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Nitrofurazone usage in food-producing animals is prohibited in most countries, including the United States. Regulatory agencies regularly monitor its use in domestic, export/import animals' food products by measuring the semicarbazide (SEM) metabolite as a biomarker of nitrofurazone exposure. However, the use of SEM is controversial because it is also produced in food naturally and thus gives false positive results. A cyano-metabolite, 4-cyano-2-oxobutyraldehyde semicarbazone (COBS), is proposed as an alternate specific marker of nitrofurazone to distinguish nitrofurazone from treated or untreated animals. A synthetic method was developed to produce COBS via metallic hydrogenation of nitrofurazone. The product was isolated and characterized by one- and two-dimensional nuclear magnetic spectroscopy (NMR) experiments, Fourier-transform infrared spectroscopy (FT-IR), and mass spectrometry. The developed synthetic procedure was further extended to synthesize isotopically labeled 4-[ 13 C]-cyano-2-oxo- [2, 3, 4- 13 C 3 ]-butyraldehyde semicarbazone. Labeled COBS is useful as an internal standard for its quantification in food-producing animals. Thus, the developed method provides a possibility for its commercial synthesis to procure COBS. This is the first synthesis of the alternate specific marker metabolite of nitrofurazone for possible usage in regulatory analysis to solve a real-world problem., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Abstract: Semicarbazide (SEM) is routinely employed as an indicator for the use of nitrofurazone, a banned antimicrobial. The validity of SEM as a nitrofurazone marker has been scrutinized because of other possible sources of the compound. Nonetheless, a U.S. trade partner rejected skin-on chicken thighs because of SEM detection and suspected nitrofurazone use. Because nitrofurazone has been banned in U.S. broiler production since 2003, we hypothesized that incidental de novo SEM formation occurs during broiler processing. To assess this possibility, raw leg quarters were collected from 23 commercial broiler processing plants across the United States and shipped frozen to our laboratory, where liquid chromatography-mass spectrometry was used to quantitatively assess for SEM. Leg quarter samples were collected at four points along the processing line: hot rehang (transfer from the kill line to the evisceration line), prechill (before the chilling process), postchill (immediately following chilling), and at the point of pack. Thigh meat with skin attached was removed from 535 leg quarters and analyzed in triplicate for SEM concentrations. The concentrations ranged from 0 to 2.67 ppb, with 462 (86.4%) of 535 samples below the regulatory decision level of 0.5 ppb of SEM. The 73 samples over the 0.5-ppb limit came from 21 plants; 53 (72.6%) of positive samples were in meat collected after chilling (postchill or point of pack). The difference in both prevalence and concentration of SEM detected before and after chilling was highly significant (P < 0.0001). These data support our hypothesis that SEM detection in raw broiler meat is related to de novo creation of the chemical during processing., (Not subject to U.S. Copyright.)

Semicarbazide (SEM) is a protein-bound nitrofurazone metabolite that is detrimental to human health. Therefore, to ensure food safety, it is necessary to detect SEM in food samples. To this end, we developed a novel electrochemical sensor to detect SEM by using a molecularly imprinted polymer (MIP) as the recognition element. Computer-aided molecular modelling was performed to guide the synthesis of the MIP, and subsequently, MIP/carboxylated single-walled carbon-nanotubes/chitosan (MIP/SWNTs-COOH/CS) was prepared as the sensing platform to develop the electrochemical sensor. The linear range of the sensor was 0.04-7.6 ng mL -1 , with a detection limit of 0.025 ng mL -1 . The sensor was successfully applied to detect SEM in four different real samples, with recoveries ranging from 83.16% to 93.40%. The results indicated that the fabricated electrochemical sensor can be widely applied to detect SEM in the environment and in agri-food products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Abstract: Semicarbazide (SEM) is the U.S. Food and Drug Administration's official marker for nitrofurazone use in food animals. The U.S. Department of Agriculture Food Safety and Inspection Service conducted a study to evaluate the source of SEM that was identified by a U.S. trading partner in a subset of chicken samples presented for inspection, even though nitrofurazone has been banned from use in U.S. food-producing animals since 2002. The study design included analyses to detect and quantify total and bound SEM in chicken collected from the eight U.S. establishments that were associated with the reported detection of SEM. Samples were collected immediately following evisceration, chilling, and cutting carcass into parts (cut-up). Although antimicrobial interventions (processes to reduce pathogen concentrations) are typically used at all three of these processing steps, the product contact time during chilling is significantly longer (hours versus seconds) than during evisceration and cut-up. In addition, parts were analyzed after 0, 10, 20, and 30 days of frozen storage. No postevisceration samples tested positive for SEM; however, most samples collected postchilling and after cut-up tested positive. The absence of SEM in postevisceration samples and detection in the subsequent postchilling samples and after the cut-up samples suggest that the detection of SEM in the sampled products is not indicative of preharvest nitrofurazone use and may be a result of postharvest processing in these establishments., (Published 2020 by the International Association for Food Protection. Not subject to U.S. Copyright.)

