Liquid chromatographic determination of three sulfonamides in animal tissue and egg

Sulfonamides are widely used as a feed additive in animal production in Japan. The present paper is a determination of 3 sulfonamides: sulfamethazine (SMZ), sulfamonomethoxine [SMX, 4-amino-N-(3-methoxypyrazinyl)-benzenesulfonamide], and sulfadimethoxine (SDX) in animal tissue and egg by liquid chromatography (LC). Tissues were extracted with acetonitrile and fat was removed by liquid/liquid partition. The sulfonamides were purified by an ODS cartridge column; then each compound was separated by an ODS LC column and detected at 268 nm. Quantification levels were 0.02 ppm for SMZ and SMX, and 0.04 ppm for SDX; detection limits were 0.01 ppm for SMZ and SMX, and 0.02 ppm for SDX. Calibration curves were linear between 2 and 40 ng for SMZ and SMX, and between 4 and 80 ng for SDX. Recoveries from muscle and egg samples spiked with 1-2 micrograms/10 g were 81-98%.     

Environmental fate of two sulfonamide antimicrobial agents in soil

Veterinary antimicrobial agents have been detected in a number of environmental samples, including agricultural soils. In this study, we investigated the persistence and sorption of the sulfonamides sulfamethazine (SMZ) and sulfachloropyridine (SCP) in soil and their potential effects on soil microorganisms. The sulfonamides dissipated more rapidly from the silt loam soil as compared to the sandy soil. Average half-lives of SMZ and SPC among the two soils were 18.6 and 21.3 days, respectively. The presence of liquid swine slurry (5% v/w) decreased sulfonamide persistence in the silt loam soil. The lower persistence of the antimicrobials in liquid swine slurry-amended soil was likely due to higher microbial activity, as compared to unamended soil, and/or to the greater bioavailability of the sulfonamides to degrading microorganisms, as estimated by sorption isotherms. Concentrations of SMZ and SPC up to 100 microg g-1 had no effect on antimicrobial degradation rates and soil microorganisms. These studies suggest that higher sulfonamide concentrations would be necessary to affect the main processes controlling their environmental fates in soil, but at the concentrations normally found in the environment, there would be little or no effects.     

Sorption and transport of sulfonamides in soils amended with wheat straw-derived biochar: effects of water pH,coexistence copper ion,and dissolved organic matter

Purpose

Sulfonamides are widely used for the prevention and treatment of bacterial infections, hard-degraded contaminants distributed in the environment if they are discharged into the soil and water. Biochar could probably influence the geochemical behavior of ionized antibiotics in the soils.

Materials and methods

To determine the sorption/desorption of three representative sulfonamides (SAs) in soils amended with biochar, we investigated the effects of water pH, Cu²⁺, and dissolved humic acid on the sorption of sulfamethoxazole (SMX), sulfamethazine (SMZ), and sulfadiazine (SD) onto two different soil samples (S1 pH?=?5.13 and S2 pH?=?7.33) amended with wheat straw-derived biochar (size 0.5～0.6 mm).

Results and discussion

Batch experiments showed that the sorption/desorption isotherms of SAs on soil with/without biochar followed the Freundlich model. The biochar had a strong adsorption potential for SMX, SMZ, and SD both in S1 and S2 at low water pH. Except for SMX, the presence of Cu²⁺ inhibited the sorption of SMZ and SD through competing hydrophobic adsorption region in soils. HA suppressed the sorption of three sulfonamides in soil S2 by electrostatic repulsion under alkaline condition. The soil leaching column experiments showed the SA transport in soils, and S1 and S2 amended with biochar (0.5 and 1.0 wt%) brought about 12–20 % increase in SMX, SMZ, and SD retention compared to the untreated soil.

Conclusions

The results indicated that the presence of biochar effectively mitigated the mobility of ionized antibiotics such as SMX, SMZ, and SD in soils, which helps us reconsider the potential risk of antibiotics in the environment.

