CN101430325A - Fast color development method for azolmetazin relict - Google Patents

Abstract

The invention provides a method for rapidly developing the color of sulfamethazinum residues in plasma. By taking advantage of the particular structure of sulfamethazinum, color development reaction between the sulfamethazinum and (N-alpha-naphthyl) ethylenediamine dihydrochloride is carried out in under acidic condition to obtain a colored diazo coupling product, wherein the preceding drawing can be referred to; and a spectrophotometer can be used for determining the residual quantity of the sulfamethazinum in the plasma according to gradation of color generated in the reaction. Only 15 min to 20min is needed by the whole process and the average recovery rate can be up to 78 percent; the detection method is rapid, precise and reliable and can be used for on site plasma sorting.

Description

Fast color development method for azolmetazin relict

One, technical field

The present invention relates to a kind of animal blood detection method, particularly a kind ofly can be fast detect the residual method of livestock products blood plasma herbal medicine (sulfadimidine) by colour developing.

Two, technical background

Along with agricultural and Developing of Animal Industry, the usable range and the consumption thereof of feed addictive constantly increase, thereby have improved the output of animal husbandry.Particularly the antibiotics feed addictive is improving aspect the breeding performonce fo animals and prophylactic effect is that other any feed addictive is all incomparable.Yet a large amount of, the long-term veterinary drug that uses in feed causes veterinary drug residual in livestock products inevitably.The livestock products veterinary drug residue is harm humans health not only, also influences the livestock products quality simultaneously, causes economic loss.

The antibiotic residues in animal-derived food problem is a current realistic problem of paying close attention to the most, has caused the attention of people's height.Sulfadimidine belongs in the veterinary drug scope that China allows to use now, has set up the national standard detection method, but has also existed vacancy in quick on-the-spot residue detection, sees following table for details.

The comparison sheet of several detection methods commonly used

This just objectively requiring to improve traditional detection method, seeks a kind of detection method fast, accurately, makes testing result more accurately, reliably.

Three, summary of the invention

The invention provides a kind of fast color development method for azolmetazin relict, utilize the special construction of sulfadimidine,, judge its residual quantity size, have and detect quick, accurate, reliable characteristics according to shade with some material generation chromogenic reaction.

Specific as follows:

A kind of fast color development method for azolmetazin relict, set up the chemical diazonium-coupling reaction of sulfadimidine, the sulfadimidine of different residual concentrations is through after reacting in the blood plasma, present different colors, by spectrophotometer, just can draw the residual concentration of sulfadimidine in the blood plasma, realize that the residual live body of sulfadimidine detects.

Sulfadimidine is white or micro-yellow powder, odorless, and substantially tasteless is soluble in hydrochloric acid, and aqueous slkali is dissolved in hot ethanol, water insoluble and ether.Its molecular structure is:

From the sulfadimidine structural drawing, as can be seen, amino is an active group, at first will consider to start with from the singularity of amino when carrying out chromogenic reaction, and azo-compound generally all has color.This reaction can generate the diazonium-coupling product of scarlet, and sulfadimidine and hydrochloride naphthodiamide can generate coloured diazonium-coupling product under acid condition:

(scarlet azo-compound)

Wherein, the proportioning of sodium nitrite and blood plasma should be in the 0.05mg/ml-0.3mg/ml scope.The coupling product that forms is carried out the full scan of visible light wave range in ultraviolet-visible spectrophotometer, and in the visible region, formed conjugates has characteristic absorption peak, and it is maximum, and to detect wavelength be 545nm.The pH value scope of reacting required acid condition is 2.0-2.5.

Effect detection of the present invention:

(1) mensuration of the recovery

Get 5.0ml blood plasma in the 50ml centrifuge tube, add 10 μ l, 50 μ l, 100 μ l concentration respectively and be 100 μ g/ml, standard operation liquid, making the sulfadimidine concentration in the blood plasma is 1.0 μ g/ml, 5 μ g/ml, three interpolation levels of 10 μ g/ml, each level is established three repetitions, according to 1.3 pre-treating methods of determining, its recovery and the coefficient of variation see the following form.

The recovery of standard addition and the coefficient of variation of table sample

As seen from the above table, on 1.0 μ g/g, 5.0 μ g/g, three interpolation levels of 10.0 μ g/g, the recovery scope of sulfadimidine is respectively in the blood plasma: 79.8%～81.3%, 81.6%～83.1%, 78.15%～81.23%, average recovery rate is more than 79.0%, and the coefficient of variation is below 2.0%.

(2) establishment of lowest detectable limit

Get 5ml blood plasma, add an amount of SM2 standard stock solution, make concentration become 0.05 μ g/ml, 0.1 μ g/ml, 0.5 μ g/ml, 1.0 μ g/ml, 5.0 μ g/ml, 10.0 μ g/ml series plasma concentration, handle according to 1.3 definite methods, measurement result shows, drug concentration in the blood plasma is proportional with the color that is generated, when concentration during greater than 1.0 μ g/ml, directly with the naked eye just can discern, when its concentration during less than 1.0 μ g/ml, can select 721 spectrophotometers for use, the detectable concentration of this moment can reach 0.1 μ g/ml.

