Sources, Toxicity and Detection Techniques of Semicarbazide: A Review

TIAN Xiuhui, LI Huanjun, HAN Dianfeng, WEN Fenggong, LIU Huan, LIU Ge, PENG Kaixiu, GONG Xianghong, LIU Xin, WANG Weiyun, YU Haixia, and XU Yingjiang,

Sources, Toxicity and Detection Techniques of Semicarbazide: A Review

TIAN Xiuhui1), LI Huanjun1), HAN Dianfeng1), WEN Fenggong4), LIU Huan2), LIU Ge2), PENG Kaixiu2), GONG Xianghong1), LIU Xin1), WANG Weiyun1), YU Haixia3), and XU Yingjiang1),*

1)Shandong Marine Resource and Environment Research Institute, Shandong Key Laboratory of Marine Ecological Restoration, Yantai 264006, China 2) College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306,China 3)Ocean College, Zhejiang University, Zhoushan 316021, China 4) Chengyang Branch, Qindao Municipal Bureau of Ecology and Environment, Qindao 266000, China

Semicarbazide has become an important pollutant in the environment, but there was no comprehensive literature on its advances. In this review, the source, biotoxicity and detection methods are summarized. Among the sources is the material of chemical reaction, a metabolite of nitrofurazone, food processing (production by azobisformamide and hypochlorite treatment), endogenous substances,. Semicarbazide is demonstrated to be accumulative and reproductive toxicity, mutagenicity and genotoxicity, endocrine disruptors, and neurotoxicity. There are still many controversies on the effect of genotoxicity and no clear result on the carcinogenic mechanism. HPLC-MS/MS is the mainstream of the detection methods. Other methods, including HPLC, immunoassay method, biosensor method, electrochemical detection, capillary electrophoresis technique, and spectral techniques mainly, have become less and less in recent years. On all these counts, to reduce the content of semicarbazide in the environment, the key is to control the use of nitrofurazone as the banned drug effectively. Meanwhile the toxicity data should be supplemented to reveal its toxic mechanism.

semicarbazide; pollutant; source; toxicity; detection methods

1 Introduction

Semicarbazide, also known as carbamoylhydrazine, is soluble in water easily but insoluble in ether or ethanol. Semicarbazide has been used to produce drugs such as nitrofurazone, nitrofurantoin, carbazochrome, hydroprednisone and hydrocortisone, and to determine compounds such as aldehydes and ketones (Tarek., 1986; Srinivasan., 1992). Semicarbazide can bind to hydroxylions, which extends into a network structure, showing stability in structure (Nardelli., 1965; Roul., 1987).

Semicarbazide is known to be a carcinogenic hydrazine and classified as Group 3 by International Agency for Research on Cancer (IARC) in 1987 (Parodi., 1981). Semicarbazide can be transmitted into the human bodythe food chain, inducing diseases such as anemia, neuritis, and liver necrosis, and may cause inevitable damage to the eyes or even DNA if long term intake (Toth, 1975, 2000). Research has shown that semicarbazide may be introduced in food processing in addition to the use of nitrofurazone (Beatriz and Anklam, 2005). Semicarbazide was detected in canned foods, such as baby foods and jams. Semicarbazide was even found in flour in European Union (Szilagyi and Calle, 2006). Due to the biotoxicity and the transmissionfood chain, semicarbazide has become an important pollutant in the environment and food (Gao., 2014). In recent five years,there was no comprehensive literature on the advances of semicarbazide. So this review has summarized the source, toxicity, and detection methods mainly in order to provide a reference for further study of semicarbazide.

2 Main Sources of Semicarbazide

2.1 Material of Chemical Reaction

Semicarbazide is used for the determination and synthesis of aldehydes, ketones and cyanates commonly (Vázquez and Albericio, 2006; Pouramiri and Kermani, 2017), and served as a high nitrogen ligand for N-alky- lation reactions (Lesniak., 2013). Moreover, semicarbazide can be used to synthesize energetic complexes and gas generators (Hron and Jursic, 2014; Pieczonka., 2014), as well as to identify noble metals (.., Pt, Ir, and Au,.) (Mathew., 1996; Ita and Offiong, 1999). Materials synthesized by semicarbazide have been applied in optical switches and modulators, data storage devices, optical information processing, and data storage widely (Ma., 2015; Raja., 2017).

