence of some variables (concentrations of semiconductor, ozone gas and pharmaceuticals and pH) on the removal of pharmaceuticals, total polyphenol content (TPC) and total organic carbon (TOC) was investigated. A discussion on the possible routes of pharmaceutical and intermediates (as TPC and TOC) elimination has been developed. Thus, OFX TiO2/UVA degradation mechanism seems to develop through the participation of non-hydroxyl free radical species. Furthermore, the presence of OFX inhibits the formation of hydroxyl radicals in the photocatalytic process. The most effective processes were those involving ozone that lead to complete disappearance of parent compounds in less than 30 min for initial pharmaceutical concentrations lower than 2.5 mg L(-1). In the ozonation systems, regardless of the pH and the presence of TiO2, pharmaceuticals are degraded through their direct reaction with ozone. Photocatalytic ozonation was the most efficient process for TPC and TOC removals (≥ 80% and ≥60% elimination after 2 h of treatment, respectively) as well as in terms of the ozone consumption efficiency (1, 5.5 and 4 mol of ozone consumed per mol of TOC mineralized, at pH 4, 7 and 9, respectively). Weakly acid conditions (pH 4) resulted to be the most convenient ones for TPC and TOC removal by photocatalytic ozonation. This was likely due to formation of hydroxyl radicals through the ozonide generated at these conditions.

Environmental monitoring study of selected veterinary antibiotics in animal manure and soils in Austria.

LC-MS/MS was used for determination of selected tetracyclines, sulfonamides, trimethoprim, and fluoroquinolones in manure samples of pig, chicken and turkey, as well as arable soils fertilized with manure. Recoveries from spiked samples ranged from 61 to 105%. Method quantification limits were set to 100 microg/kg for all substances. Analysis of 30 pig manure, 20 chicken and turkey dung, and 30 lyophilized soil samples taken in Austria revealed that in pig manure up to 46 mg/kg chlortetracycline, 29 mg/kg oxytetracycline and 23 mg/kg tetracycline could be detected. As representatives of the group of sulfonamides, sulfadimidine in pig manure and sulfadiazine in chicken and turkey dung were detected in significant amounts (maximum concentration, 20 and 91 mg/kg, respectively). Enrofloxacin was particularly observed in chicken and turkey samples. Positive detection of chlortetracycline, enrofloxacin, and ciprofloxacin, in soil samples should be outlined as most important results of this study.

Determination of isomeric N-oxide metabolites of some substituted 2,4-diaminopyrimidines by reversed-phase ion-pair high-performance liquid chromatography.

Reversed-phase ion-pair high-performance liquid chromatography has been investigated for the separation, detection, identification and quantitation of the isomeric 1N- and 3N-oxide metabolites of metoprine, pyrimethamine and trimethoprim. A rapid and sensitive analytical method for the simultaneous determination of the isomeric 1N- and 3N-oxides of metoprine and pyrimethamine was devised for in vitro metabolic studies by optimisation of mobile phase pH, pairing-ion concentration, secondary-ion concentration and percentage organic modifier.

[In vivo kinetics of cotrimoxazole in alveolar macrophages]. / Cinétique in vivo du cotrimoxazole dans les macrophages alvéolaires.

The aim of this work was to study the kinetic of intramacrophage penetration of cotrimoxazole in guinea pigs which had received 100 mg/kg of sulfamethoxazole and 20 mg/kg of trimethoprim after a single intraperitoneal injection. 30 minutes, 1, 3, 6 and 24 hours after this injection an intra-cardiac blood sample was taken and pulmonary lavage was performed immediately after sacrificing the animal by cervical cord dislocation. The level of trimethoprim and sulfamethoxazole was measured in each sample by high performance liquid chromatography (HPLC). An estimation of the dilution of the supernatant was obtained by comparing the supernatant glucose with the serum glucose. The serum kinetics of trimethoprim and sulfamethoxazole progressed in a parallel fasion with time with a maximal concentration at 30 minutes (for trimethoprim: 6.7 +/- 0.9 micrograms/ml and for sulfamethoxazole 176.1 +/- 16.2 micrograms/ml). On the other hand their penetration capacity was different in the supernatant and in the alveolar macrophages: the maximal concentrations were obtained after one hour in the supernatant and after 3 hours in the cellular extract and were respectively for trimethoprim 0.43 +/- 0.07 microgram/ml and 20.9 +/- 8.06 micrograms/ml of intramacrophage water and for sulfamethoxazole 1.86 +/- 0.24 micrograms/ml and 23.8 +/- 12.7 micrograms/ml of intramacrophage water. A concentration around six times greater was noted for the trimethoprim inside the cells compared with serum and was only 0.25 time for sulfamethoxazole. On the other hand the supernatant/serum ratio showed a greater concentration for trimethoprim (4 to 10) than for sulfamethoxazole (0.6 to 1).(ABSTRACT TRUNCATED AT 250 WORDS)

