Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 11, 1850–1860, http://dx.doi.org/10.1080/19440049.2014.953012

Semicarbazide – from state-of-the-art analytical methods and exposure to toxicity: a review Wen-Rui Tian, Ya-Xin Sang and Xiang-Hong Wang* Department of Food Science and Technology, Agricultural University of Hebei, Baoding, China (Received 6 May 2014; accepted 4 August 2014) This review assesses the state of the art concerning semicarbazide (SEM). Originally, SEM was primarily detected as a nitrofurazone veterinary metabolite, but over time scientists gradually found that azodicarbonamide in sealed cans and flour could also lead to the generation of SEM. This discovery makes the study of SEM particularly interesting. At present, an increasing number of researchers are investigating the toxicity of SEM and developing more and better analytical methods for the determination of SEM. In recent years, many researchers have focused on exposure from different foods, the public awareness of hazards and analytical detection methods for SEM in different foods. Although there have been significant achievements, these results have not been summarised in a review. The exposure from different foods, toxicity and methods of detection for SEM are comprehensively reviewed here. This review will provide not only others with a better understanding of SEM but also background information to facilitate future research. Keywords: semicarbazide; exposure; toxicity; sample preparation; determination

Introduction Semicarbazide (SEM) was originally studied as a result of the abuse of the veterinary drug nitrofurazone, an inexpensive and highly effective drug for the treatment of bacterial diseases in farm animals and aquaculture. Nitrofurazone has been extensively used in intensive farming for many years, but due to concerns raised regarding the human health risks associated with the use of these drugs and the lack of sufficient safety data, nitrofurazone was banned in the European Union in the 1990s. The methods developed for the detection of abuse originally aimed at the parent drugs themselves. However, as the parent drugs are very quickly metabolised in the living organism, the usefulness of these methods was rather limited. However, SEM, a metabolite of nitrofurazone, forms a more stable protein conjugate with proteins than the compound in the original drug. Thus, SEM can be used to detect nitrofurazone as its metabolite and measure whether nitrofurazone abuse has occurred (Mulder et al. 2007; Maranghi et al. 2009). A review article published in 2005 gave an overview of the information concerning the presence of SEM in food and the analytical methods available at that time (de la Calle & Anklam 2005). Likely sources of SEM in food matrices are summarised and discussed, and detailed information is given about the analytical methods used to determine SEM. However, the determination methods included in this review focused only on the use of LC-MS. Li et al. (2010) summarised the potential sources of SEM in food, finding that in addition to being a *Corresponding author. Email: [email protected] © 2014 Taylor & Francis

nitrofurazone metabolite, residues can be also found in the environment, food packaging, food processing procedures and flour-improving agents, which can also lead to SEM contamination in food. Zou et al. (2011) studied the original nitrofurazone drug and natural metabolites as residues in animal tissues using various detection techniques, including LC-UV, LC-MS, LC-MS/MS and ELISA, to detect the presence of these compounds and identify the minimum detection limit, comparing the scope, principles, characteristics and research progress of a variety of methods. However, in these papers, SEM was detected as a nitrofurazone metabolite, or the SEM detection method was not described in full. We believe that there is a lack of understanding of and concern about SEM among the public; thus, this review discusses the presence of SEM, toxicity, and detection and analysis methods to provide an integrated narrative to facilitate future research work and expand public knowledge of SEM (de Souza et al. 2005; Stolker & Brinkman 2005; Hong 2009; Pan et al. 2011). Analytical methods Modern residue methods generally include two parts: sample pre-treatment and measurement. Most drug residues utilise the physicochemical properties of various samples for the development of the extraction, purification, concentration and other pre-treatment steps. Thus, pre-treatment processes often play a major role in analysis. The most commonly used extraction methods are integrated into purification methods: the oscillation method

Food Additives & Contaminants: Part A and Soxhlet extraction, ultrasonic extraction, liquid–liquid extraction, column chromatography and thin-layer chromatography, among others. The sample preparation methods differ by determination method and the properties of different foods.

Sample preparation Solvent extraction Solvent extraction methods used for veterinary drug residues are divided into four categories: single-solvent extraction; mixed-solvent extraction; and extractions using alkali- or acid-buffered solutions. Multi-residue methods commonly use organic solvent extraction. When performing spectral analysis, the use of aqueous extracts can reduce the number of sample transfers. In the cases of GC, GC-MS, HPLC and LC-MS, only organic solvents are used for extraction. In acidic environments, SEM in a matrix of bound proteins undergoes dissociation followed by acid hydrolysis by the extracting agent from the extraction solution. For economic reasons, the pre-treatment of a sample in which the matrix is simple and has little effect on the measurement usually performed using liquid–liquid extraction. To obtain the most accurate results, the extraction solvent should be chosen carefully. During the experiment, the choice of extraction solvent directly affects the recovery. To obtain the highest possible recovery, we must choose a solvent with a high extraction efficiency. Yu et al. (2008) reported that biological samples, which often contain a high content of fat, proteins, pigments, etc., occasionally undergo emulsification during the extraction process. This emulsification ultimately affects the measurement result and can be minimised by pH buffering, salt or ionic strength adjustment using the acid–ethanol extraction method, the addition of perchloric acid or trichloroacetic acid, and freeze-drying to remove proteins as well as fat and fat emulsions. In 2011, Tao Yan-fei (2011) selected acidified ethanol, acidified acetonitrile and acidified methanol solvent and used the rapid solvent extraction method for extraction, finding that 1:1 methanol/ trichloroacetic acid (0.5 mol l–1) solution was the best extraction solvent in terms of recovery.

