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.

A simple and high-throughput method for multiresidue and multiclass quantitation of antimicrobials in pangasius (Pangasionodon hypophthalmus) fillet by liquid chromatography coupled with tandem mass spectrometry.

The development and validation of a throughput method for the determination of 25 antibacterial drugs (two ß-lactams, eight quinolones, two macrolides, five sulfonamides, trimethoprim, four tetracyclines and three amphenicols) in pangasius fish muscle by LC-MS/MS were performed. A simple, efficient and fast extraction procedure was developed using acetonitrile and a 0.1 M EDTA solution as solvents for extraction. All compounds were determined in a single run, and chromatographic separation was achieved using a Zorbax SB C18 column with a mobile phase comprised of purified water +0.1% formic acid (A) and acetonitrile +0.1% formic acid (B) in a linear gradient program. The method was validated aαording to the requirements of European Decision 2002/657/EC. To quantify the analytes, matrix-matched analytical curves were constructed with spiked blank tissues and showed linearity (r2) higher than 0.99. For all analytes, the precision and accuracy were determined at the levels of 3 ng/g (low), 10 ng/g (low-middle), 50 ng/g (high-middle) and 100 ng/g (high). The precision (CV%) was lower than 18.6% and the accuracy (determined as recovery) was between 65% and 119%. The limit of quantitation was 3.0 ng/g, with the exception of chloramphenicol, which was 0.3 ng/g, and amoxicillin and doxycycline, which were 10 ng/g. The method was successfully applied to analyze pangasius muscle samples from Vietnam available at the Brazilian retail market, and 5 out of 40 samples showed the presence of low-residue levels of enrofloxacin and, consequently, must be considered out of conformity. It is recommended that competent authorities should avoid the commercialization of pangasius fillet contaminated with residues of this veterinary drug.

An LC-ESI-MS/MS method for residues of fluoroquinolones, sulfonamides, tetracyclines and trimethoprim in feedingstuffs: validation and surveillance.

An increasing concern about food safety has been observed over the years. The presence of drugs residues in food is one of the major subjects of research in food safety. Feedingstuffs can be responsible for carryover into the food chain of residues of several drugs. This paper describes the development, validation and application of a fast and simple method for analysis of 24 antibiotic residues in feedingstuffs for cattle, pigs and poultry. Analytes include compounds from different antimicrobials classes, such as sulfonamides (sulfadiazine, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, sulfachlorpyridazine, sulfadoxine, sulfadimethoxine, sulfizoxazole, sulfamerazine and sulfathiazole), fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin, danofloxacin, difloxacin, sarafloxacin, flumequine, nalidixic acid and oxolinic acid), tetracyclines (tetracycline, doxycycline, oxytetracycline and chlortetracycline) and trimethoprim. Samples were extracted with methanol:water (70:30) 0.1% formic acid, followed by clean-up steps using centrifugation, low-temperature purification (LTP) and ultracentrifugation. Instrumental analysis was performed using liquid chromatography coupled to tandem mass spectrometry. Chromatographic separation was achieved using a C18 column and a mobile phase composed of acetonitrile and water, both with 0.1% formic acid. Validation parameters such as limit of detection (LOD), limit of quantification (LOQ), selectivity, linearity, accuracy, precision, decision limit (CCα) and detection capability (CCß) were determined and meet the adopted criteria. LOD and LOQ were set to 30 and 75 µg kg-1, respectively. Inter-day precision were in the range from 4.0 to 11.1%, and linearity provides values of r2 above 0.95 for all analytes. The optimised method was applied to the analysis of more than 1500 real samples within the period 2012-2017. Non-compliant results were discussed and classified in terms of analytes, feed types and target species. Multivariate analysis of the data was performed using principal component analysis.

Antibiotic pollution in the Katari subcatchment of the Titicaca Lake: Major transformation products and occurrence of resistance genes.