[Study of the in vivo penetration of cotrimoxazole in alveolar macrophages]. / Etude de la pénétration in vivo du cotrimoxazole dans les macrophages alvéolaires.

Kinetic of cotrimoxazole was studied in serum, alveolar macrophages and BAL fluid from guinea pigs receiving sulfamethoxazole (SMX, 100 mg/kg) and trimethoprim (TMP, 20 mg/kg). Guinea pigs were killed by cervical dislocation 30 min, 1 h, 3 h, 6 h and 24 h after intraperitoneal injection. Lung lavage was performed to obtain alveolar macrophages and BAL fluid. TMP and SMX levels were assayed using high-performance-liquid chromatography. Highest SMX levels were obtained in serum at 30 min, in BAL fluid at 1 h and in alveolar macrophages at 3 h. Mean SMX/TMP ratios (30 min, 1 h, 3 h) was 26.5 +/- 0.8 in serum, 3.76 +/- 1.8 in BAL fluid and 1.15 +/- 0.02 in alveolar macrophages.

Sulfaprim inyección: formulación / Inyectable sulfaprim: formulation

Se confeccionaron varias formulaciones inyectables que contienen la combinación de sulfametoxazol y trimetoprim en la proporción 5:1, y se logra una formulación estable. La principal dificultad encontrada durante el desarrollo del trabjo fue la eliminación de la presencia de cristales formados en las ampolletas durante su almacenamiento. Se discuten las distintas variantes. Se realizaron, además, valoraciones químicas y cromatográficas a diferentes muestras de más de 2 años de fabricadas. Se recomienda la utilización de una de las formulaciones con 3 años como fecha de vencimiento. Se reportan los ensayos biológicos y clínicos como satisfactorios

Pharmacodynamic evaluation of ofloxacin and trimethoprim-sulfamethoxazole in vaginal fluid of women treated for acute cystitis.

Vaginal colonization with Escherichia coli is an integral step in the development of acute cystitis, and persistent vaginal coliform colonization may also be a predisposing step to recurrent urinary tract infections. For this reason, we evaluated antibiotic concentrations in the vaginal fluid, serum, and urine and the vaginal colonization by E. coli of 56 women receiving either ofloxacin (200 mg orally twice a day) or trimethoprim-sulfamethoxazole (TMP-SMX) (160/800 mg orally twice a day) for the treatment of acute cystitis. Ofloxacin and trimethoprim both penetrated into vaginal fluid to a considerably greater extent than sulfamethoxazole. Among 33 patients given ofloxacin, the concentration of the drug in vaginal fluid during one dosage interval ranged from 1.6 to 21.6 micrograms/ml. In 21 women given TMP-SMX the range of drug concentrations in vaginal fluid was 2.6 to 32.5 micrograms/ml for TMP and 1.0 to 6.2 micrograms/ml for SMX. Treatment with both ofloxacin and TMP-SMX remarkably reduced vaginal colonization by E. coli during and up to 30 days after therapy. For the ofloxacin-treated women, eradication of vaginal E. coli was associated with a high ratio of drug concentration in vaginal fluid to that in serum. We conclude that ofloxacin and TMP both achieve high concentrations in vaginal fluid and are equally successful in eradicating E. coli from the vagina.

Crystal structure of human dihydrofolate reductase complexed with folate.

