INTRODUCTION:

The World Health Organization (WHO) defines counterfeit drugs as unreal products produced by illegal methods without making any concern for patient health and safety¹. While a counterfeit food is one that is manufactured without consideration for protection, consistency, or efficiency; it may contain the correct ingredients, but its origin is unknown; it may be lacking certain important ingredients, or it may have been replaced by less expensive ones. Actually, food that is counterfeit can be dangerous to your health^2-5. Milk has long been known as a food that contains a high proportion of dietary ingredients, making it ideal for infants, teenagers, and adults.

In fact, adulteration of milk is, unfortunately, a major problem all over the world. As, Melamine, a nitrogen-rich compound, is used to artificially boost the protein content of milk and dairy products⁶. The chemical structure of melamine is shown in Fig. 1. Melamine is a trimer of cyanamide, with a 1,3,5-triazine skeleton. It has a role as a xenobiotic metabolite. It derives from a cyanamide. It is a conjugate base of melamine^7,8. As a matter of fact, melamine is sometimes added illegally to food products in order to increase the content of protein, Standardized tests, such as the Dumas and Kjeldahl tests, are appreciated Protein by measuring the nitrogen content, so that it can be misled by adding nitrogen-rich compounds such as melamine⁹. The World Health Organization’s International Agency for Research on Cancer has concluded that there is ample evidence for the carcinogenicity of melamine in laboratory animals when it induces bladder calculi. In humans, there is insufficient proof of carcinogenicity^10,11. In fact, more than 50,000 babies were hospitalized in China in 2008, with six deaths, after consuming melamine-tainted milk powdered baby food². Melamine and cyanuric acid absorbed into the bloodstream accumulate and form large numbers of round, yellow crystals, which block and destroy renal cells, according to the Food and Drugs Administration (FDA). Furthermore, melamine has a Lethal Dose (LD50) of more than 3g\kg of body weight, which is comparable to standard table salt. The maximum melamine limit has been set at 1mg by the Codex Alimentarius⁶.

Figure 1: Structure of melamine

A literature survey showed several types of research for the determination of melamine in milk with different methods¹², where in China, HPLC-DAD method was used for the determination of melamine in milk¹³. Moreover, pasteurized milk, powder milk, infant milk, cheese and coffee mate were tested for the determination of melamine by using HPLC method^14–19. Furthermore, national monitoring and surveillance data showed the presence of melamine in dietary products in New Zeland, Canada, America, and the United Kingdom in a study that used LC-MS/MS²⁰. GC-MS methods were used for the determination of melamine as well as cyanuric acid in dietary supplements^21-27. In addition, a developed method was used for detecting the milk contaminants like toxicity of melamine and classified it based on data spectroscopy and optical fiber sensor²⁸. In one hand, there is a lot of method for the determination of melamine with the nanoparticle technique^29-31. On the other hand, Infra-Red spectroscopy is one of the most important methods that is used for the determination of melamine in milk^32-34. The current study presents a new Infra-Red method for the quantitative determination of melamine in powdered milk. The developed method was applied to all powdered milk available in the local market and compared to standard milk and standard melamine. In this respect transmission FT-IR spectroscopy has ability to analyze samples with high sensitivity, specificity and capability to serve as ‘‘fingerprint’’ technique^34,35,36. The developed method is rapid, simple, and less expensive in comparison with HPLC methods. The developed method can easily detect the presence of melamine in children’s dry milk.

MATERIALS AND METHODS:

Apparatus and Software:

Fourier transforms Infra-Red (form FT/IR 4200) Jasco- japan, connected to a personal computer loaded with Spectra Analysis.

Materials:

Standard Melamine [Purity of 99.9%, analytical grade (Sigma-Aldrich, St. Louis, Mo, USA)]. Standard Milk Obtained from national supply laboratories, Potassium Bromide [Purity of 99.7%, analytical grade (Sigma-Aldrich, St. Louis, Mo, USA)].