This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including semicarbazide (SEM), 1-aminohydantoin (AHD), 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ) in gelatin Chinese medicine. A C 18 column with the mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ were found to be 0.2 µg/kg, 0.3 µg/kg, 0.2 µg/kg and 0.2 µg/kg, whereas their limit of quantification were 0.6 µg/kg, 0.8 µg/kg, 0.6 µg/kg and 0.5 µg/kg. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was purified through dispersive solid phase extraction using 1000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent, and then further purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the clean-up procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in water: acetonitrile (95:5, v/v). The recoveries of these nitrofuran metabolites under the 1 µg/kg, 2 µg/kg and 10 µg/kg spiking levels were between 77.4% and 95.6%. These metabolites after the extraction were stable at 4 °C for 24 h. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This research aims to analyze the biosynthetic pathway of endogenous semicarbazide (SEM) in shrimps using Litopenaeus vannamei as the model target. To achieve this objective, the content of SEM in L. vannamei throughout the whole growth cycle was monitored under the strict control of external environmental interference. Experimental results showed that SEM was found in the shrimp shell at all stages, with its content decreasing first and then increasing, and no SEM was detected in the shrimp muscle of each growth stage. This indicated that endogenous SEM in L. vannamei was derived from the shrimp shell. At the same time, the content of amino acids in the shrimp shell and the corresponding substances involved in the urea cycle in the entire growth cycle of shrimp were monitored. The correlation analysis between them and the changes in the SEM content in shrimp showed that arginine had the largest correlation coefficient (0.952) with the changes in the SEM content. The main substances of the urea cycle may be related to the production of SEM. In combination with the water environmental test of high ammonia nitrogen, it was presumed that the formation of endogenous SEM was related to the amidine group of arginine and amide structure of citrulline and urea. Arginine, citrulline, and urea in the urea cycle of L. vannamei eventually produced SEM via an oxaziridine intermediate under the action of hydrogen peroxide and ammonia, and a standardized reaction test was conducted to verify the hypothesis and, thus, provided a new idea for future endogenous SEM research.

A novel molecularly imprinted stir bar (MI-SB) for sorptive extraction of semicarbazide (SEM) was prepared in present paper. The coating of the stir bar was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, dynamic adsorption and static adsorption tests. The saturated adsorption of MI-SB was about 4 times over that of non-imprinted stir bar (NI-SB). The selectivity of MI-SB for SEM was much better than NI-SB. A method to determine SEM was established by coupling MI-SB sorptive extraction with HPLC-UV. The liner range was 1-100ng/mL for SEM with a correlation coefficient of 0.9985. The limit of detection was about 0.59ng/mL, which was below the minimum required performance limit of SEM in meat products regulated by European Union. The method was applied to the determination of SEM in fish samples with satisfactory results., (Copyright © 2018 Elsevier B.V. All rights reserved.)

That intense demand for both high sensitivity and high reliability has been a key factor strengthening the surface enhanced Raman spectroscopy (SERS) in the analytical application, particular in the hyphenation with pre-concentration technique. Credible data acquisition and processing is very dependent on the stable and uniform performance of SERS-active substrate. Here, a reliable and uniform ultrathin-layer Au was proposed for protecting the porous Ag fiber (porous Ag@Au) and applied in the solid phase microextraction coupled with SERS. The Au layer was carefully deposited on porous Ag surface to form the uniform film by a galvanic-free displacement reaction. This coating endowed the substrate with high oxidation-resistance under heating and good durability in the atmosphere condition. The extraction and SERS performance of Nitrofurazone and Semicarbazide were investigated on this fiber, the bands at 1350 cm -1 and 1387 cm -1 were selected as the characteristic peaks for quantitative determination, respectively. This robust and sensitive substrate provide the high enhancement factor of 1.3 × 10 6 and low LOD of 5.1 ppb for the extraction and identification of Nitrofurazone compounds. Importantly, this work develops a versatile strategy for rapid detection of prohibited antibiotic and its marker residue in a complex matrix., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Semicarbazide, a toxic food contaminant, widely exists in food products and it originates from the thermal degradation of a food additive of azodicarbonamide or a metabolite of nitrofurazone abused in meat specimens. Many previous methods for semicarbazide determination usually required expensive instruments, difficult-to-prepare monoclonal antibodies, and a long operation time. In this study, a high-performance liquid chromatography method was developed for the rapid determination of trace semicarbazide coupling with a nucleophilic substitution reaction firstly using 4-nitrobenzoyl chloride as derivatization reagent. The derivatization reaction was mild at room temperature for 1 min in neutral solution. Then, semicarbazide derivative was separated and quantified by high-performance liquid chromatography with ultraviolet detection under optimal separation conditions at λ max = 261 nm. The proposed method offered the detection limit of 1.8 μg/L and was successfully applied for the rapid determination of trace semicarbazide in flour products. Semicarbazide in positive real samples could be actually found and quantified in the range of 0.47-7.53 mg/kg. The recoveries were 76.6-119% with relative standard deviations of 0.5-9.1% (n = 3). This developed method was rapid, reliable, and convenient for the determination of trace semicarbazide in food., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A visualized microarray sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplexed approach, metabolites of four main nitrofuran antibiotics can be detected simultaneously. Individual antigens were spotted onto 96-well plates. An indirective competitive assay format, with visualized signal response, was employed. An extraction method, based on derivatization with 2-nitrobenzaldehyde and partition into ethyl acetate, was used for screening. The limits of detection were 0.10, 0.04, 0.04, and 0.10ngg -1 for 3-amino-5-morpholino-2-oxazolidone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), and 1-aminohydantoin (AHD), respectively. The recovery rate ranged from 78% to 93% for the four targets. In addition, this method was easy to operate with low detection cost and fast speed. This microarray method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety monitoring., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Practical solutions for multiple antibiotic determination in food are required by the food industry and regulators for cost-effective screening purposes. This study describes the feasibility in development and preliminary performance of a novel multispot nanoarray for antibiotic screening in honey. Using a multiplex approach, the metabolites of the four main nitrofuran antibiotics, including morpholinomethyl-2-oxazolidone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 1-aminohydantoin (AHD) and chloramphenicol (CAP), were simultaneously detected. Antibodies specific to the five antibiotics were nano-spotted onto microtitre plate wells and a direct competitive assay format was employed. The assay characteristics and performance were evaluated for feasibility as a screening tool for antibiotic determination in honey to replace traditional ELISAs. Optimisation of the spotting and assay parameters was undertaken with both individual and multiplex calibration curves generated in PBS and a honey matrix. The limits of detection as determined by the 20% inhibitory concentrations (IC 20 ) were determined as 0.19, 0.83, 0.09, 15.2 and 35.9 ng ml -1 in PBS, 0.34, 0.87, 0.17, 42.1 and 90.7 ng ml -1 in honey (fortified at the start of the extraction), and 0.23, 0.98, 0.24, 24.8 and 58.9 ng ml -1 in honey (fortified at the end of the extraction) for AMOZ, AOZ, CAP, SEM and AHD respectively. This work has demonstrated the potential of multiplex analysis for antibiotics with results available for 40 samples within a 90-min period for antibiotics sharing a common sample preparation. Although both the SEM and AHD assay do not show the required sensitivity with the antibodies available for use to meet regulatory limits, with further improvements in these particular antibodies this multiplex format has the potential to show a reduction in cost with reduced labour time in combination with the high-throughput screening of samples. This is the first 96-well spotted microtitre plate nanoarray for the semi-quantitative and simultaneous analysis of antibiotics.

