• Milk samples contained prohibited nitrogenous contaminants.
• Imported infant milk samples showed the highest detection frequency.
• An imported infant milk sample showed melamine content above the acceptable Maximum Residue Limit.
• The exposure risk of melamine and cyromazine among the general population is acceptable.

   Milk is regarded as a wholesome food due to its high nutritional value and ease of digestion, therefore, attracting the interest of consumers. The safety of milk is a major concern for public health since it is rich in proteins and lipids, making it an superior solvent for most chemicals (Qin et al., 2020). Moreover, milk is a source of nutrients for infants since it is an alternative to human breast milk, hence, it should be healthy and free of residues whose levels could pose an undesirable risk to consumers, determining its heavy regulation (García et al., 2012; Yang et al., 2022). The contamination of milk and milk products with specific nitrogen-containing compounds has been reported as a way to raise the apparent nitrogen level and thus their protein levels. Studies have revealed that melamine is being deliberately added to dilute raw milk to falsely enhance its protein content (Ionescu et al., 2023). Consequently, numerous tons of ingredients and finished milk products have been recalled worldwide due to food safety, ethical, and legal issues (Andersen et al., 2011). Continuous consumption of milk adulterated with melamine and its analogues results in the bioaccumulation of triazine residues in the body (Dorne et al., 2013). Elevated occurrences of renal failure and kidney stones among infants have been widely reported in China, Taiwan, and the United States from 2008 onward (Abedini et al., 2021). The illness was traced to the contamination of infant milk with melamine and its structural analogues, including cyanuric acid, ammeline, and ammelide, which have been reported in the urine of infants (Guo et al., 2020; Melough et al., 2020; Sathyanarayana et al., 2019; Shi et al., 2020). To ensure the wholesomeness of the food supply system and public health, surveillance is encouraged (Abernethy and Higgs, 2013).
   Melamine is a nitrogen-rich tripolycyanamide compound applied in kitchenware, laminates, adhesives, coatings, flame retardants, fertiliser mixtures, and plastics (Zhu and Kannan, 2019). Further, melamine may result in food production due to the breakdown of cyromazine, a popular veterinary drug in animal husbandry. As direct food additives, melamine, its degradation, or metabolic products are not permitted. Melamine traces can be observed in foods because of fertilizer products that contain melamine, as well as breakdown products from cyromazine, a veterinary drug (Viñas et al., 2012). However, the presence of triazine compounds in milk is classified as either baseline or adulteration. The presence of concentrations that are the result of regular use or improper application of melamine-containing materials is referred to as baseline contamination, while adulteration refers to deliberately adding, using, or misusing substances that can break down into melamine. (Nasution and Suyanto, 2022). Baseline levels should typically be <1 mg/kg and not regarded generally as health risk (Lad and Aparnathi, 2017). Illegal additives in foods, including milk, create challenges for food safety, thus putting greater responsibility on producers and government regulatory agencies.
   Several analytical methods, including Raman scattering infrared imaging and band ratio, Fourier Transform Infrared (FTIR) spectroscopy, other infrared spectroscopy techniques, mass spectrometry methods, and capillary zone electrophoresis have been applied for the accurate detection of melamine and its analogues in milk and other food matrices (Guo et al., 2014; Huang et al., 2016; Botelho et al., 2015; Lim et al., 2016; Kunzelmann et al., 2018; Wang et al., 2017). The aforementioned state-of-the-art analytical techniques and instruments are not readily available in Nigeria and, in most cases, yield  only qualitative results. Abedini et al. (2021), however, reported the accurate use of High-Performance Liquid Chromatography (HPLC) for the analysis of melamine in milk and related products. 
   This paper modifies the Solid Phase Extraction (SPE) and HPLC-Diode Array Detection (DAD) method for the quantification of cyromazine (N-cyclopropyl-1,3,5-triazine-2,4,6-triazine) and melamine (1,3,5-triazine-2,4,5-triamine) in local and imported infants and evaporated milk products in Nigeria. The study is urgent since Nigeria imports milk from countries including China, the United States of America (USA), Italy, the Netherlands, and France, where melamine contamination has been reported in infant and evaporated milk at as high as 2,560 mg/kg (FAO, 2008).
   Regrettably, Nigeria fails to have an established limit for melamine or cyromazine in infant and evaporated milk. There is no data or literature on the occurrence and detection of melamine, cyromazine, and other nitrogen-based additives in milk available in the Nigerian market. Therefore, it is not  widely known about the wholesomeness of milk in the Nigerian market. The impact of food contamination in Nigeria has been underestimated due to underreporting and a lack of conclusive links between food contamination and illness or death. Consequently, efforts are required to improve food safety by avoiding the hazards of chemical mixtures and their effects. An all-inclusive analysis of food products, including milk, is needed to prevent the occurrence of prohibited additives, and their associated health effects. Our objective is to identify and quantify cyromazine and its metabolite melamine in local and imported evaporated and infant milk samples, and to assess the risk to the Nigerian population from melamine and cyromazine exposure through milk consumption alone, excluding all other food items.

Materials and methods
Equipment, chemicals, and reagents

   The used equipment in the study includes a sonicator bath (Elmasonic PH/20, BioLogics Inc., Manassas, USA), a vortex mixer (Vortex-Genie^® 2 mixer, Sigma-Aldrich, USA), and a centrifuge (Centrifuge-34b18, Thermo Scientific, Swedesboro, USA). Waterbath (Memmerth WTB50, Schwabach, Germany), Nalgene LDPE sample bags (Thermo Scientific, Massachusetts, USA), and syringe filters (Acrodisc syringe filters, GHP membrane, 25 mm by 0.45 μm, Sigma-Aldrich, St. Louis, MO, USA). The reagents were melamine 99% (Sigma-Aldrich, Missouri. USA.), cyromazine, HPLC grade MeOH, CH₃CN, and NH₄OH solution (Merck Life Scientific Industries, Darmstadt, Germany), HCOOH (90%) and CCl₃COOH were purchased from M and B (May and Baker; England). Water was purified using the Milli-Q system, and SPE cartridges (Agilent Technology, California, USA).