Solid-phase extraction (SPE) In SPE, a solid adsorbent is applied to a liquid sample to adsorb the target compound from the sample matrix, followed by the separation of interfering compounds and elution to achieve separation and enrichment of the target compound. SPE can be used in complex samples for the minor or trace extraction of target compounds as well as for purification or enrichment. It is currently the most prevalent pre-treatment method for veterinary drug residue

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analysis. SPE-specific products for sample extraction, concentration and purification are very good, offering a variety of chemical properties, sorbent types and sizes. SPE cartridges are often basic alumina, C18, silica gel or SCX columns. SPE also known as liquid–solid extraction (LSE), is based on the principle of LC, yielding fast and efficient sample preparation. The basic process is as follows. First, the biological sample solution is added to the column packed with the stationary phase. Analytes are retained by adsorption, distribution, ion exchange and other mechanisms and then eluted with an appropriate solvent to achieve separation, purification and enrichment. Compared with LLE, SPE introduces fewer impurities, does not induce emulsification, provides a high extraction efficiency, is appropriate for biological samples and allows rapid sample pre-treatment, making it suitable for volatile drugs, which are often thermally unstable. Recently, SPE and HPLC analysis techniques, such as the chromatographic determination of drugs and their metabolites in biological samples, have been increasingly used domestically and internationally. Based on the properties of SEM, the most popular columns are HLB, C18 and neutral alumina columns, among others. Sample preparation techniques Due to its low molecular weight of approximately 75 Da, the detection of SEM suffers from very serious interference by the matrix, providing low sensitivity. Furthermore, SEM is highly polar, and its retention time is very short. Thus, the derivatisation of 2-nitrobenzaldehyde to standard products is used to increase the ionisation efficiency of the metabolites. The general procedure for the extraction of SEM is as follows. First, 50 ml of the sample are placed in a brown polypropylene centrifuge tube and subject to hydrochloric acid hydrolysis. After adding the derivatising agent, the internal standard working solution is added, followed by homogenisation and 1 min of vortexing. The solution is set in a water bath oscillator at 37 ± 1ºC in the light for 16 h. The tubes are removed, and the solution allowed to cool to RT. Hydrochloric acid is then supplemented with 1 vol. of phosphate-buffered saline solution, and the pH is adjusted to 7.2–7.3 using different concentrations of HCl and NaOH. An appropriate amount of ethyl acetate is added, mixed and then centrifuged, and the upper ethyl acetate solution is added to a 100-ml heart-shaped flask. Ethyl acetate is then added, and the extraction step is repeated. The extracts are combined and subjected to rotary evaporation in a 40ºC water bath to dryness. The mobile phase is concentrated by heating to constant volume, and the filtration membranes are assayed. Due to the widespread presence of SEM in food, the establishment of appropriate sample pre-treatment methods for SEM extracted from food as well as its precise

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determination is important. The sample pre-treatment extraction method must be chosen based on the characteristics of the sample of interest. Meat, fish, shrimp Fish and meat are complex matrices, which may affect the detection of SEM. Complex matrices can lead to the detection of impurities and peak broadening; thus, SPE is used in the purification and elution steps to exclude unnecessary interference. Depending on the experiment, SPE cartridges appropriate for SEM include HLB, C18 and SDB-L, among others (Hua-Lin 2006; Yuan et al. 2011; Valera-Tarifa et al. 2013). Flour products SEM is found in flour products as a result of the use of azodicarbonamide as an additive. Thus, the pre-treatment method must be able to accommodate reaction products, generated by-products and products bound tightly to the surface. To this end, one article proposes a process opposite to that described previously: the use of 2:1 methanol– water to dissolve some of the SEM, shock horizontal shaking extraction for 30 min, centrifugation for 5 min, removal of the supernatant and acid hydrolysis or another derivatisation treatment (Qian 2011). Dairy products Dairy products are rich in minerals, protein, vitamins and other nutrients. However, precisely because such products contain protein, the determination of SEM can be difficult. To address this issue, the following procedure is used to remove the protein: acid hydrolysis followed by the customary pre-treatment using the appropriate concentrations of hydrochloric acid and trichloroacetic acid. Our experiments reveal that TCA is the most effective for the removal of various proteins in acid solution. Processed food and eggs Considering the wide variety of processed foods, the sample processing method must be chosen based on the particular foodstuff of interest. For coffee, tea, milk drinks and other products rich in fat, but not butter or eggs, after the ethyl acetate is evaporated from the mobile phase, the appropriate amount of acetonitrile saturated with n-hexane should be added. Jams and other fruit product matrices are complex, mainly because of the production process and the addition of additives to improve their longevity. The same experimental treatment as used for meat products, namely, the use of SPE followed by purification, is recommended (Stastny et al. 2009).

Qualitative and quantitative analysis Currently, SEM analytical methods mainly include HPLC, LC-MS and immunoassay.