     

Quantification of ptaquiloside and pterosin B in soil and groundwater using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The carcinogenic compound ptaquiloside is produced by bracken fern (Pteridium aquilinum L.). Ptaquiloside can enter the soil matrix and potentially leach to the aquatic environment, and methods for characterizing ptaquiloside content and fate in soil and groundwater are needed. A sensitive detection method has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analyzing ptaquiloside and its transformation product pterosin B. Detection limits are 0.19 microg/L (ptaquiloside) and 0.15 microg/L (pterosin B), which are 300-650 times better than previously published LC-UV methods. Sequential soil extractions are made using 5 mM ammonium acetate for extraction of ptaquiloside, followed by 80% methanol extraction for pterosin B. Groundwater samples are cleaned-up and preconcentrated by a factor of 20 using solid-phase extraction. The LC-MS/MS method enables quantification of ptaquiloside and pterosin B in soil and groundwater samples at environmentally relevant concentrations and delivers a reliable identification because of the structure-specific detection method.     

磺胺甲噁唑单克隆抗体的研制及免疫金标试纸检测方法的建立

用磺胺甲噁唑人工免疫原（BSA-SMZ）免疫BALB/C小鼠（Mus muscalus）,细胞融合技术筛选抗磺胺甲噁唑单克隆抗体（SMZmAb）杂交瘤细胞,体内诱生腹水法制备SMZ mAb,并鉴定其免疫学特性;应用SMZmAb研制SMZ残留快速检测金标试纸（SMZ-strip）,并测定其性能。结果表明,筛选出3株杂交瘤细胞,其中最好的3G6株间接ELISA效价细胞培养上清为1∶8.1×102,腹水为1∶6.4×105,亲和常数（Ka）为3.07×109 L/mol,对SMZ的半数抑制浓度（IC50）为12.4 μg/L,与磺胺嘧啶的交叉反应率（CR%）为2.84%,与其它磺胺类药物无交叉反应;SMZ-strip目测检测限为6 μg/L;其敏感性与竞争ELISA试剂盒相当,符合率为100%;SMZ-strip具有快速、敏感、特异、简便等特点,适合于SMZ残留快速检测的推广应用。     

Development and validation of an optical SPR biosensor assay for tylosin residues in honey

In recent years there has been an increase in the use of tylosin in apiculture as bacterial brood diseases become resistant to oxytetracycline. Confirmatory mass spectrometry based methods have been developed but up until now there has been no complementary screening method available capable of sub 10 microg kg(-1) detection limits. In this paper the development and validation of a screening method using optical biosensor technology is presented. The honey was first dissolved in a phosphate buffer and following solid-phase extraction (SPE) cleanup was analyzed using a Biacore Q instrument. Using the criteria specified in European Commission Decision 2002/657/EC for qualitative screening methods, the detection capability (CCbeta) of the method was determined to be 2.5 microg kg(-)(1). Honey samples containing trace residue levels of tylosin were analyzed by both the biosensor screening method and a LC-MS/MS confirmatory procedure; the results were in good agreement.     

Accelerated solvent extraction and confirmatory analysis of sulfonamide residues in raw meat and infant foods by liquid chromatography electrospray tandem mass spectrometry

This paper describes a new method for the rapid extraction and unequivocal confirmation of 13 sulfonamides (SAs) in raw meat and infant foods. The highly automated extraction procedure is based on accelerated solvent extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a confirmatory analysis. After 1 g of food matrix was blended with 2 g of C18 as a solid support material, the mixture was packed into the extraction cell and the SAs were extracted with 10 mL of hot water at 160 degrees C and 100 atm; 100 microL of the extract was directly injected into the LC-MS system. The analytes were ionized in an electrospray interface operating in the positive ion mode and were identified by selecting two multireaction monitoring transitions, which guaranteed method specificity. Typical recoveries from crude meat and baby food samples ranged from 70 to 101% at a fortification level of 100 ppb, corresponding to the maximum residue limits established by the European Union and the U.S. Food and Drug Administration. The interday method precision was less than 8.5%, and the limits of detection were below 2.6 ppb. This study has taken matrix-induced suppression of ionization into account, by comparing standard and matrix-matched calibration curves. Four of the 13 monitored SAs have been detected in some baby foods and raw meat samples, bought from Roman supermarkets and butchers' shops, using the described methodology.     