Numerous derivatives of semicarbazide present antimicrobial activity and can be used for the treatment of trypanosomes, tumors, and antibiotic therapy (Chandra and Sangeetika, 2004; Prakash., 2012; Mahmoodi., 2016; Bandyopadhyay., 2017). Semicarbazide derivatives can improve the lipophilicity of certain compounds with bioactivity (Brondani., 2007). Additionally, semicarbazide is also functioned as a bradykinin B1 receptor antagonist in medicine (Schaudt and Ezischinsky, 2010; Fedorov., 2011). Moreover, they are often acted in the separation of endocrine hormones and essential oils in the biochemical and pharmaceutical research and production (Bondebjerg., 2005; Obaleye., 2011; Chinnasamy., 2012). Semicarbazide of different purpose might enter the environment, ultimately bring hidden dangers to food safety if accumulated by organisms (Tian, 2017).

2.2 Metabolism of Nitrofurazone

Semicarbazide is regarded as the signature metabolite of nitrofurazone, a banned veterinary drug (Vass., 2008). Nitrofurazone was synthesized by 5-furan and se- micarbazide in the 1850s, as one human drug originally and for the treatment of gastrointestinal diseases and skin infections in animals lately, also applied to cattle, pigs, poultry, and fish, as well as to treat bacterial infections by the bee (Khong., 2004; Erdur., 2008). Nitrofurazone was metabolized rapidly with the half-life of only a few hours in animals, inducing to the track and detection impossible (Cooper., 2005). It is evidenced that semicarbazide protein-bound could exist for a long time in animals stably (Johnston., 2015; Tittlemier., 2007), which was detected in both pigs and chickens after treatment by nitrofurazone (Cooper and Kennedy, 2005; Mccracken., 2005) (Fig.1).

Fig.1 Structures of nitrofurazone and semicarbazide.

Nitrofurazone was banned in European Union in 1995. Its residue limit of semicarbazide as the signature metabolite was set to 1.00μgkg?1in 2003, and banned in the United States in 2002, in Japan Positive List in 2005, in the list of banned veterinary drugs and other chemical compounds in food animals in China in 2003 (Federal, 2002; The European Parliament and the Council of the European Union, 2003; Yamamoto., 2009).

2.3 Food Processing

2.3.1 Production by azobisformamide

2.3.1.1 Foaming agent

Azodicarbonamide, ADA or ADC for abbreviation, foaming agent, is often served as a plastic gasket for metal lids during packaging. ADA is decomposed to biurea of nonvolatile under high temperature, which can generate semicarbazide by thermoal decomposition (Ah- renholz and Neumeister, 1987). To verify the thermal de- composition of ADA, semicarbazide was detected in the direct extract of gasket treated by hot water without derivatization. When treated for 30min, semicarbazide was produced higher than 180℃ only, and the maximum reached 220℃ (Prakash., 1975).

A total of 107 samples of children’s foods, contained in glass jars sealed with plastic gaskets, were monitored in 11 European countries, illustrating that contents were related to the type of the foods (fruit, vegetable, meat and their combinations). It was inferred that semicarbazide produced by ADA could be transferred into foods (Szilagyi and Calle, 2006). In 2003, the European Food Safe- ty Authority (EFSA) issued the risk assessment report, declaring that ADA can produce semicarbazide, and recommending that food manufacturers should replace the package as soon as possible (European Food Safety Authority, 2003). To eliminate semicarbazide from ADA, European Union issued the 2004/1/EU directive on January 6, 2004, which stipulated the prohibition of ADA as a foaming agent from August 2, 2005, and requested the use of its alternatives (Official Journal of the European Union, 2004).

2.3.1.2 Flour additive

The residue limit of ADA as a flour additive is different globally, which is allowed and the maximum amount 45 mgkg?1in the United States, China, and Canada, whereas prohibited in European Union, Singapore, and Australia (European Food Safety Authority, 2005). ADA can be converted to biurea in wet and acidic conditions and then to semicarbazide and urazole under high temperatures (Becalski., 2006). After artificial addition in wet flour, semicarbazide was detected in all flour, with a reaction yield of 0.10%. It was believed that ADA was decomposed into biurea as stable intermediate firstly, then decomposed to semicarbazide during the whole process(Pereira., 2004). The reaction mechanism inferred is shown in Fig.2.

