A new and simple method to determine trace levels of sulfonamides in honey by high performance liquid chromatography with fluorescence detection

A novel method for the simultaneous analysis at trace level of sulfonamides (sulfaguanidine, sulfanilamide, sulfacetamide, sulfathiazole, sulfapyridine, sulfachloropyridazine, sulfamerazine, sulfameter, sulfamethazine, sulfadoxine, sulfadiazine, sulfamonomethoxine, sulfadimethoxine) in honey is described. Methanol has been used in the sample treatment step to avoid the emulsion formation and to break the N-glycosidic bond between sugars and sulfonamides. The determination is carried out by liquid chromatography in gradient elution mode, with fluorescence detection after the on-line pre-column derivatization with fluorescamine. The influence of parameters such as the mobile phase composition, column temperature, pH or injection volume, on the separation has been taken into account and the derivatization step has also been optimized. Recoveries of the compounds on spiked honey samples ranged from 56% for sulfadoxine to 96% for sulfacetamide, with relative standard deviations below 10%. The quantitation limits are between 4 and 15 ng g⁻¹.     

Simultaneous determination of 17 sulfonamides and the potentiators ormetoprim and trimethoprim in salmon muscle by liquid chromatography with tandem mass spectrometry detection

A simple, robust method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 17 sulfonamides [sulfanilamide (SNL), sulfacetamide (SAA), sulfaguanidine (SGD), sulfapyridine (SPY), sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethoxazole (SOZ), sulfamoxole (SXL), sulfisoxazole (SXZ), sulfamethizole (SML), sulfamethazine (SMZ), sulfamethoxypyridazine (SMP), sulfamonomethoxine (SMM), sulfachloropyridazine (SCP), sulfaquinoxaline (SQX), and sulfadimethoxine (SDM)] and 2 potentiators [ormetoprim (OMP) and trimethoprim (TMP)] in fish tissue has been developed. The analytes were extracted from homogenized fish tissue with water-acetonitrile (50 + 50). The extract was clarified by centrifugation and a portion defatted with hexane. The analytes were partitioned into chloroform and evaporated to dryness. The redissolved residue was applied to a C18 reversed-phase column with a water-acetonitrile (0.1% acetic acid) gradient. All of the compounds were completely separated and detected in <10 min at 30 degrees C using LC/MS/MS. Standard curves were linear over the range of 0.02 to 5 ng injected. The limit of detection varied from 0.1 ng/g for SMZ and OMP to 0.9 ng/g for SXL and SOZ. Recoveries varied from 100% for SDM, SOZ, and SQX and 85% for SMR, OMP, and TMP to approximately 30% for SAA. Relative standard deviations for repeat analysis varied from 4% for SMZ and SCP to 23% for SAA.     

Determination of sulfonamides in milk after precolumn derivatisation by micellar liquid chromatography

A simple method to identify and determine six sulfonamides (sodium sulfacetamide, sulfamethizole, sulfaguanidine, sulfamerazine, sulfathiazole and sulfamethoxazole) in milk by micellar liquid chromatography (MLC) is reported. The assay makes use of a precolumn diazotisation-coupling derivatisation including the formation of an azo dye that can be detected at 490 nm. Furthermore, the use of MLC as an analytical tool allows the direct injection of non-purified samples. The separation was performed with an 80 mM SDS-8.5% propanol eluent at pH 7. Analysis times are below 16 min with a complete resolution. Linearities (r > 0.9999), as well as intra- and inter-day precision (below 2.7%), were studied in the validation of the method. The limits of detection and quantification ranged from approximately 0.72 to 0.94 and 2.4 to 3.1 ng mL⁻¹, respectively. The detection limit was below the maximum residue limit established by the European Community. Finally, recoveries in spiked milk samples were in the 83-103% range.     

Comparative study of sample preparation methods; supported liquid membrane and solid phase extraction in the determination of benzimidazole anthelmintics in biological matrices by liquid chromatography-electrospray-mass spectrometry