Semicarbazide (SEM), an industrial raw material and the marker residue of nitrofurazone as a veterinary drug, has become a new type of marine pollutant. A standard method (ultra-performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS) was used to analyze SEM in seawater, sediment, and shellfish. A series of sections and stations were set up in radical distribution in western Laizhou Bay, with six voyages and 150 monitoring samples. The concentrations of SEM in seawater and shellfish were 10 -11 and 10 -10 kg/L, respectively, and no SEM was detected in the sediment. Distribution characteristics at each state, temporal and spatial trends, multivariate analyses, and the causes were analyzed to assess the pollution level, which aimed to offer a database for drafting the national baseline values of SEM in seawater and sediment in future. The data obtained could be used for integrated watershed management of marine environment and economic activities for constructing a blue economic zone of Shandong Peninsula in China., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Semicarbazide is a widespread food contaminant that is produced by multiple pathways. However, the toxicity of semicarbazide to human health remains unclear. Using a highly accurate and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry method, we identified and quantitated in this study for the first time the DNA and RNA adduct of semicarbazide in DNA/RNA isolated from the internal organs of semicarbazide-exposed rats. The analysis revealed a dose-dependent formation of the adducts in the internal organs of the semicarbazide-dosed rats and with the highest adduct levels identified in the stomach and small intestine. Furthermore, results showed significantly higher levels of the RNA adduct (4.1-7.0 times) than that of the DNA adducts. By analyzing DNA/RNA samples isolated from rat organs in semicarbazide-dosed rats at different time points postdosing, the adduct stability in vivo was also investigated. These findings suggest that semicarbazide could have exerted its toxicity by affecting both the transcription and translation processes of the cell.

Nitrofurazone is an antibiotic with carcinogenic properties. Efforts by regulatory authorities to control nitrofurazone from agricultural foods are an important public health measure that have, to some extent, been undermined by widespread use amongst laboratories of the unreliable marker metabolite semicarbazide. This work confirms what has long been suspected, namely that powdered dairy products that are initially free of semicarbazide develop semicarbazide under storage conditions such as occur normally across commercial supply chains. The low ng g(-)(1) levels of semicarbazide formed in this way are insufficient to present any food safety hazard. That such development of a marker metabolite is demonstrated to occur by innocent means effectively invalidates the use of semicarbazide as a marker metabolite for powdered dairy products, and exacerbates the regulatory need for a more suitable analytical methodology. In milk, unlike meat, nitrofurazone is known to remain stable and thus available for analysis in the intact form, rather than necessitating any use of a metabolite or fragment. However, no previous methodology that was capable of achieving the stringent European minimum required performance limit of 1 ng g(-)(1) when using intact nitrofurazone had been described for milk. This work describes a specific methodology using LC-MS/MS for milk and milk powder; it achieves detection of intact nitrofurazone (as well as furazolidone, furaltadone and nitrofurantoin) to levels well below 1 ng g(-)(1). Laboratories will no longer need to use semicarbazide as an unreliable marker metabolite for the analysis of nitrofurazone in dairy products, paving the way for regulatory authorities to better control nitrofurazone abuse with greater confidence.

Semicarbazide (1) is a widespread genotoxic food contaminant originating as a metabolic byproduct of the antibiotic nitrofurazone used in fish farming or as a thermal degradation product of the common flour additive azodicarbonamide. The goal of this study is to develop a simple and sensitive high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method for the detection of compound 1 in food products. In comparison to existing methods for the determination of compound 1, the reported method combining online precolumn derivatization and HPLC-FLD is less labor-intensive, produces higher sample throughput, and does not require the use of expensive analytical instruments. After validation of accuracy and precision, this method was applied to determine the amount of compound 1 in fish and bread samples. Comparative studies using an established liquid chromatography coupled with tandem mass spectrometry method did not yield systematically different results, indicating that the developed HPLC-FLD method is accurate and suitable for the determination of compound 1 in fish and bread samples.

Semicarbazide (SEM) is a side-chain metabolite of the antibiotic drug nitrofurazone (NFZ) and is employed as a conclusive marker for the use of banned NFZ. Recent studies have shown that SEM in aquatic crustaceans can be derived natively or from other sources. The presence and distribution of endogenous SEM within aquatic crustaceans is examined in this paper, which finds that the SEM content varies amongst the muscle, shell, and viscera of various prawn and crab species within the range of 0.35-26.62 ng g(-1). The effects of heating and hypochlorite treatment on SEM levels were examined. The results indicate that thermal processing introduced a more significant impact, resulting in a maximum SEM value of 15.48 ng g(-1) in a sample of shell of Portunus trituberculatus crab, while SEM levels in muscle samples were not affected by the duration of heating. Though 6% active chlorine treatment led to SEM production ranging between 39.9 and 196.4 ng g(-1) in muscle samples from various crustaceans, SEM is unlikely to originate from hypochlorite or chlorine in practice where there are limits to actual chlorine in sanitation water and facilities. 5-Nitro-2-furaldehyde (NF) was proposed as a selective marker to differentiate between endogenous SEM and NFZ-derived SEM in seafood.