Fig.2 Mechanism of reaction between biurea and semicarbazide.

2.3.2 Hypochlorite treatment

Semicarbazide can be producedHofmann reaction at high pH if treated by sodium hypochlorite (Bendall, 2009). Also, semicarbazide can be generated when hypochlorite is used for disinfection during the breaking of the eggshell. Additionally, semicarbazide has been detected in shrimp, chicken, soybean, egg white powder, milk, red algae, gelatin, carrageenan, locust bean gum, and starch after treatment by hypochlorite (Hoenicke., 2004). Research has revealed that semicarbazide was detected in 27 out of 28 samples of birds nest bottled of ready to eat, due to the use of sodium hypochlorite in the processing after excluding the source of nitrofurazone or ADA (Xing., 2012). Semicarbazide has also been detectedinseaweed for the production of carrageenan, which requiring the addition of hypochlorite solution containing 0.050%–0.10% active chlorine for bleaching treatment generally (Antonopoulos., 2004).

2.3.3Thermal processing

A certain amount of semicarbazide can be produced in different processing, such as thermal processing, affecting food content and distribution (Becalski., 2004; Kwon, 2017). High temperature and humidity were critical to the four different types of flour products (steamed bun, bread, fried food, and noodle). The amount in external was 2.26 times higher than the internal of steamed bun, and 3.58 times of bread. It was indicated that the content in the external was higher than internal. All above proved that the amount was associated with the processing closely (Ye., 2011).

Semicarbazide was detected in wild,where ozone replaced hypochlorite for disinfection. It was proposed that semicarbazide might be produced during the process finally, eliminating the possibility of hypochlorite or nitrofurazone (Saari and Peltonen, 2004). Re- search showed that carrageenan could also introduce se- micarbazide in the process of eggs (Hoenicke., 2004). Semicarbazide can be produced under thermal treatment of egg pow- der, with a larger amount under oxic than anoxic conditions. It was presumable that oxygen might play an important role in the whole process (Gatermann., 2004).

2.4 Endogenous Substances

In addition to the sources above, semicarbazide exists in some animals and plants naturally. Some animal-de- rived products may contain semicarbazide or components produced by semicarbazide. Semicarbazide was detected in wild shrimp, with the detection rate over half. Additionally, the content was over standard in the shell seriously, exhibiting no correlation between content and location (Mccracken., 2013). As concentrated to crustaceans mainly, semicarbazide might be a natural component of the shell (Van Poucke., 2011).

Another example is drone pupa that has attracted wide attention because of semicarbazide residue over standard, becoming a puzzle restricting the exports in China. Research has indicated that semicarbazide may be related to the increased amount of chitin during the growth stage lately(Jia., 2014). It was proposed that semicarbazide was related to the increased arginine before or during honey production significantly(Crews, 2014).

2.5 Summary of the Main Sources of Semicarbazide

At present, semicarbazide has been found in different samples. In addition to the use of nitrofurazone, food packaging, flour improvers, and food processing have all been the sources of semicarbazide. In view of this, higher requirements are put forward for the pollution prevention of semicarbazide in the environment. To reduce the content of semicarbazide, the key is to control the use of nitrofurazone as the banned drug effectively. As an endogenous substance, semicarbazide can be deeply explored and discussed in the future research, and the factors causing production can be furtherly studied through physical and chemical means.

3 Toxicity of Semicarbazide

3.1 Accumulative and Reproductive Toxicity

Semicarbazide can alter specific important organs and tissues (.., epiphyseal cartilage, ovary, uterus, testis, spleen, adrenal gland, thyroid gland, thymus, and pancreas) in Sprague-Dawley rats and possess an adverse effect on the reproductive system. For example, semicarbazide can trigger insufficient mineralization of epiphyseal cartilage in both male and female rats. Meanwhile chromatin concentrated primary and secondary oocytes appeared in the ovary (with a relationship between dose and effect), and the thickness of the endometrium declined in females. The diameter of the testicular tube was decreased, indicating a bad correlation with the concentration, and no changes in the sperm structure or sperm formation process in males (Maranghi., 2009).

