test method
Determination of melamine in animal food by gas chromatography-mass spectrometry
On-line detection of melamine content by spectral quadrupole
Determination of residual melamine in feed by ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry
Determination of melamine in feed by RP-HPLC
Determination of melamine in high protein food by high performance liquid chromatography-diode array method
Determination of melamine in feed by high performance liquid chromatography
Determination of melamine in feed by HPLC-quadrupole mass spectrometry
Determination of melamine in pet food by solid phase extraction-high performance liquid chromatography
Analysis of melamine in pet food by liquid chromatography -MSMS
Determination of melamine residues in feed by liquid chromatography-tandem mass spectrometry
Determination of melamine in animal food by gas chromatography-mass spectrometry
Attachment: Example of melamine detection method
Instruments and conditions
High performance liquid chromatograph; Diode array detector with detection wavelength of 240nm and column temperature of 40℃.
( 1)agelavenustiltmasb 18(4.6×250mm)； Buffer: 10mM citric acid, 10mM sodium heptane sulfonate; Mobile phase: buffer solution: acetonitrile = 85:15; Flow rate: 1.0 ml/min.
(2)agelavenustiltmasb 8(4.6×250mm)； Mobile phase: buffer: acetonitrile = 85:15; Buffer: 10mM citric acid, 10mM sodium octane sulfonate, adjusted to pH 3.0, flow rate:1.0ml/min;
Ion exchange solid phase extraction column AgelaClearnertTMPCX
Reagents and samples
Pet feed samples (provided by the Feed Supply Center of the Ministry of Agriculture); Methanol and acetonitrile are provided by Beijing Aigeer Technology Co., Ltd.; Ammonia, lead acetate and trichloroacetic acid are all purchased from Beijing Chemical Reagent Company. Melamine standard, citric acid and sodium octane sulfonate (Sigma); Methanol is chromatographically pure, while others are chemically pure.
experimental method
1, sample pretreatment method
(1) preparation of standard sample:
Take 50 mg of melamine standard, dissolve it in 20% methanol to 50 ml to obtain 1000ppm standard solution, and dilute it to the required concentration with the extract (0. 1% trichloroacetic acid) when using.
(2) Extraction:
Weigh 5g feed sample, add 50m l 0. 1% trichloroacetic acid extract, mix well, add 2mL2% lead acetate solution, and ultrasonic for 20min.
Then transfer part of the solution to a 10mL centrifuge tube, centrifuge at 8000rpm/min 10min, and take 3mL supernatant to a mixed cation exchange column (PCX).
(3) Purification (PCX column, 60mg/3ml):
A) Activation and equilibrium: 3 ml of methanol and 3 ml of water.
B) Loading: adding 3 ml of extractive solution.
C) Washing: 3 ml of water; 3mL methanol; Discard the eluent and drain the column.
D) Elution: Elute 5 ml of 5% ammoniated methanol (v/v). (Preparation of 5% ammoniated methanol: 5mL ammonia water +95mL methanol).
E) concentration: 50℃, dried with nitrogen, 20% methanol/water to 2mL, and then analyzed by HPLC or GC/MS after derivatization.
2. Melamine was put on file.
2. 1 HPLC-UV detection method for melamine
Melamine is a strong polar compound, which is poorly retained on the traditional reversed-phase C 18 chromatographic column. Only by ion pair reagent chromatography can it be well retained and separated. According to the detection method of melamine by the US Food and Drug Administration (FDA) and the melamine detection method published by the Ministry of Agriculture of China, good separation effect can be obtained by using Agela)ASB series hydrophilic chromatographic columns:
(a) chromatographic column: venus ilasb 84.6×250mm；; Standard: FDA method; Mobile phase: buffer: acetonitrile = 85:15; Buffer: 10mM citric acid, 10mM sodium octane sulfonate, adjusted to pH 3.0, flow rate:1.0ml/min; Column temperature: 40oC wavelength: 240 nm
(b) chromatographic column: venus ilasb-c184.6× 250 mm; Standard: the standard method promulgated by the Ministry of Agriculture of China; Buffer: 10mM citric acid, 10mM sodium heptane sulfonate; Mobile phase: buffer solution: acetonitrile = 85:15; Flow:1.0ml/min; Column temperature: 40℃; Wavelength: 240 nm
The recovery rate (mg/L) of blank standard addition is 0.0116% 0.1108% 0.592% 296%.
2.2 detection method of melamine by liquid chromatography-mass spectrometry
In the HPLC-UV method published by FDA, ion pair reagents are added to the mobile phase, which limits the use of LC-MS method. However, if there is no ion pair reagent chromatography, the retention of melamine on the traditional C 18 column is poor, and it cannot be separated and quantified well [3].
Based on this problem, Igel Technology Company independently developed a new method, which can effectively retain and separate reagents without ion pairs by using Agela)ASB series hydrophilic chromatographic columns. Therefore, the mobile phase in this method does not contain ion pair reagents and can be used for mass spectrometry detection.
Compared with "Updating the HPLC-UV of Milla developed by DFCC" published by FDA in April FDA2007, this method greatly reduced the minimum detection limit (MSD:0.5 ppm；; Uv: 2 ppm), which improves the detection sensitivity.
By this method, good spectrograms were obtained in ASB-C84.6× 250 mm and ASB-C 184.6× 250 mm samples respectively.
Buffer:10mmh4ac; Mobile phase: buffer:: ACN = 95: 5; Flow:1.0ml/min; Sampling volume: the sample was dissolved with 70% acetonitrile to about 65438 0 mg/ml, diluted with acetonitrile to 0.65438 0 mg/ml, and injected with 65438±00uL；; Column temperature: 40℃; Wavelength: 240 nm
Results and discussion
1, cation exchange column (PCX)
Melamine is weakly alkaline (weakly cationic compound), so cation exchange column should be generally selected in the purification process. Mixed cation exchange column (PCX) has two mechanisms of cation exchange and reverse adsorption by bonding sulfonic acid group (-SO3H) to polar polymer polystyrene/divinylbenzene (PEP) adsorbent, and has the following advantages:
A) Two different solutions (water/buffer solution with a certain pH value and organic solvent) can be used to make the sample cleaner and improve the detection sensitivity.
B) Good batch repeatability.
C) High recovery rate and good reproducibility, even if the column is dried.
2. Advantages of LC-MS method:
The detection process of (1) is simple: melamine can be well retained and separated without adding ion-pair reagents, thus avoiding the tedious process of preparing ion-pair mobile phase.
(2) detection sensitivity is improved: there is no ion pair reagent, which can be used in mass spectrometry detector, greatly reducing the minimum detection limit (MSD:0.5 ppm；; Ultraviolet: 2ppm).
(3) Reducing the detection cost: No ion-pair reagents are used, and expensive ion-pair reagents are no longer needed to be purchased, thus reducing the detection cost.
(4) Prolonging the service life of the chromatographic column: avoiding the influence of using ions to reduce the service life of the chromatographic column.
(5) The chromatographic column used in this method is universal: regardless of the FDA method, the standard method issued by the Ministry of Agriculture of China, or the LC-MS method developed by our company, good test results can be obtained by using Agela)ASB series hydrophilic chromatographic columns, providing customers with a variety of choices.
A person from the National Food Quality Supervision and Inspection Center said that in the existing national standard milk powder testing, protein, fat, bacteria and other tests are mainly carried out. Melamine is a chemical raw material, which is not allowed to be added to food, so the existing standards will not have corresponding content. In other words, melamine is not a routine test item. Under normal circumstances, few people would think of testing it.