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       Melamine is a recognized food contaminant that may be present in certain food categories both accidentally and intentionally. The aim of this study was to verify the detection and quantification of melamine in infant formula and milk powder. A total of 40 commercially available food samples, including infant formula and milk powder, from different regions of Iran were analyzed. The approximate melamine content of the samples was determined using a high-performance liquid chromatography-ultraviolet (HPLC-UV) system. A calibration curve (R2 = 0.9925) was constructed for the detection of melamine in the range of 0.1–1.2 μg mL−1. The limits of quantification and detection were 1 μg mL−1 and 3 μg mL−1, respectively. Melamine was tested in infant formula and milk powder and the results showed that melamine levels in the infant formula and milk powder samples were 0.001–0.095 mg kg−1 and 0.001–0.004 mg kg−1, respectively. These values ​​are in line with EU legislation and the Codex Alimentarius. It is important to note that the consumption of these milk products with reduced melamine content does not pose a significant risk to consumer health. This is also supported by the results of the risk assessment.
       Melamine is an organic compound with the molecular formula C3H6N6, derived from cyanamide. It has very low solubility in water and is approximately 66% nitrogen. Melamine is a widely used industrial compound with a wide range of legitimate uses in the production of plastics, fertilizers, and food processing equipment (including food packaging and kitchenware)1,2. Melamine is also used as a drug carrier for the treatment of diseases. The high proportion of nitrogen in melamine can lead to misuse of the compound and imparting the properties of protein molecules to food ingredients3,4. Therefore, adding melamine to food products, including dairy products, increases the nitrogen content. Thus, it was erroneously concluded that the protein content of milk was higher than it actually was.
       For every gram of melamine added, the protein content of food will increase by 0.4%. However, melamine is highly soluble in water and can cause more serious harm. Adding 1.3 grams of melamine to liquid products such as milk can increase the milk’s protein content by 30%5,6. Although melamine is added to animal and even human foods to increase protein content7, the Codex Alimentarius Commission (CAC) and national authorities have not approved melamine as a food additive and have listed it as hazardous if swallowed, inhaled, or absorbed through the skin. In 2012, the World Health Organization’s (WHO) International Agency for Research on Cancer listed melamine as a Class 2B carcinogen because it may be harmful to human health8. Long-term exposure to melamine may cause cancer or kidney damage2. Melamine in food can complex with cyanuric acid to form water-insoluble yellow crystals that can cause damage to kidney and bladder tissue, as well as urinary tract cancer and weight loss9,10. It can cause acute food poisoning and, in high concentrations, death, especially in infants and young children.11 The World Health Organization (WHO) has also set the tolerable daily intake (TDI) of melamine for humans at 0.2 mg/kg body weight per day based on CAC guidelines.12 The US Food and Drug Administration (US FDA) has set the maximum residue level for melamine at 1 mg/kg in infant formula and 2.5 mg/kg in other foods.2,7 In September 2008, it was reported that several domestic infant formula manufacturers had added melamine to milk powder to increase the protein content of their products, resulting in milk powder poisoning and triggering a nationwide melamine poisoning incident that sickened more than 294,000 children and hospitalized more than 50,000. 13
       Breastfeeding is not always possible due to various factors such as the difficulties of urban life, illness of the mother or child, which leads to the use of infant formula to feed infants. As a result, factories have been established to produce infant formula that is as close as possible to breast milk in composition14. Infant formula sold in the market is usually made from cow’s milk and is usually made with a special mixture of fats, proteins, carbohydrates, vitamins, minerals and other compounds. In order to be close to breast milk, the protein and fat content of the formula varies, and depending on the type of milk, they are fortified with compounds such as vitamins and minerals such as iron15. Since infants are a sensitive group and there is a risk of poisoning, the safety of milk powder consumption is of vital importance to health. After the melamine poisoning case among Chinese infants, countries around the world have paid close attention to this issue, and the sensitivity of this area has also increased. Therefore, it is especially important to strengthen the control of infant formula production in order to protect the health of infants. There are various methods for detecting melamine in food, including high-performance liquid chromatography (HPLC), electrophoresis, sensory method, spectrophotometry and antigen-antibody enzyme-linked immunosorbent assay16. In 2007, the US Food and Drug Administration (FDA) developed and published an HPLC method for the determination of melamine and cyanuric acid in food, which is the most effective method for determining melamine content17.