Immunosorbent assay (IA) The IA technique is based on antigen and antibody specificity analysis of a reversible binding reaction. The immune response involves the stereochemistry, charge, hydrogen bonding, and dipole of the antigen and antibody molecules. IA is generally coupled with conventional chemical analysis techniques, providing unmatched selectivity and high sensitivity. This technique is very suitable for the analysis of trace components in complex matrices, such as the detection of drug residues in food in a wide range of applications (Tang et al. 2009).

ELISA methods At present, the existing literature (domestic and international) on SEM immunological detection methods is mainly limited to the enzyme-linked immunosorbent assay (ELISA). ELISA analysis is specific to the antigen–antibody reaction and enzyme catalysis, efficiently incorporating an immunoassay technique, providing high accuracy, higher reaction sensitivity, high specificity, relatively low technical requirements, a shorter detection time and the ability to analyse large quantities within a short time as well as screening of large numbers of samples simultaneously. Cooper et al. (2007) reported the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, with a cross-reactivity of 1.7% with NFZ and a negligible cross-reactivity with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CCβ) of 0.25 μg kg–1 when a threshold of 0.21 μg kg–1 is applied to the selection of samples for confirmation (lowest observed: 0.25 μg kg–1 fortified sample, n = 20), thus satisfying the European Union’s minimum required performance limit for nitrofurans of 1 μg kg–1. NFZ-incurred muscles containing SEM at 0.5–5.0 μg kg–1 as determined by LC-MS/MS all positively detected by this ELISA protocol, which is also applicable to egg and chicken liver. Monoclonal antibodies (McAb) were produced to detect SEM, a metabolite of nitrofurazone used as a marker residue in animal food production. A carboxyphenyl

Food Additives & Contaminants: Part A derivative (CPSEM) of SEM was synthesised following derivatisation with 4-carboxybenzaldehyde (CBA). CPSEM was purified by recrystallisation and conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) as an immunogen or coating antigen, respectively. Hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes from mice immunised with CPSEM-BSA. Hybridomas 1F10 and 4F2 secreting antibodies against CPSEM were obtained and subcloned. Ascites of monoclonal antibodies were prepared by injecting 1 × 106 cells of hybridoma 1F10 into mice abdomen. McAb obtained from hybridoma 1F10 was highly specific for CPSEM and had no cross-reaction with various nitrofurazone metabolites or a range of veterinary drugs. The sensitivity of McAb to SEM was 0.01 ng ml–1, and the IC50 value was 1.3 ng ml–1 (SEM in the form of CPSEM). McAb 1F10 is suitable for the development of an immunoassay for SEM with sufficient sensitivity for monitoring nitrofurazone residues. In 2009, Yao et al. (2009) and coworkers synthesised hapten (CP-SEM) from SEM (SEM) and the aldehyde acid (CP). The hapten was conjugated to carrier proteins to immunise Bal b/C mice, followed by the application of the hybridoma technique to establish stable hybridomas secreting anti-SEM strains. Through the analysis of different antibody combinations and optimisation, SEM was prepared by a monoclonal antibody. Compared with the established test, the ELISA method for SEM detection is indirectly competitive, with a sensitivity of 0.1 ng ml–1 and an average recovery of 98.47%. ELISA provides high sensitivity and specificity, low cost and relatively simple sample preparation. At present, commercial kits have been listed for the detection of SEM domestically and internationally. The actual testing method often uses the immunoassay method as a screening method, followed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a confirmatory method. Fluorescence polarisation immunoassay (FPIA) The FPIA method is based on the difference between the degree of fluorescence polarisation of a fluorescentlabelled antigen and the antigen–antibody conjugate. FPIA is a competitive method for directly measuring concentrations in dilute solutions. Shen et al. (2009) prepared antibodies using a novel FPIA method for the nitrofurazone metabolite SEM. Through the design and synthesis of a new SEM hapten, CEPSEM, after coupling to a carrier protein to immunise New Zealand white rabbits, good polyclonal antibodies were prepared with high affinity and specificity. Furthermore, the fluorescent tracer CEPSEM-HDF was designed and synthesised to establishment the SEM FPIA technique. Using the optimised conditions of a tracer concentration of 0.5 nmol l–1 and an antibody dilution of

