The research advancement for analytical methodology of melamine and related analogues in environment and foods

Sun Hanwen, Wang Lixin, Ge Xusheng
(College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding 071002, China)

Abstract Melamine and related analogues as the environmental contaminants have caused the attention in the world. This paper summaries the source of melamine and related analogues, and reviews the research advancement of analytical methodology of melamine and related analogues in environment and foods. This review is helpful to providing direction and reference for the food safety monitoring and quality evaluation.

In 2007, numerous pet foods in the United States were recalled after dogs and cats consuming the products suffered, and investigators discovered that waste material from the pet food manufacturing process contaminated with melamine and/or cyanuric acid had been added to hog and chicken feeds. In 2008, there has been a problem with contamination of infant formula with melamine and cyanuric acid in China. Melamine and related analogues occur as an environmental contaminant and are considered to be released from melamine resin, fertilizer, herbicide and insecticide products into feed and foods through degradation [1]. Melamine might cause urolithiasis and bladder cancer[2]. Melamine could form insoluble complexes with cyanuric acid. This could lead to crystallization and subsequent tissue injury[1,3]. Melamine as the environmental contaminants has caused the attention in the world. It is important to monitor melamine and related analogues.
A series of analytical techniques have widely applied for the detection and determination of melamine and related analogues in feeds and foods. The research advancement of analytical methodology of melamine and cyanuric acid is reviewed to provide helpful direction for analytical design, pharmacokinetic study and reasonable diet.

    Melamine is synthesized from urea at 0.1 MPa and about 390°C with silica gel as activator. Melamine and its salts have been used in the production of melamine-formaldehyde resins for surface coatings laminates, and in the formulation of flame retardants for polymeric materials. Melamine is a metabolite of cyromazine, an approved insecticide used on a broad range of vegetable crops. Ammeline is also produced from 2-substituted diamino-s-triazine compounds such as herbicides[4,5], and from cyromazine such as insecticide[6]. Melamine has been found in a variety of foods from food-contact materials. Melamine and related analogues occur as an environmental contaminant and are considered to be released from melamine resin, fertilizer, herbicide and insecticide products into feed and foods through degradation.
    Melamine and related analogues could be released from melamine resin, fertilizer, herbicide and insecticide products into feed and foods through thermal degradation[7,8], hydrolysis[9-11], biodegradation[6,12-14] or photolysis[6,12, 15-17] under the feasible conditions. Degradation system for melamine and related analogues is shown in Fig. 1.

Fig. 1 Degradative pathway for melamine and related analogues

Melamine is produced by the thermal condensation of dicyandiamide at over 300°C. During this process, some deammonia condensation derivatives, including melame and meleme, are produced[3]. These melamine derivatives such as melamemeleme, ammeline and ammelide are also produced during the production of melamine as by-products[4].
Cyromazine could be hydrolized at pH≤2 to be melamine. Melamine manufacture by hydrolysis produces ammeline, ammelide and cyanuric acid by hydrolysis under acidic conditions [5,6]. The dissociation constants pKa are 5.0 for melamine, 4.5 and 9.4 for ammeline, 1.8, 6.9, and 13.5 for ammelide, and 6.9 for cyanuric acid. Alkaline hydrolysis of melamine produces ammeline and ammelide[7].
Cyromazine in tomatoes and chard samples could not be biodegradated, and could be degradated through de-alkyl process to be melamine[1,8]. The melamine is gradually metabolized via the same pathway by certain microorganisms to ammeline, ammelide and cyanuric acid[9]. Biodegradation of melamine formaldehyde by bacterial strain MF-1 was confirmed by the decrease in dissolved oxygen, release of ammonia, and detection of intermediate metabolites during biodegradation. Melamine, cyanuric acid, and biuret were detected as intermediate metabolites in the culture filtrate, suggesting that biodegradation of MF by strain MF-1 proceeds via successive deamination reactions of melamine to cyanuric acid[10].
Cyromazine could be degradated by photocatalysis to be melamine [1,8]. Atrazine was metabolized to hydroxyatrazine, polar metabolites, and carbon dioxide [11]. The metabolism of atrazine and 2-hydroxyatrazine by the rat and the dehalogenation of atrazine and its metabolites as a bacterial degradation process had been found[1,12]. Many years ago, the degradation of atrazine, an s-triazine type herbicide, was shown to lead cyanuric acid under UV/H2O2/TiO2 treatment[13]. The oxidation of melamine under UV-irradiation in the presence of H2O2 proceeds step by step leading to ammeline, ammelide, and finally to cyanuric acid. The toxicity of the photocatalyzed solutions was higher than the initially found for melamine. due to the intermediates generated during photo-oxidation by the UV/H2O2/TiO2 treatment.

