CN103293039A - Method for quickly obtaining positive material containing AOZ (3-amino-2-oxazolidinone) minced shrimp natural matrix in laboratory - Google Patents

Abstract

The invention discloses a method for quickly obtaining a positive material containing an AOZ (3-amino-2-oxazolidinone) minced shrimp natural matrix in a laboratory. The method comprises the following steps of: performing medicated bath on live shrimp, obtaining the positive material, and performing treatment on the medicated shrimp and the positive material. According to the method disclosed by the invention, field operation is not required, and the operation is simple, convenient and time-saving. By applying the method to national capacity verification practices, a produced quality control sample is uniform and stable, the consistency in appearance state (non-lyophilized powder) and internal combination state (in-vivo metabolism natural combination) with routine test samples can be simultaneously ensured, and the possible inconsistency of the two in operation steps and extraction efficiency in analysis can be further avoided.

Description

Obtain the method that contains the rotten natural basal body positive material of AOZ shrimp in the laboratory fast

Technical field

The invention belongs to the analytical chemistry field, be specifically related to obtain fast in a kind of laboratory the method that contains the rotten natural basal body positive material of AOZ shrimp.

Background technology

Because itrofurans medicine and metabolin thereof have sizable toxicity and spinoff, can induce the organism gene mutation, the inducing action that the teratogenesis tire is arranged, and can bring out cancer, many developed countries such as China and European Union and area forbidding itrofurans medicine require must not detect such residue in animal derived food in edible animal.But indivedual culturists still violate a ban and are used in aquatic products such as fish, shrimp.In animal body, the metabolism of itrofurans medicine is fast, its metabolin then can retain the long period, thereby metabolin is as the mark residue of such medicine.The itrofurans medicine is residual to be one of higher frequency test item such as China outlet aquatic products.The furazolidone of one of itrofurans medicine, its metabolin are 3-amino-2-oxazolones (AOZ).

The residue detection laboratory needs the quality control sample for control detects quality.Desirable quality-control sample should be apparent condition (as non-freeze-dried powder) and inherent bonding state (as being the internal metabolism natural combination) natural basal body sample consistent with daily test sample all, in order to avoid cause the possible inconsistent of the two operation steps in analysis and extraction efficiency.The candidate material that natural basal body typically refers to quality-control sample is taken at natural matrix, does not comprise being taken at " compound or the element that add known quantity in matrix material ".Latter's (term) in GB/T 15000.3-2008 " rule and the statistical methods of standard model work guide rule 3 standard model definite values " is called " reinforced (spiking) ".Yu Kongjie etc. once were reported in the preparation of open-air breed pond Chinese medicine bath method acquisition positive material and contained AOZ shrimp meat natural basal body standard model (RM), but did not see and obtain the report that contains the rotten natural basal body positive material of AOZ shrimp method fast in the laboratory.For quick many contents levels of preparation natural basal body quality-control sample is used for presiding over " checking of aquatic products Furaxone metabolite (AOZ) detectability " planning item (project implementation is pressed for time and requires to provide many contents levels for sample) of implementing CNCA (CNCA), this paper has attempted the natural basal body positive material that dipping shrimp alive in the laboratory obtains the appropriate level level fast.

Summary of the invention

The object of the present invention is to provide and obtain the method that contains the rotten natural basal body positive material of AOZ shrimp in a kind of laboratory fast, need not field work, easy saving time.Through being used for implementing national proficiency testing practice, made quality-control sample is uniform and stable, guaranteed that simultaneously apparent condition (non-freeze-dried powder) is all consistent with daily test sample with inherent bonding state (internal metabolism natural combination), in order to avoid cause the possible inconsistent of the two operation steps in analysis and extraction efficiency.