Supported liquid membrane (SLM) and solid phase extraction (SPE) have been applied as clean-up and/or enrichment techniques for a mixture of five benzimidazole anthelmintics compounds, namely albendazole, fenbendazole, mebendazole, oxibendazole, and thiabendazole. Two biological matrices, mainly urine and milk, and ultra high purity (UHP) water were spiked with a mixture of these five compounds. Waters Oasis^® MCX and International Sorbent Technology (IST) HCX SPE sorbents were used. The liquid membrane used for clean-up and/or enrichment of these compounds was 5% tri-n-octylphosphine oxide (TOPO) dissolved in n-undecane/di-n-hexyl ether (1:1). The SLM extraction efficiencies and SPE percentage recoveries ranged between 60 and 100%. The detection limits (DLs) for different benzimidazole compounds by SPE/LC-ES-MS for thiabendazole, oxibendazole, and albendazole was 0.1 ng/L, for fenbendazole and mebendazole was 1 and 10 ng/L, respectively. Similarly, the detection limits of SLM/LC-ES-MS for thiabendazole, oxibendazole, and albendazole was 0.1 ng/L and for fenbendazole and mebendazole was 1 ng/L. The results of optimization of various parameters of the SLM method are reported.     

Trace determination of sulfonamides residues in meat with a combination of solid-phase microextraction and liquid chromatography-mass spectrometry

An integrated method of combining solid-phase microextraction (SPME) with liquid chromatography-mass spectrometry (LC-MS) was evaluated for determination trace amount of sulfonamides in meat products. Eight commonly used sulfonamides, sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethazine (SMT), sulfamonomethoxine (SMMX), sulfamethoxazole (SMXZ), sulfaquinoxaline (SQX) and sulfadimethoxine (SDMX), were investigated in this study. Chromatography was performed on a C₁₈ reversed-phase column using an isocratic acetonitrile in water as the mobile phase. Fiber coated with a 65 μm thickness of polydimethylsiloxane/divinylbenzene (PDMS/DVB) was used to extract sulfonamides at optimum conditions. Analytes were desorbed with static desorption in an SPME-HPLC desorbed chamber for 15 min and then determined by LC-MS. The detection limits of these sulfonamides in pork were from 16 μg kg⁻¹ (SMT) to 39 μg kg⁻¹ (SMMX). According to the analysis, the linear range was from 50 to 2000 μg kg⁻¹ with relative standard deviation (R.S.D.s) value below 15% (intra-day) and 19% (inter-day). The proposed method was tested by analyzing meats from a local market for sulfonamides residues. Some sulfonamides in our study were detected in the meat samples. The concentration of these residual sulfonamides ranged from 66 μg kg⁻¹ (SDZ) to 157 μg kg⁻¹ (SQX) in a chicken sample. The results demonstrate that the SPME-LC-MS system is highly effective in analyzing trace sulfonamides in meat products.     

Determination of 10 sulfonamide residues in meat samples by liquid chromatography with ultraviolet detection

A liquid chromatography (LC) method is described for the simultaneous determination of 10 commonly used sulfonamide drug residues in meat. The 10 sulfonamide drugs of interest were sulfadiazine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethizole, sulfamonomethoxine, sulfachloropyridazine, sulfadoxine, sulfadimethoxine, and sulfaquinoxaline. The residues were extracted with acetone-chloroform (1 + 1). Sulfonamides were quantitatively retained in the extracting solution and afterwards eluted from a cation-exchanger solid-phase extraction cartridge with a solution of methanol-aqueous ammonia. The solution was dried, reconstituted with 5 mL methanol and filtered before analysis by LC-ultraviolet using a C18 column with a mobile phase gradient of potassium dihydrogen phosphate buffer, pH 2.5, and methanol-acetonitrile (30 + 70, v/v). The method was applied to cattle, swine, chicken, and sheep muscle tissues. The validation was performed with a fortified cattle meat sample at level of 100 ppb, which is the administrative maximum residue limit for sulfonamides in the European Union. The limit of quantitation for all sulfonamides was between 3 and 14 ppb. Recovery was evaluated for different meat matrixes. The mean recovery values were between 66.3% for pork meat samples and 71.5% for cattle meat samples.     

Hollow fiber supported ionic liquid membrane microextraction for determination of sulfonamides in environmental water samples by high-performance liquid chromatography