Semicarbazide gave rise to an inhibitory effect on body weight and food intake for Wistar Hannover GALAS rats. Based on the histopathology, semicarbazide led to a disordered arrangement of chondrocytes and deformation of knee, thorax, and tail obviously. Loss of bone mass and roughening of the thin edge of the thoracic aorta were observed in the highest concentration group. All the above demonstrated that the toxicity was manifested in the skeleton, cartilage, and aorta of rats mainly. Meanwhile, no carcinogenicity was shown in different concentrations, and no difference between the groups significantly (Takahashi., 2009).

Semicarbazide can lower the concentration of endogenous 17β-estradiol, inhibiting the maturation of the ovary in female zebrafish eventually (Yu., 2015). In addition, semicarbazide can alter the testicular morphology in male zebrafish and lower the testicular somatic index, reducing ability of reproductive regulatory ultimately (Yu, 2017).

3.2 Mutagenicity and Genotoxicity

Early studies were conducted on the mutagenicity of semicarbazide in the 1970s and 1980s. It was evidenced that semicarbazide can be similarly reacted with DNA and chromosomes to hydrazine or hydroxylamine, resulting in mutations in the chromosomes of locust (L.) spermatocytes (Bhattacharya, 1976), as well as distortion of the spinal cord in African clawed frog () embryos (Schultz., 1985). Taking mammals as example, semicarbazide can reduce levels of DNA and RNA in the lung and liver of females considerably, suggesting potential carcinogenicity and mutagenicity (De., 1983). Based on gradual derivation, it may be that semicarbazide can react with Cu (II), producing carbamoyl radicals that causing DNA damage (Hirakawa., 2003).

Semicarbazide can change the frequency of sister chromatids in human hematopoietic lymphocytes culturedmarkedly, putting resultant micronuclear rate of bone marrow polychromatic erythrocytes higher than the control group extremely, following chromosomal distortion (Vlastos., 2010). At the same dose, semicarbazide can increase hemangiomas in the females, however not established in males (Toth., 1975).

It was demonstrated that semicarbazide showed no genotoxicity in two types of male mice (Balb/C and CBA). There was no significant difference in the micronuclear rate of bone marrow polychromatic erythrocytes between the test and control group (Abramsson-Zetterberg and Svesson, 2005). Subsequently, it was found that the incidence of tumors did not differ among the groups significantly, and concluded that semicarbazide showed no carcinogenicity in Wistar Hannover GALAS rats (Takahashi, 2014).

The reaction kinetics of semicarbazide and DNA was analyzed by high precision instruments combined with biological approaches. It was proved that the genotoxicity was generated through bonded DNA adducts covalently (Fig.3). Meanwhile, it was the first time to identify and quantify semicarbazide-DNA and semicarbazide-RNA adducts in rat viscera using isotope dilution liquid chromatography tandem mass spectrometry with high accuracy and sensitivity. The data indicated that both DNA and RNA adduct exhibited a dose-dependence in organs of rats, with the highest level in the stomach and small intestine, and RNA adducts were higher than DNA by 4.10 to 7.00 times approximately. Furthermore, the stability of the adducts was investigatedfrom various organs of the rats at different times after administration. All the above indicated that semicarbazide might exert its toxicity by affecting the transcription process in cells (Wang., 2016).

Fig.3 Covalent bond process of semicarbazide with DNA.

3.3 Endocrine Disruptors

Semicarbazide plays a role in endocrine disruption, which was assessed in male and female Sprague-Dawley rats according to different doses. Semicarbazide delayed vaginal opening in females and foreskin separation in males. Meanwhile, the estrogen level of serum was reduced in a dose-dependent manner in females. Additionally, the serum level of dehydrotestosterone was also reduced in males, but no dose-response. Moreover, semicarbazide delayed sexual maturation in females and males markedly and affected the development of sexual organs to some extent. As an endocrine disruptor, semicarbazide showed multiple effects, presenting an anti-estrogen effect in females and adopting different disrupting mechanisms for females and males. However, the mechanism was unclear till now. One proposed was that semicarbazide can disrupt the hormone secretion process of the hy- pothalamic ituitary onadal axis by inhibiting γ-amino- butyric acid production (GABA) and antagonizing N- methyl-D-aspartate (NMDAR)(Maranghi., 2010).

The mechanism of endocrine disruption was experimented with in, displaying a disrupting effect on the thyroid gland and elevated the concentrations of 3,5,3’-triiodothyronine (T3) and thyroxine (T4), which might be similar to Sprague-Dawley rats aforementioned. Semicarbazide affected GABA synthesis and hormone secretion process of the hypothalamus ituitary hyroid axis mainly (Yue., 2017).