    2.1 Gas chromatography/Liquid chromatography

    Chromatographic trace analysis was used for the determination of guanidine, substituted guanidines and s-triazines in wastewater[18]. The activated carbon adsorption method and GC/MS was applied for the determination of trace 2,4,6-triamino-1 ,3,5-triazine in wastewater[19]. Gas chromatography (GC) after trimethylsilylation[20] and high-performance liquid chromatography (HPLC)[21] had been reported as analytical methods for melamine and its three hydrolytic products. Although HPLC shows a broad peak and low sensitivity for melamine and the separation of some peaks is incomplete. HPLC is more attractive than GC because no preliminary derivatisation procedures are required.
    Sugita et al.[22] describes a sensitive HPLC method for the determination of melamine and its hydrolytic products, ammeline, ammelide and cyanuric acid. The method was used to analyse leachate solutions obtained from melamine tableware with detection limit of 0.02 ppm. Cyanuric acid (CA) and chlorinated isocyanurates are standard ingredients in formulations for household bleaches, industrial cleansers, dishwasher compounds, general sanitizers, and chlorine stabilizers. Cantú et al. [23] developed a method for the determination of CA using HPLC with UV detection at 213 nm. It was proved that analysis at the lower pH range of 6.8-7.1 was inadequate due to CA keto?enol tautomerism, and at pHs of <6.8 there were substantial losses in analytical sensitivity. In contrast, pHs of >7.4 proved more sensitive but their use was rejected because of CA elution at the chromatographic void volume and due to chemical interferences. The complex equilibria of chlorinated isocyanurates and associated species were suppressed by using reductive ascorbic acid to restrict the products to CA. UV, HPLC-UV, and electrospray ionization mass spectrometry techniques were combined to monitor the reactive chlorinated isocyanurates and to support the use of ascorbic acid. The resulting method is reproducible and measures CA in the 0.5-125 mg/L linear concentration range with a method detection limit of 0.05 mg/L in water. The chlorinated salts of cyanuric acid were found an important role in recreational swimming pool waters. Recommended levels of the cyanuric acid stabilizer were in the 10-100 mg/L concentration range according to the National Swimming Pool Foundation. Cantú et al.[24] developed two isocratic HPLC methods with UV detection employing phenyl and porous graphitic carbon columns and phosphate buffer eluents (pH 6.7 and pH 9.1, respectively) to accurately measure cyanuric acid in swimming pools with method detection limits of 0.07 (phenyl) and 0.02 mg/L (porous graphitic carbon). Eleven pool water samples were fortified with 4.8-50.0 mg/L stabilizer, and the average recovery was 99.8%. Statistical analysis on the relative precisions of the two methods indicated equivalence at the 0.05 critical level.
    An air sampling and liquid chromatographic separation method for melamine was described [25]. Collection efficiency on 13-mm glass fibre filters was determined. No breakthrough was detected under a contamination level of 0.4 mg/m3 for 40-1 air samples. The detection limit for a 60-L air sample at 215 nm is 450 ng/mg. Retention behaviour of melamine on four different reversed-phase columns was described. Results of electrochemical detection studies are discussed.
    For the analysis of cyromazine and melamine in Soil,Yokley et al.[26] described the LC-UV and GC-MS method. Soil samples were extracted twice via mechanical shaking, each time with 70% acetonitrile-30% 0.050M ammomium carbonate for 30 min. An aliquot portion of the pooled extracts is subjected to strong cation exchange (SCX) purification on AG 50W-X4 resin. Final analysis was accomplished using LC-UV detection at 214 nm. Confirmatory analyses was performed using GC-MSD in the selected ion monitoring (SIM) mode. The limit of quantification (LOQ) was 10 mg/kg when using LC-UV for the analysis of cyromazine and melamine. The LOQ was 10 mg/kg when using GC-MSD for confirmatory analyses. The method passed an Independent Laboratory Validation (ILV) as per U.S. EPA FIFRA Subdivision N.
    Meleme, one of thermal decomposition products of melamine, was analyzed satisfactorily by high-performance cation-exchange chromatography using 20 mM phosphate buffer as eluent. The elution behavior of meleme and melamine at various pH values (pH 2.0-6.0) was examined and the determination was accomplishes simultaneously using photodiode array UV-Vis detection[27]. However, melame could not be analyzed satisfactorily in the system under the same conditions. Since melame was eluted more slowly than melamine and meleme, it was probably strongly adsorbed on the stationary phase.