A comprehensive global database on semicarbazide (SEM) in foodstuffs and food ingredients is presented, with over 4000 data collected in foods such as seafood (crustaceans, fish powders), meat (beef, chicken powders), dairy products (e.g. raw milk, milk powders, whey, sweet buttermilk powder, caseinate, yoghurt, cheese), honey and other ingredients. The results provide evidence that the presence of SEM in certain dairy ingredients (whey, milk protein concentrates) is a by-product of chemical reactions taking place during the manufacturing process. Of the dairy ingredients tested (c. 2000 samples), 5.3% showed traces of SEM > 0.5 µg/kg. The highest incidence of SEM-positive samples in the dairy category were whey (powders, liquid) and milk protein concentrates (35% positive), with up to 13 µg/kg measured in a whey powder. Sweet buttermilk powder and caseinate followed, with 27% and 9.3% positives, respectively. SEM was not detected in raw milk, or in yoghurt or cheese. Of the crustacean products (shrimp and prawn powders) tested, 44% were positive for SEM, the highest value measured at 284 µg/kg. Fish powders revealed an unexpectedly high incidence of positive samples (25%); in this case, fraudulent addition of shellfish shells or carry-over during processing cannot be excluded. Overall, the data provide new insights into the occurrence of SEM (for dairy products and fish powders), substantially strengthening the arguments that SEM in certain food categories is not a conclusive marker of the use of the illegal antibiotic nitrofurazone.

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.

Azodicarbonamide is widely applied in the food industry as a new flour gluten fortifier in China, Canada, the United States, and some other countries, whose metabolites of biurea and semicarbazide hydrochloride are reaction products during baking. In this study, IR, Raman and surface-enhanced Raman scattering (SERS) spectra of azodicarbonamide, biurea, and semicarbazide hydrochloride have been studied, and vibrational bands have been assigned on the basis of density functional theory (DFT) calculations. The calculated Raman spectra were in good agreement with experimental Raman spectra. The SERS method coupled with active gold substrates has also been applied for detection of the three chemicals with pure water as solvent, with the limit of detection of this method being as low as 10 μg/mL (less than 45 μg/mL). These results showed that azodicarbonamide and its metabolites could be detected by the vibrational spectra technique, which might be applied as a powerful tool for the rapid detection on these species derived from agents added to flour., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

A direct ELISA was established for the fast detection of semicarbazide (SEM) using a novel biotin derivative. Without a tedious extraction procedure, as low as 0.07 μg L(-1) of SEM could be detected reproducibly. This assay has better recovery and accuracy than competitive ELISA.

A liquid chromatography-mass spectrometry method to detect semicarbazide and lactose semicarbazone in milk was developed as part of a programme to set up methods for detecting the economically motivated adulteration of raw milk with nitrogen-containing compounds. The detection of semicarbazide was hampered by that fact that this compound tended to give broad, poor intensity peaks in the hydrophobic interaction chromatographic method employed. When spiked into milk at levels of 20-200 ppm, semicarbazide either partially or completely reacted with the matrix, which both increased the limit of detection of the method and made the setting of a threshold by using low level spikes almost impossible. Thus using lactose semicarbazone as a marker for semicarbazide addition to milk was investigated. Lactose semicarbazone was detected in semicarbazide-spiked milk, and its identity was confirmed by fragmentation analysis and comparison with the synthesised compound. The level of lactose semicarbazone correlated with the amount of semicarbazide added to the milk, and the acidic conditions employed in the extraction method appeared to enhance the sensitivity of detection by driving the semicarbazone-forming reaction towards completion. Thus lactose semicarbazone can be used as a marker for the addition of semicarbazide to milk; however, both compounds should be monitored during surveys looking for the semicarbazide adulteration of milk., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods., (Copyright © 2013 Elsevier B.V. All rights reserved.)

There is evidence that semicarbazide (SEM), a marker for the banned nitrofuran nitrofurazone, can arise from other, unrelated sources. Recently, Belgium rejected 54 consignments of Bangladeshi freshwater shrimp (Macrobrachium rosenbergii), following a laboratory decision to test meat and exoskeleton combined. To study the possible natural occurrence of SEM in wild shrimp, samples were collected and analysed from 29 sites across Bangladesh. SEM (<1.0 μg/kg) was detected in ∼65% of meat samples. However, SEM concentrations were approximately 100 times higher in the exoskeleton, and were unrelated to sampling location, strongly suggesting natural occurrence. In meat, most SEM was surface-associated. When the shrimp was shelled, some of the epidermal layer (which synthesises new exoskeleton) remained with the shell and some remained with the meat--leading to differing levels of natural SEM on the shrimp surface. This has implications for the use of SEM and the analytical strategy used to control nitrofuran use., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

An exposure assessment to nitrofuran residues was performed for three human populations (adults, teenagers and children), based on residue analyses of foods of animal origin (liver, honey, eggs and aquaculture) covering the 2-year period 2009-2010. The occurrence of nitrofuran metabolites in food on the Irish market was determined for the selected period using the data from Ireland's National Food Residue Database (NFRD) and from results obtained from the analysis of retail samples (aquaculture and honey). Laboratory analyses of residues were performed by methods validated in accordance with Commission Decision 2002/657/EC regarding performance of the analytical method and interpretation of results. Semicarbazide (SEM) was the contaminant most frequently identified and its content ranged from 0.09 to 1.27 μg kg(-1). SEM is currently used as a marker of nitrofuran abuse, but it may also occur from other sources. The presence of nitrofuran metabolite 3-amino-2-oxazolidinone (AOZ) was detected in two aquaculture samples (prawns) at 1.63 and 1.14 μg kg(-1), but such a low number of positive cases did not present sufficient data for a full AOZ exposure assessment. Therefore, the evaluation of exposure was focused on SEM-containing food groups only. Exposure assessments were completed using a probabilistic approach that generated 10 iterations. The results of both the upper- and lower-bound exposure assessments demonstrate that SEM exposure for Irish adults, teenagers and children from selected food commodities are well below EFSA-estimated safe levels.