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1/100, the following results were obtained: an IC50 value of 47.9 μg l–1, a detection limit of 8.3 μg l–1 and a linear range of 15.8–145.7 μg l–1. FPIA methods are simple and low cost, with a high sample throughput, easily realised automatic control and on-site rapid screening, among other characteristics suitable for the detection of small to medium-sized molecular substances. It is commonly used in the determination of the concentration of the drug hapten. Colloidal gold immunochromatography (ICA) ICA is an immunogold chromatography technique involving solid-phase film immunoassay that has increasingly attracted the attention of researchers. This technique is widely used for the rapid detection of drugs and toxins, proteins, nucleic acids, hormones and pesticides. Xiang et al. (2009) created a colloidal gold immunochromatographic strip to detect SEM in meat-based food residues. The active ester method was used to cross-link the SEM derivative CPSEM to bovine serum albumin (BSA), yielding the complete antigen CPSEM-BSA. The ammonium sulfate precipitation of purified monoclonal antibody, labelled anti-CPSEM, was used to restore trisodium citrate. The colloidal gold was prepared by optimising the C/T lines and gold-labelled antibody working concentration allowed the detection of SEM via a colloidal gold immunochromatographic test strip with a minimum detection limit of 0.72 ng ml–1. Although a weak nitrofurazone cross-reaction was observed, no cross-reactivity was found with other congeners. Tang et al. (2009) prepared composite anti-SEM derivative (CPSEM) antibody–colloidal gold singlestranded thiolated DNA (anti-CPSEM-Au-ssSHDNA), establishing a new high-sensitivity immunoadsorption biological barcode detection method for SEM. Ascites were used to prepare monoclonal anti-CPSEM. Next, saturated ammonium sulfate was purified and then coupled to trisodium citrate using a reduction method with a surface of 20-nm colloidal gold particles. The two conjugates prepared. In the antibody–Au coupling products based on thiolated DNA, based on competitive binding of ELISA and PCR ELISA, the enzyme technology produces signals that can be amplified DNA, establishing a new bio-barcode immunosorbent assay method for the detection of SEM with an adsorption sensitivity of 8 pg ml–1. The GICA method (ICA) is quick and easy and has high sensitivity, specificity, accuracy and stability, allowing it essentially to meet the commercial testing requirements. High-performance liquid chromatography (HPLC) HPLC is a chromatographic separation and detection technique associated with the highly sensitive quantitative

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analysis of mixtures. It is coupled with UV, fluorescence and differential refractive index detectors. Ultraviolet (UV) and diode array detectors have been used to detect the metabolites of nitrofurazone, including SEM (Xu et al. 2012). In 2008, the Chinese Ministry of Agriculture determined nitrofurazone metabolite residues by HPLC in aquatic media using a trichloroacetic acid–methanol solution and ethyl acetate as the extraction derivative on an SPE column after purification. LC-UV detection and an external standard were used. This method has a limit of 0.5 μg kg–1, a recovery of over 70% and an RSD of less than 15%. In 2009, Chumanee et al. (2009) reported the synthesis of derivatives of metabolites from furazolidone, furaltadone, nitrofurazone and nitrofurantoin using a new derivatising reagent, 2-naphthaldehyde (NTA). The reaction product was used in liquid chromatography with diode array detection (LC-DAD) for the determination of protein-bound metabolites of nitrofurazone in shrimp followed by two liquid–liquid extraction steps. Derivatives of nitrofurazone metabolites are well separated from NTA, which remains in the extract upon separation on a ChromSpher 5 Pesticide (250 × 4.6 mm, 5 mm) column at 40 ºC with a gradient of acetonitrile/5 mM ammonium acetate (pH 7.5) as the mobile phase and DAD detection at 308 nm (except for the naphthyl derivative of 1-aminohydantoin, which was detected at 310 nm). The high absorption of these derivatives allowed for the first time the simultaneous screening of these metabolites in shrimp at 1 mg kg–1 using LC-DAD. The method was validated using blank shrimp fortified with all four metabolites at 1, 1.5 and 2 mg kg–1. The recoveries were > 86% with RSDs of < 14% for all four metabolites. Comparison of the LC-DAD and APCI-MS/MS methods shows very good agreement for shrimp samples. LC-MS analysis UV detection requires the identification of a specific wavelength for detection, and the sample pre-treatment demands are very strict. MS detection does not suffer from these shortcomings, its detection limit is lower than that of UV detectors. Thus, MS detection has certain advantages for the determination of drug residues in food. The UV absorption of SEM is weak, so LC-MS analysis is more widely used for SEM detection. At present, many countries have applied LC-MS/MS for the detection of nitrofurazone drugs and their metabolites as a confirmatory method. For example, in December 2006, the Ministry of Agriculture adopted LC-MS/MS as the standard method for the determination of SEM metabolite residues in food of animal origin. In 2005, a confirmatory method based on isotope dilution LC-MS/MS was developed by Mottier et al. (2005) for the low-level determination of SEM residues in meat. The procedure entails an acid-catalysed release of protein-bound metabolites, followed by their in situ

