September 13th, 2008
Melamine Sample Preparation and Latest LC-MS Detection: ASB Hydrophilic Column
Sample Preparation and Latest LC-MS Detection Method for Melamine Developed by Beijing Aijieer Technology Co., Ltd.: Sample Preparation and Latest Detection Method of ASB Hydrophilic Column Melamine Zhang Junyan, He Lvxing (Beijing Aijieer Technology Co., Ltd., Beijing, 100085)
Abstract Melamine is an important chemical material. It is commonly used in the manufacture of melamine resin. It is a commonly used fireproof material in the construction industry. It was originally irrelevant to the food and feed industries, but several incidents in the United States caused the death of feed pets. connect together. After investigation, it was found that these imported feeds contained a certain concentration of melamine. In response, the U.S. Food and Drug Administration (FDA) required feed manufacturers to provide melamine test reports. Therefore, the melamine incident also caused an explosion of detection methods in the field of analysis. , Acer Technology Co., Ltd. has a high sensitivity, quickly developed a superior detection method, this article will be discussed in detail.
Keywords melamine, sample preparation, LC-MS
1 Introduction The melamine incident became a hot topic in the society. In March 2007, the United States recalled a large number of melamine-contaminated pet feeds. This was caused by pet food killing cats and dogs. According to incomplete statistics, there are tens of thousands of pets in the United States alone who have died from the consumption of toxic feed in North America. There are numerous complaints. U.S. Food and Drug Administration survey shows that the urine crystals of recovered pet foods and dead animals Melamine was found in kidney cells. The researchers also found that there was a higher concentration of melamine in the wheat gluten supplements used to recover pet foods. Although no reports of animal deaths or adverse reactions have been reported in China, the toxicity of melamine is somewhat controversial. However, melamine is not a feed material and is not a feed additive that the country allows. Some illicit manufacturers add melamine mainly to increase the apparent protein content of the product, and melamine is widely added to starch, gluten, and protein powder, resulting in not only feed manufacturers, but also other food factories that require melamine detection. To ensure the safety of their products.
In this paper, the solid phase extraction method was used to pretreat the sample and compare the different detection methods, including the FDA published detection method [1] on the analysis of melamine.
Melamine (melamine) referred to as triamine, scientific name trinitrotriazine, alias melamine, cyanuramide, triamide, molecular formula: C3N6H6, C3N3 (NH2) 3 . Molecular weight: 126.12, is an important nitrogen heterocyclic organic chemical raw materials [2]. Melamine is weakly alkaline and can react with various acids to form melamine salt. In a strong acid or strong alkali solution, melamine is hydrolyzed and the amine group is gradually replaced by a hydroxy group to produce cyanuric acid diamide, cyanuric acid monoamide and cyanuric acid. Melamine reacts with aldehydes to form addition compounds. The melamine is reacted with aldehydes to form a resin. The melamine resin is a versatile material that is fire-resistant, heat-resistant, and highly stable. It is used in the production of plastics, kitchen utensils, fire-retardant fibers, commercial filters, glues, and flame retardants. Some Asian countries are also used to make fertilizers.
2 Materials and reagents
2.1 Instruments and Conditions
Agilent 1100 High Performance Liquid Chromatograph (Agilent, USA); Diode Array Detector (DAD), Detection Wavelength 240 nm, Column Temperature: 40°C.
(1) Agela VenusilTM ASB C18 (4.6×250 mm); Buffer: 10 mM citric acid, 10 mM sodium heptane sulfonate; Mobile phase: Buffer solution: Acetonitrile=85:15; Flow rate: 1.0 mL/min.
(2) Agela VenusilTM ASB C8 (4.6×250 mm); mobile phase: buffer: acetonitrile=85:15; buffer: 10 mM citric acid, 10 mM sodium octane sulfonate, adjusted to pH 3.0; flow rate: 1.0 mL/min ;
Ion Exchange Solid Phase Extraction Column Agela ClearnertTM PCX (Beijing Aijieer Technology Co., Ltd.)
2.2 Reagents and samples of pet feed samples (provided by the Feed Supply Center of the Ministry of Agriculture); methanol and acetonitrile were provided by Beijing Aijieer Technology Co., Ltd.; ammonia water, lead acetate, and trichloroacetic acid were purchased from Beijing Chemical Reagent Company; melamine standards, Citric acid, sodium octane sulfonate (Sigma); methanol was chromatographically pure, others were chemically pure.
3 experimental methods
3.1 Sample Preparation Methods
(1) Standard sample preparation:
Take 50mg of melamine standard, dissolve to volume with 20% methanol to 50mL to get 1000ppm standard solution. When using, dilute to the desired concentration with extract (0.1% trichloroacetic acid).
(2) Extraction:
Weigh 5g of feed sample, add 50ml of 0.1% trichloroacetic acid extract, mix thoroughly, add 2ml of 2% lead acetate solution and sonicate for 20min. Then take a portion of the solution and transfer it to a 10 mL centrifuge tube. Centrifuge at 8000 rpm/min for 10 min. Take 3 mL of the supernatant and mix the cation exchange cartridge (PCX).
(3) Purification (PCX cartridge, 60mg/3mL):
a) Activation and balance: 3mL methanol, 3mL water
b) Loading: Add 3mL extract
c) Rinse: 3 mL of water; 3 mL of methanol; discard the eluent and drain the cartridge.
d) Elution: elution with 5 mL of 5% ammoniated methanol (v/v). (Formulation of 5% ammoniated methanol: 5 mL ammonia + 95 mL methanol).
e) Concentrate at 50°C, blow dry with nitrogen, 20% methanol/water to 2 mL, HPLC analysis or post-derivatization GC/MS analysis.
