A Rapid and Space-Saving Method for Determining Melamine in Milk Under Organic Solvent–Free Conditions

Melamine is an organic base compound rich in nitrogen and is widely used in plastics (for example, dishware) and adhesives. Recently, there have been reports from several countries of melamine contamination of a variety of food products, including products containing milk and milk-derived ingredients from China (1). According to the World Health Organization (WHO), in China, where adulteration has occurred, water has been added to raw milk to increase its volume. As a result of this dilution the milk has a lower protein concentration. Companies using the milk for further production (for example, of powdered infant formula) normally check the protein level through a test measuring nitrogen content. Adding melamine increases the nitrogen content of the milk and therefore its apparent protein content (2). Significant exposure to melamine may result in bladder and kidney stones, which, in turn, may result in acute or chronic renal failure and, in rare cases, death. Health Canada has decided to adopt the WHO's new recommended tolerable daily intake (TDI) level of 0.2 mg/kg b.w./day and has reassessed its maximum tolerable limit for melamine in food products containing milk to a maximum of 2.5 ppm (3). Milk is a very important and basic food because it is highly nutritious, inexpensive, and readily available. Monitoring the presence of melamine in milk is, therefore, an important means of guaranteeing food safety.

In response to recent expansion in the internal food trade, the development of international standardized methods to determine chemical residues in foods is essential to guarantee equitable international trade in these foods and to ensure food safety for consumers. The optimal harmonized method for routine monitoring of chemicals in foods should be simple, quick, economical, and cause negligible harm to the environment and analyst.

Previous high performance liquid chromatography (HPLC) techniques combined with a selective detection technique for determining melamine in foods including milk have a crucial drawback: All of the methods consume organic solvents in the HPLC and LC–mass spectrometry (MS)-MS mobile phases as well as for extraction and deproteinization in sample preparation (4–6). Thus, the risk associated with these solvents extends beyond direct implications to human health by affecting the ecosystem in which we all reside. Additionally, the disposal of waste organic solvents through incineration has steadily increased over the past decade and costs large amounts of money. Thus, eliminating organic solvents is an important goal in terms of protecting the environment, human health, and the economy (7,8).

This article describes a rapid and inexpensive technique to strictly monitor melamine residue in milk without organic solvent consumption.

Experimental

Reagents and apparatus: A melamine standard and octanesulfonic acid (sodium 1-octanesulfonate) as a reagent for ion-pairing were purchased from Wako Pure Chem. Ltd. (Osaka, Japan). Distilled water was of HPLC grade (Wako). A stock standard solution of melamine was prepared by dissolving the melamine standard in water to a concentration of 100 µg/mL. Working mixed standard solutions of the compound were prepared by diluting the stock solution with water. A handheld ultrasonic homogenizer (model HOM-100, 2-mm i.d. probe, Iwaki Glass Co., Ltd., Funabashi, Japan), a microcentrifuge (Biofuge fresco, Kendo Lab. Products, Hanau, Germany), and a Microcon Ultracel YM-3 (3000 normal molecular weight limit, 0.5-mL maximum volume; Millipore, Bedford, Massachusetts) centrifugal ultrafiltration device were used in the sample preparation. The following five types of nonpolar sorbent columns (250 mm × 4.6 mm, 5-µm d_p) with their guard columns (5 mm × 4.6 mm) for HPLC were used: Column A: Wakosil 5TMS (C1) (Wako); Column B: Mightysil RP-4GP (C4) (Kanto Chemical Co., Inc., Tokyo, Japan); Column C: Lichrospher 60 RP-selectB (C8) (Merck, Darmstadt, Germany); Column D: Mightysil RP-18 (C18) (Kanto); Column E: Inertsil ODS-4 (C18) (GL Science, Tokyo, Japan) (Table I).