An increasing number of studies pointed out the ubiquitous presence of medical residues in surface and ground water as well as in soil compartments. Not only antibiotics can be found in the environment but also their transformation products about which little information is generally available. The development of bacterial resistance to antibiotics is particularly worrying as it can lead to sanitary and health problems. Studies about the dissemination of antibiotics and associated resistances in the Bolivian Altiplano are scarce. We provide baseline information on the occurrence of Sulfamethoxazole (SMX) and Trimethoprim (TMP) antibiotics as well as on the most common human SMX transformation products (TP) and on the occurrence of sulfonamide resistance genes. The studied water and soil compartments presented high levels of antibiotic pollution. This situation was shown to be mainly linked with uncontrolled discharges of treated and untreated wastewaters, resulting on the presence of antibiotics in the Titicaca Lake. SMX TPs were detected in surface waters and on soil sampled next to the wastewater treatment plant (WWTP). SMX resistance genes sulI and sulII were widely detected in the basin hydrological network, even in areas unpolluted with antibiotics. Mechanisms of co-selection of antibiotic- and metal- resistance may be involved in the prevalence of ARG's in pristine areas with no anthropogenic activity and free of antibiotic pollution.

Green method by diffuse reflectance infrared spectroscopy and spectral region selection for the quantification of sulphamethoxazole and trimethoprim in pharmaceutical formulations.

An alternative method for the quantification of sulphametoxazole (SMZ) and trimethoprim (TMP) using diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS) and partial least square regression (PLS) was developed. Interval Partial Least Square (iPLS) and Synergy Partial Least Square (siPLS) were applied to select a spectral range that provided the lowest prediction error in comparison to the full-spectrum model. Fifteen commercial tablet formulations and forty-nine synthetic samples were used. The ranges of concentration considered were 400 to 900 mg g-1SMZ and 80 to 240 mg g-1 TMP. Spectral data were recorded between 600 and 4000 cm-1 with a 4 cm-1 resolution by Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS). The proposed procedure was compared to high performance liquid chromatography (HPLC). The results obtained from the root mean square error of prediction (RMSEP), during the validation of the models for samples of sulphamethoxazole (SMZ) and trimethoprim (TMP) using siPLS, demonstrate that this approach is a valid technique for use in quantitative analysis of pharmaceutical formulations. The selected interval algorithm allowed building regression models with minor errors when compared to the full spectrum PLS model. A RMSEP of 13.03 mg g-1for SMZ and 4.88 mg g-1 for TMP was obtained after the selection the best spectral regions by siPLS.

Feasibility of amlodipine besylate, chloroquine phosphate, dapsone, phenytoin, pyridoxine hydrochloride, sulfadiazine, sulfasalazine, tetracycline hydrochloride, trimethoprim and zonisamide in SyrSpend(®) SF PH4 oral suspensions.

The objective of this study was to evaluate the feasibility of 10 commonly used active pharmaceutical ingredients (APIs) compounded in oral suspensions using an internationally used suspending vehicle (SyrSpend(®) SF PH4 liquid): (i) amlodipine, (as besylate) 1.0mg/mL; (ii) chloroquine phosphate,15.0 mg/mL; (iii) dapsone, 2.0 mg/mL; (iv) phenytoin, 15.0 mg/mL; (v) pyridoxine hydrochloride, 50.0 mg/mL; (vi) sulfadiazine, 100.0 mg/mL; (vii) sulfasalazine, 100.0 mg/mL; (viii) tetracycline hydrochloride, 25.0 mg/mL; (ix) trimethoprim, 10.0 mg/mL; and (x) zonisamide, 10.0 mg/mL. All suspensions were stored both at controlled refrigeration (2-8 °C) and controlled room temperature (20-25 °C). Feasibility was assessed by measuring the percent recovery at varying time points throughout a 90-day period. API quantification was performed by high-performance liquid chromatography (HPLC-UV), via a stability-indicating method. Given the percentage of recovery of the APIs within the suspensions, the expiration date of the final products (API+vehicle) was at least 90 days for all suspensions with regard to both the controlled temperatures. This suggests that the vehicle is stable for compounding APIs from different pharmacological classes.

Fast simultaneous determination of trimethoprim and sulfamethoxazole by capillary zone electrophoresis with capacitively coupled contactless conductivity detection.