       Melamine concentrations in infant formula measured using a new infrared spectroscopy technique ranged from 0.33 to 0.96 milligrams per kilogram (mg kg-1). 18 A study in Sri Lanka found melamine levels in whole milk powder to range from 0.39 to 0.84 mg kg-1. In addition, imported infant formula samples contained the highest levels of melamine, at 0.96 and 0.94 mg/kg, respectively. These levels are below the regulatory limit (1 mg/kg), but a monitoring programme is needed for consumer safety. 19
       Several studies have examined the levels of melamine in Iranian infant formulas. About 65% of the samples contained melamine, with an average of 0.73 mg/kg and a maximum of 3.63 mg/kg. Another study reported that the level of melamine in infant formula ranged from 0.35 to 3.40 μg/kg, with an average of 1.38 μg/kg. Overall, the presence and level of melamine in Iranian infant formulas have been assessed in various studies, with some samples containing melamine exceeding the maximum limit set by regulatory authorities (2.5 mg/kg/feed).
       Considering the huge direct and indirect consumption of milk powder in the food industry and the special importance of infant formula in feeding children, this study aimed to validate the detection method of melamine in milk powder and infant formula. In fact, the first objective of this study was to develop a rapid, simple and accurate quantitative method for detecting melamine adulteration in infant formula and milk powder using high performance liquid chromatography (HPLC) and ultraviolet (UV) detection; Secondly, the objective of this study was to determine the melamine content in infant formula and milk powder sold in the Iranian market.
       The instruments used for melamine analysis vary depending on the food production location. A sensitive and reliable HPLC-UV analysis method was used to measure melamine residues in milk and infant formula. Dairy products contain various proteins and fats that may interfere with melamine measurement. Therefore, as noted by Sun et al. 22, an appropriate and effective cleanup strategy is necessary before instrumental analysis. In this study, we used disposable syringe filters. In this study, we used a C18 column to separate melamine in infant formula and milk powder. Figure 1 shows the chromatogram for melamine detection. In addition, the recovery of the samples containing 0.1–1.2 mg/kg melamine ranged from 95% to 109%, the regression equation was y = 1.2487x − 0.005 (r = 0.9925), and the relative standard deviation (RSD) values ​​ranged from 0.8 to 2%. The available data indicate that the method is reliable in the studied concentration range (Table 1). The instrumental limit of detection (LOD) and limit of quantification (LOQ) of melamine were 1 μg mL−1 and 3 μg mL−1, respectively. In addition, the UV spectrum of melamine exhibited an absorption band at 242 nm. The detection method is sensitive, reliable and accurate. This method can be used for routine determination of melamine level.
       Similar results have been published by several authors. A high-performance liquid chromatography-photodiode array (HPLC) method was developed for the analysis of melamine in dairy products. The lower limits of quantification were 340 μg kg−1 for milk powder and 280 μg kg−1 for infant formula at 240 nm. Filazzi et al. (2012) reported that melamine was not detected in infant formula by HPLC. However, 8% of the milk powder samples contained melamine at a level of 0.505–0.86 mg/kg. Tittlemiet et al.23 conducted a similar study and determined the melamine content of infant formula (sample number: 72) by high-performance liquid chromatography-mass spectrometry/MS (HPLC-MS/MS) to be approximately 0.0431–0.346 mg kg−1. In a study conducted by Venkatasamy et al. (2010), a green chemistry approach (without acetonitrile) and reversed-phase high-performance liquid chromatography (RP-HPLC) were used to estimate melamine in infant formula and milk. The sample concentration range was from 1.0 to 80 g/mL and the response was linear (r > 0.999). The method showed recoveries of 97.2–101.2 over the concentration range of 5–40 g/mL and the reproducibility was less than 1.0% relative standard deviation. Furthermore, the observed LOD and LOQ were 0.1 g mL−1 and 0.2 g mL−124, respectively. Lutter et al. (2011) determined melamine contamination in cow’s milk and milk-based infant formula using HPLC-UV. Melamine concentrations ranged from < 0.2 to 2.52 mg kg−1. The linear dynamic range of the HPLC-UV method was 0.05 to 2.5 mg kg−1 for cow’s milk, 0.13 to 6.25 mg kg−1 for infant formula with a protein mass fraction of <15%, and 0.25 to 12.5 mg kg−1 for infant formula with a protein mass fraction of 15%. The LOD (and LOQ) results were 0.03 mg kg−1 (0.09 mg kg−1) for cow’s milk, 0.06 mg kg−1 (0.18 mg kg−1) for infant formula <15% protein, and 0.12 mg kg−1 (0.36 mg kg−1) for infant formula 15% protein, with a signal-to-noise ratio of 3 and 1025 for LOD and LOQ, respectively. Diebes et al. (2012) investigated melamine levels in infant formula and milk powder samples using HPLC/DMD. In infant formula, the lowest and highest levels were 9.49 mg kg−1 and 258 mg kg−1, respectively. The limit of detection (LOD) was 0.05 mg kg−1.