For achieving the above object, the present invention adopts following technical scheme:

Obtaining the method that contains the rotten natural basal body positive material of AOZ shrimp in the laboratory fast may further comprise the steps:

(1) live in the laboratory the obtaining of shrimp dipping and positive material

The dipping experiment condition: filling tap water about 40 cm are dark in the dipping experiment pool, get furazolidone tablet pulverize, calculate and take by weighing furazolidone medicine end by the AOZ initial concentration of dip, add water 100 mL, mixing; Inject the dipping experiment pool, the medicated bath pools that obtain required AOZ initial concentration dip are standby; 5 kg do not have furazolidone pollution shrimp alive directly to be shifted in the medicated bath pools, and picks up counting; During dipping, connect uninterruptedly oxygenation in the dip of emulsion tube with pneumatic pump; Placing the indoor of medicated bath pools is free key joint indoor air temperature to make 20 ℃ of basic maintenances;

The dipping preliminary experiment: with reference to open-air shrimp pond dipping experiment experience, AOZ has maximal value in 4 h after dipping～6 h shrimp bodies, selects dipping 5 h as the dipping time; The shrimp body has different AOZ content behind the medicine of the dip initial concentration of identical dipping asynchronism(-nization), and is proportionate; Adopt single horizontal dip initial concentration to carry out preliminary experiment, to survey the relation that obtains behind the medicine AOZ content and dip initial concentration in the shrimp body under this experiment condition;

Dipping is also obtained positive material: the hypothesis of pressing shrimp body AOZ content and the straight line correlation of dip initial concentration behind the medicine according to the preliminary experiment result, according to the rotten AOZ content of the shrimp of expection gained and after considering irradiation sterilization suitably loss percentage determine the dip initial concentration; Carry out the dipping experiment, obtain the positive material of desired AOZ contents level;

(2) processing of medicine shrimp and positive material

Shrimp decaptitates to peel off after freezing execution and gets meat ,-20 ℃ of freezing preservations; Freezing state shrimp sample is transferred to 4 ℃ of refrigerator defrostings is placed in the refiner, fully homogenate; Mix all sample slurries; Then, getting packs in right amount bounces bag and is placed on and bounces in the formula homogenizer, press the frequency of 180/min, bounces to take off behind 2 min to rub at every turn and starches sample in mixing bag, bounces again, bounces so repeatedly three times; Bounce back slurry sample and be mixed in the drum, again artificial mixing; Get every part of 25 g of rotten sample PE film inner bag of packing into, put the outer bag of the PA/CPP composite membrane of anti-the boiling again; With vacuum sealer the outer bag that installs rotten sample is sealed mouth; Stick the sample label of band numbering; With pack, seal, the sample of good sign send Compton, Fujian Province irradiation technique company limited to carry out irradiation sterilization, irradiation dose is 12 kGy; Sterilization back sample places-20 ℃ of refrigerators to store.

Remarkable advantage of the present invention is: the present invention need not field work, easy saving time.Through being used for implementing national proficiency testing practice, made quality-control sample is uniform and stable, guaranteed that simultaneously apparent condition (non-freeze-dried powder) is all consistent with daily test sample with inherent bonding state (internal metabolism natural combination), in order to avoid cause the possible inconsistent of the two operation steps in analysis and extraction efficiency.

Embodiment

1 material

1.1 material

1.1 material and reagent

Penaeus Vannmei (Penaeusvanammi) adopts commercially available shrimp alive, about 55 tails of every 500g.Commercially available medical furazolidone sheet, detecting furazolidone content through high performance liquid chromatography is 6%; The dipping water, tap water.

