Recent developments in the detection of melamine

Recent developments in the detection of melamine 1 first-author †E-mail:liuyuan@jaas.ac.cn †E-mail:liuyuan@jaas.ac.cn liuyuan@jaas.ac.cn 2 1 1 1 Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing 210014, China Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing 210014, China Michigan State Universities, East Lansing 48824-1212, USA Michigan State Universities, East Lansing 48824-1212, USA In recent years, there were two reported outbreaks of food borne illness associated with melamine. The presence of melamine and its related compounds in milk, feed, and other foods has resulted in the need for reliable methods for the detection and accurate quantification of this class of contaminants. The sample pretreatment for melamine in a complex matrix usually involves a liquid extraction by a polar solvent, followed by a further clean-up with solid phase extraction. Analyses of melamine and related compounds are commonly carried out by liquid or gas chromatographic methods conjugated with mass spectrometry. Other innovative screening methods, which use antibodies, molecularly imprinted polymers, capillary electrophoresis, and gold nanoparticles, are also used to develop assays and biosensors to melamine. However, many of these methods have been hindered by matrix effects, the solubility of melamine-cyanuric acid complex, and background contamination. This article reviews recent developments for detecting melamine and discusses future directions. Melamine Detection Confirmation methods Screening methods Sample pretreatment Introduction 1 In recent years, there were two reported outbreaks of food borne illness associated with melamine-contaminated food. In March 2007, pet food ingredients contaminated with melamine and its analogues ammeline, ammelide, and cyanuric acid (Fig. 1 1 ) resulted in a major outbreak of renal disease and associated deaths in cats and dogs in the United States. As a result, several major companies have recalled more than 5 300 pet food products (FDA, 2009b 2009b ). In September 2008, Sanlu and 21 other diary companies in China were selling milk and infant formula that had been contaminated with melamine, which leads to kidney stones and renal failure. By November 2008, nearly 300 000 people had become ill, and six infants died. The situation has since become an international health scare (Look Back '08:10 Big Events in CHINA (II), 2008 2008 ). It was determined that melamine had been deliberately added to pet food and raw milk to give the false appearance of a high level of protein. Melamine alone is of low toxicity; however it is able to form an insoluble complex with cyanuric acid, which can lead to kidney toxicity. The World Health Organization (WHO) has recommended the tolerable daily intake (TDI) for melamine to be 0.2 mg melamine per kg of body mass. Meanwhile, maximum residue levels (MRLs) for melamine have also been set worldwide. In China and the US, the MRL for infant formula has been set at 1.0 mg/kg and at 2.5 mg/kg for milk and other milk products, while in Europe, the Food Safety Authority has set the limit to 2.5 mg/kg for all products containing greater than 15% milk (WHO, 2009b 2009b ). Considering the presence of melamine in milk and animal feeds, and their possible contamination in other foods, there is a need for establishing sensitive and reliable methods that are capable of screening samples and confirming the presence and quantities of melamine and other metabolites. This article aims to review the recent detection methods for melamine and its related compounds in different matrices. Sample pretreatment 1 The strategies of sample pretreatment depend on the different matrixes and the selective abilities of detection methods. Melamine (2,4,6-triamino-1,3,5-triazine; C 3 H 6 N 6 ; relative molecular weight ( M W ): 126.12) is a small molecular compound with a polar compound. The sample pretreatment for melamine usually involves liquid extraction by a polar solvent, and some complex matrixes also require further clean-up with solid