The association of trimethoprim and sulfamethoxazole is a very effective with antibiotic properties, and commonly used in the treatment of a variety of infections. Due to the importance in diseases treatment of humans and also of animals, the development of methods for their quantification in commercial formulations is highly desirable. In the present study, a rapid method for simultaneous determination of these compounds using CE with capacitively coupled contactless conductivity detection was developed. A favorable working region for both analytes was from 12.5 to 200 µmol/L (linear responses with R > 0.999 for N = 5). Other parameters calculated were sensitivity (1.28 ± 0.10/1.45 ± 0.11) min/(µmol L), RSD (4.5%/2.0%), and LOD (1.1/3.3) µmol/L for trimethoprim and sulfamethoxazole, respectively. Under this condition, the total run time was only 2.6 min. The proposed method was applied to the determination of trimethoprim and sulfamethoxazole in commercial samples and the results were compared to those obtained by using a HPLC pharmacopoeia method. This new method is advantageous for quality-control analyses of trimethoprim and sulfamethoxazole in pharmaceuticals samples, because it is rapid and precise. Moreover, it is less laborious and demands minimum amounts of reagents in comparison to the recommended method.

A multidimensional high performance liquid chromatography method coupled with amperometric detection using a boron-doped diamond electrode for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk.

The development and validation of a multidimensional HPLC method using an on-line clean-up column coupled with amperometric detection employing a boron-doped diamond (BDD) electrode for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk are presented. Aliquots of pre-prepared skim-milk samples were directly injected into a RAM octyl-BSA column in order to remove proteins that otherwise would interfere with milk analysis. After exclusion of the milk proteins, SMX and TMP were transferred to the analytical column (an octyl column) and the separation of the compounds from one another and from other endogenous milk components was achieved. SMX and TMP were detected amperometrically at 1.25V vs. Ag/AgCl (3.0molL(-1) KCl). Results with good linearity in the concentration ranges 50-800 and 25-400microgL(-1) for SMX and TMP, respectively, were obtained and no fouling of the BDD electrode was observed within the experimental period of several hours. The intra- and inter-assay coefficients of variation were less than 10% for both drugs and the obtained LOD values for SMX and TMP were 25.0 and 15.0microgL(-1), respectively.

Simultaneous quantification of sulfamethoxazole and trimethoprim in whole egg samples by column-switching high-performance liquid chromatography using restricted access media column for on-line sample clean-up.

This work reports the application of restricted access media (RAM) column, in a multidimensional configuration, for simultaneous analysis of sulfamethoxazole (SMX) and trimethoprim (TMP) in whole eggs with ultraviolet detection. The proteins were partially precipitated by adding 0.5 mL of acetonitrile into 1.0 mL of blended egg followed by centrifugation. The supernatant was injected (250 microL) directly into the multidimensional system. At the first dimension, a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 mm x 46 mm I.D., Luna silica, 10 microm particle size and 100 A pore size), was used for extraction and concentration of the analytes and at second dimension, an octadecyl column (150 mm x 46 mm I.D., Luna silica, 10 microm particle size and 100 A pore size), for analysis. The developed method showed good selectivity, accuracy and precision for quantification of these different compounds in eggs, and the limits of quantification were 80 ng/mL, for both compounds. The validated method is reliable and sensitive for monitoring residues in whole eggs samples and thus, to determine withdraw period for laying hens using veterinary medicine having SMX-TMP combination.

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.

High-performance liquid chromatography method for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk using an on-line clean-up column.

A bidimensional HPLC method for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk has been developed and validated. After centrifugation, aliquots (150 microl) of milk samples were directly injected to a column-switching HPLC system. At the first step a RAM octyl-BSA column was employed to automatically remove proteins that otherwise would interfere with milk analysis. The mobile phase 0.01 M phosphate buffer pH 6.0:acetonitrile (95:5, v/v) was used in the first 5 min for the elution of milk proteins and then 0.01 M phosphate buffer pH 6.0:acetonitrile (83:17, v/v) for transfer SMX and TMP to the analytical column. The separation of SMX and TMP from one another and from other remaining milk components was performed on an octyl column using the mobile phase 0.01 M phosphate buffer pH 5.0:acetonitrile (82:18, v/v), which were detected by UV at 265 nm. The calibration graphs were linear in the concentration ranges of 25-800 ng/ml and 50-400 ng/ml for SMX and TMP, respectively. The intra- and inter-assay coefficients of variation were less than 15% for both drugs. The validated method was applied to the analysis of milk samples of twelve (two groups of six) cows after administration (intramuscular or subcutaneous) of a single recommended therapeutic dose of the SMX-TMP combination.