Analyrical reagent is: water, ultrapure water; Acetonitrile, methyl alcohol, ethyl acetate, chromatographically pure; Anhydrous sodium sulfate, hydrochloric acid, NaOH, sodium chloride are analyzed pure; The derivating agent o-nitrobenzaldehyde, Fluka company; Furazolidone standard items, 3-amino-2-oxazolidinyl ketone (AOZ) standard items, AOZ-D4 standard items, Sigma company; AOZ, AOZ-D4 standard solution: standard reserving solution: take by weighing nitrofuran metabolite standard items AOZ(AOZ_D4 respectively) (accurately to 0.1mg), respectively with methyl alcohol dissolving and be transferred in the 100 mL volumetric flasks, with methanol constant volume to scale, this solution concentration is 100.0 μ g/mL, preservation below 4 ℃ places tool plug reagent bottle in 4 ℃ of stored refrigerated, and storage life is 6 months.The standard intermediate liquid: draw standard reserving solution 1.00 mL of above-mentioned nitrofuran metabolin respectively, with methanol constant volume to 100 mL, concentration is 1.00 μ g/mL.

Wrappage: nylon/polypropylene (PA/CPP) compound membrane bag of anti-the boiling, 120 mm * 110 mm, thickness 0.06 mm is outer packaging bag; No. 4 tygon (PE) film valve bag, 120 mm * 85 mm, thickness 0.04 mm cuts off from sealing as inner bag.

1.2 instrument and equipment

Efficient liquid phase liquid chromatograph, Agilent 1100 types; The liquid chromatography-tandem mass spectrometry instrument, Waters UPLC/Premier, charged spray ionization (ESI) source; Air bath vibration shaking table, Shanghai fine jade spy; The vortex oscillator, German IKA; Hydro-extractor, ANKE TDL-5,5000 r/min, Anting Scientific Instrument Factory, Shanghai; 5000 r/min; Ultrasonic cleaner, Kunshan Ultrasonic Instruments Co., Ltd.; The agitating type refiner, Denmark FOSS 2094 types; Bounce homogenizer, Stomacher 3500 types, Britain Seward; Vacuum packing machine, SC-300A type, Anxi County three and tea machinery factory; Incubator, KB720 type, German Binder; Co60-gamma-ray irradiation device, design capacity 500,000 Curie, Compton, Fujian Province irradiation technique company limited; Refrigerator-freezer, BC/BD-718A, Qingdao Haier Special Electric Freezer Co., Ltd; Dipping experiment pneumatic pump, fishing booth board ACO-002 type, ZheJiang Sunsun Industrial Co., Ltd.; The dipping experiment pool, commercially available plastic square bucket, the about 67cm * 46cm of lower outer diameter, high 61cm.

1.3 AOZ detects in the shrimp

Carry out sample pre-treatments by GB/T21311-2007 " nitrofurans medicament metabolite residue quantity measuring method high performance liquid chromatography-tandem mass method in the animal derived food ", adopt liquid chromatography-tandem mass spectrometry to detect.

Chromatographic condition: Acquity UPLC BEH C18 post, 55 mm * 2.1 mm, 1.7 μ m; Column temperature: 35 ℃; Phase: the A that flows is 5 mmol/L ammonium acetates+0.2% aqueous formic acid, and B is methyl alcohol, gradient elution, 0～1.0 min, A is 90%, 1.0～3.5 min, and A is changed to 20%, 3.5～4.0 min by 90%, A is changed to 10% and keep 0.3 min by 20%, 4.3～4.5 min, and A is changed to 90% by 10%; Flow velocity: 0.3 mL/min; Sample size: 10 μ L.

Mass spectrum condition: ESI source, positive ion mode; Taper hole voltage: 0.6kV; Source temperature: 120 ℃; Desolventizing temperature: 350 ℃; The retention time of Furaxone metabolite, parent ion and daughter ion see Table 1, adopt d4-AOZ as marking inner mark method ration in the isotope.