3.4 Neurotoxicity

Semicarbazide interfered with the transmission of nerve signals and led to relevant behaviors abnormal by antagonizing NMDAR and inhibiting glutamic acid decarboxylase (GAD) (Santos., 2008). Semicarbazide has been evidenced to be an inhibitor of mediated effect between GAD and GABA. The model on neuroendocrine regulation in organisms was shown in Fig.4, which has been verified in the reproductive the system of male zebrafish, causing disorder through GABA in males (Yu., 2017). Semicarbazide can enhance the spontaneous behaviors by inhibiting synthesis of GABA, for example, increasing the number of erections and prolonging the grooming of Sprague-Dawley rats (Maranghi., 2009). Moreover, semicarbazide antagonized NMDAR and affected its pathway of signal transduction, which might result in symptoms such as epilepsy and senile dementia ultimately (Qin., 1996).

Fig.4 Neuroendocrine regulation model of semicarbazide.

3.5 Summary of the Toxicity of Semicarbazide

At present, the toxicity is mainly concentrated in accumulative and reproductive toxicity, mutagenicity and genotoxicity, endocrine disruptors, and neurotoxicity. There have been many researches on the toxicity of semi- carbazide, especially on the effect of genotoxicity, but there are still many controversies. There is no clear conclusion on the carcinogenic mechanism till now. As a residual substance of food additives and veterinary drug, semicarbazide poses a certain threat to the human health. Therefore, attention should be paid to its existence. The extent of its harm should be determined through scientific toxicology and safety evaluation. This will bring great so- cial benefits to the improvement of human health and quality of life.

4 Detection Techniques of Semicarbazide

4.1 High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS)

Leitner firstly established the HPLC-MS/MS by optimizing the pretreatment, chromatographic condition, and mass spectrometry, a theoretical foundation for detecting semicarbazide (Leitner., 2001). The product was purified by solid-phase extraction with a polystyrene adsorbent, achieving the recovery of higher than 90.0%. The target derivatized was scanned in the positive ion mode and quantified in the multiple reaction monitoring modes (MRM) of triple quadrupole mass spectrometry. The limit of detection (LOD) reached 0.50μgkg?1. Most methods following have been extended and improved based on this theory. The key step of the derivatization reaction of se- micarbazide, with 2-nitrobenzaldehyde, is shown in Fig.5.

Fig.5 Reaction of derivatization of semicarbazide with 2-nitrobenzaldehyde.

To improve the accuracy of quantification, stable isotope internal standards are used in HPLC-MS/MS commonly, despite the high price. The pretreatment mainly includes the following steps: hydrolysis, derivatization (2-nitrobenzaldehyde), extraction (hydrochloric acid and ethyl acetate mainly) and purification (Liquid-liquid extraction or solid phase extraction). Table 1 summarizes the literatures on HPLC-MS/MS.

4.2 High Performance Liquid Chromatography (HPLC)

The pretreatment is similar to HPLC-MS/MS: hydrolysis, derivatization, extraction and purification. After pretreatment, fluorescent detector, photodiode array (PDA), and ultraviolet visible detector (UV) can be used for dete- ction. Most samples analyzed are foods, such as fish and bread (Wang and Chan, 2016), flour (Wei., 2017), shrimp (Fernando., 2015), instant noodles, and meat products mainly (Li., 2015).

Besides, a polymeric stir bar imprinted molecularly was prepared, and the sensitivity can be improved through specific adsorption. Combined with HPLC-UV, the stir bar was applied in the flesh. The LOD reached 0.59ngmL?1and RSD less than 10.0% (Tang., 2018). HPLC-PDA was performed in animal feed, extracted with ammonium acetate solution, and purified on a Sep-Pack NH2solid-phase extraction column. The LOD was 200.0μgkg?1. Although consistent with HPLC-MS/MS, the sensitivity was much lower (Barbosa., 2007).