Analysis of beta-lactam antibiotics in incurred raw milk by rapid test methods and liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A recently developed confirmatory LC-MS method has been applied to the quantification of five major beta-lactam antibiotics in suspect raw bovine milk samples that gave a positive response with receptor-based (BetaStar) and rapid microbial inhibitory screen tests (Delvotest SP). In total, 18 presumptive positive raw milk samples were reanalyzed; 16 samples showed traces of antibiotic residues that could be identified and quantified by the LC-MS method, ranging from the limits of confirmation up to 38 microg/kg. Of the positive samples, only five (approximately 30%) were found to be violative of EU maximum residue limits. The most frequently detected antibiotic residues were cloxacillin and penicillin G, the former often in combination with amoxicillin or ampicillin. This study compares the results obtained by the three methods on identical samples and addresses how these relate to certain criteria such as sensitivity and selectivity. Furthermore, the limitations of the LC-MS method and the potential impact of the presence of frequently more than one residue in the same milk sample on the response of the rapid test methods are discussed.     

Rapid and sensitive determination of sulfonamide residues in milk and chicken muscle by microfluidic chip electrophoresis

A new, rapid, and sensitive method was proposed for the determination of sulfonamide residues in milk and chicken muscle samples by microchip electrophoresis with laser-induced fluorescence detection. Separation of fluorescamine-labeled sulfonamides was accomplished by using a buffer containing 5 mmol/L boric acid and 1% (w/v) polyvinyl alcohol (PVA). The pH, amount of PVA, and concentration of boric acid in the running buffer were found to have great influence on the separation. By optimizing these conditions, the separation of four sulfonamides, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, and sulfanilamide, was achieved within 1 min with limits of detection (S/N = 3) of 0.2-2.3 μg/L, which are well below the maximum residue limit. The proposed method also exhibited very good repeatability; the relative standard deviations for both within-day and between-day measurements were ≤3.0%. With a simplified sample pretreatment protocol, fast determination of sulfonamides in real samples was successfully performed with standard addition recoveries of 93.3-100.8 and 82.9-92.3%, respectively.     

Development of an enzyme-linked immunosorbent assay for the pyrethroid insecticide cyhalothrin

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of the pyrethroid insecticide cyhalothrin. Three haptens with an amine or propanoic acid terminus were synthesized and then conjugated with bovine serum albumin to give immunogens. Eight polyclonal antisera produced by rabbits were screened for titers and affinity using three different coating antigens. The antiserum CWB-C had the highest affinity with cyhalothrin and a low affinity with fenvalerate, fenpropathrin, deltamethrin, and fluvalinate. The half-maximum inhibition concentration for cyhalothrin was 37.2 microg/L, and the limit of detection was 4.7 microg/L. The recoveries of different concentrations of cyhalothrin (0.1-2500 microg/L) from fortified tap water, well water, and wastewater samples as determined with the ELISA were 81-114%.     

Development Method for Extracting and Analyzing Antibiotic and Hormone Residues from Treated Wastewater Sludge and Composted Biosolids