The retention time of table 1 furazolidone, parent ion and daughter ion

Annotate: underscore is the quota ion fragment

Shrimp dipping and positive material obtains 1.4 live

1.4.1 dipping experiment condition: about 40 cm of filling tap water are dark in dipping experiment pool (empty bucket).Get furazolidone tablet pulverize, calculate and take by weighing an amount of furazolidone medicine end by the AOZ initial concentration of dip, add about 100 mL of water, mixing; Inject the dipping experiment pool, the medicated bath pools that obtain required AOZ initial concentration dip are standby.5 kg do not have furazolidone pollution shrimp alive directly to be shifted in the medicated bath pools, and picks up counting.During dipping, connect uninterruptedly oxygenation in the dip of emulsion tube with pneumatic pump.Placing the indoor of medicated bath pools is free key joint indoor air temperature to make 20 ℃ of basic maintenances.Behind dipping times 5 h, pick up the medicine shrimp.

1.4.2 dipping preliminary experiment

With reference to open-air shrimp pond dipping experiment experience, AOZ has maximal value in 4 h after dipping～6 h shrimp bodies, selects dipping 5 h as the dipping time; The shrimp body has different AOZ content behind the medicine of the dip initial concentration of identical dipping asynchronism(-nization), and is proportionate.Adopt single horizontal dip initial concentration to carry out preliminary experiment, to survey the relation that obtains behind the medicine AOZ content and dip initial concentration in the shrimp body under this experiment condition.It is 35.6 ng/L that this paper selects preliminary experiment dip initial concentration x, taking by weighing through the high performance liquid chromatography detection level is that 6% furazolidone medicine end, 0.073 g prepares the dipping experiment condition by 1.3.1, buy 5 kg and live shrimp by 1.3.1 dipping 5 h, gained medicine shrimp through freezing execution, decaptitate and peel off, stir into the rotten and homogeneous of shrimp.Detect the rotten AOZ content of gained shrimp.

1.4.3 dipping is also obtained positive material

The hypothesis of pressing shrimp body AOZ content and the straight line correlation of dip initial concentration behind the medicine according to the preliminary experiment result, according to the rotten AOZ content of the shrimp of expection gained and after considering irradiation sterilization suitably loss percentage determine the dip initial concentration.Carry out the dipping experiment, obtain the positive material of desired AOZ contents level.Be desirably in the shrimp gruel of 2 different content levels in the scope of 2 μ g/kg-8 μ g/kg, selecting the dip initial concentration is 13.6ng/L( ) and 22.4 ng/L( ).Take by weighing content respectively and be furazolidone medicine end 28 mg and each portion of 46 mg of 6%, prepare two medicated bath pools that dip Furanzolidon-containing initial concentration in the pond is about B pond 13.6 ng/L, C pond 22.4 ng/L respectively by 1.4.1.Two parts of each 5 kg are not had furazolidone pollution shrimp alive be transferred to respectively in B, the C medicated bath pools, carry out dipping by 1.4.1.Pick up the medicine shrimp behind dipping 5 h.The medicine shrimp that B, C pond pick up be labeled as respectively B, C(hereinafter to be referred as be B shrimp, C shrimp).The gruel of detection gained shrimp contained AOZ content after homogeneous became the rotten also irradiation sterilization of shrimp respectively.

1.5 the processing of medicine shrimp and positive material

B shrimp, C shrimp are respectively by following step process: decaptitating after freezing execution peels off gets meat ,-20 ℃ of freezing preservations.Freezing state shrimp sample is transferred to 4 ℃ of refrigerator defrostings is placed in the refiner, fully homogenate.Mix all sample slurries.Then, getting packs in right amount bounces bag and is placed on and bounces in the formula homogenizer, press the frequency of 180/min, bounces to take off behind 2 min to rub at every turn and starches sample in mixing bag, bounces again, bounces so repeatedly three times.Bounce back slurry sample and be mixed in the drum, again artificial mixing.Get every part of about 25 g of rotten sample PE film inner bag of packing into, put the outer bag of the PA/CPP composite membrane of anti-the boiling again.With vacuum sealer the outer bag that installs rotten sample is sealed mouth.Stick the sample label of band numbering.With pack, seal, the sample of good sign send Compton, Fujian Province irradiation technique company limited to carry out irradiation sterilization, irradiation dose is 12 kGy.Sterilization back sample places-20 ℃ of refrigerators to store.