conversion into 2-nitrobenzaldehyde (NBA) imine-type derivatives. Liquid–liquid extraction and clean-up on a polymeric SPE cartridge are then performed, followed by LC-MS/MS analysis using positive electrospray ionisation (ESI), employing MRM of three transition reactions for each compound. Reliable quantification is obtained using one deuterated analogue per analyte (d4-NBA derivative) as an internal standard (IS). Validation of the method in chicken meat was conducted following European Union criteria for the analysis of veterinary drug residues in foods. The decision limit (CCα) and detection capability (CCβ) were 0.20 and 0.34 μg kg–1, respectively, which are below the minimum required performance limit (MRPL) set at 1 μg kg–1 by the European Union. Bock et al. (2007) reported an already well-described method for the determination of SEM, which was adapted to the needs of our laboratory and checked for its robustness regarding sample conditions and the processing step. Using the same data, the method was validated and the measurement uncertainty was estimated. All criteria and requirements of Commission Decision 2002/657/EC were fulfilled. The CCα determined was 0.7 μg kg–1, and the CCβ was 0.88 μg kg–1. The measurement uncertainty was estimated as 17% taking into account the effects of the matrix, a fortification level of ≤ 0.5 μg kg–1, time and sample preparation influences. In 2008, an LC-ESI-MS/MS method for the analysis of metabolites of nitrofurazone in raw milk was developed by Radovnikovic et al. (2011). The samples were obtained by the hydrolysis of protein-bound drug metabolites, derivatisation with 2-nitrobenzaldehyd (2-NBA) and clean-up extraction liquid–liquid with ethyl acetate. LC separation was achieved using a Phenomenex Luna C18 column. The mass spectrometer was operated in MRM mode using positive ESI. The method validation was performed according to the criteria laid down in Commission Decision No. 2002/657 EC. The validation included the determination of the linearity, repeatability, within-laboratory reproducibility, accuracy, decision limit (CCα) and detection capability (CCα). The calibration curves were linear, with typical R2 values higher than 0.991. The coefficient of variation (CV, %) was below 9.3%, and the accuracy (RE, %) ranged from −7.0% to 7.0%. The CV within-laboratory reproducibility was lower than 9%. CCα and CCα were 0.26 and 0.34 μg kg–1, respectively, and were thus below the minimum required performance limit (MRPL) set at 1 μg kg–1 by the European Union. Liang et al., (2009) reported a detection limit of 0.5 μg kg–1 for SEM using LCMS by adapting the national standard methods and industry standard method (SN/T1627-2005) for the determination of nitrofurazone metabolites in aquatic products. Li et al. (2009) investigated the azodicarbonamide decomposition of nitrofurazone metabolites in related studies using LC-MS/MS with ESI positive ion mode, MRM and an internal standard for the quantitative determination of the SEM content. The LOQ was 0.5 μg kg–1, recoveries were 85–94%, and the RSD of measurement was less than 9.4% (n = 5). Radovnikovic et al. (2011) developed and validated an UHPLC-MS/MS method

Food Additives & Contaminants: Part A for the pre-slaughter determination of SEM in animal plasma (bovine, ovine, equine and porcine). This method was proposed as an alternative method for on-farm surveillance. Plasma samples were derivatised with 2-nitrobenzaldehyde and subsequently extracted with organic solvent. The extracts were concentrated and then analysed by UHPLC-MS/MS. The method was validated according to Commission Decision 2002/657/EC. Inter-species recovery for SEM was 57%. CCα was calculated from within-laboratory reproducibility experiments as 0.071 μg kg–1. In addition, the assay was applied to plasma samples taken from pigs treated with furazolidone. Yu et al. (2013) focused on the detection of SEM by LC-MS/MS in samples of water and sediment slurries of aquaculture ponds. The results indicated that SEM could be simultaneously detected in the water and sediment. The LODs of SEM were 0.4–0.6 μg ml–1 in water and the sediment slurries. The sorption extents of SEM in sediment slurries were 51.0–54.5%. The recoveries of SEM were 94.7– 102.3% and 92.3–98.1% in the water and the sediment slurries, respectively. Adding NaCl significantly increased the extraction efficiencies of the NFM derivatives. These methods can potentially screen numerous aquatic environment samples of various salinities (He et al. 2009). Thus, LC-MS analysis technology provides high sensitivity and specificity for rapid analysis and detection of nitrofurazone drugs and their metabolites in confirmatory methods. In recent years, such detection methods have been widely studied and applied to nitrofurazone residues of drugs and their metabolites. However, the equipment is expensive, with complicated operation and high operating costs. Thus, many grassroots laboratories and testing units cannot afford to use this technique (Zhao et al. 2011).

Other detection methods Sensor technology Biosensor technology has been used to develop high-specificity, low-cost and highly efficient analytical techniques and has been applied to the detection of SEM. O’Mahony et al. (2011) developed a chemiluminescence-based biochip array sensing technique and applied it to the screening of honey samples for residues of banned nitrofurazone antibiotics. Using a multiplex approach, SEM could be detected simultaneously. Individual antibodies specific towards the metabolites were spotted onto biochips. A competitive assay format with chemiluminescent response was employed. The method was validated in accordance with European Union legislation 2002/657/EC, 2002 and assessed by comparison with the UHPLC-MS/MS method applied to 134 honey samples of worldwide origin. A similar extraction method based on extraction of the analytes on Oasis™ SPE cartridges followed by derivatisation with nitrobenzaldehyde and partitioning into ethyl acetate was used for both screening and LC-MS/MS methods.