Currently, a number of food producers use hypochlorite to bleach food and inhibit the growth of bacteria, preserving the food. Because the presence of high amounts of nitrogen could result in the formation of semicarbazide (SEM), the bleaching process could be one of the predominant sources of SEM in food. To investigate this, we selected instant bottled bird's nest as an example of a food that is bleached in its production. SEM was detected in 27 of 28 instant bottled bird's nest samples. The levels of SEM detected mostly fell in the range of 5 to 50 μg/kg, which accounted for 75% of all samples measured. The SEM detected in the instant bottled bird's nest was found to have originated neither from the use of the antimicrobial agent nitrofurazone nor from azodicarbonamide, which is used as a blowing agent in gaskets used to seal the metal lid of the bottle. Instead, it could have originated from the bleaching process used in the preparation of the nests. Additionally, human exposure to SEM via consumption of instant bottled bird's nest for five subgroups of the population was estimated. Sensitivity analysis suggested that concentration of SEM in food is the most significant parameter governing human exposure via consumption of SEM-containing food.

The method of MacMahon and Lohne for analysis of nitrofuran metabolites in shrimp was optimized to streamline the extraction processes and the LC analysis. This revised method includes 16 h of mild acid hydrolysis/derivatization followed by ethyl acetate extraction and analysis by LC/MS/MS in the atmospheric pressure chemical ionization mode. This revised method was validated in shrimp for concentrations of 0.25 to 2.0 ng/g. The LOQ was 0.25 ng/g for all metabolites. The LOD was 0.052 nglg for 1-aminohydantoin (AHD), 0.206 ng/g for 3-amino-2-oxazolidinone (AOZ), 0.108 ng/g for semicarbazide (SC), and 0.062 ng/g for 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). The spike recoveries with RSD into negative matrix at 1 ng/g were 100.2% (3.2%) for AHD, 102.5% (1.0%) for AOZ, 103.7% (2.3%) for SC, and 104.0% (3.3%) for AMOZ. The spike recoveries at 1 ng/g into unknown samples (n=108) containing varied levels of nitrofuran metabolites were 112.6% (25.7%) for AHD, 108.1% (12.1%) for AOZ, 103.0% (12.0%) for SC, and 100.3% (6.9%) for AMOZ. Interday precision with samples containing incurred AOZ concentrations of 0.92 to 17.8 ppb performed over a year was 10.4% RSD. The method is accurate and precise for determining nitrofuran concentrations in the edible tissue of shrimp.

Azodicarbonamide, as a bleaching agent and improving agent, is a permitted food additive in certain countries and can be determined by high-performance liquid chromatography. However, it partially degrades with the heat of processing to form trace amounts of semicarbazide, which shows carcinogenicity and also has been shown to cause tumors. The concentration of semicarbazide in azodicarbonamide-treated flour was determined by isotope dilution ((13)C, (15)N(2)-semicarbazide) liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS). The quantification was obtained utilizing the homologous internal standard. The limits of detection were 1 mg/kg for azodicarbonamide and 0.5 × 10(-3) mg/kg for semicarbazide. The rates of recovery were 82.3-103.1% for azodicarbonamide and 72.4-116.5% for semicarbazide. This study prepared four different types of flour products to investigate the variation of semicarbazide. The concentration of semicarbazide in all types of flour products is higher than that in flour, and the concentration of semicarbazide in outside of flour products is slightly higher than that in the inside. As the problem of food safety hazard aggravates daily, we should be more concerned about food security and human health.

In the past year there has been an increased incidence in Belgium of cases of positive semicarbazide (SEM) tests in imported freshwater Macrobrachium rosenbergii prawns, seemingly indicating the possible abuse of nitrofurazone, a banned antimicrobial agent. This was in contrast to all other European countries where no significant increase in SEM-positive samples was detected. A possible explanation for this discrepancy between Belgium and the other European Union member states could be the fact that only in Belgium were whole prawns (meat + shell) analyzed for the presence of tissue-bound metabolites of nitrofurans, whereas in the other countries only the edible part (meat) of these prawns was analyzed. To investigate the possible natural occurrence of SEM in freshwater prawns, an animal trial was set up. In this experiment two groups of 10 juvenile M. rosenbergii, previously raised under standardized laboratory conditions, were stocked into two separate aquaria, a control group under reference conditions (no addition of nitrofurazone) and a group exposed to a daily dose of 50 mg of nitrofurazone L(-1) of culture water. Results of this animal trial proved that SEM naturally occurs in M. rosenbergii prawns but that at the current minimum required performance limit (MRPL) no tissue-bound SEM can be found in the meat of nontreated animals. In addition to this animal trial, commercial samples of other crustacean species, the shell and meat of which were analyzed separately, were also analyzed for the presence of SEM.

The release of the substances from food packaging into food triggers the problem for food safety and is the matter of particular interest in the European Commission (EC) and Member States. The problems concerning the presence of these substances in the food which result from their migration from food contact materials, and the EC activities to discover the causes of this phenomenon including an assessment of possible health impact are presented in this article. The risk assessment is provided by the European Food Safety Authority which is obliged to issue a scientific opinion on expected heath effects. Food contact articles which do not conform to the food safety provisions are reported to the Rapid Alert System for Food and Feed (RASFF). The health hazards due to the presence in food of the semicarbazide (SEM), photoinitiators (isopropylthioxantone (ITX), benzophenone compounds), primary aromatic amines (PAAs) and bisphenol A (BPA) were discussed and the relevant European Commission actions aiming at consumers health protection as a reaction to reduce the expected risk were also presented in this article.