Extraction and analysis methods have been developed for the detection of the following four antibacterial agents and two natural estrogens in treated municipal wastewater sludge and commercial compost: sulfamethoxazole (SMX), sulfadimethoxine (SDM), tetracycline (TET), oxytetracycline (OXY), estrone (E1), and 17??-estradiol (E2). The antibiotics and estrogens were extracted from secondary sludge and mixed compost using ultrasonic solvent extraction. Citric acid (pH?4.7) and methanol were used as extraction buffer, followed by tandem-solid-phase extraction cleanup, strong anion exchange?+?hydrophilic?Clipophilic balance for antibiotics and CarboPrep/NAX for estrogens. For quantification, two different methods were employed, using HPLC?CMS/MS, with an electrospray ionization source for antibiotics and an atmospheric-pressure chemical ionization source for estrogens. Recoveries were 11?C31% for the sulfonamides (SMX and SDM) and tetracyclines (TET and OXY) and 30?C59% for the estrogens (E1 and E2) over the entire method. Limits of detection for the extraction method were in the nanogram per gram range for dry weight sludge and compost samples. Neither of the two sulfonamide antibiotics was detected in secondary sludge or mixed compost samples. Estrogens were found in compost in amounts of 160?±?65?ng/g (E1) and 21?±?3?ng/g (E2), but not in sludge. The tetracyclines, as well as what is believed to be the 4-epimer of OXY, were found in both sludge and compost in amounts of 1.57?±?0.67 and 2.95?±?0.42???g/g (TET), 0.56?±?0.12 and 6.51?±?0.52???g/g (OXY), and 7.60?±?1.68 and 1.35?±?0.24???g/g (4-epi-OXY), respectively. These results indicate that sorption-prone compounds are not removed during the wastewater treatment process and can persist through sludge digestion and that the composting process does not sufficiently eliminate these particular contaminants. Thus, biosolids (even composted) are an additional source of drug residues leaching into the environment, and it must be considered while using biosolids as fertilizer.     

Determination of isopropyl thioxanthone (ITX) in fruit juices by pressurized liquid extraction and liquid chromatography-mass spectrometry

A rapid LC-MS method, which compares different mass analyzers-single quadrupole, ion trap, and triple quadrupole-was developed for the quantitative determination of isopropyl thioxanthone (ITX) in fruit juices. ITX, a photoinitiator in UV-cured inks that can reach foods from the cartons for beverages in which they are used, was extracted from fruit juice samples with acetone/hexane (50:50) using pressurized liquid extraction. This method gave detection limits of 3, 3, and 0.01 microg/L and quantification limits of 10, 10, and 0.05 microg/L using single quadrupole, ion trap, and triple quadrupole, respectively. Five replicate fortifications of different fruit juices at the quantification limit gave recoveries oscillating between 68 and 72% with CV varying between 14 and 18%. This is the first report of a positive mass spectrometric identification and quantification of ITX in fruit juice samples packed in TetraPack. The sensitivity and specificity of the LC-MS/MS analysis using the triple quadrupole enables it to be the method of choice for risk assessment and monitoring. The method was applied to apricot, orange, peach, apple, grape and pineapple, and cherry and strawberry juices and to fruit nectars from Spain and Italy, and the ITX was detected in the range of 0.05-0.78 microg/L.     

Multiresidue determination of pesticides in soil by gas chromatography-mass spectrometry detection

An analytical multiresidue method for the simultaneous determination of various classes of pesticides in soil was developed. Pesticides were extracted from soil with ethyl acetate. Soil samples were placed in small columns, and the extraction was carried out assisted by sonication. Pesticides were determined by gas chromatography with electron impact mass spectrometric detection in the selected ion monitoring mode. Spiked blank samples were used as standards to counteract the matrix effect observed in the chromatographic determination. Pesticides were confirmed by their retention times, their qualifier and target ions, and their qualifier/target abundance ratios. Recovery studies were performed at 0.2, 0.1, and 0.05 microg/g fortification levels of each pesticide, and the recoveries obtained ranged from 87.0 to 106.2% with a relative standard deviation between 2.4 and 10.6%. Good resolution of the pesticide mixture was achieved in approximately 41 min. The detection limits of the method ranged from 0.02 to 1.6 microg/kg for the different pesticides studied. The developed method is linear over the range assayed, 25-1000 microg/L, with determination coefficients >0.999. The proposed method was used to determine pesticide levels in real soil samples, taken from different agricultural areas of Spain, where several herbicides and insecticides were found.     