By using ionic liquid as membrane liquid and tri-n-octylphosphine oxide (TOPO) as additive, hollow fiber supported liquid phase microextraction (HF-LPME) was developed for the determination of five sulfonamides in environmental water samples by high-performance liquid chromatography with ultraviolet detection The extraction solvent and the parameters affecting the extraction enrichment factor such as the type and amount of carrier, pH and volume ratio of donor phase and acceptor phase, extraction time, salt-out effect and matrix effect were optimized. Under the optimal extraction conditions (organic liquid membrane phase: [C₈MIM][PF₆] with 14% TOPO (w/v); donor phase: 4 mL, pH 4.5 KH₂PO₄ with 2 M Na₂SO₄; acceptor phase: 25 μL, pH 13 NaOH; extraction time: 8 h), low detection limits (0.1–0.4 μg/L, RSD ≤ 5%) and good linear range (1–2000 ng/mL, R² ≥ 0.999) were obtained for all the analytes. The presence of humic acid (0–25 mg/L dissolved organic carbon) and bovine serum albumin (0–100 μg/mL) had no significant effect on the extraction efficiency. Good spike recoveries over the range of 82.2–103.2% were obtained when applying the proposed method on five real environmental water samples. These results indicated that this present method was very sensitive and reliable with good repeatabilities and excellent clean-up in water samples. The proposed method confirmed hollow fiber supported ionic liquid membrane based LPME to be robust to monitoring trace levels of sulfadiazine, sulfamerazine, sulfamethazine, sulfadimethoxine and sulfamethoxazole in aqueous samples.     

A confirmatory method for the simultaneous extraction,separation, identification and quantification of Tetracycline,Sulphonamide, Trimethoprim and Dapsone residues in muscle by ultra-high-performance liquid chromatography–tandem mass spectrometry according to Commission Decision 2002/657/EC

A rapid confirmatory multi-residue method for the analysis of tetracyclines, sulphonamides, trimethoprim and dapsone by UPLC-MS/MS is described. The method is able to quantify and confirm the following 19 compounds, oxytetracycline, tetracycline, chlortetracycline, doxycycline, sulfadiazine, sulfathiazole, sulfapyridine, trimethoprim, sulfamerazine, sulfamethizole, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachlorpyridazine, dapsone, sulfamethoxazole, sulfisoxazole, sulfaquinoxaline and sulfadimethoxine. Samples are extracted with 0.1 M EDTA and acetonitrile, which is then evaporated under a stream of nitrogen and reconstituted in water. Following centrifugation and filtering, an aliquot is analysed by UPLC-MS/MS using positive electrospray ionisation and multiple reaction monitoring. The method is deemed rapid as all analytes are extracted by a single extraction technique, with no solid-phase extraction clean up required. Validation is according to Commission Decision 2002/657/EC and was carried out for bovine, porcine, ovine and poultry species. Specificity, recovery, repeatability, reproducibility, CCα and CCβ data is presented.     

Determination of sulfonamide residues in the tissues of food animals using automated precolumn derivatization and liquid chromatography with fluorescence detection

A liquid chromatographic method for the determination of sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfadoxine, sulfaethoxypyridazine, sulfamethazine, sulfaquinoxaline, and sulfathiazole residues in the muscle, liver, and kidney of food animals using sulfapyridine as internal standard is reported. Tissues are extracted using a modified version of AOAC Official Method 983.31 (Sulfonamide Residues in Animal Tissues). The sample extract is reconstituted in pH 3.0 buffer-acetonitrile (60 + 40) and filtered into an autosampler vial. Using a programmable autosampler of a liquid chromatograph, a portion of the sample is derivatized precolumn with fluorescamine. The sulfonamide derivatives are separated by liquid chromatography using a C18 column with a mobile phase of 0.02M phosphoric acid-acetonitrile (60.5 + 39.5) and detected by fluorescence (excitation, 405 nm; emission, 495 nm). The method was applied to swine and cattle muscle, liver, and kidney; sheep and horse muscle and kidney; and chicken muscle and liver. The mean values for samples fortified with sulfonamides at levels between 0.05 and 0.2 microg/g agreed within 96-99% of spiked levels, with coefficients of variation ranging from 4-10%. The limit of detection (LOD) for all sulfonamides was 0.01 microg/g, with the exception of sulfaquinoxaline, for which the LOD was 0.015 microg/g.     

Determination of eight sulfonamides in bovine kidney by liquid chromatography/tandem mass spectrometry with on-line extraction and sample clean-up