The crystal structure of recombinant human dihydrofolate reductase with folate bound in the active site has been determined and the structural model refined at 0.2-nm resolution. Preliminary studies of the binding of the inhibitors methotrexate and trimethoprim to the human apoenzyme have been performed at 0.35-nm resolution. The conformations of the chemically very similar ligands folate and methotrexate, one a substrate the other a potent inhibitor, differ substantially in that their pteridine rings are in inverse orientations relative to their p-aminobenzoyl-L-glutamate moieties. Methotrexate binding is similar to that previously observed in two bacterial enzymes but is quite different from that observed in the enzyme from a mouse lymphoma cell line [Stammers et al. (1987) FEBS Lett. 218, 178-184]. The geometry of the polypeptide chain around the folate binding site in the human enzyme is not consistent with conclusions previously drawn with regard to the species selectivity of the inhibitor trimethoprim [Matthews et al. (1985) J. Biol. Chem. 260, 392-399].

Penetration of trimethoprim and sulfamethoxazole into cysts in a patient with autosomal-dominant polycystic kidney disease.

This study examines the causes for the therapeutic failure of trimethoprim-sulfamethoxazole in a patient with infected cysts caused by a sensitive strain of Escherichia coli. We determined the concentration of trimethoprim and sulfamethoxazole in eight cysts (four proximal, four distal) following therapeutic nephrectomy in a patient treated eight days with trimethoprim-sulfamethoxazole in appropriate doses. In four proximal cysts, mean trimethoprim level was 16.1 +/- 0.8 micrograms/mL with mean sulfamethoxazole level of 94.7 +/- 13.0 micrograms/mL. In distal cysts, mean trimethoprim level was 227.8 +/- 16.8 micrograms/mL with mean sulfamethoxazole level of 9.7 +/- 3.6 micrograms/mL. Serum peak and trough trimethoprim concentrations were 9.8 micrograms/mL and 5.4 micrograms/mL with peak and trough sulfamethoxazole concentrations of 136.0 micrograms/mL and 65.0 micrograms/mL. Significant WBC counts were present in seven cysts, three proximal and four distal. All three proximal cysts were sterile; in contrast, the four distal cysts grew the same strain of E coli isolated from the blood and urine of this patient. The infection resolved following nephrectomy. We conclude that the failure of trimethoprim-sulfamethoxazole to eradicate the infection was caused by the inability of sulfamethoxazole to enter distal cysts in sufficient concentration for the synergistic effect commonly seen with trimethoprim and sulfamethoxazole in combination. Treatment of cyst infections with trimethoprim-sulfamethoxazole should probably be avoided in instances when the organism is resistant to trimethoprim alone.

Assessment of antimicrobial penetrance into the pancreatic juice in humans.

The penetrance of mezlocillin, metronidazole and trimethoprim-sulfamethoxazole into the pancreatic juice of humans was measured in ten patients convalescing from acute pancreatitis at the time of endoscopic retrograde cholangiopancreatography. Therapeutic levels were obtained in the serum for all three antimicrobial agents; simultaneously aspirated nonbile stained pancreatic juice contained therapeutic levels of metronidazole and trimethoprim-sulfamethoxazole. Mezlocillin was not present in a therapeutic level in any patient with nonbile stained pancreatic fluid.

Concentration of antibiotics in simple renal cysts.

Patients with infected renal cysts are known to respond poorly to antibiotic therapy. We evaluated the serum, cyst fluid and urine levels of 3 antibiotics in 4 patients with simple renal cysts. Despite excellent urine and serum concentrations gentamicin was detected in the cyst of only 1 of the 3 patients studied. Neither sulfamethoxazole nor trimethoprim was detected in the cyst of 1 patient despite adequate plasma levels. These data help explain the poor medical response of such patients and support the concept of early surgical intervention.

An analytical approach to the quantitation of known drugs in human biological samples by HPLC.

Although many HPLC methods are available in the literature only a fraction of these are applicable to the analysis of known drugs in human biological fluids. This paper presents the favoured approach of a laboratory involved in the quantitative assay of drugs in man for the subsequent study of pharmacokinetics and bioavailability.