    An ion chromatographic method was used for the determination of isocyanuric acid, ammelide, ammeline and melamine in crude isocyanuric acid using an Omnipac PCX 500 column, 100 mM potassium chloride-200 mM hydrochloric acid-5% acetonitile solution as mobile phase and a UV detector at 215 nm[28]. Melamine cyanurate is a salt of melamine and cyanuric acid, which is commonly used as a flame retardant in nylon polymer. Free cyanuric acid and/or melamine in nylon polymer can cause processing problems in molded applications, resulting in brittle parts with low ductility. Hamilton et al.[29] developed an analytical methods for extraction of free cyanuric acid and melamine from nylon 6/6,6 co-polymer using an octanesulfonic acid/isopropanol solution. An ion exclusion chromatography method with ultraviolet spectroscopic detection has been developed to quantify free cyanuric acid in the nylon extractant. Extraction efficiency of 88-133% and limit of detection of 0.4 ppm was obtained for free cyanuric acid in nylon polymer using this method. A liquid chromatography method with ultraviolet spectroscopic detection has been developed to quantify free melamine in the nylon extractant. Extraction efficiency of 76-90% and limit of detection of 2.8 ppm was realized for free melamine in nylon polymer using this method. These new analytical techniques have been successfully utilized to quantify free melamine and cyanuric acid in nylon 6/6,6 co-polymer.
    2.2 Capillary electrophoresis
    Like HPLC, capillary zone electrophoresis (CZE) has been successfully coupled to MS detection for the analysis of a wide range of significantly different types of compounds, ranging from inorganic metal ions to protein complexes. Other advantages of CE over HPLC include low solvent consumption, minimal sample size required, increased mass sensitivity, and separation efficiency[30]. The CZE allowing a characterization step on top of the sensitive mass detection, results in a powerful tool for the rapid analysis of complex mixtures by CE-MS. Nielen and van de Ven[31] analyzed melamine resins by CZE-MS, utilizing a precoated capillary to reverse the capillary surface charge allowing the use of a low pH carrier electrolyte without diminishing the EOF. A low separation efficiency was obtained with this method (four components were identified) and although the capillary was precoated, the run time was not particularly fast (35 min).
    A CZE method for the determination of the major components of (methoxymethyl)melamine resins, with quantitative analysis of unreacted melamine using electrospray ionization-mass spectrometry (ESI-MS) was presented[32]. Using a low background electrolyte pH, components are separated according to their charge/ionic radius ratio with a distinctly different separation selectivity compared to the HPLC methods commonly employed in melamine-resin analysis. The use of a time-of-flight mass spectrometer (TOF-MS) was concluded to be necessary, as the complex samples studied required maximum sensitivity and resolution, which is clearly superior for TOF-MS detectors over their quadrupole counterparts. A standard curve of free melamine was determined with an R2 = 0.999 over a concentration range of an order of magnitude. This method offers the unique separation selectivity of CZE as well as a quicker analysis time, especially for dimers compared to the HPLC methods. Recently, an improved method based on CZE coupled to either ion-trap mass spectrometry or quadrupole- TOF-MS for the analysis of melamine-formaldehyde condensates was presented[33]. Employing a formic acid-based electrolyte containing 50% acetonitrile and MS detection up to 13 compounds could be determined in lab-made resins, synthesized by mixing formaldehyde and melamine in different ratios ranging from 1:1.5 to 1:4. The use of quadrupole-time-of-flight mass spectrometry for detection allowed the assignment of molecular formulas for all 13 substances with high accuracy.
    2.3 Vibrational Spectroscopy
    Conventionally, the content of free melamine in MF resins is determined by liquid chromatography (LC) after extraction with water. This method, however, can only be applied to the determination of free melamine in soluble resins, there is a serious limitation. Scheepers et al.[34]have shown that Raman spectroscopy can be used to determine the free melamine content in melamine-formaldehyde resins. In contrast to the conventional liquid chromatography method, the Raman spectroscopic method can be directly applied to cured resins without sample preparation. The necessary assignments of bands were supported by theoretical analysis of Raman frequencies. In addition, Raman imaging was used to determine the homogeneity of the free melamine content in cured melamine-formaldehyde resins.

Recently, melamine was found to have contaminated the feed of multiple food production species leading to concern over the ability to establish an appropriate withdrawal interval and protect the safety of the food supply.
