CN102608109B - Thiocyanate radical detecting kit and application thereof - Google Patents

Abstract

The invention discloses a thiocyanate radical detecting kit, which is used for detecting thiocyanate radical content of fresh milk. The kit comprises a ready-to-use standard liquid, a sensitizer, a precipitator, a color development liquid and a needle-type filter. The kit has the advantages of simplicity, quickness, high sensitivity, good stability, low price and the like, and is suitable for field batch detection.

Description

Thiocyanate radical detection kit and application thereof

Technical field

The present invention relates to a kind of thiocyanate radical detection kit and application thereof.

Technical background

In " the non-edible material from soybeans of the illegal interpolation of possibility in food and food additives kind list (first) of easy abuse " that the Ministry of Public Health issues, in clear stipulaties milk and milk products, sodium thiocyanate belongs to illegal interpolation material.Excess free enthalpy, can cause acute and chronic poisoning.Calomel poisoning shows as eye and upper respiratory tract irritation, and severe intoxication shows as the loss of consciousness, the encephaledema that often occurs together and respiratory failure.Chronic toxicity mainly suppresses transhipment and the thyroid hormone synthesis of iodine.Especially larger risk is existed to the intelligence of fetus and baby and nervous system development.Can be effectively antibacterial, fresh-keeping after mixing sodium thiocyanate in raw milk or milk powder, be one of the illegal interpolation material at illegal milk family.

At present, sodium thiocyanate detection method mainly contains: (1) chromatography comprises the chromatography of ions and chromatography of ions-mass spectrometry.These methods are highly sensitive, and mass spectroscopy sensitivity can reach 0.10 μ g/L, can as confirmation method.The fertilizer method that the Ministry of Public Health is recommended is the chromatography of ions, the method detection sensitivity 0.1mg/kg.But these expensive equipment, working sample length consuming time, detects a sample more than one hour.Be not suitable for enterprise and grass-roots unit's Site Detection.(2) spectrophotometric development process, first, utilizes the protein in trichloroacetic acid precipitation milk, reacts according to thiocyanate radical and ferric ion the principle taken on a red color, and 460nm detects absorptance.Sensitivity 1-2mg/kg.Due to thiocyanate radical and ferric ion complex compound instability, the method requires to add in ferric ion 10-20 minute and detects, and is restricted during batch detection.

Summary of the invention

Technical matters to be solved by this invention is to provide thiocyanate radical detection kit and the application thereof of a kind of applicable enterprise and grass-roots unit's Site Detection and batch detection.

The technical scheme that the present invention takes:

A kind of thiocyanate radical detection kit, comprises titer, enhanced sensitivity liquid, precipitation agent, nitrite ion, syringe filter; Described titer is sodium thiocyanate solution or the potassium thiocyanate solution that thiocyanate radical concentration is respectively 0,0.5,1.0,2.5,5.0,7.5,10.0 mg/L; Described enhanced sensitivity liquid is the sodium dodecylsulphonate of 10g/L-200g/L, lauryl sodium sulfate or sodium dodecyl benzenylsulfonate aqueous solution; Described precipitation agent is 10%-50%(W/V) trichloroacetic acid solution; Described nitrite ion is 10%-50%(W/V) ferric salt solution.

Described trivalent iron salt is ferric trichloride, ferric nitrate or iron sulfate.

Described kit is for measuring the content of fresh milk thiocyanate radical, and concrete assay method is as follows:

(1) sample pre-treatments

Get the enhanced sensitivity liquid that a certain amount of fresh milk adds 10% volume, mixing, then add isopyknic precipitation agent with fresh milk, mixing, centrifugal 6000rpm/min, 5min, filter by described syringe filter;

(2) develop the color

Get a certain amount of filtrate, add the nitrite ion of 10% volume, mixing;

(3) detect

The absorbance of 456nm determination step (2) gained solution,

(4) get the titer with described fresh milk equivalent respectively, measure absorbance by the described method in step (1)-(3), draw absorbance-thiocyanate radical content standard curve, calculate the content of thiocyanate radical in described fresh milk sample according to described typical curve.

The Cleaning Principle of kit of the present invention is: thiocyanate radical and ferric ion react and take on a red color, and adds a certain amount of surfactant and can improve its sensitivity and stability.Fresh milk is with after sour precipitating proteins, centrifugal, mistake filters fat and precipitation, and filtrate adds the ferric ion of equivalent, and 456nm measures absorbance, in the absorbance recorded and sample, the content of thiocyanate radical is proportional, according to the content of thiocyanate radical in typical curve calculation sample.