Sample used:

Sky milk (batch number: BT 2301213), Mark milk (BT 297), Halibna (BT 905), Halibna (BT 905), Jena (BT 521), Verde campo (BT 229), Nido (BT 908661), Alpen (BT 40411677), Kikose (BT 908661), Brinto milk (BT 220), Nan (BT 814557), Anchor (BT1819086), Rinolac (BT 290065).

Samples preparation:

The solid preparing method was followed by the disk method, where the potassium bromide was dried at 105° C for an hour. No pre-treatment was required for the samples. All milk samples were weighed precisely, then KBr was powdered and smoothed with a mortar and pestle to reduce the particle size, then 2 mg of the milk sample was mixed with 200mg KBr at the ratio (KBr 100:1 Sample) %, the sample was mixed well to ensure the homogeneity of the mixture, then the powdered sample mixture was pressed using a template for the device, a transparent disk free of scratches was obtained (using a press), and then it was placed with an infrared spectroscopy device to be measured.

Procedures:

Several analyzes were conducted for several samples of milk powder for adults and infants, some of which were nationally bottled and some imported from abroad. Measurements were performed with a separation resolution of 1-4cm in the range of 400-4000 cm^-1 using potassium bromide. The samples and standards (milk-melamine) were analyzed in the form of potassium bromide granules to record their individual FT-IR spectra. The infrared spectrum of melamine is characterized by absorption bands in the regions 3000-3500 cm^-1 and 1300-1700 cm^-1 due to the stretching and bending of the existing amine groups. The characteristic absorption bands are listed with their interpretation in Tab. 1. The absorption band at 814 cm^-1 characterizes the out-of-plane bending pattern of the 1,3,5 triazine ring of melamine. This band was absent in the milk powder spectrum and therefore this band was selected to draw the standard curve of melamine.

Table 1. The interpretation of melamine bands

Wave number (cm^-1)	Interpretation
3420-3470	NH2 stretching
3330	NH2 asymmetric stretching
3130	NH2 symmetric stretching
1655	NH2 deformation
1560	Triazine stretching
1440	Triazine semicircle stretching
1027	C-N stretching
814	Triazine out of plane binding

Table 2. Standard samples used in melamine calibration curve

Sample No	Milk weight (g)	Melamine weight (g)	Melamine concentration in the sample (%)
M1	0.15	0	0
M2	0.1	0.01	0.09
M3	0.6	0.01	1.6
M4	0. 3	0.01	3.2
M5	0.14	0.01	6.7
M6	0.1	0.05	33.3
M7	0.08	0.07	46.7
M8	0.06	0.09	60
M9	0.05	0.1	66.6

Validation of the method:

A series of 9 calibration standards covering the range of 0.09–33.3% MEL were mixed with KBr to make a pellet of 100 mg by total weight to ensure homogeneity and the required ratio of the constituents. Validation of IR spectroscopy was evaluated by standard addition of samples analyzed in triplicate.

Synthetic milk samples were prepared by spiking known amount of MEL separately to make the concentrations of each sample of 10, 15, 25% for infant powder milk.

The LOD and limit of quantification (LOQ) of the proposed method were measured by selecting the band area at low concentrations of standards, until the component-related signal disappeared. The LOD and LOQ were calculated by the following formulae:

LOD= 3 x SD x -------

LOQ= 10 x SD x -------

Where SD is the standard deviation, C is the concentration of analyte and M is the mean band area.

The precision of the method is expressed through the relative standard deviation (RSD%) values of the results according to the proposed method. The precision was determined on the same day (intra-day) at a rate of three measurements per day, immediately after preparation, then at an interval of three hours, and with a frequency of (n = 5), and during three consecutive days (inter-day), at a rate of one measurement every day, with a frequency of (n = 5), and we present the results in the Tab .3.

