CN102621252A

CN102621252A - Pretreatment method for detecting nitrofuran metabolites in aquatic product

Info

Publication number: CN102621252A
Application number: CN2012100708904A
Authority: CN
Inventors: 陈劲星
Original assignee: FUJIAN CCIC-FAIRREACH FOOD SAFETY TESTING CO LTD
Current assignee: FUJIAN CCIC-FAIRREACH FOOD SAFETY TESTING CO LTD
Priority date: 2012-03-19
Filing date: 2012-03-19
Publication date: 2012-08-01

Abstract

The invention discloses a pretreatment method for detecting nitrofuran metabolites in an aquatic product. The method comprises the following steps of: weighing a sample to be detected, dissolving, derivating, extracting, and performing chromatographic separation. A constant temperature oscillator, a reduced pressure rotary evaporator, a silica gel chromatographic column and a high speed refrigerated centrifuge are used in the method. A sample solution prepared by the method can be directly detected by liquid chromatography-tandem mass spectrometry. Compared with the conventional national standard method, the pretreatment method has the advantages of low cost, high repeatability and the like, is simply and effectively operated, is high in operability, is expected to be popularized and applied to environmental protection industry, commodity inspection, entry-exit inspection and quarantine and the like on a large scale, and is obvious in economic and social benefits.

Description

The pre-treating method that the nitrofuran metabolin detects in the aquatic products

Technical field

The invention belongs to the analytical chemistry field, more specifically relate to the pre-treating method that the nitrofuran metabolin detects in a kind of aquatic products.

Background technology

Itrofurans medicine (Nitrofurans) is the extensive pedigree antibiotic with 5-nitrofuran basic structure of synthetic; They act on the microbial enzyme system; Suppress acetyl coenzyme A; Disturb the metabolism of microorganism carbohydrate, common gram-positive bacteria, negative bacterium are all had effect, mainly be applicable to enterobacterial infection and protozoosis.Itrofurans is to photaesthesia; Metabolism is rapid in vivo; Because former medicine and residual in animal body metabolic product thereof have teratogenesis, mutagenesis and carcinogenic danger, from security consideration, developed country such as America and Europe and China have successively issued the ban that bans use of such veterinary drug.So in the detection of food security, detect the nitrofuran metabolin.After the entry to WTO, antibiotic residual quantity detects one of the important technology index that becomes in world's meat trade and technology barriers in the animal derived food.The aquatic products of China outlet European Union once were detected residues of veterinary drug such as containing furazolidone, nitrofurazone like fishes and shrimps etc., and this has seriously restricted the outlet of China's aquatic products.

Reported a series of detection methods that in samples such as fish, shrimp, shellfish, detect the nitrofuran metabolite in recent years, wherein the report with ELISA is maximum, and has some commercial kits to occur.Yet, the shortcoming of ELISA also clearly, a general kit can only detect a kind of nitrofuran metabolite, and the cost of kit is higher.Therefore, to the detection of nitrofuran metabolite, method in common remains behind 2-nitrobenzaldehyde derivatization and measures with the liquid chromatography-tandem mass spectrometry method in the world at present, and the key of this method is the pretreatment technology of sample.

Summary of the invention

The object of the present invention is to provide the pre-treating method that the nitrofuran metabolin detects in a kind of aquatic products, the testing sample after this method is handled can directly detect with the liquid chromatography-tandem mass spectrometry method.This pre-treating method is compared with existing national standard method, has effective, low, the high repeatability and other advantages of cost simple to operate, has very strong operability.

For realizing above-mentioned purpose, the present invention adopts following technical scheme:

The pre-treating method that the nitrofuran metabolin detects in a kind of aquatic products may further comprise the steps:

(1) takes by weighing 2.00 g (being accurate to 0.01 g) aquatic products in 50 mL centrifuge tubes;

(2) in above-mentioned centrifuge tube, add 0.2 mol/L hydrochloric acid, 15 mL and 0.2wt% o-nitrobenzaldehyde-acetonitrile solution 0.5 mL, fully vortex shakes up, at 37 ℃ of following constant-temperature shaking 14 ~ 16 h;

(3) centrifuge tube is taken out, suitably pH to 7.0～7.5 are regulated with the pH damping fluid in the cooling back;

(4) in centrifuge tube, add 20 mL ethyl acetate, add a cover oscillation extraction 3min, at the centrifugal 5min of 3500r/min, with plastic suction pipe supernatant is drawn in the 150mL heart bottle, 40 ℃ the decompression rotary evaporation is extremely dried down;

(5) with 2 mL acetonitrile water (volume ratio is 1:9) solution and the saturated normal hexane wash-out of 2 mL acetonitriles, eluent takes off layer clear liquid with 0.25 μ m filtering with microporous membrane in the layering of 15000r/min high speed centrifugation, and gained filtrating is liquid to be measured.

Described aquatic products comprise fish, shrimp, crab.

PH damping fluid in the said step (3) is a phosphate buffer.

Extraction solution in the said step (4) is an ethyl acetate, and extraction times is 1 time.

Remarkable advantage of the present invention is:

(1) derivating agent in national standard method dissolves with dimethyl sulfoxide; The present invention can effectively reduce the solvent composition (because acetonitrile can be used as eluant, eluent in the back) in the sample then with the acetonitrile dissolving, helps improving the elute effect of subsequent step.