3.1 Gas chromatography–mass spectrometry (GC-MS)
The U.S. Food and Drug Administration (FDA) developed a procedure for screen animal feeds in the presence of melamine and some related compounds by derivatization before GC-MS[35]. Samples are extracted using a mixture of acetonitrile/water/diethylamine and the analytes are subsequently converted to trimethylsilyl derivatives for analysis. This procedure has been evaluated using dry protein materials (wheat gluten, rice protein, corn gluten, and soy protein), wet and dry pet foods, and dry animal feeds. It is anticipated that the method will also be applicable to a variety of other matrices. This method for melamine, ammeline, ammelide and cyanuric acid should be regarded as interim. Because of the need to rapidly provide this information, the method has not undergone the rigorous internal and external validation required for an official method. This procedure provides a general guide for the sample preparation and analysis of wheat gluten and pet food matrices for melamine using GC/MS. The performance of the method may change when different equipment and supplies are used or when different sample matrices are encountered.
Based on FDA method, GC/MS was used to analyze for melamine and related compounds cyanuric acid, ammelide, and ammeline. Several changes were made to this method to optimize performance. The analytes were easily identified by reliable retention time matching and mass spectra. Analyses of high and low spike levels in dry cat food were successful as matrix components did not coelute with compounds of interest. GC after trimethylsilylation was reported as analytical methods for melamine and its three hydrolytic products[36]. Capillary GC separation coupled with mass spectrometry was used for detection of cyromazine and its metabolite melamine [37]. confirmatory method was presented for the determination of melamine in different animal food products by GC-MS[38]. Samples are extracted using a mixture of methanol/water/triethylamine and the analytes are subsequently converted to trimethylsilyl derivatives for analysis, and benzoguanamine was employed as the internal standard. The linear range was 0.1-50.0 mg/L. The recoveries were in the range of 82.0-105.6% and the RSDs were no more than 5.8%. The limit of detection (LOD) was about 0.1 mg/g.
3.2 Liquid chromatography (LC)
A LC method was developed for the determination of melamine in wheat gluten. After being extracted by acetonitrile and water from samples melamine was separated on a Kromasil 10025, 250 mm ×416 mm, C8 column with acetonitrile and sodium 1-heptanesulfonate (0.01 mol/L, pH 3.3, 15:85, by volume) detected by ultraviolet detector. The average recoveries were 91- 96% for high, middle and low concentration levels( n = 8) . The RSD was 0.05-10.2% ( n = 8) and the LOD (S /N = 3) was 65 mg/L[39]. A method was reported for the determination of melamine in feedstuff using Symmetry C18 stainless column and liquid chromatography-ultraviolet detection at 236 nm. The RSD is less than 3. 0% and the working curves of melamine were in the linear range 1 mg/L–100 mg/L[40]. An analytical method for determination for melamine in vegetable materials by LC using PR18(4.6×250 mm, 5 mm), with water-ammonia- perchloric acid as mobile phase and UV detection at 240 nm was developed[41]. Samples were extracted by blending with water. The extract was purified by adding potassium hexaoyan oferrate (II) trihydrate and zinic acetate dihydrate to precipitate protein. The LOD was 0.5 mg/kg and the average recoveries were 70.2–80.2 % at the concentration of 1.0-4.0 mg/kg.
The thermal decomposition of melamine produces melame and meleme, while alkaline hydrolysis of melamine produces ammeline and ammelide. These four melamine derivatives were determined by high-performance cation-exchange chromatography using phosphate buffer as the eluent[42]. The elution behavior during the isocratic and gradient elutions was examined and the determination was simultaneously achieved using photodiode array UV-Vis detection. An isocratic elution with 50 mM phosphate buffer (pH 2.5) seemed most suitable for the rapid and quantitative analysis. Three types of gradient elutions involving phosphate- and NaCl-concentrations, and pH also showed satisfactory separations for the melamine derivatives.
3.3 Liquid chromatography–mass spectrometry(LC-MS)
A LC-MS method was established for the determination of melamine in feeds and food additives with the detection limits of 0.01 mg/L[43]. LC-MS/MS method for determination of melamine in feeds and food additives was developed[44]. The sample was extracted and separated on a Kromasil-C18 column (416 mm×250 mm, 5mm) with acetonitrile–0.1% formic acid (5:95, v/v) as the mobile phase at flow rate 0.4 mL/min, and determined by (+) ESIMS/MS. The parent ion of (+) ESIMS was m/z 127, and its daughter ions of MS/MS m/z 85, 109 were selected as qualitative. The TIC of MS/MS was selected as quantitative of melamine. The method proved to be simple and accurate, and has been applied to determination of melamine in feeds and food additives.
A sensitive method was developed for the determination of melamine residues in five exported feeds by LC-MS/MS[45]. Test sample was extracted with water-acetonitrile (1:1, v/v). The extract was centrifuged and the supernatant was determined and confirmed by LC- MS/MS. Mobile phase in LC was water-acetonitrile (80:20, v/v). Electro- spray ionization source was applied. The determination ion pairs were 127.2 /85.2 and 127.2 /68.2, and the confirmation ion pairs were 127.2/ 85.2. The LOD was 0.2 mg/kg.