Simultaneous determination of binary mixtures of trimethoprim and sulfamethoxazole or sulphamethoxypyridazine by the bivariate calibration spectrophotometric method.

The bivariate calibration algorithm was applied to the spectrophotometric simultaneous determination of trimethoprim (TMP), sulfamethoxazole (SMX) or sulphamethoxypyridazine (SMP) binary mixtures in pharmaceutical and veterinary products. The results obtained were compared with those from derivative spectrophotometry. The statistical evaluation of the method bias showed that the proposed procedure is comparable with commonly used first-derivative spectrophotometry. However, the advantage of bivariate calibration is its simplicity, due to the minimal spectra manipulation when compared with derivative techniques.

Second derivative spectrophotometric determination of trimethoprime and sulfamethoxazole in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD).

An easy and rapid second-derivative spectrophotometric method for the simultaneous analysis of trimethoprime (TMP) and sulfamethoxazole (SM) is described. These drugs have been used as antibacterial against a wide spectrum of organisms and combinations of these drugs are commonly used for the treatment of a variety of infections. The most advantageous approach of this method is the use of HP-beta-CD, which allows to improve the performance of the second-derivative ultraviolet spectrophotometry. For both compounds, a shift of the absorption bands and variations of their intensity were observed. The calibration graphs were linear in the concentration range of TMP (1.92-19.2 microg ml(-1)) and SM (1.60-16.5 microg ml(-1)), the correlation coefficient for the calibration graphs was better than 0.9994 and the precision was satisfactory (CV%< 4.96) in HP-beta-CD solutions. The proposed method was successfully applied to the assay of commercial tablets. The results were compared to those obtained by second-derivative ultraviolet spectrophotometry in the absence of HP-beta-CD. Thereby, the details of the statistical treatment of the analytical data are also presented.

Depletion study of trimethoprim and sulphadiazine in milk and its relationship with mastitis pathogenic bacteria strains minimum inhibitory concentrations (MICs) in dairy cows.

Time-related concentrations in milk of a combination of trimethoprim-sulphadiazine (TMP-SDZ) intramammary formulated infusion and its relationship with pathogenic bacteria strains minimum inhibitory concentrations (MICs) isolated from clinical mastitis cows were analysed. The MICs study was performed for Escherichia coli, Staphylococcus aureus and Streptococcus sp. strains. The SDZ concentrations in milk were analysed using high-performance liquid chromatography (HPLC) and TMP using a microbiological assay. Ten lactating cows milked three times daily were used in the time-concentration studies of TMP-SDZ. Milk samples (approximately 20 mL) from the treated mammary quarters were taken at 6, 12, 24, 30 and 36 h after first administration. In order to define the withdrawal time, milk samples from the treated mammary quarters were taken at 24, 36, 48, 72, 84 and 96 h, after finishing the therapy. The MICs fluctuated between 1 and 8 microg/mL. Effective therapeutic concentrations lasted for 36 h when intramammary infusion was repeated three times every 12 h. No TMP was detected in milk for 24 h after finishing therapy. Milk SDZ concentrations were below 0.1 microg/mL in all treated cows after 84 h finishing therapy. At 96 h after finishing therapy, no SDZ milk concentrations were found in six animals, although four animals of the experimental group still had concentrations of 0.07 microg/mL.

The multicomponent automated dissolution system: an alternative in the development and pharmaceutical analysis of generic polydrugs.

The necessity of assuring the quality of polydrugs, especially those with low aqueous solubility and in vivo absorption, has led to the development and evaluation of new techniques that can reduce the time and cost of analysis. This study examines the efficiency and accuracy of an automated dissolution system, fitted with an integrated multicomponent detector, for analysis of generic polydrugs using multiple linear regression (MLR). Trimethoprim and sulphamethoxazole were chosen as model drugs for this study and comparison was made with a conventional analysis based on HPLC. Both analytical systems under study gave reproducible and accurate results. Analysis of variance showed that there was no significant statistical difference between the methods of analysis, nor any statistical difference between the measured amounts of drug in the three different formulations. We have demonstrated that low cost instrumentation coupled with MLR data processing provides entirely satisfactory drug analysis to standard at least as good as that achieved using HPLC and provides an opportunity to reduce the time and analysis cost of other generic formulations.

Sulfaprim inyección. formulación / Inyectable sulfapri: 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

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