phase extraction (SPE) (WHO, 2009a 2009a ). The solution for melamine extraction includes a mixture of acetonitrile/water/diethylamine (50:40:10, v/v) solution in feed (Squadrone et al., 2010 2010 ), kidney (Filigenzi et al., 2008 2008 ), and milk (Miao et al., 2009 2009 ), and trichloroacetic acid solution (some coupled with lead acetate) in milk or milk-based products (Ibáñez et al., 2009 2009 ; Sun H.W. et al., 2010 2010 ) and eggs (Xia et al., 2009 2009 ). Some have also reported using methanol (Venkatasami and Sowa, 2010 2010 ) or hydrochloric acid (Wei et al., 2010 2010 ) to extract melamine from matrixes. The organic solutions or acids are used to extract melamine and precipitate protein in the samples to reduce the interference caused by the sample matrix. Diethylamine is used to maintain the extraction solvent at an alkaline pH to prevent melamine and cyanuric acid from forming an insoluble salt of melamine cyanurate (Miao et al., 2009 2009 ). Some tissue samples also require dichloromethane (Andersen et al., 2008 2008 ) or hexane (Karbiwnyk et al., 2009 2009 ) to act as a defatted reagent. Mixed-mode cation exchange solid-phase extraction cartridges are the most popular cleaning-up method for melamine samples. However, some highly selective methods (Yang S.P. et al., 2009 2009 ) or nondestructive methods (Mauer et al., 2009 2009 ) do not require any pretreatment before detection. Confirmation methods 1 Some recent publications of instrumental methods for analysis of melamine and related compounds are summarized and compared in Table 1 1 . LC: liquid chromatography; MS: mass spectrometry; HPLC: high performance liquid chromatography; DAD: diode array detection; UV: ultraviolet; GC: gas chromatography; MEL: melamine: CYA: cyanuric acid; CYRO: cyromazine; AML: ammeline; AMD: ammelide; LOD: limit of detection; SPE: solid phase extraction; MCX: mixed-mode cation exchange sorbent for bases; RP: reverse phase; PCX: polymer based cation exchange cartridge; HILIC: hydrophilic interaction chromatography Method Matrix Analyst LOD (mg/kg) Comments Reference LC-MS/MS Human urine MEL, CYA 0.00066 Cation-exchange solid-phase extraction Panuwet et al., 2012 2012 LC-MS/MS Rat plasma, liver, kidney, spleen, bladder, and brain MEL 0.00560–0.0083l Evaluated matrix effect of biological samples; background contamination was observed Wu et al., 2009 2009 LC-MS/MS Milk-based products and other food and beverage products MEL 0.01–0.10 Ion-pair liquid chromatography; no SPE clean-up step Ibáñez et al., 2009 2009 LC-MS/MS Milk and dairy products MEL, CYRO 10 Purified by Waters Oasis MCX column Feng et al., 2008 2008 LC-MS/MS Kidney tissue MEL, AMD, AML, CYA 0.092–0.140 Validated by analysis of samples fortified with MEL-CYA powder; multi-residue analysis; polar RP column Filigenzi et al., 2008 2008 LC-MS/MS Catfish, trout, tilapia, salmon, and shrimp MEL 0.0032 Hydrophilic interaction chromatography; MEL was determined in fish dosed with MEL and a combination of MEL and CYA Andersen et al., 2008 2008 LC-MS/MS Porcine muscle MEL 0.0017 Separated by ether-linked phenyl column Filigenzi et al., 2007 2007 LC-MS Catfish, pork, chicken, and pet food MEL, CYA 0.01 Ion exchange solid phase clean-up Varelis and Jeskelis, 2008 2008 HPLC-DAD Milk and diary products MEL 0.035–0.110 Separated by Nucleosil C 8 column Filazi et al., 2012 2012 HPLC-DAD Liquid milk MEL 0.018 Purified by Cleanert PCX-SPE cartridges; separated by C 18 column Sun H.W. et al., 2010 2010 HPLC-DAD, LC-MS/MS Plant origin protein powder MEL 10 and 0.5 HPLC-DAD for screening; LC-MS/MS for confirmation Ding et al., 2008 2008 HPLC-DAD Rice protein concentrate feeds MEL, AMD, AML, CYA 55–113 Separated by polar selectivity column; eight validation data Muñiz-Valencia et al., 2008 2008 HPLC-UV Infant formula MEL 0.1 Acetonitrile-free; Kromasil C 18 column Venkatasami and Sowa, 2010 2010 HPLC-UV Diary products MEl 0.003 HILIC; NH 2 column Zheng et al., 2012 2012 HPLC-UV Cereal flours MEL, AMD, AML, CYA 5–90 Multi-residue analysis; the formation of MEL-CYA was prevented by alkaline