An improved LC-MS/MS method for the determination of semicarbazide in whole egg is described. Waters OASIS-MCX cation exchange purification cartridges increased the sensitivity for analysis by LC-MS/MS. The validation study was carried out according to criteria and requirements of Commission Decision 2002/657/EC for confirmatory analysis and provided the data as follows: The correlation coefficient for the matrix calibration curve, in the range of 0-5 microg kg(-1), was r=0.9968. The detection capability and decision limit, measured according to ISO11843-2, were CCalpha=0.20 microg kg(-1) and CCbeta=0.25 microg kg(-1). Repeatability (CVSr) and within-laboratory reproducibility (CVSwr) determined for the concentration levels of 0.2, 0.5 and 1.0 microg kg(-1) SEM ranged from 11.9 to 5.7% and 11.8 to 6.3%, respectively. The validated method was applied to investigate SEM stability in incurred materials (egg homogenates) during long-term storage at -20 degrees C and 4 degrees C. The study proved by a two-sampling test that SEM at levels of 17. 7, 1.2, 10.6 and 0.47 microg kg(-1) was stable for up to 12 months.

Detecting illegal nitrofurazone treatment of food-producing animals has been undermined by the reliance on semicarbazide as a marker metabolite for analysis because, during processing, semicarbazide forms in a variety of foods not exposed to nitrofurazone by previously unexplained pathways. In this study, formation of semicarbazide was examined under dairy product processing conditions. Hypochlorite dosed into milk on an industrial scale, at concentrations extreme for unintentional residues, produced monochloramine, but, without pH adjustment, hypochlorite alone did not generate semicarbazide. Dosed hypochlorite and peroxyacetic acid each generated low ng g(-1) levels of semicarbazide in milk (and whey) only when used in conjunction with localised high pH conditions, which can occur during both anion exchange and neutralisation. Dosed hydroxyurea generated semicarbazide at any pH, with greatest levels formed at high pH. Detecting nitrofurazone abuse can be better accomplished by analysis either for nitrofurazone itself or for the marker metabolite 5-nitro-2-furaldehyde, rather than semicarbazide.

The reaction between 2-hydrazinopyridine and ethylisothiocyanate produced 4-ethyl-1-(pyridin-2-yl) thiosemicarbazide (HEPTS). Its reaction with copper fluoride, chloride, bromide, acetate, nitrate, perchlorate, sulfate, carbonate, hydroxide and copper metal produced 15 Cu(II) complexes. The copper metal is easily oxidized in aqueous-ethanol solution of HEPTS giving [Cu2(EPTS)(H2O)3(OH)3]EtOH. Different complexes for the same anion were synthesized by controlling the heating time. Characterization by elemental, thermal, magnetic and spectral (electronic, IR, mass and ESR) studies showed the formation of mono-, di-, tri- and tetra nuclear complexes. The room temperature solid state ESR spectra of the complexes show an axial spectrum with dx2-y2 ground state, suggesting distorted tetragonal geometry around Cu(II) center. The kinetic and thermodynamic parameters for the different decomposition steps in the complexes were calculated. HEPTS and its Cu(II) complexes showed high activity against gram negative bacteria; [Cu3(EPTS)2(EtOH)2Br4] has more activity.

An enzyme-linked immunosorbent assay (ELISA) method is described for the semi-quantitative determination of semicarbazide (SEM), the marker residue for the banned nitrofuran drug, nitrofurazone, in chicken eggs. The sample homogenate is subjected to acid hydrolysis and derivatisation with o-nitrobenzaldehyde, followed by ethyl acetate/hexane extraction and detection by ELISA. The ELISA procedure has been validated using 0.3, 1.0 and 3 microg kg(-1) of SEM in fortified samples. Detection capability (CC(ss)) was based on the acceptance of 5% false compliant results for a given concentration level according to Commission Decision 2002/657/EC and was determined to be 0.3 microg kg(-1) with a respective limit of detection of 0.13 microg kg(-1). A validated LC-MS/MS method was used for the analysis of incurred egg samples and the results compared with ELISA. A good correlation between the results obtained from ELISA and LC-MS/MS within the concentration range 0.12-20.3 microg kg(-1) was observed in samples collected from chickens fed with a medicated ration of nitrofurazone (r = 0.992, n = 14). Validated ELISA enabled reliable monitoring of SEM levels in eggs collected from incurred chickens over a 90-day period.

Nitrofuran antibiotic residues in food continue to be of international concern. The finding of sources of semicarbazide (SEM), other than through the misuse of nitrofurazone, present a challenge to the use of SEM as a definitive marker residue for this drug. Detection of intact (parent) nitrofurazone would avoid confusion over the source of SEM residues. Broiler chickens were fed sub-therapeutic nitrofuran-containing diets and their tissues were analysed for parent compounds and metabolites by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS). Depletion half-lives in muscle were longer for tissue-bound metabolite residues, 3.4 days --3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) -- to 4.5 days (SEM), than total metabolite residues, 2.0 days (AOZ) to 3.2 days (SEM). Metabolite concentrations were higher in eyes than in muscle. Metabolite half-lives in eyes ranged from 8.5 days (1-aminohydantoin (AHD)) to 20.3 days (SEM). Nitrofuran parent compounds were also detected in eyes. Furaltadone was detected in single eyes after 21 days' withdrawal of a 6 mg kg(-1) furaltadone diet. When 50 eyes from broilers containing metabolites in muscle close to the 1 microg kg(-1) minimum required performance level (MRPL) were pooled into single samples, 1.2 ng of furazolidone and 31.1 ng of furaltadone were detected, but nitrofurazone was not detected due to the long depletion half-life of SEM in muscle. Further studies are required to improve LC-MS/MS nitrofurazone sensitivity and refine the sample size necessary to use nitrofurazone detection in pooled eyes as a complement to SEM detection in muscle.

Azodicarbonamide, an approved food additive, is commonly used as a flour additive and dough conditioner in the United States and Canada. A number of researchers have clearly established a link between the use of azodicarbonamide and semicarbazide contamination in commercial bread products. However, all of these studies have primarily focused on the final baked product and have not extensively investigated the processing and conditions that affect the final semicarbazide levels. In this study, a previously developed method for measuring free semicarbazide in bread was applied to dough samples during the mixing and kneading process. Additionally, flour and bread samples were spiked with biurea or azodicarbonamide to help elucidate semicarbazide formation pathways. The results showed that semicarbazide was not formed as a byproduct of azodicarbonamide decomposition to biurea, which occurs upon the addition of water. Indeed, semicarbazide was not detected after room temperature or elevated temperature dough maturation, but only after baking. It was concluded that although azodicarbonamide is the initial starting material, semicarbazide formation in bread occurs through a stable intermediate, biurea.