Table 3. Result of the precision

The repeatability (intra-day)
P C %	Measured concentration %
P C %	1	RSD%	2	RSD%	3	RSD%
6	6.08	0.13	6.06	0.23	6.05	1.85
8	8.18	0.24	8.18	0.17	7.95	1.41
10	10.16	0.33	10.16	0.14	9.90	1.38
The repeatability (inter-day)
P C %	Measured concentration %
P C %	Day1	RSD%	Day2	RSD%	Day3	RSD%
6	6.06	0.23	6.06	0.33	6.08	0.14
8	8.18	0.17	8.08	0.18	8.24	0.27
10	10.16	0.14	10.20	0.14	10.04	0.13
PC: prepared concentration

RESULT AND DISCUSSION:

The detection principle is based on the difference in the infrared spectroscopy of melamine and milk and on the presence of a non-interfering specific absorption peak at 814 cm^-1 for melamine. Fig. 2 shows IR spectrum of standard melamine.

Figure 2: IR spectrum of standard melamine

Initially, a standard curve was drawn that correlates the concentration of melamine in milk with the intensity of absorption at 814 cm^-1, and Tab. 2 shows the samples prepared for this purpose. The standard curve shape of melamine is shown in fig. 3, and as the figure shows, the relationship between melamine concentration and the absorption intensity is linear. Therefore, the percentage of melamine in adulterated milk can be determined through the infrared spectrum of the milk sample using the intensity of absorption at 814 cm^-1 through the following relationship:

(0.025 + Y)

X% = ------------------------------ X 100

(0.049)

Figure 3: Calibration curve of standard melamine

Before the method was applied to the detection of MEL in real samples, three different samples infant powder were spiked with MEL concentrations of 10,15 and 25 % (with three similar measurements) to determine the recovery and accuracy of the method, the results of which are shown in Table 4. The relative recoveries ranged from 99.50–100.60% with relative standard deviations (RSD) from 1.38–1.90%.

Table 4. Result of the determination of melamine in powder milk

Added%	Found%	Recovery%	RSD n=3%
10	9.92	99.50	1.90
15	14.97	99.66	1.49
25	25.05	100.60	1.38

A standard at the levels that produced a signal distinguishable from the baseline noise was used to determine the LOD based on a signal to noise ratio of 3. The LOD and LOQ were 0.009% and 0.09%, corresponding to 90 and 900 mg.kg^-1, respectively.

The results showed a good precision of the applied method, as on the same day the relative standard deviation value did not exceed 1.85%, while during three consecutive days the relative standard deviation value did not exceed 0.33%.

Twelve samples were analyzed and their infrared spectra were recorded. Fig. 4 shows their spectra. The infrared spectra show that there is no melamine in these samples and the results are listed in Tab. 5.

Figure.4 Infrared spectra of milk samples

CONCLUSION:

A simple, cost-effective and environmentally friendly analytical method that is used to detect the presence of melamine in powder milk by infrared spectroscopy. In this paper, melamine standard was prepared and its infrared spectrum was read, and twelve local samples of infant formula were analyzed. And it appeared in all samples that they did not contain melamine (negative) after reading their spectra. A small portion of what may be adulterated with melamine was analyzed. The next work will cover a larger number of milk samples of different origin, production and packaging.

CONFLICT OF INTEREST:

The authors declare that they have no conflicts of interest.

ACKNOWLEDGMENTS:

The current study was supported by Al-Watanyia Private University.

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Received on 27.03.2024 Revised on 13.09.2024

Accepted on 24.12.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):656-660.

DOI: 10.52711/0974-360X.2025.00097

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sample	Sky milk	Mark milk	Halibna	Jena	Verde cambo	Nido	Alpen
Melamine	-	-	-	-	-	-	-
Sample	Alpen	Kikose	Brinto Milk	Nan	Anchor	Rinolac
Melamine	-	-	-	-	-	-
(-): negative.