Table I: Physical and chemical specifications of the reversed-phase columns* used and chromatographic melamine separation obtained under the HPLC condition ranges examined†

HPLC: The HPLC system included a model PU-980 pump and DG-980-50 degasser (both from Jasco Corp., Tokyo, Japan), as well as a model SPD-M10A VP photodiode array (PDA) detector (Shimadzu Scientific Instruments, Kyoto, Japan). The analytical column was a 250 mm × 4.6 mm, 5-µm d_p Mightysil RP-4GP column (Kanto Chemical Co., Inc., Tokyo). The isocratic mobile phase was 0.05 mol/L octanesulfonic acid and the flow rate was 1.0 mL/min. The PDA detector scanned over the range of 190–300 nm, detecting melamine at 202 nm (a maximum for melamine). The column temperature was maintained at 40 °C. The injection volume was 20 µL.

Sample preparation: An accurately weighed 0.2-mL sample (pasteurized cow's milk) was transferred to a microcentrifuge tube and homogenized with the ultrasonic homogenizer for 30 s with 0.6 mL of water. After homogenization, the capped tube was centrifuged at 12,000g for 5 min. A 100-µL portion of the supernatant liquid was placed into an Ultracel YM-3 centrifugal ultrafiltration device and centrifuged at 5000g for 5 min. The ultrafiltrate was injected into an HPLC system.

Results and Discussion

The main aim of this work was to develop a technique for determining melamine levels in milk without using any organic solvents.

Sample preparation: The present sample preparation procedure realized a space-saving extraction and easy purification of melamine in a short time while completely eliminating the consumption of organic solvent. The procedure resulted in a high recovery rate and high reproducibility with considerable cost savings compared to established methods.

HPLC conditions: To achieve the separation with a 100% aqueous mobile phase and optimize a faster separation, the author tested five types of reversed-phase columns. Table I lists the physical-chemical specifications of the columns. This study used water or 0.05 mol/L octanesulfonic acid as the isocratic aqueous mobile phase and examined column temperatures from 25 to 45 °C, HPLC flow rates ≥0.8 mL/min, and HPLC retention times ≤15 min. Because the HPLC separations were performed serially, the time per run was critical for routine residue monitoring. The short run time also affected the method-development time. The five columns were compared with regard to the separation between melamine and its interfering peaks. The chromatographic separation obtained under the condition ranges examined also are presented in Table I

Columns A, B, and D had difficulty separating melamine and the interfering milk extract throughout the examined condition ranges. No melamine was eluted from Columns C or E. The complete separation of melamine and interference peaks, a symmetrical peak, and a short retention time were achieved with Column B using an isocratic mobile phase of 0.05 mol/L octanesulfonic acid with a flow rate of 1.0 mL/min and a column temperature of 40 °C. This HPLC analysis achieved optimal separation in <5.5 min without the need for a gradient system to improve the separation or pre-column washing after the analysis. The elevated column temperature, 4% carbon content in the column, and ion-pair action of the octanesulfonic acid mobile phase used here were necessary to obtain the findings described. From the data shown in Table I, it is difficult to prove the critical parameter in the column with regard to the retention of melamine and its peak form.

Figure 1 displays typical chromatograms for milk samples obtained following the procedure developed here. The data shown in the figure demonstrate that the present method can provide quantitation and identification of melamine by HPLC, with the PDA detector set at 202 nm (the maximum absorbance for melamine). The present HPLC system easily confirmed the peak identity of the target compound. Melamine was identified in a milk sample by its retention time and absorption spectrum. The melamine spectrum obtained from the sample is practically identical to that of the standard. The present HPLC system did not require the use of mass spectrometry, which is very expensive and is not available in a lot of laboratories for routine analysis, particularly in developing countries.

Figure 1: Chromatograms obtained from the HPLC system for a blank milk sample (upper profile) and a milk sample spiked with melamine (1 µg/mL) (lower profile). The PDA detector was set at 202 nm. Peak 1 = melamine (retention time = 5.1 min).

Method validation: The present method was qualified in terms of analytical performance parameters calculated according to guidance from the United States Food and Drug Administration (9). Table II summarizes the main method validation parameters.

