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Determination Of Melamine In Aquatic Products By SPE-HPLC

Melamine, a nitrogen-containing heterocyclic organic compound, can damage the urinary system, including the kidneys, in the human body. Due to the defects in the traditional protein content detection method of food and feed, illegal merchants often add melamine to aquatic feed to falsify the protein index, leading to melamine residues in aquatic products and posing a serious threat to food safety. At present, there is an urgent need for a rapid, accurate, and reliable detection method for melamine in aquatic products. The solid-phase extraction-high-performance liquid chromatography (SPE-HPLC) method established by relevant researchers has become an effective analytical method for detecting melamine in aquatic products, thanks to its high separation efficiency, good purification efficiency, and accurate quantitative results.

This article will detail the principles, experimental procedures, key parameters, and application effects of this method, providing a reference for the quality supervision and detection of aquatic products.

Basic Principles Of Determination Of Melamine In Aquatic Products By SPE-HPLC

SPE-HPLC combines the purification advantages of solid-phase extraction with the separation and detection advantages of high-performance liquid chromatography, forming a complete analytical process: “extraction-purification-separation-quantification”.
  • Solid Phase Extraction (SPE): Adopting a mixed cation exchange solid phase extraction column, based on the dual retention mechanism of ion exchange and reversed-phase adsorption, it can effectively remove interfering substances such as proteins and lipids in aquatic product samples, and selectively retain melamine, realizing the purification and enrichment of the target substance.
  • High Performance Liquid Chromatography (HPLC): Using a C18 chromatographic column for separation, with ion-pair reagent buffer and acetonitrile as the mobile phase, the ultraviolet detector is used to detect at a specific wavelength. Based on the retention time of melamine, qualitative analysis is performed, and quantitative analysis is carried out using the external standard method by establishing a linear relationship between peak area and mass concentration.
The combination of the two technologies compensates for the deficiency that direct HPLC detection is easily compromised by the complex matrix of aquatic products, thereby greatly improving the accuracy and sensitivity of melamine detection.

Experimental Materials and Reagents for SPE-HPLC

The selection of instruments and reagents is the basis for ensuring the smooth progress of the detection experiment. The main instruments and key reagents required for the SPE-HPLC method for determining melamine in aquatic products are as follows:

Core Detection Instruments

The experiment needs to be equipped with high-precision analytical instruments and sample pretreatment equipment, including an American Varian Prostar 230 high-performance liquid chromatograph (with ultraviolet detector), a German Sigma 3K30 centrifuge, a high-speed homogenizer, an American solid phase extractor, a Tianjin MTN2800W nitrogen blower, a Kunshan KQ-250E ultrasonic cleaner, etc. All instruments must be calibrated and maintained regularly to ensure accurate test results.

Key Reagents and Columns

  • Chromatographic reagents: Chromatographically pure methanol, acetonitrile, analytical grade ammonia water (25%~28%), 1% trichloroacetic acid solution, 2% lead acetate solution, 5% ammonia methanol solution, 20% methanol aqueous solution, etc., all reagents need to meet the chromatographic analysis requirements to avoid background interference.
  • Ion-pair reagent buffer: Prepare by weighing 2.16 g sodium heptanesulfonate and 2.10 g citric acid, dissolving in water, adjusting the pH to 3.0, and bringing to a constant volume; this is the key mobile-phase component for improving the separation of melamine.
  • Solid phase extraction column and filter membrane: Aijie mixed cation exchange solid phase extraction column (60 mg, 3 mL), 0.45 μm organic phase filter membrane, which is the core material for sample purification.
  • Standard solution: Melamine standard product (Sigma) is used to prepare a standard stock solution (1.0 mg/mL), standard intermediate solution (100 μg/mL), and standard working solution (1, 5, 10, 25, 50 μg/mL) with 20% methanol solution, which is used for drawing a standard curve and quantitative calculation.

Determination Of Melamine In Aquatic Products By SPE-HPLC Experimental Procedure

The experimental operation is divided into three key steps: sample pretreatment (extraction + purification), setting chromatographic analysis conditions, and sample determination. Each step has strict operational specifications to ensure experiment repeatability.