Adopt phosphate buffer when (2) transferring pH=7-7.5 in the present invention, more help acid adjustment; And national standard method is directly transferred pH with NaOH.

(3) it is concentrated directly to get this layer with high speed centrifugation behind the 20 mL ethyl acetate extractions one time in the flow process; And national standard method extracts 2 times, each consumption 40mL, and the extraction back concentrates after using NaCl solution washing, ethyl acetate layer to cross anhydrous sodium sulfate.Because the object distribution ratio is big, flow operations method simple possible can effectively reduce pre-treatment step, shortens analysis time.

Description of drawings

Fig. 1 is the pre-treating method process flow diagram that the nitrofuran metabolin detects in the aquatic products of the present invention.

Fig. 2 is the MRM spectrogram of the derivative products of Furaxone metabolite (AOZ).

Fig. 3 is the MRM spectrogram of the derivative products of AMOZ (AMOZ).

Fig. 4 is the MRM spectrogram of the derivative products of Cistofuran metabolite (AHD).

Fig. 5 is the MRM spectrogram of the derivative products of Furacilin metabolite (SEM).

Embodiment

Described aquatic products comprise fish, shrimp, crab.

The o-nitrobenzaldehyde compound method of 0.2wt% is following in the said step (2): take by weighing 160 milligrams of o-nitrobenzaldehydes, after specpure acetonitrile dissolving, be settled to 10 milliliters with congener acetonitrile.

PH damping fluid in the said step (3) is that concentration is that 1.0 moles every liter, pH are 7.4 phosphate buffer.

0.25 μ m miillpore filter in the step (5) is the organic phase filter membrane.

Embodiment 1

Following examples combine accompanying drawing to narrate concrete principle of work of the present invention:

The concrete operations flow process of the pre-treating method that the nitrofuran metabolin detects in the said aquatic products of the present invention is as shown in Figure 1.At first be taking by weighing, dissolving of testing sample.At 37 ℃ of following constant-temperature shaking 14 ~ 16 h the nitrofuran metabolin of sample is derived then.Next using 1.0 moles every liter, pH is that 7.4 phosphate buffer is regulated pH value of solution to 7.0～7.5 after deriving, and with ethyl acetate the nitrofuran metabolic product after deriving is extracted then.This operation will make the nitrofuran metabolic product in the sample separate with complicated sample matrix.Be further purified through acetic acid ethyl ester extract with preparative chromatography more at last, obtain directly to advance the product that performance liquid chromatographic column carries out separation detection.

Concrete steps:

(1) takes by weighing 2.00 g (being accurate to 0.01 g) roast eel sample in 50 mL centrifuge tubes;

(2) in above-mentioned centrifuge tube, add 0.2 mol/L hydrochloric acid, 15 mL and 0.2% o-nitrobenzaldehyde solution, 0.5 mL, fully vortex shakes up, at 37 ℃ of following constant-temperature shaking 14 ~ 16 h;

(5) with 2 mL acetonitrile water (1:9) solution and the saturated normal hexane wash-out of 2 mL acetonitriles, eluent takes off layer clear liquid with 0.25 μ m filtering with microporous membrane in the layering of 15000r/min high speed centrifugation, the test that promptly is available on the machine of gained filtrating.

The precision correlation data:

Figure 2012100708904100002DEST_PATH_IMAGE002

Fig. 2 is the MRM spectrogram of the derivative products of Furaxone metabolite (AOZ).Fig. 3 is the MRM spectrogram of the derivative products of AMOZ (AMOZ).Fig. 4 is the MRM spectrogram of the derivative products of Cistofuran metabolite (AHD).Fig. 5 is the MRM spectrogram of the derivative products of Furacilin metabolite (SEM).Can find out that from Fig. 2-Fig. 5 the MRM spectrogram of the derivative products of four kinds of nitrofuran metabolins is all noiseless.

The above is merely preferred embodiment of the present invention, and all equalizations of doing according to claim of the present invention change and modify, and all should belong to covering scope of the present invention.

Claims

1. the pre-treating method that the nitrofuran metabolin detects in the aquatic products, it is characterized in that: described pre-treating method may further comprise the steps:

(1) takes by weighing 2.00 g aquatic products in 50 mL centrifuge tubes;

(5) use mixed solution and the 2 mL acetonitriles saturated normal hexane wash-out of the volume ratio of 2 mL acetonitriles and water as 1:9, eluent takes off layer clear liquid with 0.25 μ m filtering with microporous membrane in the layering of 15000r/min high speed centrifugation, and gained filtrating is liquid to be measured.

2. the pre-treating method that the nitrofuran metabolin detects in the aquatic products according to claim 1, it is characterized in that: described aquatic products comprise fish, shrimp, crab.

3. the pre-treating method that the nitrofuran metabolin detects in the aquatic products according to claim 1, it is characterized in that: the pH damping fluid in the said step (3) is a phosphate buffer.

4. the pre-treating method that the nitrofuran metabolin detects in the aquatic products according to claim 1, it is characterized in that: the extraction solution in the said step (4) is an ethyl acetate, extraction times is 1 time.