conditions Ehling et al., 2007 2007 GC-MS/MS Milk and milk products MEL, AMD, AML, CYA 0.002 Multi-residue analysis Miao et al., 2009 2009 GC-MS Milk products MEL 0.0003 Zirconia hollow fiber sorptive microextration; simple and solvent free pretreatment Li et al., 2009 2009 GC-MS Liquid milk and milk powder MEL 0.01 Coupled column separation; no derivatization step Xu et al., 2009 2009 GC-MS Eggs MEL 0.01 SPE pretreatment Xia et al., 2009 2009 Liquid chromatographic (LC) methods 2 In most laboratories, melamine and its metabolites are routinely separated by LC and detected by selective techniques. Tandem mass spectrometry (MS/MS) provides the highest degree of selectivity, followed by single-stage MS, diode array detection (DAD), and lastly ultraviolet (UV) absorption. An interim method for the determination of melamine and cyanuric acid residues in foods using LC-MS/MS was proposed by the Food and Drug Administration (FDA) at levels of 0.25 mg/kg in infant formula and other dairy-containing food products or ingredients (FDA, 2009a 2009a ). In 2008, a national standard method for rapid determination of melamine in raw milk using LC-MS/MS has been issued in China with the limit of detection (LOD) of 50 μg/kg (AQSIQ and SAC, 2008c 2008c ). In the early studies, an analysis method was developed as a degradation product of the cyromazine (CYRO) and S -triazine herbicides. Since the 2007 pet food recall, LC method has been more widely applied to detect melamine in feed samples. Due to the polar features of melamine, it is difficult to obtain sufficient retention time by the traditional C 18 column (Ehling et al., 2007 2007 ). Many methods utilize ion-exchange (Varelis and Jeskelis, 2008 2008 ), ion pair chromatography (Ibáñez et al., 2009 2009 ), or hydrophilic interaction chromatography (HILIC) (Andersen et al., 2008 2008 ; Heller and Nochetto, 2008 2008 ) to overcome these issues. Considering the potential for high concentrations of melamine and cyanuric acid to form crystals in tissues, it is necessary to detect melamine and its relate compounds in combined crystals (Filigenzi et al., 2008 2008 ). Dane and Cody ( 2010 2010 ) also designed a method by using direct analysis in real time (DART) MS to eliminate the interference from 5-hydroxymethylfurfural with direct analysis of melamine in powdered milk. Gas chromatographic (GC) methods 2 GC is another common confirmation method used to analyze melamine and related compounds, since the instruments are relatively cheap to maintain and use. However, since melamine is a strong polar substance, it usually needs an extra derivatization step with three methyl silanized reagents in a sample pretreatment phase. In 2007, the FDA developed a GC-MS screening and confirmation method for the presence of melamine, ammeline, ammelide, and cyanuric acid (Smoker and Krynitsky, 2008 2008 ). In 2008, the Chinese government issued two national standards to determine melamine in raw milk, diary products (AQSIQ and SAC, 2008b 2008b ), and plant products (AQSIQ and SAC, 2008a 2008a ) by GC-MS and GC-MS/MS methods. Miao et al. ( 2009 2009 ) described a method to simultaneously determine the presence of melamine, ammelide, ammeline, and cyanuric acid in milk and milk products by GC-MS/MS. Xu et al. ( 2009 2009 ) determined melamine in milk products by GC-MS with a coupled capillary column system with a limit detection of 0.01 mg/kg. In addition, hollow fiber sorptive extraction (HFSE) followed by GC-MS was developed for the determination of melamine residues from milk products (Li et al., 2009 2009 ). Screening methods and other techniques 1 In most cases, the type of method selected will depend on cost, labor, and the available facilities. Alternatives to costly chromatographic methods include cheaper screening methods, which have sufficient sensitivity and selectivity for melamine and have also been developed. Immunological assays, capillary electrophoresis (CE), molecularly imprinted polymer (MIP), and some visual detection are the most popular screening methods for melamine. These methods are compared