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The biochip array method was capable of detecting SEM below the reference point for action of 1 μg kg–1. The detection capability was below 0.9 μg kg–1 for SEM. The IC50 values could be 2.19 μg kg–1. This biosensor method possesses the potential to be a fit-for-purpose screening technique in the area of food safety. Jin et al. (2013) used L-cysteine (Cys) and chitosan (Chi) cross-linked to form a functional polymer composite with active groups of thiol and amino groups (denoted as ChiCys) under the coupling reagents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydrosuccinimide (EDC/ NHS). It was then modified on the surface of a glassy carbon electrode (GCE). The active groups in the Chi-Cys composite can adsorb gold nanoparticles by self-assembly. The electrochemical immunosensor can be fabricated using a gold nanoparticle-functional chitosan biological composite film as a matrix to immobilise the monoclonal antibody against SEM (SEM-McAb). SEM residues were detected using electrochemical impedance spectroscopy. Under the optimised conditions, the relative change in impedance was proportional to the logarithmic value of SEM concentration in the range of 1.0 to 1.0 × 104 ng ml–1 (r = 0.9998) with a detection limit of 1.0 ng ml–1. The specificity, reproducibility, stability and accuracy of the proposed impedimetric immunosensor were also evaluated. The propose immunosensor was economical, efficient and potentially applicable in the detection of SEM in food samples. Spectrometric detection technology Xie et al. (2013) studied the IR, Raman and surfaceenhanced Raman scattering (SERS) spectra of SEM hydrochloride and assigned the vibrational bands based on density functional theory (DFT) calculations. The calculated Raman spectra were in good agreement with the experimental Raman spectra. The SERS method coupled with active gold substrates has also been applied for the detection of the three chemicals with pure water as the solvent, with a limit of detection of as low as 10 μg ml–1 (less than 45 μg ml–1). These results showed that SEM could be detected using vibrational spectroscopy, a potentially powerful tool for the rapid detection of these species derived from flour additives. SEM occurrence in food products To date, SEM has been found in various foods and has many different sources. In addition to the use of nitrofurazone drugs (of which it is a metabolite) food packaging, flour improving agents and food processing operations are potential sources of SEM contamination in food. In 2013, the report of the twentyfirst session of the Codex Committee on Residues of Veterinary Drugs in Food (Codex 2013/26-RVDF 2013) has shown that semicarbazide is not a unique indicator of nitrofurazone use and low levels can be associated.

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Animal-derived food Animal-derived food refers to food of animal origin, including poultry, meat, eggs, aquatic products, milk and dairy products, which are the primary sources of protein, fat, minerals and vitamins A and B for the human body. With the improvement of living standards, meat, eggs, milk and dairy products greatly enrich people’s diets but have also suffered from varying levels of concern about the dangers of excessive chemical drug residues in animalderived food. Due to its highly toxicity and carcinogenic effects, nitrofurazone has aroused attention in countries all over the world. Although most of the world has prohibited the use of nitrofurazone in veterinary drugs, because of its efficacy and price advantages, farmers continue to use it without authorisation. Thus, in animal-derived food, one of the main sources of SEM is the abuse of nitrofurazone in veterinary drugs. The majority of testing laboratories treat SEM as the metabolite of nitrofurazone. In these places, SEM is used as a standard to identify the excessive use of the veterinary drug nitrofurazone from food samples (Xu and Xu 2007; Yi-ping 2008). At the present, an increasing number of studies are finding that the presence of SEM may not exclusively be the result of the use nitrofurazone; instead, it may also be inherent in the food itself, such as honey or bee pupae. Male pupae and its lyophilised powder is a new development in bee products in recent years; its export volume has increased on a yearly basis, and its market prospects are broad. However, residues of nitrofurazone antibiotics exceeding the standards, especially nitrofurazone metabolites such as SEM, are frequently found. Zhou et al. (2008) conducted an experiment to determine the causes of SEM levels exceeding the standards. They found that in bee colonies not using any nitrofurazone drugs, besides SEM, the levels of nitrofurazone metabolites did not exceed 0.12 μg kg–1. The SEM content in the drone pupa was lowest at 14 days of age (0.35 μg kg–1). With increased aging, the SEM content increased significantly, with the highest content found at 24 days of age (up to 15.6 μg kg–1). On the other hand, pre-cooking using 50 mg kg–1 sodium hypochlorite disinfectant also led to the detection of SEM in meat. Thus, SEM may inherently exist in animal products (Zhou et al. 2008).

Aquatic food Aquatic food refers to plants and animals obtained from marine and freshwater fisheries and their processed products and can be categorised into fish, shrimp, crab and shellfish. Currently, the world’s annual output of aquatic products is 1.2 million tonnes. The presence of drug residues in aquaculture is a major threat to human health and has led to widespread concern in society and the

government. Efforts to control aquaculture drug residues aim to accommodate human health needs as well as the sustained and healthy development of aquaculture and aquatic products to satisfy the needs of international trade competition. According to FAO statistics, since the 1970s, approximately 75% of the world’s output of aquatic products has been processed after sales. SEM is found in many forms of seafood. The source of SEM in aquatic products is relatively complicated and can be divided into four categories: naturally occurring, resulting from pollution from the sea, resulting from the processing of aquatic products and use as disinfectant, the last of which includes the abuse of nitrofurazone (Xu et al. 2010; Tian et al. 2013). When residual disinfectant is introduced into aquatic products, it can produce SEM. The SEM content and disinfectant concentration and action are directly proportional to the time and contact area. Therefore, it is important to remove residual disinfectant as much as possible in the cleaning processes. After SEM was first detected in aquatic food, many researchers have studied SEM in aquatic products, exploring its origin, bioaccumulation and elimination rule; its content in the body and the surrounding environment; and the SEM pollution conditions of some regions. The results showed that in shellfish aquatic products, shell SEM content is high, but almost no SEM has been found in the meat, the living body of shrimp shells, shrimp shells or the water in the shells. Thus, the shell is the source of SEM in shellfish aquatic products. Shellfish and other organisms have SEM enrichment capabilities that vary by species. Clam SEM content is closely related to the water environment. Clams can absorb water and accumulate a small amount of SEM in the body. When transferred to clean seawater, clams can eventually eliminate SEM residues from their bodies. The SEM inside the clam obeys a similar elimination rule: an initially rapid elimination followed by a long plateau in the SEM content (Ni et al. 2012; Xing et al. 2012; Hassan et al. 2013; McCracken et al. 2013; Yu et al. 2013). Flour products Flour is indispensable in the modern diet. A regular on the dining table, flour products form a crucial part of many foods, including bread, noodles, dumplings and hamburgers. It exists so widely that safety of flour additives is particularly important to human food safety. For decades, mankind continuously pursued flour quality improvement, and accompanied by the emergence of additives, food taste has been greatly improved. However, as mentioned above, the flour improvement agents added to improve the taste may or may not be safe. As a new type of flour additive, azodicarbonamide is popular and accepted in the world today. If it can be safely used in food, it is the ideal substitute for potassium

