CN103487529A

CN103487529A - Method for detecting residual amount of semi carbazide in flour and flour products by high performance liquid chromatography

Info

Publication number: CN103487529A
Application number: CN201310480062.2A
Authority: CN
Inventors: 宋薇
Original assignee: PONY TEST TECHNOLOGY (TIANJIN) CO LTD
Current assignee: PONY TEST TECHNOLOGY (TIANJIN) CO LTD
Priority date: 2013-10-15
Filing date: 2013-10-15
Publication date: 2014-01-01

Abstract

The invention discloses a method for detecting the residual amount of semi carbazide in flour and flour products by a high performance liquid chromatography. The method comprises the following steps: extracting the semi carbazide in the flour and the flour products by vibration or ultrasonic treatment through a methanol water solution or an acetonitrile water solution; after centrifuging an extracting solution, adopting 9-hydroxylxanthene as a derivation agent to derivate under an acidic condition; separating deviated products by a chromatographic column which takes octadecyl silane as a filling agent; detecting by a fluorescence detector; and quantifying by using an external standard method. The method has the characteristics of accuracy in qualification, simple steps, high accuracy, low detection limit and the like, and is applicable to detecting the residual amount of the semi carbazide in the flour and the flour products.

Description

A kind of method of semicarbazides residual quantity in high effective liquid chromatography for measuring flour and Flour product

Technical field

The present invention relates in the food safety detection detection method of semicarbazides residual quantity in a kind of flour and Flour product, be specifically related to the method for semicarbazides residual quantity in a kind of high effective liquid chromatography for measuring flour and Flour product.

Background technology

Semicarbazides (semicarbazide, SEM) has another name called the carbamyl hydrazine, chemical formula (molecular formula): CH ₅n ₃0, it is a kind of white crystals, easy deliquescence, water-soluble absolute ethyl alcohol and the ether of being insoluble to, for the preparation of the photochromic dyestuff on electrothermal sensitive recording paper, also for medicine, the intermediate of the organic syntheses such as agricultural chemicals, for the production of nitrofurazone, furantoin, Adenaron, the medicines such as prednisolone, hydrocortisone.Also for measuring aldehyde, ketone reagent.

Semicarbazides (semicarbazide in animal derived food, SEM) metabolin usually used as nitrofurazone (nitrofurazone) detects in residue of veterinary drug, it has suspicious mutagenesis and carcinogenicity, and the residual quantity that most countries is defined in animal derived food must not detect.The detection that European Union formulates is limited to 1.0 μ g/kg, and the Positive List System of Japan's implementation in 29 days Mays in 2006 has stipulated that new detection is limited to 0.5 μ g/kg.Recent study finds, semicarbazides source be not limited only to Furacoccid metabolism in animal body and produce, gluten reinforcing agent azodicarbonamide (azodicarbonamide is called for short ADA) hydrolysis and high temperature pyrolysis reaction also produce semicarbazides.Azodicarbonamide can be converted into fast nonvolatile matter biruea (hydrazodicarbonamide) and urazole (urazole) under moistening condition, and under acid condition, the two can slowly generate semicarbazides through heat treated.

In the Flour production process, azodicarbonamide can increase mouthfeel and the chewiness of flour.Flour product is after high temperature, damp and hot processing, and azodicarbonamide wherein can be converted into semicarbazides, long-term edible can the health generation of human body harm.

At present, in China GB2760-2011 " food security national standard food additives Application standard " regulation wheat flour, the maximum use amount of azodicarbonamide is 0.045g/kg.Now country do not have in flour and Flour product semicarbazides coherent detection standard and the requirement of limiting the quantity of.GB2760-2011 " food security national standard food additives Application standard " food industry processing aid list and SN/T2360.25-2009 " import and export food adjuvant inspection procedure the 25th part: food industry processing aid " all do not carry out stipulating to the semicarbazides in food.

In current food, the examination criteria of semicarbazides content has: GB/T22987-2008 " furaltadone in milk and milk powder, nitrofurazone, the mensuration Liquid Chromatography-Tandem Mass Spectrometry of furantoin and Furaxone metabolite residual quantity ", GB/T18932.24-2005 " furaltadone in honey, nitrofurazone, the mensuration Liquid Chromatography-Tandem Mass Spectrometry of furantoin and Furaxone metabolite residual quantity ", No. 1077 bulletin-2-2008 of the Ministry of Agriculture " the mensuration high performance liquid chromatography of itrofurans metabolite residue amount in aquatic products ".