in Table 2 2 . FPIA: fluorescence polarization immunoassay; ELISA: Enzyme-linked immunosorbent assay; MIPES: molecularly imprinted polymer-electrochemical sensor; MIPCD: molecularly imprinted polymer-chemiluminescence detection; CE: capillary electrophoresis; MS: mass spectrometry; UV: ultraviolet; CZE: capillary zone electrophoresis; DAD: diode array detection; NP: nanoparticle; MEL: melamine; CYA: cyanuric acid; AML: ammeline; AMD: ammelide; LOD: limit of detection Method Matrix Analyst LOD (mg/kg) Comments Reference FPIA Milk MEL 0.0093 Fluorescence polarization immunoassay Wang et al., 2011 2011 ELISA Animal muscle tissues MEL, CYA 0.0018 (MEL); 0.0045 (CYA) Hapten synthesis Liu et al., 2010 2010 ELISA MEL standard MEL 0.0026 Hapten synthesis Lei et al., 2010 2010 ELISA Dog food MEL 0.009 Commercial kit Garber, 2008 2008 MIPES Milk MEL 8.82×10 −5 High sensitivity Li et al., 2011 2011 MIPCD Liquid milk and milk powder MEL 0.02 The 96-well micro-plate was modified with sol-gel film Yu et al., 2009 2009 CE-MS Milk powder MEL, AML, AMD, CYA 0.0022‒0.0194 Mass spectrometric detection Huang et al., 2012 2012 CE-UV Infant formula MEL 0.0005 and 0.0092 Two on-line preconcentration steps Tsai et al., 2009 2009 CZE-DAD Egg, dairy products and pet feed MEL, AML, AMD, CYA 0.045‒0.342 Without solid phase extraction step Xia et al., 2010 2010 Ag NP MEL Raw milk 0.292 Label-free silver nanoparticles as a probe Ping et al., 2012 2012 Au NP Milk MEL 0.006 Based on the 18-crown-6 ether functionalized Au NP Kuang et al., 2011 2011 Au NP Milk MEL 0.04 Colorimetric visual detection, label-free Au NP Wei et al., 2010 2010 Microfluidic device Milk products MEL 0.23 No sample pretreatment, UV detection Zhai et al., 2010 2010 Enzyme-linked immunosorbent assay (ELISA) 2 ELISA is a good screening method used for large amounts of samples because of its simplicity and low cost. Liu et al. ( 2010 2010 ) and Lei et al. ( 2010 2010 ) separately reported the synthesis of different haptens and production of polyclonal antibodies to melamine, the half maximal inhibitory concentration (IC 50 ) values of the two types of antibodies are 13 and 70.6 ng/ml, respectively. Wang et al. ( 2011 2011 ) developed a fluorescence polarization immunoassay (FPIA) for the determination of melamine in milk with the LOD of 9.3 ng/ml. In addition, commercial ELISA kits based on melamine antibodies are also available (Garber, 2008 2008 ; Kim et al., 2008 2008 ). The sensitivities of many ELISA methods are equivalent or even superior to LC methods. However, most of the antibodies show cross reactivity to the structure of related compounds of melamine; therefore, the positive samples need be rechecked by a confirmation method. Molecularly imprinted polymer (MIP) 2 In recent years, MIP has been used to select melamine in the pretreatment step due to its molecular recognition functions. Yang H.H. et al. ( 2009 2009 ) prepared MIPs with high affinity to melamine and used them to selectively extract melamine in dairy products and feed samples. Yu et al. ( 2009 2009 ) also prepared a 96-well micro-plate modified with molecularly imprinted melamine sol-gel film, by which the highly selective and high throughput chemiluminescence detection of melamine was achieved. The linear range of detection for melamine was between 0.1‒50 mg/kg, with a lower detection limit of 0.02 mg/kg. Capillary electrophoresis (CE) 2 Melamine can also be separated using CE, which is an efficient and cost-effective analytical method for small molecules with many advantages, including minimum sample and reagent consumption, and fast separation speed. The pretreatment steps for melamine in milk and other matrices are quite simple since they do not require the SPE step after extraction. However, if the method was coupled with the commonly used UV detection, the detection sensitivity and reproducibility would become a problem (Tsai et al., 2009 2009 ). Visual detection 2 Melamine could decrease the stability of citrate-stabilized gold nanoparticles (Au NPs), thus causing dramatic and visible color changes and