4.3 Immunoassay Method

The pretreatment of ELISA includes the following steps generally: hydrolysis, derivatization, extraction and purification. In order to save cost and time, liquid-liquid extraction is generally used. Semicarbazide was analyzed in chicken muscle, obtaining a sensitivity of 0.25μgkg?1, which was lower than the European Union limit and consistent with HPLC-MS/MS, no false-negative in eggs or liver (Cooper., 2007). At different levels, semicarbazide added was performed in pig tissues and children’s foods, with recoveries of 89.1%–105.3% and RSD of 6.70%–15.5%, and LOD of 0.11μgkg?1–0.30μgkg?1. The results validated were consistent with HPLC-MS/MS, and could be applied for the screening of samples (Vass., 2008).

Anti-semicarbazide monoclonal antibodies were prepared, reaching a sensitivity of 0.010μgL?1, with the half-maximal inhibitory concentration of 1.30μgL?1, and no cross-reaction (Gao., 2007). Colloidal gold immunochromatography assay was performed in meat, LOD 0.72 ngmL?1(Tang., 2011). Semicarbazide was determined in fish, no cross-reaction among antibodies and the sensitivity reached up to 0.75ngmL?1(Wang., 2018). A gold nanoparticle-based bio-barcode immunoassay was performed and obtained a sensitivity of 8.00pgmL?1, 25 times higher than ELISA (Tang., 2011).

4.4 Biosensor Method

Biosensors possesses good sensitivity generally. The biosensor eliminated the need for cumbersome extraction with organic reagents. In the presence of 50.0μgL?1interfering components, no cross-reaction was observed, and LOD was 0.10μgL?1(Lu., 2016). A specific single antibody against semicarbazide was established on a biochip and then analyzed with a biochip analyzer. The LOD was 0.90μgkg?1, and the half-maximum inhibitory concentration was 2.19μgkg?1. These results were consistent with HPLC-MS/MS on the premise of the same extraction condition (Omahony., 2011). According to the results above, both the sensitivity and half-maximum inhibitory concentration are lower than ELISA.

Semicarbazide was determined using a novel impedance sensor based on gold nanoparticle functionalized chitosan composite membrane. The LOD was 1.00ngmL?1. This method was also evaluated in terms of stability, reproducibility, specificity, and accuracy, applied to pork, honey, salted sheep casing, salted pigskin, and shri- mp, with recovery of 82.0%–93.5% and RSD of 3.10%– 4.90%. The samples above were verified by HPLC-MS/ MS and the results were consistent (Jin., 2013).

A novel visualized microarray sensing technique was developed that visualized the signal response in honey using a multiplexing method. The signal response was intuitive and LOD of 0.040ngg?1. The result indicates that the 96 well plates visualized microarray has good applicability in the detection of honey in comparison with ELISA. Although the time for the preparation required is identical to HPLC-MS/MS or ELISA generally, the speed is increased markedly, indicating great potential in scree- ning (Li., 2017).

Table 1 Determination of semicarbazide in different samples using HPLC-MS/MS

4.5 Electrochemical Detection

According to the voltammetric behavior at the electrode, the parameters affecting the volt-ampere response were determined, including supporting electrolyte, pH, accumulation time, and accumulation potential. The peak current was proportional to the concentration, and LOD was 1.00μmolL?1. Semicarbazide was determined in tap water spiked, with recovery of 92.3%–104.7% and RSD of 3.60%–6.60% (Zhang., 2014). The irOx-based modified electrode was used as an electrochemical sensor. Separation and determination were enabled in a wide range of pH and verified the multiple potential interferents (.., glucose, proline, nitrate, chloride, oxalic acid, and ethanol) did not affect the response at the electrode. The linear range reached up to three orders of magnitude, with a correlation coefficient greater than 0.995, and LOD 0.75μgL?1. Given the low sensitivity, these methods are rarely used (Casellaand Contursi, 2015).

4.6 Capillary Electrophoresis Technique

The parameters were investigated, such as the concentration ratio of borate and phosphate in the buffer, pH of the running electrolyte, and the voltage. It was found that surfactant sodium deoxycholate obtained the greatest influence on the resolution. After separated on a C18column, the migration time showed good reproducibility, and the LOD was 0.40μgmL?1. Additionally, recovery was conducted with shrimp as a substrate and was very low, only 50.0% (Wickramanayake, 2006).