       Javaid et al. reported that melamine residues in infant formula were in the range of 0.002–2 mg kg−1 by Fourier transform infrared spectroscopy (FT-MIR) (LOD = 1 mg kg−1; LOQ = 3.5 mg kg−1). Rezai et al.27 proposed a HPLC-DDA (λ = 220 nm) method to estimate melamine and achieved a LOQ of 0.08 μg mL−1 for milk powder, which was lower than the level obtained in this study. Sun et al. developed an RP-HPLC-DAD for the detection of melamine in liquid milk by solid phase extraction (SPE). They obtained a LOD and LOQ of 18 and 60 μg kg−128, respectively, which is more sensitive than the current study. Montesano et al. confirmed the effectiveness of the HPLC-DMD method for the assessment of melamine content in protein supplements with a limit of quantification of 0.05–3 mg/kg, which was less sensitive than the method used in this study29.
       Undoubtedly, analytical laboratories play an important role in protecting the environment by monitoring pollutants in various samples. However, the use of a large number of reagents and solvents during analysis may result in the formation of hazardous residues. Therefore, green analytical chemistry (GAC) was developed in 2000 to reduce or eliminate the adverse effects of analytical procedures on operators and the environment26. Traditional melamine detection methods including chromatography, electrophoresis, capillary electrophoresis, and enzyme-linked immunosorbent assay (ELISA) have been used to identify melamine. However, among the numerous detection methods, electrochemical sensors have attracted much attention due to their excellent sensitivity, selectivity, rapid analysis time, and user-friendly characteristics30,31. Green nanotechnology utilizes biological pathways to synthesize nanomaterials, which can reduce the generation of hazardous waste and energy consumption, thereby promoting the implementation of sustainable practices. Nanocomposites, for example, made from environmentally friendly materials, can be used in biosensors to detect substances such as melamine32,33,34.
       The study shows that solid-phase microextraction (SPME) is used effectively due to its higher energy efficiency and sustainability compared to traditional extraction methods. The environmental friendliness and energy efficiency of SPME make it an excellent alternative to traditional extraction methods in analytical chemistry and provide a more sustainable and efficient method for sample preparation35.
       In 2013, Wu et al. developed a highly sensitive and selective surface plasmon resonance (mini-SPR) biosensor that utilizes the coupling between melamine and anti-melamine antibodies to rapidly detect melamine in infant formula using an immunoassay. The SPR biosensor combined with an immunoassay (using melamine-conjugated bovine serum albumin) is an easy-to-use and low-cost technology with a detection limit of only 0.02 μg mL-136.
       Nasiri and Abbasian used a high-potential portable sensor in combination with graphene oxide-chitosan composites (GOCS) to detect melamine in commercial samples37. This method showed ultra-high selectivity, accuracy, and response. The GOCS sensor demonstrated remarkable sensitivity (239.1 μM−1), a linear range of 0.01 to 200 μM, an affinity constant of 1.73 × 104, and an LOD of up to 10 nM. Moreover, a study conducted by Chandrasekhar et al. in 2024 adopted an eco-friendly and cost-effective approach. They used papaya peel extract as a reducing agent to synthesize zinc oxide nanoparticles (ZnO-NPs) in an eco-friendly method. Subsequently, a unique micro-Raman spectroscopy technique was developed for the determination of melamine in infant formula. ZnO-NPs derived from agricultural waste have demonstrated potential as a valuable diagnostic tool and a reliable, low-cost technology for monitoring and detecting melamine38.
       Alizadeh et al. (2024) used a highly sensitive metal-organic framework (MOF) fluorescence platform to determine melamine in milk powder. The linear range and lower detection limit of the sensor, determined using 3σ/S, were 40 to 396.45 nM (equivalent to 25 μg kg−1 to 0.25 mg kg−1) and 40 nM (equivalent to 25 μg kg−1), respectively. This range is well below the maximum residue levels (MRLs) set for the identification of melamine in infant formula (1 mg kg−1) and other food/feed samples (2.5 mg kg−1). Fluorescent sensor (terbium (Tb)@NH2-MIL-253(Al)MOF) demonstrated higher accuracy and more precise measurement capability than HPLC39 in detecting melamine in milk powder. Biosensors and nanocomposites in green chemistry not only enhance detection capabilities but also minimize environmental hazards in line with sustainable development principles.