The all reagent of kit of the present invention is the working fluid configured, can direct control, low for equipment requirements, can quick, sensitive, detect fresh milk in a large number.Before precipitating proteins of the present invention, in fresh cow milk, add the anionic surfactant of doses, make sensitivity reach 0.5 mg/kg, improve the stability of sample in testing process, after reaction terminates, in 0-2 hour, product stable, is applicable to grass-roots unit or testing agency's batch detection.

Accompanying drawing explanation

Fig. 1 is the thiocyanate radical dose-response curve of kit standard liquid of the present invention.

Embodiment

Embodiment 1: the composition of kit and detection method

1. the composition of kit

1.1, titer 7 bottles: potassium rhodanide is put into 80 DEG C of drying in oven 2 hours, accurately takes dried potassium rhodanide 1.6732g in 1000ml volumetric flask, constant volume, and mixing, obtains 1000ppm thiocyanate radical standard reserving solution; Get thiocyanate radical Standard Stock solutions 1 mL, be placed in 100 mL volumetric flasks, add water to scale, this solution, containing thiocyanate radical 10 mg/L, is thiocyanic acid standard intermediate liquid; Pipette 0,0.5,1.0,2.5,5.0,7.5,10.0 mL thiocyanic acid standard intermediate liquids, with water constant volume in 10 mL volumetric flasks, concentration is respectively 0,0.5,1.0,2.5,5.0,7.5,10.0 mg/L;

1.2, enhanced sensitivity liquid 1 bottle: concentration is 10g/L-200g/L sodium dodecyl sulfate aqueous solution;

1.3, precipitation agent 1 bottle: concentration is 10%-50%(W/V) trichloroacetic acid solution;

1.4, nitrite ion 1 bottle: concentration is 10%-50%(W/V) ferric chloride aqueous solutions;

1.5, syringe filter one.

Kit storage temperature is 4 DEG C.

2. detection method

2.1, Specification Curve of Increasing

Seven kinds of each 3ml of thiocyanate radical standard solution in correct amount Example 1.1, respectively add enhanced sensitivity liquid 300 μ l respectively, mixing.Then respectively add precipitation agent 3ml, respectively add nitrite ion 300 μ l after mixing, mixing, 465nm measures absorbance, draws thiocyanate radical-light absorption value typical curve, calculates regression equation.

2.2, sample pre-treatments

Accurately measure each 3ml of fresh milk, add sensitizer working fluid 300 μ l respectively.Mixing.Respectively add precipitation agent 3ml, mix latter 6000 revs/min centrifugal 5 minutes.Take out, get supernatant syringe filter and filter.

2.3 colour developing

Get filtrate 3ml, add developer 300 μ l, mixing, 465nm measures absorbance.

2.4. interpretation of result

According to typical curve regression equation, thiocyanate radical content in calculation sample.

Embodiment 2 kit sensitivity determination

1, enhanced sensitivity liquid: 10g/L sodium dodecyl sulfate aqueous solution;

2, precipitation agent: 30%(W/V) trichloroacetic acid solution;

3, nitrite ion: 25%(W/V) ferric chloride aqueous solutions.

Seven kinds of each 3ml of thiocyanate radical standard solution in correct amount Example 1 respectively, each standard solution 3 repetition.Respectively add enhanced sensitivity liquid 300 μ l, mixing.Then, respectively add precipitation agent 3ml, respectively add nitrite ion 300 μ l after mixing, mixing, 465nm measures absorbance.Drawing standard curve, calculates regression equation.

Table 1 kit sensitivity determination

Concentration of standard solution mg/L	0	0.5	1.0	2.5	5	7.5	10
								Absorbance	0	0.014±0.0009	0.029±0.0010	0.072±0.0006	0.146±0.0006	0.222±0.0012	0.295±0.0035

According to above Plotting data typical curve, see Fig. 1.The method sensitivity is 0.5mg/L, calculates regression equation, R ²=0.9999.