The development of a direct competitive enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies specific for semicarbazide (SEM) is described. Molecular modelling of the hapten mimics and other key components of the assay system was conducted to explain antibody properties in relation to hapten design. The small aliphatic molecule SEM was coupled to 3-carboxybenzaldehyde to produce carboxyphenyl-SEM (CPSEM), for the generation of specific antibodies. Five rabbits produced antibodies against NPSEM (used in direct and indirect ELISA formats) exhibiting a 50% binding inhibition level (IC(50) values) of 0.06-2.28 microgL(-1) in assay buffer for SEM. The most sensitive indirect assay based on the antibody MVK39 showed a high dynamic range providing a linear readout in the range of 0.01-0.2 microgL(-1). Antibody MVK31 (IgG) allowed specific SEM detection at an IC(50)=0.14 microgL(-1) in direct ELISA and was evaluated using solvent extracted SEM-spiked porcine and baby food samples. Recovery levels determined from fortified samples (0.5, 1.0, 1.5, 5, 10 and 20 microgkg(-1)) of porcine and baby food ranged from 82.9 to 105.3%, respectively, with a coefficient of variation less than 15.5%. Respective detection capability and threshold of the assay for porcine muscle, set on the basis of acceptance of no false negative results, was 0.3 and 0.11 microgkg(-1).

Nitrofuran antibiotics cannot be used in food production within the European Union because of their potential health risks to consumers. The recent discovery of their widespread use in global food industries and the finding of semicarbazide in baby food as a result of packaging contamination have focused attention on the toxicity and stability of these drugs and their metabolites. The stability of the nitrofuran marker residues 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) were tested. Muscle and liver of nitrofuran treated pigs were cooked by frying, grilling, roasting and microwaving. Between 67 and 100% of the residues remained after cooking, demonstrating that these metabolites are largely resistant to conventional cooking techniques and will continue to pose a health risk. The concentration of metabolites in pig muscle and liver did not drop significantly during 8 months of storage at -20 degrees C. Metabolite stock and working standard solutions in methanol were also stable for 10 months at 4 degrees C. Only a 10 ng ml(-1) solution of SEM showed a small drop in concentration over this extended storage period.

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed--all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been 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, cross-reactivity being 1.7% with NFZ and negligible 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 (CCbeta) of 0.25 microg kg(-1) when a threshold of 0.21 microg kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 microg kg(-1) fortified sample, n=20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 microg kg(-1). NFZ-incurred muscles (12) containing SEM at 0.5-5.0 microg kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver.

Recently doubts have arisen on the usefulness of semicarbazide as marker residue for the illegal use of the antibiotic nitrofurazone (NFZ) in aquaculture and poultry production. Most notably azodicarbonamide (ADC) has been implicated as an alternative source of semicarbazide. ADC is used in some countries as a dough conditioner at concentrations up to 45 mg kg(-1). The use of ADC-treated flour or dough in coated or breaded food products may generate false non-compliant results in the analytical method for nitrofurazone metabolites, which is currently in use. During the dough preparation process ADC is largely reduced to biurea, which can be considered as an appropriate marker residue of ADC. Thus far no methods have been published for the determination of biurea in food commodities. Due to its polar nature it is very difficult to generate sufficient retention on conventional C18 HPLC columns. With a TSK amide HILIC type column good retention was obtained. A straightforward extraction-dilution protocol was developed. Using a mixture of dimethyl formamide and water biurea was nearly quantitatively extracted from a variety of fresh, coated and processed products. Mass spectrometric detection was performed with positive electrospray ionisation. The sensitivity and selectivity of the mass spectrometer for biurea was very good, allowing detection at concentrations as low as 10 microg kg(-1). However, in some extracts severe ion suppression effects was observed. To overcome the implications of ion suppression on the quantitative performance of the method an isotopically-labelled biurea internal standard was synthesized and incorporated in the method. The method developed can be used effectively in nitrofurazone analysis to eliminate the risk of false non-compliant results due to the presence of azodicarbonamide-treated components in the food product.

An interlaboratory validation study was conducted according to harmonized protocols to evaluate the effectiveness of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of semicarbazide (SEM) in fresh whole egg and in an industrially processed whole egg powder. The sample was extracted with hydrochloric acid and derivatized with 2-nitrobenzaldehyde, with 1,2-[(15)N2(13)C]SEM as the internal standard. The extract was neutralized and purified on a solid-phase extraction cartridge. SEM was determined by reversed-phase LC with detection by MS/MS. Five fresh egg samples, of which 3 were obtained from hens fed nitrofurazone (NFZ), one was spiked with SEM at 50 microg/kg and one was a blank sample, and 5 industrial whole egg powder samples, of which 3 were spiked with fresh whole egg from hens fed NFZ, one was spiked with SEM at 350 microg/kg, and one was a blank sample, were sent to 15 laboratories in 10 different European countries. Results were obtained from 12 participants. Average recoveries of SEM from the fresh egg and the egg powder samples were 105.3 and 121.3%, respectively. The relative standard deviation for repeatability (RSDr) ranged from 2.9 to 9.3%, and the relative standard deviation for reproducibility (RSDR) ranged from 22.5 to 38.1%. The method showed acceptable within- and between-laboratory precision for both matrixes, as evidenced by the HorRat values, at the target levels for the determination of SEM.