Table II: Method validation data for melamine-fortified milk samples

The accuracy and precision (average recoveries and their relative standard deviations [RSDs]) are well within the method acceptable criteria for residue analysis established by the United Nations Food and Agriculture Organization's and the World Health Organization's Codex Alimentarius Commission: average recoveries of 70–110% with RSDs < 20% when the spiked level for the analyte is 0.01–0.1 µg/mL, and average recoveries of 80–110% with RSDs < 15% when the spiked level is ≥0.1 µg/mL (10). In terms of selectivity, the target spectrum from the milk sample was practically identical to that of the standard. Because of the satisfactory purification and the high absorbance of melamine, the PDA detector was able to detect melamine at trace levels. The time and budget required for the analysis of a single sample were <20 min and approximately US $6.10 as of January 20, 2011, respectively. For sequential analyses, a batch of 24 samples could be analyzed in 3 h.

Residue Monitoring in Commercial Milk

Milk was purchased from a number of convenience stores in Osaka, Japan, and used as real milk samples and analyzed using the proposed method. No samples contained detectable concentrations of melamine. The chromatograms were free from interference.

Conclusions

In conclusion, the present study describes an easy, space-saving, and organic solvent–free method for determining melamine in milk. The entire procedure, which has a negligible impact on the environment and humans (that is, because there is no solvent consumption), has a short analysis time (<20 min/sample), is inexpensive (approximately US $6.10 per sample), and reproducible (with recoveries ≥88.3% with RSDs ≤3.2%). The method validation data demonstrate the reliability of the method. Given these results, this procedure is proposed as an international standardized method for the routine monitoring of melamine in milk.3

Naoto Furusawa is with the Grad-uate School of Human Life Science, Osaka City University, Osaka, Japan. The author can be contacted via e-mail at furusawa@life.osaka-cu.ac.jp

References

(1) Health Canada. Melamine (web page, accessed January 17, 2011). http://www.hc-sc.gc.ca/fn-an/securit/chem-chim/melamine/index-eng.php#who

(2) World Health Organization, Melamine and Cyanuric acid: Toxicity, Preliminary Risk Assessment and Guidance on Levels in Food, 25 September 2008; updated 30 October 2008. http://www.who.int/foodsafety/fs_management/Melamine.pdf

(3) Health Canada. Questions and Answers – Melamine. (web page, accessed January 17, 2011). http://www.hc-sc.gc.ca/fn-an/securit/chem-chim/melamine/qa-melamine-qr-eng.php#8

(4) S. Tittlemier, Health Canada, Background Paper on Methods for the Analysis of Melamine and Related Compounds in Foods and Animal Feeds, Ottawa, Ontario, 1–4 December 2008. http://www.who.int/foodsafety/fs_management/Melamine_1.pdf

(5) Health Canada, Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, LPS-005. Determination of Melamine in Various Milk-Containing Products by Liquid/Liquid Extraction and Cation Exchange Solid Phase Extraction to Prepare Samples, and Electrospray Positive Ionization Liquid Chromatography Tandem Mass Spectrometry to Quantitate Melamine. Ottawa, Ontario, November 2008. http://www.hc-sc.gc.ca/fn-an/alt_formats/hpfb-dgpsa/pdf/pubs/melamine_milk-lait-eng.pdf

(6) M. Rambla-Alegre, J. Peris-Vicente, S. Marco-Peiró, B. Beltrán-Martinavarro, and J. Esteve-Romero, Talanta 81, 894–900 (2010).

(7) P.T. Anastas and J.C. Warner (Eds), Green Chemistry: Theory and Practice (Oxford University Press, Oxford, United Kingdom, 1998).

(8) T. Yoshimura, T. Nishinomiya, Y. Homda, and M. Murabayashi, Green Chemistry – Aim for the Zero Emission of Chemicals (Sankyo Publishing Co. Ltd. Press, Tokyo, Japan, 2001).

(9) United States Food and Drug Administration, Guidelines for Submitting Samples and Analytical Data for Methods Validation, Rockville, Maryland (1987); http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm123124.htm

(10) United Nations Food and Agriculture Organization's and World Health Organization, Codex Alimentarius. Standard CAC/GL 67, Model Export Certificate for Milk and Milk Products. 2008. http://www.codexalimentarius.net/web/standard_list.do?lang=en