The embodiment 3 kit recovery and precision measure

Gather nipple milk a, through sulfate by ion chromatography thiocyanate radical content 2.04mg/L, as reference liquid, maybe the thiocyanate radical standard reserving solution configured is added in reference liquid by dilute with water, its final concentration is made to reach 0.51,1.02,2.04,2.54,5.04 and 10.04 mg/L, each concentration 6 repetition, titer is with 1.1 in embodiment 1.The preparation of enhanced sensitivity liquid, precipitation agent and nitrite ion is with embodiment 2.According to kit measurement method, measure thiocyanate radical content in each sample, and calculate the recovery and the coefficient of variation.Result is as follows:

The table 2 kit recovery, the coefficient of variation measure

Concentration of standard solution mg/L	0.51	1.02	2.54	5.04	10.04
						Recovery %	104.85±4.19	90.53±4.06	98.89±0.73	101.03±3.73	95.49±2.20
The coefficient of variation (cv%)	4.22	5.18	1.26	0.75	2.53

In sensing range, the recovery is greater than 90%, is less than 110%, and the coefficient of variation is less than 10%, meets national standard.

Embodiment 4: kit detects time stability

Gather commercially available plain chocolate 4 parts, titer is with 1.1 in embodiment 1.The preparation of enhanced sensitivity liquid, precipitation agent and nitrite ion is with embodiment 2.According to kit measurement method, measure thiocyanate radical content in each sample.After adding developer mixing, measured once every 30 minutes, measure four times altogether, calculating mean value and the coefficient of variation.

Table 3 kit detects time stability

Sample number into spectrum	1	2	3	4
					Mean value (mg/L)	2.68±0.04	2.77±0.04	3.04±0.08	3.20±0.08
The coefficient of variation (cv%)	1.79	1.54	2.66	2.5

Result shows, and in two hours, the coefficient of variation is less than 5%, and measurement result is stablized, and kit of the present invention is applicable to batch detection.

Embodiment 5 is fallen apart cylinder Ruzhong thiocyanate radical content detection

Gather loose cylinder breast 4 parts, every part is repeated 3 times, according to kit measurement method, measures thiocyanate radical content in each sample.Wherein, titer is with 1.1 in embodiment 1, and enhanced sensitivity liquid is 200g/L sodium dodecyl sulfate aqueous solution, and precipitation agent is 50%(W/V) trichloroacetic acid solution, nitrite ion is 50%(W/V) ferric chloride aqueous solutions.Result is as follows:

Sample number into spectrum	1	2	3	4
					Thiocyanate radical content (mg/L)	2.154±0.05	1.67±0.02	3.22±0.04	3.73±0.06

Thiocyanate radical content detection in embodiment 6 nipple milk

Gather nipple milk 6 parts, every part is repeated 3 times, according to kit measurement method, measures thiocyanate radical content in each sample.Wherein, titer is with 1.1 in embodiment 1.Enhanced sensitivity liquid is 120g/L sodium dodecyl sulfate aqueous solution, and precipitation agent is 10%(W/V) trichloroacetic acid solution, nitrite ion is 10%(W/V) ferric chloride aqueous solutions.Result is as follows:

Claims (3)

1., for measuring a thiocyanate radical detection kit for fresh milk thiocyanate radical content, it is characterized in that it comprises titer, enhanced sensitivity liquid, precipitation agent, nitrite ion, syringe filter; Described titer is sodium thiocyanate solution or the potassium thiocyanate solution that thiocyanate radical concentration is respectively 0,0.5,1.0,2.5,5.0,7.5,10.0 mg/L; Described enhanced sensitivity liquid is the sodium dodecylsulphonate of 10g/L-200g/L, lauryl sodium sulfate or sodium dodecyl benzene sulfonate aqueous solution; Described precipitation agent is 10%-50%(W/V) trichloroacetic acid solution; Described nitrite ion is 10%-50%(W/V) ferric salt solution.

2. the thiocyanate radical detection kit for measuring fresh milk thiocyanate radical content according to claim 1, is characterized in that described trivalent iron salt is ferric trichloride, ferric nitrate or iron sulfate.

3. the application of kit described in claim 1 or 2, it is characterized in that the content measuring fresh milk thiocyanate radical, concrete assay method is as follows:

(1) sample pre-treatments

Get the enhanced sensitivity liquid that a certain amount of fresh milk sample adds its volume 10%, mixing, then add precipitation agent isopyknic with described fresh milk, mixing, centrifugal 6000rpm/min, 5min, filter by described syringe filter;

(2) develop the color

Get a certain amount of filtrate, add the nitrite ion of its 10% volume, mixing;

(3) detect

The absorbance of 456nm determination step (2) gained solution,

(4) get the titer with described fresh milk equivalent respectively, measure absorbance by the described method in step (1)-(3), draw absorbance-thiocyanate radical content standard curve, calculate the content of thiocyanate radical in described fresh milk sample according to described typical curve.