An ultraperformance LC-electrospray-MS/MS method was established for fast determination melamine residue in feeds[46]. The use of 1% trichloroacetic acid plus 10% dimethyl sulfoxide could increase recovery of melamine residue from feeds. The residue was quantified with multiple reaction monitoring (MRM) mode. The method was validated and good results were obtained with respect to precision, repeatability and spiked recovery. The method has good repeatability and high sensitivity, and can be applied for the determination of melamine residue in feeds.
3.4  Immunoassay
The immunoassay technique is widely used in clinical laboratories as the basis for decisions on diagnosis and therapy. Recent cases of adulteration with melamine have led to the need for rapid and reliable screening methods. Melamine in pet food (fortified or originally contaminated) was determined by enzyme immunoassay (EIA), LC-DAD, and ultraperformance LC-MS/MS[47]. The LOD for EIA and LC-DAD was 0.02 and 0.1 mg/mL, respectively. The linear ranges of the calibration curves for EIA and LC-DAD were 0.02-0.5 and 0.1-500 mg/mL, respectively. The coefficient of determinations (r2) of the standard curves for EIA and LC was 0.9991 and 0.9999, respectively. Coefficient of variations from both inter- and intra-assay was <9.31%, and recovery range for all concentrations was between 71 and 105%. The R2 values between the EIA and LC-DAD methods for melamine analysis of the fortified and originally contaminated samples were 0.9973 and 0.9885. The R2 values for LC-MS/MS with LC-DAD and with EIA were 0.9566 and 0.9489, respectively.
Commercial enzyme-linked immunosorbent assay (ELISA) test kits for the detection of triazines were evaluated[48]. The recently released Melamine Plate kit (Abraxis, Warminster, Pa.) displayed a limit of detection of 9 ng/mL for melamine in phosphate-buffered saline (PBS) and approximately 1 mg/mL for melamine added to dog food. Using the melamine ELISA, both extraction protocols yielded identical results with a dog food sample adulterated with melamine. The recovery of melamine spiked into gravy from dog food was app. 75%. The ELISA for melamine proved to be a useful alternative to more cumbersome methods. The Melamine plate kit is a competitive enzyme-labeled immunoassay. Melamine was extracted from a sample by blending or shaking with extraction solution. The Melamine assay has an estimated minimum detectable concentration in buffer of 10 mg/kg. The Melamine ELISA kit has been evaluated along with other commercial enzyme-linked immunosorbent assay (ELISA) test kits for the detection of triazines. The recently released Abraxis ELISA for melamine was proved to be a useful alternative to more cumbersome methods.

Melamine and its compounds also have been found in feeds of animals raised for human consumption. Because these animals were fed melamine-contaminated feeds, it is important to monitor edible tissues from these animals for melamine residues that may enter the human food supply. Melamine has been used for the adulteration of cereal flours in order to increase their apparent protein content. Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine. Analysis of melamine and related compounds in human foods is an important work for health safety. Several quantitative and confirmatory methods were reported by using LC and LC-MS/MS.
4.1 Liquid chromatography (LC)                              
The Chinese government released a government regulation (GB/T 22388-2008) that established analytical methods for melamine in raw milk and dairy products by LC-UV detection with the method LOD of 50 mg /kg. A simple and convenient LC method was proposed for the detection of the adulteration of cereal flours with all four chemicals[49]. The precipitate formation between melamine and cyanuric acid was prevented by using alkaline conditions (pH 11-12) for both standards preparation and sample extraction. The method uses matrix-matching, which involves the construction of a calibration curve on a blank (negative control) matrix, which is then used for the quantification of melamine and by-products in adulterated (positive) samples. Matrix-matching compensates for analyte losses during sample preparation, and for matrix effects. The method has been successfully applied to wheat, corn, and rice flours, and is expected to be applicable (with some modifications) to soy flour as well. The method allows for the detection of melamine, ammeline, and ammelide at approximately 5 mg/kg, and cyanuric acid at approximately 90 mg/kg in wheat flour.