       Green chemistry principles have been applied to various methods for the determination of melamine. One approach is the development of a green dispersive solid-phase microextraction method using the natural polar polymer β-cyclodextrin cross-linked with citric acid for the efficient extraction of melamine 40 from samples such as infant formula and hot water. Another method uses the Mannich reaction for the determination of melamine in milk samples. This method is inexpensive, environmentally friendly, and highly accurate with a linear range of 0.1–2.5 ppm and a low detection limit 41. Furthermore, a cost-effective and environmentally friendly method for the quantitative determination of melamine in liquid milk and infant formula was developed using Fourier transform infrared transmission spectroscopy with high accuracy and detection limits of 1 ppm and 3.5 ppm, respectively 42. These methods demonstrate the application of green chemistry principles to the development of efficient and sustainable methods for the determination of melamine.
       Several studies have proposed innovative methods for melamine detection, such as the use of solid-phase extraction and high-performance liquid chromatography (HPLC)43, as well as fast high-performance liquid chromatography (HPLC), which does not require complex pre-treatment or ion-pair reagents, thereby reducing the amount of chemical waste44. These methods not only provide accurate results for the determination of melamine in dairy products, but also comply with the principles of green chemistry, minimizing the use of hazardous chemicals and reducing the overall environmental impact of the analytical process.
       Forty samples of different brands were tested in triplicate, and the results are presented in Table 2. Melamine levels in the infant formula and milk powder samples ranged from 0.001 to 0.004 mg/kg and from 0.001 to 0.095 mg/kg, respectively. No significant changes were observed between the three age groups of infant formula. In addition, melamine was detected in 80% of milk powder, but 65% of infant formulas were contaminated with melamine.
       The melamine content in industrial milk powder was higher than in infant formula, and the difference was significant (p<0.05) (Figure 2).
       The results obtained were below the limits set by the FDA (below 1 and 2.5 mg/kg). In addition, the results are in line with the limits set by CAC (2010) and the EU45,46, i.e. the maximum permitted limit is 1 mg kg-1 for infant formula and 2.5 mg kg-1 for dairy products.
       According to a 2023 study by Ghanati et al.47, the melamine content in different types of packaged milk in Iran ranged from 50.7 to 790 μg kg−1. Their results were below the FDA permissible limit. Our results are lower than those of Shoder et al.48 and Rima et al.49. Shoder et al. (2010) found that melamine levels in milk powder (n=49) determined by ELISA ranged from 0.5 to 5.5 mg/kg. Rima et al. analyzed melamine residues in milk powder by fluorescence spectrophotometry and found that the melamine content in milk powder was 0.72–5.76 mg/kg. A study was conducted in Canada in 2011 to monitor melamine levels in infant formula (n=94) using liquid chromatography (LC/MS). Melamine concentrations were found to be below the acceptable limit (preliminary standard: 0.5 mg kg−1). It is unlikely that the fraudulent melamine levels detected were a tactic used to increase protein content. However, it cannot be explained by the use of fertilizers, relocation of container contents, or similar factors. Furthermore, the source of melamine in milk powder imported into Canada was not disclosed50.
       Hassani et al. measured the melamine content in milk powder and liquid milk in the Iranian market in 2013 and found similar results. The results showed that except for one brand of milk powder and liquid milk, all other samples were contaminated with melamine, with levels ranging from 1.50 to 30.32 μg g−1 in milk powder and 0.11 to 1.48 μg ml−1 in milk. Notably, cyanuric acid was not detected in any of the samples, reducing the possibility of melamine poisoning for consumers. 51 Previous studies have assessed the melamine concentration in chocolate products containing milk powder. About 94% of imported samples and 77% of Iranian samples contained melamine. Melamine levels in imported samples ranged from 0.032 to 2.692 mg/kg, while those in Iranian samples ranged from 0.013 to 2.600 mg/kg. Overall, melamine was detected in 85% of samples, but only one specific brand had levels above the permissible limit.44 Tittlemier et al. reported melamine levels in milk powder ranging from 0.00528 to 0.0122 mg/kg.
       Table 3 summarizes the risk assessment results for the three age groups. The risk was less than 1 in all age groups. Thus, there is no non-carcinogenic health risk from melamine in infant formula.
       Lower levels of contamination in dairy products may be due to unintentional contamination during preparation, while higher levels may be due to intentional additions. Furthermore, the overall risk to human health from consuming dairy products with low melamine levels is considered low. It can be concluded that consuming products containing such low levels of melamine does not pose any risk to consumer health52.