1.6 homogeneity, stability test

After will making sample and being distributed into unit testing sample and serial number, each concentration level sample is randomly drawed 10 samples respectively, measures twice under each sample repeat condition.Adopt one-factor analysis of variance method (F method of inspection) that assay is carried out statistical treatment.If F less than degree of freedom be (f1, f2) and the critical value F α of given level of significance α (α=0.05) (f1 f2), then shows the sample room there was no significant difference, and sample is uniform.

Carried out totally 6 stability tests of 6 timing nodes, the date is respectively May 11, May 26, May 28, June 15, July 18, August 5.Randomly draw each six parts of two horizontal samples of B, C on these dates.Detection method, testing staff, instrument, laboratory are detected identical with homogeneity.Adopt t method of inspection evaluation sample stability, t is the critical value t α (n1+n2-2)=2.14 of n1+n2-2 less than level of significance α (α=0.05) degree of freedom as a result, and showing between two mean values does not have significant difference.

1.7 implement national proficiency testing

With the national laboratory detectability checking of the sample successful implementation CNCA of above-mentioned acquisition, B sample, C sample respectively have 34 tame testing laboratories to participate in, and median is respectively 4.20 μ g/kg.6.74?μg/kg。

2 results and discussion

2.1 the selection of dipping time

With reference to open-air shrimp pond dipping experiment experience, before AOZ content reaches peak value in the shrimp body after the dipping, its AOZ content speedup is very fast, the AOZ content should be steady relatively near the peak value, and AOZ content comparatively fast descends behind the peak value, 9 h-24 h behind the medicine, it should be about the same taking in that amount that furazolidone metabolite becomes AOZ measures with AOZ degraded (and discharging in body), AOZ concentration fall off rate is mild, and 24 h change behind the water very fast to 30 h reductions of speed, and reduction of speed is gradually slow behind 30 h.As seen have before and after the peak value relatively stably, three time periods behind 9 h-24 h and the 30h behind the medicine, these three time periods are suitable drags for shrimp, to reduce content difference between individuality.Consider and save the dipping time as far as possible, also since under indoor conditions the high density dipping should not carry out for a long time, reducing shrimp mortality ratio alive, the indoor dipping experimental selection of this paper near the 5h the peak value finish dipping and drag for shrimp.

2.2 dipping preliminary experiment

Y is 13.5 μ g/kg after testing by the rotten AOZ content of 1.4.2 preliminary experiment gained shrimp.Y=0.38x is then arranged under this experiment condition.Suppose that rule of thumb irradiation sterilization has 15% loss percentage approximately, i.e. AOZ content behind the irradiation

, then

2.3 positive material is obtained in dipping

Press

Estimation dip initial concentration x.This paper expects to obtain 4.4 μ g/kg, 7.2 μ g/kg's , select dip initial concentration x to be respectively and be 13.6ng/L(

) and 22.4 ng/L(

).Be respectively B shrimp 4.61 μ g/kg, C shrimp 7.05 μ g/kg by the rotten testing result AOZ of 1.4.3 gained shrimp content.Little with the desired value difference.Irradiation sterilization effect and investigation barrier packaging effect must be verified in list of references 6.

2.4 homogeneity stability test result

The uniformity testing statistics sees Table 2.Look into critical value F0.05(9,10)=3.02.Table 2 calculates the F value of each sample all less than critical value, shows when 0.05 level of significance there was no significant difference in sample room and the sample.

Table 2 uniformity testing statistics

The stability test statistics sees Table 3.Reference value is the uniformity testing data in the table 3.Look into the two tails of t critical=2.14, table 3 calculates gained t value all less than t pair of tail critical values.Show that sample is stable.