Muniz-Valencia et al.[50] developed a simple LC method for determination of melamine and its degradation products, ammeline, ammelide, and cyanuric acid in rice protein concentrate. Because ammelide is small and highly polar molecules, insufficient retention was achieved by using traditional C18 columns. A Luna cyano column has unique polar selectivity and is one of the most stable CN phases. By using a Luna cyano column and 5 mM sodium phosphate (pH 5.0) as mobile phase the optimum separation of these compounds was achieved with UV-DAD detection at 220 nm, and resorcine as internal standard. This procedure enabled separation of these compounds with baseline resolution (values in the 2.1-10.1 range) in about 8 min. Method validation was carried out in rice protein concentrates in accordance with the European Commission decision 2002/657/EC criteria. The extraction efficiencies for these compounds were in the range 99-100% and the within-laboratory reproducibility at 1.0, 1.5, and 2.0 detection capabilities concentration levels were smaller than 5, 4, and 3%, respectively. Decision limits (CCa) were 113, 60, 55, 65 and detection capabilities (CCb) were 125, 70, 65 and 75 mg/kg for cyanuric acid, ammelide, ammeline and melamine, respectively. The proposed method was successfully applied to the analysis of other rice protein concentrates and several animal feed samples.
Crude melamine may contain several by-products, i.e. ammeline, ammelide, and cyanuric acid. The simultaneous analysis of all four chemicals is difficult because of the formation of an insoluble salt between melamine and cyanuric acid. The ion-pair LC with diode-array detection was applied for the separation and determination of metformin and five related impurities [51]. Sodium octanesulphonate (0.010M) at pH=3 was used as an ion pair agent in the aqueous component of the mobile phase. Isocratic elution with 80% acetonitrile and 20% aqueous component was applied with a flow rate of 1.2mL/min. A double end-capped, 5µm particle size Inertsil ODS-2 stationary phase (250 mm length, 4.6 mm internal diameter column) was used. The Limit of quantification (LOQ) of 35-250pg were obtained using detection at 218 nm. Retention data were used in estimating the thermodynamic parameters (enthalpy and entropy) corresponding to the mobile/stationary phase transfer.
4.2 Liquid chromatography-mass spectrometry (LC-MS)
For the determination of melamine residues, several methods had been reported. A quantitative and confirmatory method was presented to determine melamine residues in edible tissues from fish fed this contaminant[52]. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by LC-MS/MS with hydrophilic interaction and electrospray ionization in positive ion mode with an average recovery of 63.8% (21.5% relative standard deviation, n=121). Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. Melamine residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg. Incurred shrimp and a survey of market seafood products were also analyzed as part of this study. A method for the determination of melamine residue in plant origin protein powders was developed using LC-diode array detection for preliminary screening of the samples for melamine, and LC-MS/MS was used in the confirmatory of melamine[53]. Trichloroacetic acid solution was used to precipitate proteins and to dissociate the target analyte from the sample matrix. The supernatant was cleaned up with strong cation exchange column. LC-MS/MS was performed in selected ion monitoring mode with trichloroacetic acid solution as ion pair reagent. The LOD was 0.5 mg/kg. The mean recoveries were 72-82% (matrix match calibration curve). Filigenzi et al.[54] reported a new method for the analysis of melamine in porcine muscle tissue using solid-phase extraction and HPLC-MS/MS Melamine was extracted in 50% acetonitrile in water. Homogenates were centrifuged and supernatants were acidified and washed with methylene chloride. The aqueous extracts were cleaned up using mixed-mode C8/strong cation exchange SPE and then concentrated, fortified with a stable isotope-labeled analog of melamine, and analyzed by LC-MS/MS. Gradient LC separation was performed using an ether-linked phenyl column with ammonium acetate/acetic acid and acetonitrile as the mobile phase. Multiple reaction monitoring (MRM) mode of two precursor-product ion transitions for melamine and one for the internal standard was used. A five point calibration curve ranging from 50 to 2000 ng/mL of melamine in solvent was used to establish instrument response. The method was validated by analysis of seven replicate porcine muscle tissue samples fortified with 10 ng/g of melamine. The mean recovery for the seven replicates was 83% with 6.5% relative standard deviation and the calculated method LOD was 1.7 ng/g.
A triple quadrupole LC-MS-MS method was presented for the quantitative determination and confirmation of melamine residues in catfish. Catfish tissue was extracted with 50:50 acetonitrile:water and 1 N hydrochloric acid and cleaned-up using Oasis MCX solid phase extraction cartridges[55]. Extracts were analyzed by LC-MS-MS with HILIC and electrospray ionization in positive ion mode. The precursor ion for melamine was m/z 127. Two product ion transitions were monitored at m/z 85 and 68 for quantification and confirmation. Catfish tissue was fortified at 10, 25, 50, 100, and 500 mg/kg, the average recovery of melamine from fortified samples (n = 17) was 76.3 % with an RSD of 14.3 %.