A sensitive, high performance liquid chromatography/tandem mass spectrometric (i.e. mass spectrometry/mass spectrometry; LC/MS/MS) method with on-line extraction and sample clean-up for the screening and confirmation of residues of sulfonamides in kidney is described. The sulfonamides are extracted from homogenized kidney with methanol. After centrifugation of the extract, an aliquot of the extract is directly injected on the LC/MS/MS system with further extraction and clean-up of the sample on-line. Detection of the analytes was achieved by positive electrospray ionization (ESI) followed by multiple reaction monitoring. For each sulfonamide the collisional decomposition of the protonated molecule to a common, abundant fragment ion was monitored. The method has been validated for sulfadimethoxine, sulfaquinoxaline, sulfamethazine, sulfamerazine, sulfathiazole, sulfamethoxazole, sulfadiazine and sulfapyridine. Calibration curves resulting from spiked blank kidney samples at the 10-200 microg/kg level showed good linear correlation. At the level of 50, 100 and 200 microg/kg both within- and between-day precision, as measured by relative standard deviation (RSD), were less than 16%. The limits of detection (LODs) ranged from 5 to 13.5 microg/kg. The recoveries ranged from 78 to 82%. The procedure provides a rapid, reliable and sensitive method for the determination of residues of sulfonamides in bovine kidney. The advantage of this method over existing methods is its decreased sample preparation and analysis time, which makes the method more suitable for routine analysis.     

Continuous flow liquid membrane extraction: a novel automatic trace-enrichment technique based on continuous flow liquid-liquid extraction combined with supported liquid membrane

Combining the continuous flow liquid-liquid extraction (CFLLE) and supported liquid membrane (SLM) extraction, a novel aqueous-aqueous extraction technique that we termed continuous flow liquid membrane extraction (CFLME) is developed for trace-enrichment. The analyte was firstly extracted into the organic phase in the CFLLE step, then transported onto the organic liquid membrane that formed on the surface of the micro porous membrane of the SLM equipment. Finally, it passed through the liquid membrane and was trapped by the acceptor. Aspects related to CFLME were studied by using dichloromethane as liquid membrane, and sulfonylurea herbicides as model compounds. An enrichment factor of over 1000 was obtained when 10 μg l⁻¹ of MSM was enriched for 120 min by this technique. The drawbacks of only a few organic solvents can be selected as liquid membrane with a limited lifetime in SLM operation was overcome. In this CFLME method, almost all solvents that used in the conventional liquid-liquid extraction (LLE) can be adopted and the lifetime of liquid membrane is no longer a problem.     

Evaluation of carbon nanotubes as a solid-phase extraction adsorbent for the extraction of cephalosporins antibiotics, sulfonamides and phenolic compounds from aqueous solution

The adsorptive potential of carbon nanotubes (single-walled carbon nanotubes and multi-walled carbon nanotubes) for solid-phase extraction of three groups of highly polar compounds (namely cephalosporins antibiotics, sulfonamides and phenolic compounds) was tested in this article. The analytes were strongly retained by the carbon nanotubes. And acceptable recoveries were obtained with the addition of ammonium acetate into eluents. The effects of solution pH on the recoveries of the antibiotics and phenolic compounds were examined. To check the retention abilities of three groups of compounds on carbon nanotubes, fixed amount of each analyte was added to different volumes (up to 500 mL) of aqueous solution, and then extracted by the sorbents. Comparative studies showed that the carbon nanotubes were much superior to C₁₈ for the extraction of the highly polar analytes. For the cephalosporins antibiotics and sulfonamides, the carbon nanotubes showed stronger retention capability than graphitized carbon blacks, but for some of the phenolic compounds graphitized carbon blacks seemed to be more suitable, indicating different retention mechanisms of these analytes. To further assess the enrichment ability of carbon nanotubes for highly polar compounds, the solid-phase extraction method of multi-walled carbon nanotubes packed cartridge was well developed, and the sulfonamides were used as model compounds. Under the optimal procedures, the detection limits of sulfonamides were in the range of 27-38 ng L⁻¹. The spiked recoveries from several real water samples obtained for sulfathiazole and sulfadiazine ranged from 55% to 79% and 72% to 92%, respectively, while the recoveries of sulfapyridine and sulfamethazine were in the range of 85-102%.     

A multivariate multianalyte screening method for sulfonamides in milk based on front-face fluorescence spectroscopy

Screening methods are used to detect the presence of a substance or class of substances at the level of interest and are specifically designed to avoid false compliant results. They should allow the running of a high number of samples per day at a low cost under routine conditions. In this work, a rapid and simple method for the screening of six sulfonamides (sulfadiazine, SD; sulfamerazine, SMR; sulfamethazine, SMT; sulfachloropyridazine, SCP; sulfathiazole, STZ and sulfamethoxazole, SMO) in milk samples is proposed and assessed according to the criteria required by the European Regulation, Decision 2002/657/EC. The method is based on modelling front-face fluorescence emission spectra by means of partial least squares class modelling (PLS-CM). The milk samples are pre-treated with a single easy step of derivatization with fluorescamine.After confirming that the method has equal analytical sensitivity for all the six sulfonamides, it is established that the multivariate analytical sensitivity at 100 μg L⁻¹ is 37.5 μg L⁻¹ when analysing a mixture of six sulfonamides added to different brands of milk and measured in different days. In addition, the method is applied to samples from 11 commercial brands of milk. For β = 0.05, threshold value established by the Decision 2002/657/EC for this method, the probability of false non-compliance, α, is equal to 0.17, allowing the suitable screening of these six sulfonamides.     