Sample Pretreatment: Extraction and Purification

Pretreatment is the most critical step in detecting melamine powder in aquatic products, as it directly affects purification efficiency and detection accuracy.
  1. Extraction: Weigh 5 g of aquatic product sample (accurate to 0.01 g) into a 100 mL polytetrafluoroethylene centrifuge tube, add 48 mL of 1% trichloroacetic acid solution and 2 mL of 2% lead acetate solution accurately, homogenize at 11000 r/min for 30 s with a high-speed homogenizer, shake on a shaker for 30 min, and centrifuge at 5000 r/min for 10 min to obtain the supernatant. Trichloroacetic acid can precipitate proteins, and lead acetate can remove lipid interference, enabling effective extraction of melamine from the complex matrix.
  2. Purification: First activate the mixed cation exchange solid phase extraction column with 3 mL methanol and 3 mL water in turn; accurately absorb 10 mL of supernatant and pass through the column at a speed of less than 1 mL/min; wash the column with 3 mL water and 3 mL methanol successively, pump to near dryness; elute with 3 mL of 5% ammonia methanol solution, collect the eluent, blow dry with nitrogen at 50 ℃, dissolve the residue with 1 mL of 20% methanol solution, vortex and mix well, and filter with 0.45 μm organic phase filter membrane to obtain the sample solution to be tested.

Setting of Chromatographic Analysis Conditions

Optimized chromatographic conditions ensure ideal peak shape, separation efficiency, and retention time for melamine. The key parameters are set as follows:
  • Chromatographic column: Varian Polaris C18 column (5 μm, 4.6 mm×250 mm);
  • Mobile phase: Ion-pair reagent buffer: acetonitrile = 90:10 (v/v);
  • Flow rate: 1.0 mL/min; Column temperature: 30 ℃; Detection wavelength: 240 nm;
  • Injection volume: 20 μL.
Under the above conditions, the melamine target peak appears at 7.6 min, and all impurity peaks flow out within 10 min, with a good separation effect and no peak overlapping.

Sample Determination and Quantitative Calculation

Inject the prepared standard working solution and the sample solution to be tested into the high-performance liquid chromatograph; then record the peak area and retention time. Qualitatively judge whether the sample contains melamine according to the retention time; take the mass concentration of the standard working solution as the abscissa and the peak area as the ordinate to draw a standard curve, and calculate the melamine content in the aquatic product sample according to the peak area of the sample and the linear regression equation.

Application Value and Significance of the Determination Of Melamine In Aquatic Products By SPE-HPLC

Before the establishment of this method, China had issued detection standards for melamine in feed and dairy products, but there was no corresponding national standard for the determination of melamine in animal tissues such as aquatic products. The SPE-HPLC method for determining melamine in aquatic products fills this technical gap, and has the advantages of simple operation, low cost and easy popularization, which is suitable for the routine detection of melamine in aquatic products by agricultural product quality supervision and detection institutions at all levels.

In practical application, this method can quickly and accurately detect melamine residues in various aquatic products such as fish, shrimp, and crab, provide technical support for the quality supervision of the aquatic product market, effectively curb the illegal practice of adding melamine to aquatic feed, and protect consumer health. At the same time, the method can also serve as a reference for detecting melamine in other animal tissues and has significant potential for promotion and application in the field of food safety detection.

conclusion

The solid-phase extraction-high-performance liquid chromatography method established for the determination of melamine powder in aquatic products is a mature, reliable analytical method. By extracting trichloroacetic acid solution, purifying a mixed cation-exchange solid-phase extraction column, and separating and detecting melamine by HPLC under optimized conditions, the qualitative and quantitative analysis of melamine in aquatic products can be efficiently performed. The method exhibits good linearity, an appropriate detection limit, high accuracy, and good precision, and is suitable for the routine detection of melamine in aquatic products.

As people’s attention to food safety continues to grow, the detection technology for melamine in aquatic products will continue to advance. Based on this method, combining it with mass spectrometry technology can further improve detection sensitivity and qualitative accuracy, and provide more powerful technical support for the comprehensive supervision of melamine residues in aquatic products. In the future, rapid, miniaturized, and high-throughput detection methods will be the trend of development, and it is of great significance to build a more comprehensive aquatic product safety detection system.

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