cyanuric acid For the analysis of cyanuric acid in catfish, tilapia, salmon, trout and shrimp tissue, cyanuric acid was extracted from ground fish or shrimp with an acetic acid solution, defatted with hexane, and isolated with a graphitic carbon black solid-phase extraction column. Residues were separated from matrix components using a porous graphitic carbon LC column, and then analyzed with electrospray ionization in negative ion mode on a triple quadrupole mass spectrometer. Selective reaction monitoringwas performed on the [M-H]-m/z 128 ion resulting in the product ions m/z 85 and 42. Recoveries from catfish, tilapia and trout fortified with 10-100 mg/kg of cyanuric acid averaged 67% with a relative standard deviation (R.S.D.) of 18% (n=107). The method detection limit for catfish, tilapia and trout was 3.5 mg/kg. An internal standard, 13C3-labeled cyanuric acid, was used in the salmon and shrimp extractions[56]. Average recovery of cyanuric acid from salmon was 91% (RSD=15%, n=18) with a method detection limit of 7.4 mg/kg. Average recovery of cyanuric acid from shrimp was 85% (RSD = 10%, n =13) with a method LOD of 3.5 mg/kg. Cyanuric acid has gained interest as a potential degradation product of triazine herbicides, such as simazine and atrazine. The cyanuric acid was extracted from the water through a microscale liquid–liquid extraction. The extract was evaporated to dryness, and an aqueous solution of quaternary ammonium cationic surfactant was added. When injected into the electrospray mass spectrometer, the surfactant and the cyanuric acid form a mass-selective stable association complex, which could be used for confident quantification of cyanuric acid. Cyanuric acid was determined by stable association complex electrospray mass spectrometry (ESI-MS) with detection limit of 130 mg/L[57]. The cyanuric acid concentrations determined with ESI-MS were not significantly different at the 95% confidence level to those determined by conventional HPLC. A recovery of 100% from a fortified urine sample illustrates the robustness of the technique.
For the determination of melamine and cyanuric acid in infant formula, the Center for Veterinary Medicine (CVM) developed a LC triple quadrupole tandem mass spectrometry method[58], which consists of an initial extraction with 2.5% aqueous formic acid, followed by a series of filtration, centrifugation, and dilution steps. Both compounds were analyzed in the same chromatographic program using a zwitterionic HILIC LC column. Electrospray ionization was used in both the negative ion (cyanuric acid) and positive ion (melamine) modes. Two selected reaction monitoring (SRM) transitions were monitored for both compounds. The amount of compounds present was determined with a calibration curve consisting of sample extracts from infant formula fortified from 0.25 to 5 mg/g. The range of recovery from fortified infant samples (n=38) was 70-114 %, and 72-110% for cyanuric acid and melamine, respectively. The LOQ and confirmation were 0.25 mg/g for both analytes in dry infant formula. Varelis and Jeskelis synthesized the labelled internal standards [13C3]-melamine and [13C3]-cyanuric acid using the common substrate [13C3)]-cyanuric chloride by reaction with ammonia and acidified water, respectively[59]. Standards with excellent isotopic and chemical purities were obtained in acceptable yields. These compounds were used to develop an isotope dilution LC-MS method to determine melamine and cyanuric acid in catfish, pork, chicken, and pet food. The method involved extraction into aqueous methanol, liquid-liquid extraction and ion exchange solid phase clean-up, with normal phase LC in the so-called hydrophilic interaction mode. The method had a limit of detection of 10 mg/kg for both melamine and cyanuric acid in the four foods with a percentage coefficient of variation of less than 10%. The recovery of the method at this level was in the range of 87-110% and 96-110% for melamine and cyanuric acid, respectively. Recently, melamine and cyanuric acid-induced crystalluria, uroliths, and nephrotoxicity in dogs and cats were investigated[60]. This article discusses the 2007 recall of canned pet food because of concerns about adverse effects on kidney function of cats and dogs. The discovery of melamine and cyanuric acid in the foods is detailed. Case studies, including clinical, pathology, histology, and toxicology findings, are presented. An attempt is being made to identify the minerals in the uroliths and kidney tissues of affected animals. A clinicopathological study on exposed children with ultrasonographic evidence of urolithiasis was conducted.