An interlaboratory validation study funded by the European Commission, Directorate General for Health and Consumer Protection (DG SANCO), was conducted to evaluate the effectiveness of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of semicarbazide (SEM) in different types of baby food at a possible future European regulatory limit (10 ng/g). The test portion of the sample was extracted with hydrochloric acid, and the analyte was derivatized with 2-nitrobenzaldehyde, with 1,2-[15N2, 13C] SEM as an internal standard. The extract was neutralized and then purified on a solid-phase extraction cartridge. The SEM was determined by reversed-phase LC with detection by MS/MS. Apple puree, rice pudding, and meat/vegetable meal baby food materials, spiked with SEM at levels of about 3, 10, and 30 ng/g, respectively, were sent to 20 laboratories in 12 different European countries, which submitted results from 17 participants. Recoveries ranged from 88.8 to 106.1%. Based on results for spiked samples (blind pairs at 3 levels), the relative standard deviations for repeatability (RSDr) ranged from 4.2 to 6.9% and the relative standard deviations for reproducibility (RSDR) ranged from 16.6 to 24.3%. The method showed acceptable within- and between-laboratory precision for all 3 matrixes, as evidenced by HorRat values, at the target levels for the determination of SEM.

Levels of semicarbazide were determined by liquid chromatography-tandem mass spectrometry using isotope dilution ((13)C(15)N(2)-semicarbazide) methodology, and they were measured, after hydrolysis in 0.125 M hydrochloric acid and derivatization with 2-nitrobenzaldehyde, as a sum of free and bound semicarbazide. Levels of semicarbazide in 11 bakery products, which were sampled at three time intervals from the same source, varied from not detected (<1ng g(-1)) to 560 ng g(-1). In some instances, concentrations of semicarbazide varied between batches of the same product, at times more than tenfold, suggesting that the addition of azodicarbonamide to the same product is not standardized in many baking establishments.

The discovery of trace levels of semicarbazide (SEM) in bottled foods (especially baby foods) led to a consideration of the safety of this hydrazine compound by regulatory agencies worldwide. Azodicarbonamide, which is used in the jar-sealing technology known as Press On-Twist Off (or Push-Twist/PT) closures for the formation of a hermetic, plastisol seal, partially degrades with the heat of processing to form trace amounts of SEM. This review has evaluated the potential toxicological risks of resulting exposure to SEM and also the benefit of the PT technology (with azodicarbonamide) in the context of possible microbial contamination. It also considers the potential impact on infant nutrition if parents come to the conclusion that commercial baby foods are unsafe. SEM shows limited genotoxicity in vitro that is largely prevented by the presence of mammalian metabolic enzymes. Negative results were found in vivo in DNA alkaline elution, unscheduled DNA synthesis and micronucleus assays. This pattern is in contrast to the genotoxic hydrazines that also have been shown to cause tumours. Carcinogenicity studies of SEM are of limited quality, show a questionable weak effect in mice at high doses, which are not relevant to human exposure at trace levels, and show no effect in the rat. The IARC has assigned SEM as Group 3, 'Not classifiable as to its carcinogenicity to humans'. Based on estimates of exposure to infants consuming baby foods (with the assumption of SEM levels at the 95th percentile of 20 ng g(-1) in all of the consumed 'ready-to-eat' foods) compared with a no observed adverse effect level (NOAEL) in developmental toxicity studies, the margin of safety is more than 21 000. Since the risk of an adverse effect is negligible, it is clear that any theoretical risk is outweighed by the benefits of continuing use of the PT closure (with azodicarbonamide blowing agent) to ensure both the microbial integrity and availability of commercial baby foods as a valuable source of infant nutrition.

Recently, semicarbazide has been found in food in jars sealed with cap liners that were manufactured using azodicarbonamide as a blowing agent. These reports raised the concern that the use of azodicarbonamide-an approved dough conditioner-may result in semicarbazide residues in bread. To answer this question, a method based upon the previously reported liquid chromatography/tandem mass spectrometry determination of the semicarbazone of o-nitrobenzaldehyde was utilized. The method adopted for this work includes an extensive cleanup and reaction with o-nitrobenzaldehyde at pH 3.5, rather than with the widely used 0.1 M HCl, to form the semicarbazone derivative. A stable isotope dilution assay was used to determine the free semicarbazide present in the bread products. Levels of semicarbazide ranged from 10 to 1200 ppb in commercial bread products with azodicarbonamide listed among their ingredients.

A method for detection of semicarbazide (SEM) in baby food was validated. SEM was extracted with hydrochloric acid and derivatised with 2-nitrobenzaldehyde, using [15N2,13C] semicarbazide as internal standard. The extract was neutralised, purified on a solid phase extraction cartridge and SEM was determined by reversed phase LC-MS-MS. Linearity was demonstrated in the ranges from 0.1 ng ml(-1) to 1 ng ml(-1) and from 2 ng ml(-1) to 80 ng ml(-1). Matrix effects were non significant for meat-based and significant for apple and rice-based baby foods, in both ranges. Mean recoveries ranged from 87.8% to 107.2% with relative standard deviation from 0.2% to 9.1%, considering both ranges. Limits of detection and quantification were 0.1 microg kg(-1) and 0.25 microg kg(-1), respectively. The results of the validation process demonstrated the method suitability for use in food control.

This review provides an overview of the information currently available about the presence of semicarbazide (SEM) in food. Likely sources of SEM in food matrices are summarised and discussed. Detailed information is given about the analytical methods used to determine SEM; features and drawbacks associated with them are carefully evaluated. Performance criteria characterising the analytical methods discussed are also given.

Intact nitrofurazone is present in whole eyes of chickens fed varying levels of this banned antibiotic and may therefore be used as an alternative to the controversial marker residue, semicarbazide, to monitor for abuse of this drug in primary production.

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400?mg?kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Nitrofuran metabolite residues AOZ, AMOZ, AHD and SEM were detected at parts per million concentrations in retina of pigs fed therapeutic doses of nitrofuran antibiotics. Discovery of this residue depot may allow widespread technology transfer to laboratories lacking LC-MS/MS thus improving global monitoring of these drugs.