Standards            
   Standard solutions (5,000 µg/ml) of the analytes were prepared by weighing and dissolving 5 mg standard in 5 ml HCOOH:H₂O (50:50 v/v) and preserved at 4 ^oC. Working solutions were provided as standards by appropriate dilution when required.
Milk samples
   Domestic and imported evaporated and infant milk samples for infants aged 0-12 months (totalling 182) were purchased from supermarkets and stores in Ogun state, Nigeria. These samples were manufactured between August 2021 and December 2022. On arrival in the laboratory, the samples were wrapped and labelled in sample bags and stored in the freezer until extracted to maintain their integrity.
Sample extraction and cleanup
   The samples were extracted and cleaned with a modified method previously described by Srimathi et al. (2017). A 3 g sample was treated with 15 ml of 1% CCl₃COOH and 5 ml of CH₃CN in a centrifuge tube. The tubes were capped, sonicated for 10 min, and placed on a vertical shaker for another 10 min. The tubes were centrifuged for 10 min at 4,000 rpm. The supernatant was transferred to a 25 ml volumetric flask and brought to volume with 1% CCl₃COOH prior to being filtered on acid-pretreated paper. In a glass tube, 5 ml of filtrate was placed, and 5 ml of H₂O was added. A vortex mixer was applied to thoroughly mix it. The 10 ml sample solution (equivalent to 0.4 g) was loaded onto a C₁₈ SPE cartridge that had previously been preconditioned with 3 and 5 ml MeOH and H₂O, respectively. The entire effluent was discarded after a two-step wash with 3 ml of H₂O, and the cartridge was dried for 5 min. Finally, 6 ml of 5% NH₄OH in MeOH was used to elute the column. In a 45 °C water bath, the eluent was dried before filtering through a 0.45µm syringe filter, and the residue was re-suspended in a constant volume of 1 ml of the mobile phase.
Chromatographic conditions and method validation
   The extracts were quantified using an Agilent HPLC (Agilent Technology 1,200 series) equipped with a Zorbax Eclipse plus C₁₈ column (150×4.6 mm, 5 m particle size) from Agilent Technologies, Germany.  Analysis was performed in the gradient mode at a flow rate of 0.5 ml/min using double-distilled water and acetonitrile (30:70) at an injection volume of 5 μl. Analytes were measured at 214 nm with a DAD. Retention time and peak area of the chromatograms of standards were utilized for analyte quantification. The method was validated as described by Oyedeji et al. (2021).
Estimation of dietary exposure and hazard index (HI)
   A real-life scenario is presented by exposure to chemical mixtures, facilitating the assessment of cumulative risk. The exposure assessment of an individual to melamine and cyromazine depends on the availability of food consumption and anthropometric data. Dietary exposure results from the product of the contaminant concentration  in food with the amount of food consumed daily, and is usually normalised to body weight for a given individual. In this study, exposure assessment was computed according to the equation ofOyedeji et al. (2020) with slight modifications as follows:

    Eq.1

where, D_E: daily co-exposure of the two additives (μg/kg/day); Cc : concentration of the two additives (μg/kg); D_c : daily consumption of milk (g/day);BD_av  : average body weight (kg). The estimated daily milk consumption for different age categories (infants, males, and females) was obtained from the Centre for Disease Control (CDC) in the United States (CDC, 2022). Milk consumption in Nigeria for adults stands at 8 L (7,440 g) per day (Izuaka, 2021). The average body weights (97^th percentile) of infants and adults in Nigeria are 10 and 70 kg, respectively (CDC, 2001; Oyedeji et al., 2020).
The Hazard Index (HI) values were computed using the following equation:

                                                                 Eq. 2

where ADI is the acceptable daily intake (200 µg/kg body weight) for the two additives; HI<1=risk is considered acceptable; 1≤HI≤10=risk exists but does not require immediate action; HI>10=risk is considered unacceptable (Oyedeji et al., 2020).
Statistical analysis-Descriptive statistics
   For data entry and descriptive statistics, Microsoft Excel was used, and statistical analysis was performed using Sigma Plot version 14 (Systat Software, USA).

Results
   Selectivity, precision, accuracy, linearity, detection and quantification limits were determined using calibration curves obtained from least-squares linear regression analysis of peak area versus analyte concentration. The parameters of regression coefficients and linearity for melamine and cyromazine and external calibration are illustrated in Table 1.
   For the studied concentration range, the linearity was greater than 0.98. The Limits of Detection (LOD) and Limits of Quantification (LOQ) are presented in Table 1. Recovery ranged between 99 and 102% for the two analytes at different spike levels, as demonstrated in Table 2.

Centre for Disease Control and Prevention (CDC). (2001). Data table of infant weight-for-age charts. URL: https://www.cdc.gov/ growthcharts/html_charts/wtageinf.htm. Accessed 20 September 2022.

Centre for Disease Control and Prevention (CDC). (2022). How much and how often to feed infant formula. URL: https://www.cdc.gov/nutrition/infantandtoddlernutrition/formula-feeding/how-much-how-often.html#:~:text=You%20can%20start%20by%20offering,12%20times%20in%2024%20hours. Accessed 20 September 2022.

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