Determination of florfenicol amine residues in animal edible tissues by an indirect competitive ELISA

Florfenicol (FF) is a broad-spectrum antibiotic used increasingly in aquaculture, livestock, and poultry to treat diseases. To avoid using labor-intensive instrumental methods to detect residues of FF in food and food products, a simple and convenient indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method for florfenicol's major metabolite, florfenicol amine (FFA), was developed using a polyclonal antibody prepared in this study. FFA was covalently attached to carrier protein as immunogen by using the glutaraldehyde method. The antibodies obtained were characterized by an ELISA method and showed excellent specificity and sensitivity with the 50% inhibition values (IC 50) of 3.34 microg/L for FFA in PBS buffer. In the ELISA, sample extractions were performed by ethyl acetate/ammonium hydroxide (90 + 10, v/v) following combined acid hydrolysis of FF and its known metabolites. The limits of detection (LOD) calculated from the analysis of 20 known negative swine muscle, chicken muscle, and fish samples were 3.08, 3.3, and 3.86 microg/kg (mean + 3 SD), respectively. Recoveries of FFA fortified at the levels of 5, 50, 100, and 300 microg/kg ranged from 64.6 to 124.7%, with coefficients of variation of 11.3-25.8% over the range of FFA concentrations studied. Validation of the ELISA method with FFA-fortified swine muscle at the levels of 10, 50, 100, and 200 microg/kg was carried out using GC, resulting in a similar correlation in swine muscle ( r = 0.97). The results suggest that this ELISA is a specific, accurate, and sensitive method, which is suitable for use as a screening method to detect residues of FFA in animal edible tissues.     

土壤中磺胺类抗生素的检测方法优化及残留、降解研究

优化了磺胺甲基嘧啶(SM1)、磺胺二甲嘧啶(SM2)、磺胺对甲氧嘧啶(SMT)、磺胺甲噁唑(SMZ)4种磺胺类抗生素的高效液相色谱(H PLC)检测方法,分析了广州市养殖场周边土壤中磺胺类抗生素的残留特征,并进行了2种磺胺类抗生素的土壤降解试验。结果表明,4种磺胺类抗生素分别在0.10~10μg ml-1范围内线性良好,相关系数R>0.99。确定了最佳提取液为甲醇:含EDTA的Mcllvain缓冲液=1∶1(V/V),4种磺胺类药物的检测限与回收率分别为2.9~4.7μg kg-1、83.6%～90.1%。广州市18个规模化养殖场周边土壤中磺胺类抗生素污染以SM2为主,含量为1.75μgkg-1,其它3种均未被检出。土壤中磺胺类抗生素的含量总体呈随培养时间而不断下降的趋势,SMZ的降解速率大于SM2。     

Molecularly imprinted polymer online solid-phase extraction coupled with high-performance liquid chromatography-UV for the determination of three sulfonamides in pork and chicken

A selective imprinted amino-functionalized silica gel sorbent was prepared by combining a surface molecular imprinting technique with a sol-gel process for online solid-phase extraction-HPLC determination of three trace sulfonamides in pork and chicken muscle. The imprinted functionalized silica gel sorbent exhibited selectivity and fast kinetics for the adsorption and desorption of sulfonamides. With a sample loading flow rate of 4 mL min (-1) for 12.5 min, enhancement factors and detection limits for three sulfonamides ( S/ N = 3) were achieved. The precision (RSD) for nine replicate online sorbent extractions of 5 microg L (-1) sulfonamides was less than 4.5%. The sorbent also offered good linearity ( r (2) > 0.99) for online solid-phase extraction of trace levels of sulfonamides. The method was applied to the determination of sulfonamides in pork and chicken muscle samples. The prepared polymer sorbent shows promise for online solid-phase extraction for HPLC determination of trace levels of sulfonamides in pork and chicken samples.     

Disposable biosensor test for organophosphate and carbamate insecticides in milk

A highly sensitive and rapid biosensor test based on disposable screen-printed thick-film electrodes was developed, which is suitable for monitoring organophosphate and carbamate residues in foods of animal origin with increased fat contents such as milk. The wild-type enzyme was combined with three engineered variants of Nippostrongylus brasiliensis acetylcholinesterase (NbAChE), to obtain enhanced sensitivity. The sample pretreatment could be reduced to a minimum. There was no extraction or fat removal necessary. With the biosensor test paraoxon concentrations down to 1 microg/L could be detected in milk. The detection limit for carbaryl was 20 microg/L. Recovery rates for paraoxon and carbaryl in milk samples lay between 89 and 107%. Ten milk samples from local markets were tested both with the biosensor test and with standard chromatographic multiresidue methods. Two milk samples caused AChE inhibition rates of >50%. Accordingly, 4 microg/L tebufenpyrad, 4 microg/L tetraconazole, and 2 microg/L bifenthrin were detected in one of these milk samples. The other milk sample contained 2 microg/L tebufenpyrad.     