Capillary electrophoresis was used to determine ADA and semicarbazide simultaneously, conducted on a capillary column within 25min using buffer solution containing 20.0mmolL?1sodium tetraborate, 30.0mmolL?1β- cyclodextrin, 17.0% isopropyl alcohol (v/v), and 25.0mmolL?1sodium dodecyl sulfate. However, LOD failed to meet the requirement of the European Union (Chen., 2016). Semicarbazide was analyzed in aquatic products by capillary electrophoresis. The optimized conditions were as follows: the buffer solution containing 20.0mmolL?1sodium dihydrogen phosphate, 20.0mmolL?1disodium hydrogen phosphate, 80.0mmolL?1sodium deoxycholate, and 10.0% methanol, with pH 9.0. No semicarbazide was detected (Zhai., 2015).

The main limitation of capillary electrophoresis technique is that the sensitivity was so low that far from the residue requirements, thus limiting the application.

4.7 Other Spectral Techniques

The spectrophotometric method for simultaneous determination of semicarbazide and hydrazine was established by H-point standard addition method accurately. And good accuracy and precision were achieved. To verify this method, three concentrations were added to river water, with a satisfactory result, but rarely used at present(Safavi, 2003).

ADA, biurea, and semicarbazide were determined in flour by Raman spectroscopy. The vibration bands of the three analytes were calculated according to the density functional theory. Raman spectra were in good agreement with the result. When pure water as the solvent, LOD reached 10.0μgmL?1(<45.0μgmL?1), and the reproducibility also needed to be improved furtherly. The results above indicated that ADA and its metabolites can be detected by vibrational spectroscopy, but the sensitivity and accuracy are relativelylow (Xie., 2013).

4.8 Summary of the Detection Techniques

HPLC-MS/MS is the mainstream currently for the determination of semicarbazide, which requires expensive instruments, accessories, consumables and higher requirements on the operator’s professional skills. HPLC required less expensive instruments compared with HPLC-MS/MS. Impurities with retention time close to semicarbazide must be removed, otherwise false-positive result, so the pretreatment was tedious with strict requirements. With the promotion of HPLC-MS/MS, HPLC coupled with different detectors has to become less and less due to tedious pretreatment and higher LOD. Even if the accuracy and precision can meet the requirement on minimum residue limit, HPLC-MS/MS is still required for verification furtherly. ELISA is often used as a screening method only, with the advantages of sensitive reactions, high specificity, low technical requirements relatively, and short time. Immunoassays may produce false results (false positive or false negative), and HPLC- MS/MS for verification generally. However, ELISA has a wide application in some grass-root institutions not equipped with advanced instruments. Similar to ELISA, the biosensor method is used for screening of a large number of samples at one time generally, which are expected to be practical in food safety. Other methods have become less and less in recent years.

5 Conclusions

Semicarbazide has various sources, and the metabolization of nitrofurazone as a veterinary drug is not the only one. To date, semicarbazide has been found in different foods (including animal-derived and non-animal-derived) and even in the environment. Semicarbazide possesses accumulative and reproductive toxicity, mutagenicity and genotoxicity, endocrine disruptors, and neurotoxicity, while the mechanism needs to be cleared up furtherly. HPLC-MS/MS is currently the mainstream. Other methods have certain limitations and cannot meet the requirement or efficient determination; thus, there have been fewer applications in recent years.

Semicarbazide in the environment can lead to toxic food chains and threaten the safety ultimately. Semicarbazide can be accumulated in the body and cause an adverse effect. Researchers should continue to supplement the toxicity data to reveal the toxic mechanism. At the same time, the degradation law should be studied in depth. At present, there is no environmental standard for semicarbazide in water or other media at home or abroad. In order to control the ecological and the safety risk of direct oral exposure, it is urgent to carry out environmental risk and human health assessment. Researchers should streng- then the investigation and research on the source and emission, formation process and distribution, and pollution level in food and environment in the future, so as to provide basic data for the environment and food safety standards of semicarbazide.

Acknowledgements

The work was supported by the National Key R&D Program of China (No. 2017YFC1600702), the Key Lab of Marine Bioactive Substance and Modern Analytical Technique, SOA (No. MBSMAT-2019-04), the Modern Agro-Industry Technology Research System in Shandong Pro- vince (No. SDAIT-26-05), and the National Natural Science Foundation of China (No. 31901787).

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. E-mail: xuyingjiang@yeah.net

July 23, 2020;

January 29, 2021;

March 15, 2021

? Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2021

(Edited by Ji Dechun)