Sulfonamide (Sas) Schnelltestkit

A new multi-residue method for analysis of sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfaguanidine and sulfamethoxazole) in non-target feeds using high performance liquid chromatography-fluorescence detection (HPLC-FLD) and pre-column derivatization was developed and validated. Sulfonamides (SAs) were extracted from the feed with an ethyl acetate/methanol/acetonitrile mixture. The cleaning was performed on a Strata SCX cartridge. The HPLC separation was performed on a Zorbax Eclipse XDB C18 column with a mobile phase gradient system of acetic acid, methanol and acetonitrile.

 The method has been validated according to EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit, detectability, limits of detection and quantification, recovery, precision and selectivity have been determined and reasonable results obtained. Using the HPLC-FLD method, recoveries were satisfactory (79.3-114.0%), with repeatability and reproducibility ranging from 2.7-9.1% and 5.9-14.9%, respectively. The decision limit (CCα) and detectability (CCβ) were 197.7-274.6 and 263.2-337.9 µg/kg, respectively, and the limit of detection (LOD) and limit of quantification (LOQ) were 34.5-79, 5 or 41.3–89.9 µg/kg, depending on the analyte. The results showed that this analysis method is simple, fast, sensitive and suitable for routine control of feed. The limit of detection (LOD) and the limit of quantification (LOQ) were 34.5–79.5 and 41.3–89.9 µg/kg, respectively, depending on the analyte. The results showed that this analysis method is simple, fast, sensitive and suitable for routine control of feed. The limit of detection (LOD) and the limit of quantification (LOQ) were 34.5–79.5 and 41.3–89.9 µg/kg, respectively, depending on the analyte. The results showed that this analysis method is simple, fast, sensitive and suitable for routine control of feed.

introduction

Livestock production has increased significantly in recent decades, mainly due to intensive farming. Veterinary medicines are used extensively in animal husbandry for the treatment of bacterial infections and for prophylactic purposes. One group that is widely used as antibacterial drugs in both human and veterinary medicine are sulfonamides (SAs). Sulfonamides are synthetic antimicrobial compounds that are commonly used to treat respiratory, gastrointestinal, and urinary tract infections [  1  ].

  •  Sulfonamides are high-spectrum chemotherapy drugs against gram-positive and gram-negative bacteria and are used to treat infections caused by microorganisms resistant to other antibiotics. High doses of SAs can provoke strong allergic reactions, so the drugs are prescribed carefully.
  •  In veterinary practice, SAs can be incorporated into animal feed (medicated feed) as a therapeutic agent. SAs can be present in non-medicated feeds because traces of previously manufactured medicated feed can be inadvertently mixed with the first batches of the next feed when using the same production line.  The transfer of trace amounts of compounds from one manufactured batch of feed to the next is called carryover.
  •  Consequently, current feed production technologies can lead to unavoidable cross-contamination of non-medicated feed with undesirable substances, contributing to potential animal health and public health hazards.
  • In the European Union, animal feeds have to comply with several regulations set by the current legislation. The main regulations give requirements for the composition, storage, transport and use of feed.
  • Cross-contamination can occur during production and handling at the feed mill, during transport or on the farm. The carry-over of veterinary medicinal products during feed production can also lead to the contamination of non-medicated feed.
  • The use of antibiotics in feed for non-medical purposes was banned in the EU in 2006. Monitoring for the undeclared or illegal use of these antibacterial substances takes place within the framework of national feed control programs6  ].

Several analytical methods have been developed to identify and quantify sulfonamide residues in animal tissues, eggs, milk and honey.

Existing methods include high performance liquid chromatography (HPLC) with different detectors: diode array/UV detector [  1  ,  12  ,  13  ,  14  ], fluorescence [  15  ], mass spectrometry [  9  ,  16  ,  17  ], gas chromatography (GC) [  7  ] and capillary electrophoresis (CE) [  2 ]. However, only a few methods are described in the literature for the analysis of sulfonamides in medicated and non-target feed. The methods for detecting and determining sulfonamides in animal feed are based on liquid chromatography with a UV detector, diode array detectorand enzyme-linked immunosorbent assay test (ELISA)Liquid chromatography-tandem mass spectrometry methods (LC-MS/MS or UHPLC-MS/MS) have been used to validate multi-residue quantification of sulfonamides in feed.

Kimet al. developed an HPLC-UV method for the determination of sulfacetamide, sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethizole, sulfamethazine, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxin, sulfisoxazole, sulfadimethoxine, sulfaquinoxaline, sulfamethoxydiazine, sulfisomidine and sulfachloropyrazine. The recoveries achieved ranged from 78.2–105.2%, while the limit of quantification (LOQ) values ​​ranged from 46.9–150.0 µg/kg .

A method for the determination of sulfadiazine and sulfamethazine in feed was developed by Patyra et al. developed using an LC-MS/MS instrument. Mean recovery ranged from 76.0–77.4% and LOQ values ​​ranged from 106.2–174.6 µg/kg.

Gavilan et al. proposed a method for the determination of sulfaclorpiridazine, sulfadiazine, sulfametazine, sulfamethizole, sulfametoxazole, sulfametoxipiridazine, sulfapiridazine, sulfaquinoxaline, sulfatiazole and sulfadimidine in feed samples using LC-MS/MS with a mean recovery of 84–113 % .

Liu et al. proposed a method for the determination of 16 sulfonamides in feed using a solid phase extraction (SPE) technique with basic alumina cartridges and LC-MS/MS, with a mean recovery of 80.1–111.7% .

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In this study, liquid chromatography with a fluorescence detector after pre-column derivatization with fluorescamine is used for the first time for the detection and quantification of five sulfonamides in non-target feeds. This paper presents the development of a selective and sensitive method for the simultaneous analysis of five SAs using Strata-SCX strong cation exchange cartridge extraction, fluorescamine derivatization, and high-performance liquid chromatography-fluorescence detection (HPLC-FLD) analysis.  The whole process has been validated according to Commission Decision 2002/657/EC.

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