Table 3 stability test statistics

2.5 implement the median that obtains behind the national proficiency testing

The national laboratory proficiency testing [7] of having used the successful implementation of gained positive, this proficiency testing provides the test specimens of A, B, 3 contents levels of C altogether, wherein two contents level samples of B, C are this paper gained B sample, C sample (the A sample then is the original retention Quality Control in this laboratory sample, and wherein two horizontal samples are detected in each chamber of participating in the experiment).Respectively there are 34 families in the laboratory of detecting B sample, C sample, through the result add up the B sample, C sample median is respectively 4.20 μ g/kg, 6.74 μ g/kg.Compare median and desired value, the relative differences of B sample, C sample is respectively-4.5% and-6.4%.The laboratory result ASSOCIATE STATISTICS parameter of B sample, C sample sees Table 4.B sample, C sample median have been implemented to participate in proficiency testing result follow-up the benefit suspicious and laboratory that peels off and have been surveyed (measuring audit) activity as designated value.4 tame laboratories are participated in the B sample and are mended survey, and 9 tame laboratories are participated in C samples benefit and surveyed, and the result is except B sample result is suspicious, and other results are all satisfied.

Table 4 is participated in the statistical summaries of proficiency testing laboratory result

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

Claims (1)

1. obtain the method that contains the rotten natural basal body positive material of AOZ shrimp in the laboratory fast, it is characterized in that: may further comprise the steps:

(1) live in the laboratory the obtaining of shrimp dipping and positive material

The dipping experiment condition: filling tap water about 40 cm are dark in the dipping experiment pool, get furazolidone tablet pulverize, calculate and take by weighing furazolidone medicine end by the AOZ initial concentration of dip, add water 100 mL, mixing; Inject the dipping experiment pool, the medicated bath pools that obtain required AOZ initial concentration dip are standby; 5 kg do not have furazolidone pollution shrimp alive directly to be shifted in the medicated bath pools, and picks up counting; During dipping, connect uninterruptedly oxygenation in the dip of emulsion tube with pneumatic pump; Placing the indoor of medicated bath pools is free key joint indoor air temperature to make 20 ℃ of basic maintenances;

The dipping preliminary experiment: with reference to open-air shrimp pond dipping experiment experience, AOZ has maximal value in 4 h after dipping～6 h shrimp bodies, selects dipping 5 h as the dipping time; The shrimp body has different AOZ content behind the medicine of the dip initial concentration of identical dipping asynchronism(-nization), and is proportionate; Adopt single horizontal dip initial concentration to carry out preliminary experiment, to survey the relation that obtains behind the medicine AOZ content and dip initial concentration in the shrimp body under this experiment condition;

Dipping is also obtained positive material: the hypothesis of pressing shrimp body AOZ content and the straight line correlation of dip initial concentration behind the medicine according to the preliminary experiment result, according to the rotten AOZ content of the shrimp of expection gained and after considering irradiation sterilization suitably loss percentage determine the dip initial concentration; Carry out the dipping experiment, obtain the positive material of desired AOZ contents level;

(2) processing of medicine shrimp and positive material

Shrimp decaptitates to peel off after freezing execution and gets meat ,-20 ℃ of freezing preservations; Freezing state shrimp sample is transferred to 4 ℃ of refrigerator defrostings is placed in the refiner, fully homogenate; Mix all sample slurries; Then, getting packs in right amount bounces bag and is placed on and bounces in the formula homogenizer, press the frequency of 180/min, bounces to take off behind 2 min to rub at every turn and starches sample in mixing bag, bounces again, bounces so repeatedly three times; Bounce back slurry sample and be mixed in the drum, again artificial mixing; Get every part of 25 g of rotten sample PE film inner bag of packing into, put the outer bag of the PA/CPP composite membrane of anti-the boiling again; With vacuum sealer the outer bag that installs rotten sample is sealed mouth; Stick the sample label of band numbering; With pack, seal, the sample of good sign send Compton, Fujian Province irradiation technique company limited to carry out irradiation sterilization, irradiation dose is 12 kGy; Sterilization back sample places-20 ℃ of refrigerators to store.