Evaluation of fumonisin contamination in cornflakes on the Belgian market by "flow-through" assay screening and LC-MS/MS analyses

A total of 205 cornflake samples collected in Belgian retail stores during 2003-2004 were surveyed for the natural occurrence of fumonisin B1 (FB1), B2 (FB2), and B3 (FB3). These cornflake samples, originating from conventional as well as from organic production, were analyzed using an intralaboratory-validated LC-MS/MS method. Additionally, 90 cornflake samples were subjected to rapid screening using a flow-through enzyme immunoassay method to demonstrate the practicability of a screening test coupled to a validated confirmatory LC-MS/MS method for the management of food safety risks. FB(1) concentrations ranged from not detected (nd) [LOD (FB1) = 20 microg/kg] to 464 microg/kg with mean and median concentrations of respectively 104 +/- 113 and 54 microg/kg. For FB2 and FB3, the concentration ranges varied respectively from nd [LOD (FB2) = 7.5 microg/kg] to 43 microg/kg and from nd [LOD (FB3) = 12.5 microg/kg] to 90 microg/kg. Mean concentrations for FB2 and FB3 were respectively 12 +/- 8 and 21 +/- 15 microg/kg, while the median concentration was 11 microg/kg for FB2 and 19 microg/kg for FB3. From the statistical tests (chi2 and ANOVA model III), it could be concluded that the agricultural practice did not have any significant effect on the fumonisin concentrations but that the variation between different batches was significant (p < 0.0001).     

Development of a magnetic particle-based enzyme immunoassay for the determination of penoxsulam in water

A competitive enzyme-linked immunoassay (ELISA) for the quantitation of Penoxsulam [2-(2,2-difluoroethoxy)-6-(trifluoromethyl-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl))benzenesulfonamide] in ground and surface waters was developed. This immunoassay utilizes magnetic particles as the solid phase to which polyclonal rabbit anti-Penoxsulam antibodies are attached. The ELISA has an estimated detection limit of 0.17 ppb (microg/mL) of Penoxsulam in water. Specificity studies indicate that the antibody can distinguish Penoxsulam from its major metabolites and structurally similar pesticides. Interference studies indicate that the ELISA has a wide tolerance of sample pH and salinity and for compounds commonly found in surface and ground waters. The ELISA was shown to compare favorably to LC-MS/MS on ground and surface water samples (r(2) = 0.957). The various studies performed demonstrate the usefulness of the ELISA technique as a rapid and high-throughput analytical method for the cost-effective monitoring of water samples.     

Method for the analysis of triadimefon and ethofumesate from dislodgeable foliar residues on turfgrass by solid-phase extraction and in-vial elution.

Triadimefon, a fungicide, and ethofumesate, an herbicide, are commonly applied to turfgrass in the Pacific Northwest, resulting in foliar residues. A simple and rapid method was developed to determine triadimefon and ethofumesate concentrations from dislodgeable foliar residues on turfgrass. Turfgrass samples were washed, and wash water containing surfactant (a 0.126% solution) was collected for residue analysis. This analytical method utilizes a 25 mm C(8) Empore disk and in-vial elution to quantitatively determine triadimefon and ethofumesate in 170 mL aqueous samples. The analytes were eluted by placing the disk in a 2 mL autosampler vial with 980 microL of ethyl acetate and 20 microL of 2-chlorolepidine, the internal standard, for analysis by GC/MS. The method quantitation limits are 0.29 microg/L for ethofumesate and 0.59 microg/L for triadimefon. The method detection limits are 0.047 microg/L and 0.29 microg/L for ethofumesate and triadimefon, respectively. Concentrations of triadimefon and ethofumesate from dislodgeable foliar residues from a field study are reported.