3.2 HPLC detection method
3.2.1 Melamine HPLC-UV detection method
Melamine is a strongly polar compound, and it is poorly retained on traditional reversed-phase C18 columns. It requires ion chromatography reagent chromatography for good retention and separation, according to the US Food and Drug Administration (FDA) melamine detection method and China. The melamine detection method announced by the Ministry of Agriculture, using Agela ASB Series Hydrophilic Chromatography Columns, can achieve a good separation effect. The analysis chromatograms are as follows:
Figure 2 Spectra of melamine separation on a Venusil ASB column
(a) Column: Venusil ASB C8 4.6 x 250 mm; standard: FDA method; mobile phase: buffer: acetonitrile = 85:15; buffer: 10 mM citric acid, 10 mM sodium octane sulfonate, pH adjusted to 3.0; : 1.0 mL/min; column temperature: 40 oC; wavelength: 240 nm
(b) Column: Venusil ASB-C18 4.6 x 250 mm; Standard: Standard method of the Chinese Ministry of Agriculture; Buffer: 10 mM citric acid, 10 mM sodium heptane sulfonate; Mobile phase: Buffer solution: Acetonitrile = 85:15; : 1.0 mL/min; column temperature: 40°C; wavelength: 240 nm
Blank plus level (mg/L) recovery
0.01 116%
0.1 108%
0.5 92%
2 96%
From Table 1 above, it can be seen that the use of PCX column to purify the sample can get a satisfactory recovery rate. This method is more accurate and reliable than the FDA-declared pretreatment method.
3.2.2 Melamine LC-MS detection method
Due to the addition of ion-pairing reagents to the mobile phase in the HPLC-UV method published by the FDA, the use of the LC/MS method was limited; however, without the use of the ion-pair reagent chromatographic method, melamine was poorly retained on the conventional C18 column and could not be obtained. Better separation and quantification [3].
Based on this problem, Agilent Technologies has independently developed a new method using Agela ASB series hydrophilic columns, which can be effectively retained and separated without ion-pairing reagents. Therefore, the mobile phase does not contain ion-pairing reagents and can be used for mass spectrometry detection.
Compared with the “Updated FCC Developmental Melamine Quantitation (HPLC-UV)†published by the FDA in April 2007, this method greatly reduced the minimum detection limit (MSD: 0.5 ppm; UV: 2 ppm) and improved detection sensitivity.
The spectra obtained with this method on ASB-C8 4.6x250mm ASB-C18 4.6x250mm are as follows:
Figure 3 Spectroscopic analysis of melamine by LC-MS method
Buffer: 10 mM NH4AC; mobile phase: Buffer::ACN=95:5; flow rate: 1.0 mL/min; sample volume: sample was first dissolved with 70% ACN to approximately 1 mg/mL, and diluted with ACN to 0.1 mg/mL. mL, 10 uL; Column Temperature: 40°C; Wavelength: 240 nm
4 Results and Discussion
4.1 Cation Exchange Column (PCX)
Melamine is weakly alkaline (weak cationic compound), and the cation exchange column should generally be selected for the purification process. The mixed cation exchange column (PCX) has a sulfonic acid group (-SO3H) bonded to a polar polymer polystyrene/divinylbenzene (PEP) adsorbent with both cation exchange and reversed phase adsorption Mechanism and has the following advantages:
a) The sample can be cleaned with two different solutions (water/buffer solution and organic solvent with a certain pH) to improve the detection sensitivity.
b) Batch repeatability is good.
c) High recovery rate, good reproducibility, and high recovery even if the column runs dry.
4.2 advantages of LC-MS method:
(1) The detection process is simple: Without the addition of ion-pairing reagents, melamine can be well retained and separated, avoiding the complex process of preparing ions to the mobile phase.
(2) Improved detection sensitivity: No ion pair reagents can be used in mass spectrometer detectors, greatly reducing the minimum detection limit (MSD: 0.5ppm; UV: 2ppm).
(3) Reduced detection costs: Without the use of ion-pairing reagents, it is no longer necessary to purchase expensive ion-pairing reagents, which reduces the cost of detection.
(4) Extended column lifetime: Avoids the impact of using ion pair reagents to reduce column lifetime.
(5) The chromatographic column used in this method is versatile: Whether it is the FDA method, the standard method of the Ministry of Agriculture of China and the LC-MS method developed by the company, the use of the Agela ASB series hydrophilic column All can get a good test result, thus providing customers with a variety of options.
References [1] WC Andersen, SB Turnipseed, Determination of Melamine Residues in Catfish Tissue by Triple Quadrupole LC-MS-MS with HILIC Chromatography. Laboratory Information Bulletin. vol. 23, No. 4396, 2007,
[2] DW Hamilton, PA O'Neal, Analytical methods for the quantification of free melamine and cyanuric acid in Nylon 6/6,6 copolymer. J. Sep. Sci., vol.26, pp.510–514, 2003.
[3] JV Sancho, M. Ib Ìa ̃nez, Residue determination of cyromazine and its metabolite melamine in chard samples by ion-pair liquid chromatography coupled to electrospray tandem mass spectrometry. Analytica Chimica Acta. vol.530, pp. 237– 243, 2005.
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