For the analysis of melamine, cyanuric acid and related analogs, Campbell et al.[61] described a matrix-assisted laser desorption ionization/time-of-flight mass spectrometric method. (M+H)+ ions were observed for ammelide and ammeline under positive ion conditions with sinapinic acid as the matrix. With alpha-cyano-4-hydroxy-cinnamic acid as the matrix, a matrix-melamine complex was observed. However, no complex was observed with sinapinic acid as the matrix. (M-H) was observed for cyanuric acid with sinapinic acid as the matrix。
Cyromazine and melamine Cyromazine and melamine are small polar basic molecules, making their determination at residue levels complicated. Sancho et al.[62] presented a LC-ESI-MS/MS method for the determination of cyromazine and its metabolite melamine in chard samples. The method involves an extraction procedure with phosphate buffer and methanol using high-speed blender, the addition of tridecafluoroheptanoic acid as ion-pair reagent and the injection of the five-fold diluted extract on LC coupled to ESI-MS/MS. The method was validated for chard samples, spiked at 0.05 and 0.5 mg/kg. Quantification was carried out by using matrix-matched standards calibration and recoveries were satisfactory, with mean values for cyromazine of 103% and 93%, and RSD lower than 7%. In the case of melamine, recoveries were 89% and 86%, with RSD lower than 13%. A LOQ of 0.05mg/kg was obtained for both compounds, with the limit of detection below 0.01mg/kg. The method, with very little sample handling and good sensitivity, was applied to the rapid determination of low residue levels of these compounds in chards from field residue trials. All the quality controls during the analysis were satisfactory with average recoveries of 92% and 78% for cyromazine and melamine, respectively.
The consumption of food tainted by melamine, ammeline, ammelide, and cyanuric acid was implicated in numerous instances of renal failure in cats and dogs. A method was developed for the analysis of these compounds in kidney tissue using HPLC-MS/MS[63]. The analytes were extracted by homogenization of kidney tissue in 50/40/10 acetonitrile/water/diethylamine. The homogenate was centrifuged, and an aliquot of supernatant was diluted with acetonitrile, concentrated, and fortified with a stable isotope-labeled analogue of melamine. Analytes were detected using atmospheric pressure chemical ionization and multiple reaction monitoring. Quantification of positive samples was performed using the internal standard method and five-point calibration curves ranging between 50 and 1000 ng/mL of each analyte. The method was validated by analysis of replicate kidney tissue samples fortified with the individual analytes and by analysis of kidney samples fortified with melamine cyanurate powder at two different concentrations. This method was successfully used for routine postmortem diagnosis of melamine toxicosis in animals. Melamine was also detected by this method in paraffin-embedded tissue from animals suspected to have died of melamine toxicosis.
4.3 Vibrational analysis
Recently, vibrational spectroscopic methods such as Raman spectroscopy have been increasingly used as analytical techniques for evaluating food safety and quality. A surface enhanced Raman spectroscopy (SERS) in combination with SERS-active substrates was developed for the analysis of melamine concentration in wheat gluten, chicken feed, and processed foods[64]. The SERS was able to rapidly detect 0.1% melamine in wheat gluten, 0.05% in chicken feed, 0.05% in cakes, and 0.07% in noodle, respectively. A partial least squares model was established for the quantification of melamine in foods by SERS: R = 0.90, RMSEP = 0.33. In addition, SERS results were verified by HPLC analysis based on a simplified FDA method. Compared with HPLC, the SERS method is much faster and simpler, requires minimum sample preparation, but still yields satisfactory qualitative and quantitative results. These results demonstrate that it is an applicable approach to use SERS to screen foods, eliminate presumptive negative samples of melamine contamination from the sample population, and then verify presumptive positive samples using HPLC protocols. Combining these methods could provide a more rapid and cost-effective way for monitoring melamine contamination in increasingly large numbers of imported foods and feed products

    When considering regulatory actions to be taken on contaminated food products potential human health impact, availability of foods and other factors should be taken into account. Investigations into sources of melamine contamination should be carried out in all cases to the extent possible.
    There are no established regulatory limits for melamine and related triazines in any type of food. None of them should be present in foods because they are toxic at high dose exposure. LC-MS is the principal analytical method currently used by the Food and Drug Administration (FDA) for detection and quantification of melamine in foods. The limit of detection of this method is as low as 10 mg/kg. However, this standard method involves time-consuming and labor-intensive procedures, especially in terms of sample preparation and clean-up steps. In addition, it is not a cost-effective screening tool for large numbers of samples as typically encountered in imported food and feed ingredients. Therefore, it is of critical importance to develop simpler, quicker, cost-effective, and sensitive methods for melamine detection in food systems information on toxicity of melamine and analogues and on the levels of melamine compounds in edible tissues would be useful for future assessments.
    This research could carry out in the following problems: Analytical method improvement to confirm low levels in tissues, including development and dissemination of standards; Characterization of the crystals found in experimental animals and clinical cases; Basic toxicological studies in several species, especially comparative renal effects; Further examination of the relative toxicity of melamine and analogues and the toxicity of co-exposures and the determination of the role of additivity and synergism in melamine compound toxicity; and Development of biomarkers for onset of melamine compound -type renal failure for better clinical diagnoses.