       Considering the importance of food safety management in the dairy industry, especially in terms of protecting public health, it is of utmost importance to develop and validate a method for assessing and comparing melamine levels and residues in milk powder and infant formula. A simple and accurate HPLC-UV spectrophotometric method was developed for the determination of melamine in infant formula and milk powder. The method was validated to ensure its reliability and accuracy. The detection and quantification limits of the method were shown to be sensitive enough to measure melamine levels in infant formula and milk powder. According to our data, melamine was detected in most of the Iranian samples. All detected melamine levels were below the maximum allowable limits set by CAC, indicating that consumption of these types of dairy products does not pose a risk to human health.
       All chemical reagents used were of analytical grade: melamine (2,4,6-triamino-1,3,5-triazine) 99% pure (Sigma-Aldrich, St. Louis, MO); HPLC-grade acetonitrile (Merck, Darmstadt, Germany); ultrapure water (Millipore, Morfheim, France). Disposable syringe filters (Chromafil Xtra PVDF-45/25, pore size 0.45 μm, membrane diameter 25 mm) (Macherey-Nagel, Düren, Germany).
       An ultrasonic bath (Elma, Germany), a centrifuge (Beckman Coulter, Krefeld, Germany) and HPLC (KNAUER, Germany) were used to prepare the samples.
       A high performance liquid chromatograph (KNAUER, Germany) equipped with a UV detector was used. The HPLC analysis conditions were as follows: a UHPLC Ultimate system equipped with an ODS-3 C18 analytical column (4.6 mm × 250 mm, particle size 5 μm) (MZ, Germany) was used. The HPLC eluent (mobile phase) was a TFA/methanol mixture (450:50 mL) with a flow rate of 1 mL min-1. The detection wavelength was 242 nm. The injection volume was 100 μL, the column temperature was 20 °C. Since the retention time of the drug is long (15 minutes), the next injection should be made after 25 minutes. Melamine was identified by comparing the retention time and the UV spectrum peak of melamine standards.
       A standard solution of melamine (10 μg/mL) was prepared using water and stored in a refrigerator (4 °C) away from light. Dilute the stock solution with the mobile phase and prepare working standard solutions. Each standard solution was injected into the HPLC 7 times. The calibration equation 10 was calculated by regression analysis of the determined peak area and the determined concentration.
       Commercially available cow’s milk powder (20 samples) and samples of different brands of cow’s milk-based infant formula (20 samples) were purchased from local supermarkets and pharmacies in Iran for feeding infants of different age groups (0–6 months, 6–12 months, and >12 months) and stored at refrigerated temperature (4 °C) until analysis. Then, 1 ± 0.01 g of homogenized milk powder was weighed and mixed with acetonitrile:water (50:50, v/v; 5 mL). The mixture was stirred for 1 min, then sonicated in an ultrasonic bath for 30 min, and finally shaken for 1 min. The mixture was then centrifuged at 9000 × g for 10 min at room temperature and the supernatant was filtered into a 2 ml autosampler vial using a 0.45 μm syringe filter. The filtrate (250 μl) was then mixed with water (750 μl) and injected onto the HPLC system10,42.
       To validate the method, we determined the recovery, accuracy, limit of detection (LOD), limit of quantification (LOQ), and precision under optimal conditions. The LOD was defined as the sample content with a peak height three times the baseline noise level. On the other hand, the sample content with a peak height 10 times the signal-to-noise ratio was defined as the LOQ.
       The device response was determined using a calibration curve consisting of seven data points. Different melamine contents were used (0, 0.2, 0.3, 0.5, 0.8, 1 and 1.2). The linearity of the melamine calculation procedure was determined. In addition, several different levels of melamine were added to the blank samples. The calibration curve was constructed by continuously injecting 0.1–1.2 μg mL−1 of a standard melamine solution into infant formula and powdered milk samples and its R2 = 0.9925. The accuracy was assessed by repeatability and reproducibility of the procedure and was achieved by injecting samples on the first and three subsequent days (in triplicate). The repeatability of the method was assessed by calculating the RSD % for three different concentrations of added melamine. Recovery studies were performed to determine the precision. The degree of recovery by the extraction method was calculated at three levels of melamine concentration (0.1, 1.2, 2) in samples of infant formula and dry milk9,11,15.
       Estimated daily intake (EDI) was determined using the following formula: EDI = Ci × Cc/BW.
       Where Ci is the average melamine content, Cc is the milk consumption and BW is the average weight of children.
       Data analysis was performed using SPSS 24. Normality was tested using the Kolmogorov-Smirnov test; all data were nonparametric tests (p = 0). Therefore, the Kruskal-Wallis test and the Mann-Whitney test were used to determine significant differences between groups.
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