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bromate. Azodicarbonamide at low levels can undergo fast oxidation with moist wheat flour, performing better than potassium bromate, and has the dual role of bleaching and oxidation (Li et al. 2009). In an in-depth study of azodicarbonamide, its decomposition was found to release oxygen and other harmful byproducts under high-temperature hydrolysis. Studies have shown that dry azodicarbonamide-containing flour heated to 150–200ºC forms SEM. Becalski et al. (2006) tested 11 types of baked food made from flour for SEM. Ye et al. (2011) tested SEM in different types of flour products. These studies reported that the SEM contents of flour products levels varied substantially. Noonan et al. (2008) discussed the conversion of azodicarbonamide into SEM in bread making. Notably, SEM is not generated in the dough stage, being found only that stage in bread making. As a flour additive, azodicarbonamide breaks down into products including SEM under certain conditions. The main way to produce SEM from azodicarbonamide-containing flour is to subject it to high-temperature thermal hydrolysis. It has been postulated that the acid hydrolysis of biurea can form SEM with an efficiency of approximately 0.1%. Thus, azodicarbonamide is commonly used as a flour additive up to a maximum of 45 mg kg–1 flour in the United States, Canada and Asia. However, azodicarbonamide is banned as a food additive in Australia and Europe.

red alga, locust bean gum, carrageenan, gelatin, starch and glucose) and determined the total free and bound SEM. For the samples processed with 0.015–0.05% of active chlorine hypochlorite solution, the SEM content only increased significantly in the protein powder; carrageenan and starch were found to form a small amount of SEM. For the North Sea shrimp, milk, soy flakes and red algae, the SEM content did not change significantly when the active chlorine content was increased from 0.015% to 1%. The chicken, protein powder, carrageenan, locust bean gum, gelatin and starch containing 1% of active chlorine hypochlorite solution all produced SEM. When processed with 12% hypochlorite solution, the shrimp, chicken, soy flakes, red alga, carrageenan, protein powder, glucose and starch all contained SEM. Azodicarbonamide has also been widely used as a blowing agent for the production of gasket seals in food jars, and it has been connected to the elevated levels of SEM encountered in baby food products. It was shown that SEM is a minor decomposition product formed from azodicarbonamide during the heat treatment needed for the sealing of the jars. The European Union prohibited the use of azodicarbonamide as a blowing agent for cap seals that may come into contact with foodstuffs in 2005 (Hoenicke et al. 2004; de Souza et al. 2005; Szilagyi & de la Calle 2006; Zhi-Feng et al. 2009; Han et al. 2012).

Additives in processed food

Toxicity of SEM

Food processing involves complex and precise processing of different raw ingredients and additives to produce a variety of foods appropriate for different age groups, enriching people’s eating habits. These food additives change the taste and colour of food, increasing visual and taste enjoyment, but may also pose a threat to human health. After the findings of SEM in carrageenan and alerts arising through the RASFF, over 100 different samples of carrageenan have been analysed by Hoenicke et al. (2004), with 80% testing positive. The SEM levels usually ranged from < 1 to 50 mg kg–1. However, some samples yielded SEM concentrations of up to 400 mg kg–1. Nevertheless, the parent drug nitrofurazone was not detected (below 10 mg kg–1), even in carrageenan, which contained > 100 mg kg–1 SEM. Carrageenan is a food additive used as a thickening, gelling and suspending agent, e.g. in ice cream, pudding, yoghurt, fruity jelly, chocolate milk and different meat products. The detection of SEM in carrageenan might therefore be explained by its natural occurrence in the raw materials (Hoenicke et al. 2004). Sodium hypochlorite is widely used for sanitisation and disinfection in food plants and in agriculture. Hoenicke et al. 2004) used 0.015%, 0. 05%, 1% and 12% of active chlorine hypochlorite solution to analyse different samples (North Sea shrimp, chicken, milk, protein powder, soybean,

Cumulative toxicity The cumulative toxicity test results for SEM show that it provides a moderate accumulation of toxicity. SEM subchronic toxicity studies show that animal weight gain slows as the SEM content per body volume gradually increases, especially among female mice, which experienced growth stagnation or even weight loss, indicating that SEM has a dose–effect relationship. Comparing the organ coefficient, blood tests and histological examination, SEM causes damage to the heart, liver and kidneys. In SEM reproductive toxicity studies on male mice, comparing the experimental group with a negative control group, the sperm deformity rate of the high-dose group (2.7% ± 0.01%) and medium-dose group (2% ± 0.03%) were significantly elevated, indicating a certain degree of SEM reproductive toxicity. The acid phosphate activity in low-dose group mice tests (31.5 ± 2.07 IU l–1) was significantly elevated, indicating that low doses of SEM impede sperm capacitation and other activities in a dose-sensitive manner and may be an important cause of male infertility.