High Performance Liquid Chromatographic Determinations of Sulfonamides by Ionic Suppression

Abstract

A high pressure liquid chromatography procedure is reported for extraction and quantitation of 8 sulfonamides in stock solutions and in vitro plasma samples. This assay consists of a single, one-step extraction of sulfonamides from plasma and is sensitive to 10.0 ng/ml at 254 nm without additional concentration of the sample. Four sulfonamides (sulfamerazine, sulfamethazine, sulfapyridine and sulfathiazole) were separated from the plasma matrix by either mobile phase regardless of pH. The sulfonamides with the highest pKa, sulfanilamide (10.5) and sulfaguanidine (11.3), were only separable from plasma in a 50% water/50% methanol mobile phase at pH 7.45. The sulfonamide with the lowest pKa, sulfisoxazole (4.9), and its metabolite, acetylsulfisoxazole (N⁴), were separated from plasma by either mobile phase, 50/50 or 60/40 water/methanol, when acetate buffer reduced the pH to 4.00. Standard concentration curves of peak height were the most sensitive at 254 nm when a 60% water/40% methanol mobile phase at pH 4.00 was used. Sulfanilamide and sulfaguanidine were the most responsive to ultraviolet quantitation at 254 nm regardless of ionic suppression or polarity of the mobile phase.     

Extraction and preconcentration of manganese(II) from biological fluids (water, milk and blood serum) using supported liquid membrane and membrane probe methods

A supported liquid membrane (SLM) technique was investigated to extract and preconcentrate Mn(II) from water, milk and blood serum. Di-2-ethylhexyl phosphoric acid (DEHPA) with kerosene as diluent was used as a carrier in the membrane to transport Mn(II) from the donor side to acceptor side. The membrane was modified with tri-n-octylphosphine oxide (TOPO) to increase its polarity. Various parameters were investigated to optimise the extraction efficiency: pH of the donor and acceptor phase, dilution factor, donor flow rate. Scanning electron microscope images of the membranes revealed that some matrix compounds were deposited on the surface, thus limiting the extraction process. The optimum conditions found were: pH 3 in the donor phase, 0.2 M nitric acid in the acceptor phase, donor flow rate between 1.0 and 0.3 ml min⁻¹, 15% (w/v) DEPHA and 10% TOPO in kerosene as a carrier in membrane, and dilution factors of 20 times for blood serum and 30 times for milk. The extraction efficiencies were found to be low but constant and highly reproducible showing, strong dependence on sample matrix. The new SLM extraction probe was developed and optimised for Mn(II) extraction. Compared to traditional SLM configurations, this is the simplest configuration. The use of stirring allows the same sample to be extracted many times giving higher extraction efficiency and to minimise the sample size. Adsorptive stripping voltammetry (AdSV) was applied to measure Mn(II) concentration. The optimised method was used to determine the concentration of Mn(II) in water, milk and blood serum samples.     

Optimization and Validation of a Confirmatory Method for Determination of Ten Sulfonamides in Feeds by LC and UV-Diode Array Detection

A simple, rapid, and reliable multiresidue method, based on liquid chromatography (LC) and ultraviolet (UV)-diode array detection (DAD), is described for assaying ten sulfonamides (sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachloropyridazine, sulfamethoxazole, sulfaquinoxaline, and sulfadimethoxine) in feeds. The chromatographic separation is accomplished using a C18 column, eluted with a mobile phase consisting of acetate buffer, acetonitrile, and methanol. The sample preparation requires a simple extraction with chloroform/acetone and a purification step by solid-phase extraction. The analytical parameters of precision, detection and quantification limits, recovery, and ruggedness have been evaluated by a validation procedure following the European guidelines of Regulation 882/2004/EC and Decision 657/2002/EC.     