indicating the presence of melamine. Guo et al. ( 2010 2010 ) and Wei et al. ( 2010 2010 ) have reported using the label free Au NPs to detect melamine in milk with a simple sample preparation step. The aggregation of Au NPs could be visually detected after several seconds; however, many compounds could cause the aggregation of Au NPs. To increase the specificity of the method, Wei et al. ( 2010 2010 ) introduced the cyanuric acid to the detection system and used the dual signal readout to determine melamine. In addition to Au NPs, silver nanoparticles (Ag NPs) could also be used for visual detection of melamine (Ping et al., 2012 2012 ). Although, the sensitivity and specificity of visual detection are not as precise as chromatographic methods, the results can be directly observed by the naked eye, therefore offering a simple and promising method to be used for on-site screening melamine contamination in milk products or at home diagnosis. Other alternative methods 2 Some other methods by electrochemical, spectrophotometric, and nondestructive detection have also been used to develop assays or biosensors to melamine. Cao et al. ( 2009 2009 ) reported a novel electrochemical method, which used interaction between d(T) 20 and melamine. This method was applied successfully to the determination of melamine in milk products with a recovery of 95%. A spectrophotometric method based on the Mannich reaction has been reported for melamine detection. The method was tested to determine the level of melamine in a contaminated fish sample with a recovery value and LOD of 97% and 0.063 μg/ml, respectively (Rima et al., 2009 2009 ). Mauer et al. ( 2009 2009 ) also described a nondestructive and rapid method to detect melamine in infant formula powder using near- and mid-infrared spectroscopy with an LOD of 1 mg/kg with little to no sample preparation. Raman spectrometry is another rapid and cost effective method for the rapid screening of melamine with minimum sample preparation (Lin et al., 2008 2008 ; Cheng et al., 2010 2010 ). Conclusions 1 Nowadays, there are a lot of methods available to detect melamine and its related compounds (Sun F.X. et al., 2010 2010 ; Tittlemier, 2010 2010 ). They included some highly selective confirmation methods and rapid screening methods. Currently, LC and GC conjugated with mass detection are the most powerful confirmation methods to analyze melamine and related compounds. However, these methods can be expensive and time-consuming, and often require sample pretreatment to extract analytes and eliminate matrix effects. Rapid screening methods like ELISA are faster and less expensive than the traditional instrumental methods, but the positive samples from these screening methods must be confirmed by selective methods, for their lack of selective ability of mass-based detections. Newer technologies such as biosensor and nondestructive methods are also becoming available, with sufficient sensitivity and specificity to enable rapid on-site screening with or without simple pretreatment. Even so, there are some common difficulties with melamine analysis, such as the matrix effects (Wu et al., 2009 2009 ), solubility of melamine-cyanuric acid complex (Filigenzi et al., 2008 2008 ), and background contamination, and some existing detection methods still need to be validated. The future work should focus on resolving these difficulties and establishing mobile, simple, and reliable screening methods, which could be used outside the laboratories and operated by someone who is not a technician (WHO, 2009a 2009a ). Establishing protein detection methods, which are not affected by melamine and its metabolites, is another approach to prevent the risk of melamine to human (Field and Field, 2010 2010 ). References WC Andersen , SB Turnipseed , CM Karbiwnyk , SB Clark , MR Madson , CM Gieseker . Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry . J Agric Food Chem , 2008 . 56 ( 12 ): 4340 - 4347 . 18494486 DOI: 10.1021/jf800295z http://doi.org/10.1021/jf800295z . 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