Chromosome aberration toxicity Chromosome aberration and marrow cell micronucleus tests in mouse bone on different dose groups of female

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mice reveal a significant difference, indicating that SEM possesses mutagenic action. In chromosome aberration tests on bone marrow cells in mice, the aberration rate in the high-dose group (30.7%) and moderate-dose group (23.6%) was obviously higher than that of the negative control group (1.4%). Thus, the test result is positive, revealing that SEM is a chromosome-splitting agent. Embryo toxicity studies of SEM, in which each dose group was compared with a negative control group under the same experimental conditions, no significant differences were found in foetal rat average weight, body length or tail length, indicating that SEM possesses no developmental toxicity. Furthermore, the occurrence of bone and internal organ malformation in foetal rats was not significantly different than that in the negative control group, indicating that SEM has no obvious teratogenic effects under the experimental conditions. In SEM toxicity studies of the immune system on male mice, the thymus index and spleen weight are lower, indicating that SEM induces immune suppression male mice (but not female mice) (Jia 2008). Genotoxicity Vlastos et al. (2010) observed in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and in vivo micronucleus induction on rat bone marrow polychromatic erythrocytes (PCEs). Comparing the MN and SCE frequencies among the control and examined concentrations (0.5, 2.5, 5, 10 and 20 μg ml–1) via SCE analysis did not reveal statistically significant differences except, marginally, for the highest concentration (20 μg ml–1). On the other hand, oral administration of 50, 100 and 150 mg kg–1 b.w. of SEM produced a statistically significant effect on MN frequencies on Wistar rats’ bone marrow PCEs, with no dose–response pattern. Endocrine disruption Maranghi et al. (2010) studied the pleiotropic in vivo toxicological effects of SEM to explore its possible role as an endocrine modulator. The endocrine effects of SEM were assessed in vivo in male and female rats after oral administration for 28 days at 0, 40, 75 and 140 mg kg–1 b. w. pro die during the juvenile period. The vaginal opening and pre-putial separation were recorded. The concentration of sex steroid in the blood, ex vivo hepatic aromatase activity and testosterone catabolism were detected. The in vitro approach to testing the role of SEM as (anti)estrogen or N-methyl-d-aspartate receptor (NMDAR) (anti)agonist included different assays: yeast estrogenicity, MCF-7 proliferation, stimulation of the alkaline phosphatase activity in Ishikawa cells and LNCaP-based NMDAR interference assay. In vivo SEM-treated female rats showed delayed

vaginal opening at all tested doses, whereas in males preputial separation was anticipated at SEM doses of 40 and 75 mg kg–1 and delayed at 140 mg kg–1. The latter effect was probably due to the significantly decreased body weight gain observed at the higher doses in both sexes. Serum estrogen levels were dose-dependently reduced in treated females, where as dihydrotestosterone serum levels were also decreased, but a clear dose– response was not evidenced. Testosterone catabolism was altered in a gender-related way: aromatase activity was increased in males treated at 75 and 140 mg kg–1 and in females in all dose groups. In the three estradiol-competitive assays, SEM showed a weak anti-estrogenic activity, whereas in the LNCaP-based NMDAR interference assay, SEM activity resembled an MK-801 antagonist effect. SEM appeared to act as an endocrine disrupter, showing multiple and gender-specific mechanisms of action. A possible cascade mechanism of SEM on reproductive signalling pathways may be hypothesised. Such an in vivo–in vitro approach appeared to be a useful tool for revealing SEM activity on endocrine homeostasis (AbramssonZetterberg & Svensson 2005; Maranghi et al. 2010). Conclusions In recent years, an increasing number of researchers have studied SEM, a marker of nitrofurazone abuse. Although it has been used to indicate nitrofurazone for many years, false positive results are possible due to it is presence in many foods either naturally or as a result of decomposition by other substances. This study summarises the origins, toxicity and state-of-the-art in the determination of SEM. SEM in animal foods, aquatic products, flour products and processed products. Its occurrence can be attributed to four mechanisms: natural occurrence in food, pollution of the sea, the use of additives or disinfectants in the processing of food and the abuse of nitrofurazone. The sample preparation techniques differ greatly by foodstuff. Fish and meat are more complex matrices, so solid phase extraction is used to purify and elute samples. Flour products contain SEM due to the use of azodicarbonamide, which generates by-products that are tightly bound to the surface. Therefore, the first use of the opposite process, starting with 2:1 methanol–water to dissolve a portion of SEM, has been reported. Dairy products are rich in protein, leading to the normal pre-treatment involving the appropriate concentration of hydrochloric acid into trichloroacetic acid. Currently, LC-MS/MS is a commonly used method in nitrofurazone drug residue analysis. However, this technique requires complicated pre-treatment and expensive equipment. The ELISA method has broad prospects for development, being easily performed. In particular, the successful development of monoclonal antibodies against nitrofurazone residues has played a huge role. However,

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