In domestic and foreign literature in wheat flour and its products the semicarbazides detection method be mainly the Liquid Chromatography-Tandem Mass Spectrometry method.Under acid condition, sample is hydrolyzed, add derivative reagent 2-nitrobenzaldehyde solution to be derived, after ethyl acetate extracts, with Liquid Chromatography-Tandem Mass Spectrometry detected (Zhang Xianchen etc. in Ultra Performance Liquid Chromatography-series connection level Four bar MS flour nitrofurazone metabolic product semicarbazides residual quantity; Chinese Journal of Health Laboratory Technology, 2009,19 (4): 715-717).Utilize the Liquid Chromatography-Tandem Mass Spectrometry method detect in wheat flour and its products semicarbazides the residual quantity pre-treatment comparatively complicated, matrix interference is also more serious, and the Liquid Chromatography-Tandem Mass Spectrometry instrument is expensive, method is difficult for universal.

This method has been set up and has been utilized xanthydrol for derivative reagent, and high performance liquid chromatography separates, and fluorescence detector detects the method for semicarbazides residual quantity.When the method is applied to the mensuration of semicarbazides residual quantity in flour and Flour product, pre-treating method is optimized, character in conjunction with flour and Flour product matrix, utilize methanol aqueous solution or acetonitrile solution to extract semicarbazides, simplified the step of pre-treatment, improve the adding proportion of derivative reagent, obviously reduced the consumption of derivative reagent.

The assay method of semicarbazides residual quantity in flour of the present invention and Flour product, have the advantages such as step is simple, accuracy is high, detection limit is low.

Summary of the invention

The technical problem to be solved in the present invention is to select suitable organic solvent to extract the semicarbazides in flour and Flour product, reduces matrix interference, selects suitable derivative reagent simultaneously and optimizes the adding proportion of derivative reagent.

Technical matters of the present invention can be solved by the following technical programs:

1. standard operation solution preparation

With the amino urea standard reserving solution of high concentration, stepwise dilution becomes series standard solution, as 2.00 μ g/mL, and 5.00 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL, 50.0 μ g/mL.Get respectively each concentration standard of 0.5～1.0mL in the 1.5mL sample injection bottle, adding 50-300 μ L concentration is 0.02mol/L9-hydroxyl xanthene derivating agent, and 1.5mol/L hydrochloric acid solution 20-100 μ L is after the derivative 20-30min of lucifuge, to be analyzed under room temperature.

2. sample solution preparation

(1) sample extracts: accurately take 1-5g flour or pulverize after the Flour product sample in the 50mL centrifuge tube, add 10～20mL methanol aqueous solution or acetonitrile solution, concussion or ultrasonic extraction 30～60min; Wherein, methanol aqueous solution concentration is 20%～60%, and acetonitrile solution concentration is 20%～60%.Extract is centrifugal 3～10min under 6000～8000r/min condition, gets supernatant and crosses the organic filter membrane of 0.45 μ m micropore, collects filtrate;

(2) the sample extract is derivative: get above-mentioned filtrate 0.5～1.0mL, adding 50～300 μ L concentration is 0.02mol/L9-hydroxyl xanthene derivating agent, 1.5mol/L hydrochloric acid solution 20～100 μ L, and under room temperature, after the derivative 20～30min of lucifuge, sample liquid is to be analyzed.

3. standard operation solution and sample liquid are analyzed

Standard operation solution after derivative adopts the following testing conditions that separates to separate through high performance liquid chromatography with sample liquid, and fluorescence detector detects, and external standard method is quantitative, in calculation sample semicarbazides residual quantity.

High performance liquid chromatography separates testing conditions:

Instrument: high performance liquid chromatograph, be furnished with fluorescence detector;

Chromatographic column: the chromatographic column that the carbon octadecyl silane is filling agent;

Mobile phase: acetonitrile+0.02mol/L sodium acetate solution (gradient elution, the elution program of optimization is in Table 1);

Flow velocity: 1.0mL/min;

Sample size: 5～20 μ L;

Column temperature: 25～40 ℃;

Excitation wavelength: 213nm, emission wavelength: 308nm.

Table 1 liquid chromatography gradient elution program

Time/min	Acetonitrile/%	0.02mol/L sodium acetate/%
			0.00	20	80
0.05	20	80
			12.0	50	50

13.0	100	0
			20.0	100	0
22.0	20	80
			25.0	20	80

Beneficial effect of the present invention is, has set up the assay method of semicarbazides residual quantity in a kind of flour and Flour product, adopts methanol aqueous solution or acetonitrile solution to extract the semicarbazides in sample, has optimized the adding proportion of derivative reagent and hydrochloric acid solution.