Four, description of drawings

Fig. 1 is a standard working curve of the present invention---concentration absorbance graph of a relation

Five, embodiment

A kind of fast color development method for azolmetazin relict, set up the chemical diazonium-coupling reaction of sulfadimidine, the sulfadimidine of different residual concentrations is through after reacting in the blood plasma, present different colors, by spectrophotometer, just can draw the residual concentration of sulfadimidine in the blood plasma, realize that the residual live body of sulfadimidine detects.

This reaction can generate the diazonium-coupling product of scarlet, and sulfadimidine and hydrochloride naphthodiamide are can generate coloured diazonium-coupling product under 2 the acid condition in the pH value:

(scarlet azo-compound)

Wherein, the proportioning of sodium nitrite and blood plasma is 0.1mg/ml.

The coupling product that forms is carried out the full scan of visible light wave range in ultraviolet-visible spectrophotometer, and in the visible region, formed conjugates has characteristic absorption peak, and it is maximum, and to detect wavelength be 545nm.

(1) instrument

Electronic balance (AG245, PB303, PB5001), Switzerland Mei Tele company, induction amount 0.01mg; 721 spectrophotometers; Hydro-extractor (RC25C, RC3C), du pont company.

(2) preparation of solution

Standard inventory solution: accurately take by weighing 10mg sulfadimidine standard items, the water constant volume makes it the standard that concentration is 1mg/ml, and preserves in the refrigerator below 4 ℃ in the volumetric flask of 10ml.

50% trichloroacetic acid: accurately take by weighing the 50g trichloroacetic acid, be dissolved in the water of 50ml, making it concentration becomes 50% solution.

0.5% sodium nitrite: accurately take by weighing the 0.5g sodium nitrite, the water constant volume makes it concentration and is 0.5% solution in the volumetric flask of 1000ml.

The 2g/L hydrochloride naphthodiamide: accurately take by weighing the 2g hydrochloride naphthodiamide, the water constant volume makes it the solution that concentration is 2g/ml in the volumetric flask of 1000ml.

(3) disposal route of plasma sample

Add the 1ml trichloroacetic acid in the 5ml blood plasma, the centrifugal 10min of 4000r/min filters, and carries out drip washing with 2ml water again, adds the sodium nitrite of 10 μ l, leaves standstill 5min, adds the hydrochloride naphthodiamide of 1ml.

(4) preparation of standard working curve

In 6 test tubes, add 5ml blood plasma respectively, adding concentration in 6 test tubes respectively is sulfadimidine standard reserving solution 0 μ l, 25 μ l, 50 μ l, 75 μ l, the 100 μ l of 1mg/ml, and making the sulfadimidine in the blood plasma is 0 μ g/ml, 5 μ g/ml, 10 μ g/ml, 15 μ g/ml, 20 μ g/ml.As horizontal ordinate, absorbance is as ordinate with concentration, drawing standard working curve---concentration absorbance graph of a relation.

(5) detection of plasma sample

Add the 1ml trichloroacetic acid in the 5ml blood plasma, the centrifugal 10min of 4000r/min filters, and carries out drip washing with 2ml water again, adds the sodium nitrite of 10 μ l, leaves standstill 5min, adds the hydrochloride naphthodiamide of 1ml.Directly measure its absorbance down in 545nm,, just can draw the residual quantity of sulfadimidine in the blood plasma according to prepared standard working curve with spectrophotometer.

The diazo coupling of sulfadimidine is easy, quick, can be applicable to the on-the-spot rapid screening of blood plasma, the entire process process only needs 15～20min to finish, even this method adopts more easy 721 spectrophotometers, linear relationship between absorbance and the residual concentration is good, and its related coefficient can reach more than 0.98.On three interpolation levels of 1.0,5.0,10.0 μ g/g, the recovery scope of sulfadimidine is respectively in the blood plasma: 79.8%～81.3%, 81.6%～83.1%, 78.15%～81.23%, average recovery rate is more than 79.0%, and the coefficient of variation is below 2.0%.

Claims (2)

1, a kind of fast color development method for azolmetazin relict, it is characterized in that: chemical diazonium-coupling reaction of setting up sulfadimidine, the sulfadimidine of different residual concentrations is through after reacting in the blood plasma, present different colors, by spectrophotometer, just can draw the residual concentration of sulfadimidine in the blood plasma, realize that the residual live body of sulfadimidine detects.

This reaction can generate the diazonium-coupling product of scarlet, and sulfadimidine and hydrochloride naphthodiamide can generate coloured diazonium-coupling product under acid condition:

(scarlet azo-compound)

Wherein, the proportioning of sodium nitrite and blood plasma should be in the 0.05mg/ml-0.3mg/ml scope.

The coupling product that forms is carried out the full scan of visible light wave range in ultraviolet-visible spectrophotometer, and in the visible region, formed conjugates has characteristic absorption peak, and it is maximum, and to detect wavelength be 545nm.

2, fast color development method for azolmetazin relict according to claim 1 is characterized in that: the pH value scope of reacting required acid condition is 2.0-2.5.