A reliable high-performance liquid chromatography with ultraviolet detection for the determination of sulfonamides in honey

The sulfonamides are stable chemotherapeutics used against the bacterial disease affecting bees, known as American foulbrood (Bacillus larvae), so their residues could appear in the honey of treated bees. Their presence at a concentration above the limit value is a potential hazard to human health. Brazilian authorities have included in the National regulatory monitoring program, the control of the three most widely used sulfonamides in honey production, i.e., sulfathiazole, sulfamethazine and sulfadimethoxine. A method for the determination of residual sulfonamides in honey, using sulfapyridine as an internal standard has been developed, optimized and validated. Some changes were implemented on current available methodologies for the analysis of sulfonamides in honey in order to adopt such procedures to Brazilian honey samples. Sulfonamides were extracted from honey with dichloromethane after dissolution with 30% sodium chloride, and cleaned up with solid phase extraction on Florisil columns. The eluate was analyzed by high-performance liquid chromatography with ultraviolet detection. The limit of detection was determined at 3 μg kg⁻¹, 4 μg kg⁻¹ and 5 μg kg⁻¹ for sulfathiazole, sulfamethazine and sulfadimethoxine, respectively with average recoveries of 61.0% for sulfathiazole; 94.5% for sulfamethazine and 86.0% for sulfadimethoxine at the 100 μg kg⁻¹ level. As the final step of validation procedure, the analysts were submitted to a blind spiked sample prepared by the quality assurance officer which results were successfully obtained regarding recovery and deviations.     

Validation of a confirmatory method for the determination of sulphonamides in muscle according to the European Union regulation 2002/657/EC

A simple multiresidue method is described for assaying 10 sulphonamides (SAs) (sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfachlorpyridazine, sulfamethoxazole, sulfaquinoxaline and sulfadimethoxine) in muscle samples. Samples were prepared by homogenizing the tissue, extracting with ethyl acetate and cleaning up with a cation-exchange solid-phase extraction (SPE) column. The detection of analytes was achieved by HPLC-diode array detection (DAD) at 270 nm. The procedure was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit, detection capability, trueness and precision. The results of validation process demonstrate that the method is suitable for application in European Union statutory veterinary drug residue surveillance programmes.     

Simultaneous determination of 16 sulfonamides in honey by liquid chromatography/tandem mass spectrometry

A method is described for the determination of 16 sulfonamides in honey. Samples are dissolved in phosphoric acid solution (pH2), cleaned up with 2 solid-phase extraction (SPE) cartridges, an aromatic sulfonic cation-exchange cartridge and an Oasis HLB SPE cartridge, and analyzed both qualitatively and quantitatively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) under the selected conditions. Without exception, calibration curves were linear (r = > 0.995), when sulfamethizole was between 1.0 and 25.0 microg/kg; sulfacetamide, sulfapyridine, sulfadiazine, sulfachloropyridazine, sulfamethoxazole, sulfamerazine, sulfisoxazole, sulfamonomethoxine, and sulfadoxine were between 2.0 and 50.0 microg/kg; sulfamethoxypyridazine, sulfadimethoxine, and sulfathiazole were between 4.0 and 100.0 microg/kg; sulfamethazine and sulfameter were between 8.0 and 200.0 microg/kg; and sulfaphenazole was between 12.0 and 300.0 microg/kg. Average recoveries at 4 fortification levels in the range of 1.0-300 microg/kg in honey were 70.9-102.5%, and relative standard deviations were 2.02-11.52%. The limits of quantitation for the 16 sulfonamides were between 1.0 and 12.0 microg/kg, with the LC/MS/MS method.     

Validation Method for the Determination of Sulfonamide Residues in Bovine Milk by HPLC

A simple, rapid, sensitive and reliable high-performance liquid chromatographic method for the simultaneous determination of eight sulfonamides (SAs) in bovine milk was developed (sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxypyridazine, sufamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline) in bovine milk was developed. Samples were prepared by extraction with ethyl acetate and cleaning-up with an anion solid-phase extraction (SPE) column. Analytical separation was performed on an Inertsil ODS-3 column with photodiode-array detection at 270 nm under gradient condition. The whole procedure was evaluated according to the European Commission Decision 2002/657/EC. Specificity, decision limit (CCα), detection capacity (CCβ), trueness and precision were determined during the validation process. It was found that the analytes were isolated from spiked samples with good recoveries between 70.5 and 89.0%. The used analytical conditions allow to successively separating all the tested sulfonamides with good limit of detection between 0.8 and 1.5 μg L⁻¹.