The accompanying drawing explanation

Spectrogram (concentration 20 μ g/mL) after Fig. 1 semicarbazides standard substance is derivative

The negative blank face sample packet of Fig. 2 spectrogram

The negative Bread Samples mark-on spectrogram (adding scalar 100 μ g) of Fig. 3

Embodiment

Below in conjunction with specific embodiment, the present invention is described in further detail, but the present invention is not limited to specific embodiment.

The mensuration of semicarbazides residual quantity in the commercially available croissant of embodiment

1, analytical procedure

Take and pulverize rear bread sample 1.00g (being accurate to 0.0001g) in the 50mL centrifuge tube, add 10mL20% methanol aqueous solution mechanical shaking extraction 30min, the centrifugal 5min of 8000r/min, supernatant, after the organic membrane filtration of 0.45 μ m, is collected filtrate.Pipette filtrate 0.5mL in the 1.5mL sample injection bottle, add 200 μ L0.02mol/L9-hydroxyl xanthenes and 50 μ L1.5mol/L hydrochloric acid solutions, lucifuge derivatization reaction 30min, to be measured.

2, high-efficient liquid phase chromatogram condition

Instrument: the Agilent1200 high performance liquid chromatograph is furnished with fluorescence detector; Chromatographic column: the chromatographic column that the carbon octadecyl silane of take is filling agent (4.6 * 150mm, 5 μ m); Mobile phase: acetonitrile+0.02mol/L sodium acetate solution (gradient elution, elution program is in Table 1); Flow velocity: 1.0mL/min; Sample size: 10 μ L; Column temperature: 35 ℃; Excitation wavelength: 213nm, emission wavelength: 308nm.

3, standard working curve

The semicarbazides standard reserving solution stepwise dilution of 100 μ g/mL is obtained to 2.00 μ g/mL, 5.00 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL, the standard operation liquid of 50.0 μ g/mL.Get respectively the standard operation liquid 0.5mL of preparation, add 200 μ L0.02mol/L9-hydroxyl xanthene solution and 50 μ L1.5mol/L hydrochloric acid solutions, lucifuge derivatization reaction 30min under room temperature.According to above-mentioned instrument condition, measure, gained typical curve equation is: Y=12.7C-6.53, and wherein, Y is the chromatographic peak peak area, C is object concentration, coefficient R 2=0.9997.

4, Specimen Determination and mark-on reclaim result

After measured, in this commercially available croissant, semicarbazides is not detect.This commercially available croissant is carried out to recovery testu, add scalar and be respectively 50 μ g, 100 μ g, recovery of standard addition is between 79.2%-86.0%, and relative standard deviation is between 4.7%-4.9%.

Above-described embodiment is used for illustrative purposes only, and is not the restriction to patent of the present invention; It should be pointed out that for those of ordinary skill in the art, in the situation that do not break away from the present invention, conceive scope, can also make various variations and modification, these all belong to protection scope of the present invention; Therefore, all equalizations of doing with the claims in the present invention scope change and modify, and all should belong to the coverage of the claims in the present invention.

Claims

1. the method for semicarbazides residual quantity in a high effective liquid chromatography for measuring flour and Flour product, its feature comprises the steps:

(1) get a certain amount of sample and add the extraction solvent, the semicarbazides in vibration or ultrasonic extraction sample;

(2) extract, after centrifugal treating, is got supernatant and is crossed miillpore filter;

(3) get a certain amount of filtrate and add 0.02mol/L9-hydroxyl xanthene derivating agent, add a certain amount of hydrochloric acid solution, derivative under acid condition;

(4) the sample liquid after derivative separates through high performance liquid chromatography, and fluorescence detector detects, and external standard method is quantitative, calculate in flour and Flour product semicarbazides residual quantity.

2. method according to claim 1, is characterized in that, in described extraction flour and Flour product semicarbazides solvent be methanol aqueous solution or acetonitrile solution.

3. method according to claim 1, is characterized in that, the consumption of described 0.02mol/L9-hydroxyl xanthene derivative reagent is 50 μ L～300 μ L; The concentration of hydrochloric acid solution is 0.15mol/L～1.5mol/L, and the consumption of hydrochloric acid solution is 20 μ L～100 μ L.

4. method according to claim 1, is characterized in that, this method adopts high performance liquid chromatography to separate, and fluorescence detector detects.