CN102798560A - Clenbuterol matrix standard substance and preparation method thereof - Google Patents
Clenbuterol matrix standard substance and preparation method thereof Download PDFInfo
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- CN102798560A CN102798560A CN2012103187078A CN201210318707A CN102798560A CN 102798560 A CN102798560 A CN 102798560A CN 2012103187078 A CN2012103187078 A CN 2012103187078A CN 201210318707 A CN201210318707 A CN 201210318707A CN 102798560 A CN102798560 A CN 102798560A
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Abstract
The invention discloses a clenbuterol matrix standard substance and a preparation method thereof. The preparation method comprises the following steps of: uniformly mixing pig urine in which a solid substance is removed; then adding a clenbuterol standard substance to a quantity value level; uniformly mixing to obtain a mixed solution; and carrying out uniformity check on the mixed solution and then carrying out subpacking and freeze drying to obtain the clenbuterol matrix standard substance. The preparation method disclosed by the invention is scientific and reasonable and is strong in operability; the prepared matrix standard substance can be stored under the condition of -20DEGC and provides a quantity value tracing substance for detecting clenbuterol residue in daily pig urine; and according to the prepared matrix standard substance, the problem that different detection results are caused by matrix difference is solved and the monitoring of the clenbuterol residue in pig products is accurately ensured.
Description
Technical field
The present invention relates to Clenbuterol substrate standard substance and preparation method thereof.
Background technology
Clenbuterol is commonly called as " clenbuterol hydrochloride ", is a kind of receptor, activator, has to promote biological growth, obviously improve the effect of animal meat rate; Once (the HPLC method of clenobuterol hydrochloride is measured in the feed, Ying Yongfei, Wu Pinggu etc. to be widely used as feed additive for promoting growth; Feed detects, and 2006,23 (7): 54-56); But it is a succession of because of after eating the animal-derived food poisoning generation that contains " clenbuterol hydrochloride "; Clenbuterol is forbidden as feed addictive by a lot of countries in the world, and is required must not detect in the animal-derived food Clenbuterol (The Ministry of Agriculture of the People's Republic of China, MOA, animal food herbal medicine MRL [Z]; 1997, agriculture and animal husbandry is sent out (1997) No. 7).In the plant and the process of circulation, national quality testing department is gathered the urine of biopsy sample more and is carried out the speed survey screening of Clenbuterol, to guarantee the security of animal-derived food.
The chemical structural formula of Clenbuterol
The method that is used to detect Clenbuterol at present is main: enzyme linked immunosorbent assay (" animal product clenobuterol hydrochloride (clenbuterol hydrochloride) detection method progress ", Liu Guoyan, Chai Chunyan, animal science and animal medicine; 2002,19 (4): 31-34), high performance liquid chromatography (" high effective liquid chromatography for measuring pig urine in clenobuterol hydrochloride ", Su Wenzhou; Cai Yuzhi, Luo Xiaoyan, Chinese sanitary inspection magazine; 2001,1 (4): 436-437), gas chromatography mass spectrometry method (" in the biomaterial gas chromatography-mass spectrography of Clenbuterol measure ", Wu Pinggu; The analytical test journal, 2002,21 (1): 19-21) with LC-MS method (" LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver "; Kaita De Wach, Hubert De Brabander, Dirk Courtheyn; Analyst, 1998,123:2701-2705).
But, adopt different assay methods, exist evident difference between the analysis result of same sample, value is difficult to accomplish unified, is difficult to guarantee the comparability of different experiments chamber testing result.But standard substance has vital role aspect accuracy that guarantees analytical approach and the traceability.And; The difference of analyzing matrix itself makes and in analytic process, can not adopt traditional purity rubric material to calibrate simply or carry out quality control; Need to adopt the Clenbuterol substrate standard substance meeting the demands, with guarantee testing result accurately and have a traceability of value; Calibration instrument; Detection method is verified and the quality control of test experience chamber.But do not have the very effectively report of Clenbuterol substrate standard substance and preparation method thereof in the prior art, this present situation needs to be resolved hurrily.
Summary of the invention
Technical matters to be solved by this invention is, overcome the matrix effect of existing Clenbuterol detection method, testing process carried out quality control and defective such as calibration difficulties as a result, and a kind of Clenbuterol substrate standard substance and preparation method thereof is provided.This series standard material has clear and definite value and uncertainty through strict investigation and definite value, can be under-20 ℃ of conditions long preservation (at least one year), have good stability and homogeneity.
The preparation method of Clenbuterol substrate standard substance of the present invention comprises the steps: the fresh pig urine of removing behind the solid matter is mixed; Add the Clenbuterol standard substance to magnitude level; Mix again mixed solution, with this mixed solution behind uniformity testing, packing; Freeze drying again gets final product.
Among the present invention, described fresh pig urine is the conventional described fresh healthy swine urine in this area, generally is meant the urine of the live hog that directly collects, and its isolated time is less than 5 hours, Clenbuterol residual volume＜0.10ng/mL.
The detection method of described Clenbuterol residual volume; Be preferably the gold mark and detect block-regulations (quick detection test paper method), enzyme linked immunosorbent assay (ELISA), liquid chromatography tandem mass spectrometry (LC/MS/MS) or combined gas chromatography mass spectrometry (GC/MS) method, more preferably ELISA or LC/MS/MS method.But described gold mark detects the method for operating list of references of block-regulations: Shelver, W.L., Smith, D.J., 2004.J.Agric.Food Chem.52 (8), 2159-2166.
Among the present invention, described method of removing solid matter is this area conventional method, is preferably centrifugal separation or quantitative test filter paper filtering method, more preferably quantitative test filter paper filtering method.Be generally normal-temperature operation, described normal temperature refers generally to 0 ℃-40 ℃ of room temperatures.
Among the present invention, the described method of operating that mixes can be the conventional method of operating that mixes, preferred magnetic stirrer mixing.The operating conditions of described magnetic stirring apparatus is preferred: stir speed (S.S.) is 200～1000rpm, most preferably 500rpm; Mixing time is 10-40min, most preferably 30min.
Among the present invention; The form of described Clenbuterol standard substance can be the solid form or the solution form of Clenbuterol standard substance; Preferably the solution of Clenbuterol standard substance is little for guaranteeing that pig urine sample matrix changes, and more preferably adds the WS of Clenbuterol standard substance.
Among the present invention; Described magnitude level is the concentration of Clenbuterol solution; Preferably not can be: X1, X2, X3, X4, X5 with the ng/mL score; Wherein X1=0～0.30, X2=0.30～0.80, X3=0.50～2.00, X4=1.50～8.00, X5=5.00～15.00, and meet the following conditions: X1＜X2＜X3＜X4＜X5; More preferably X1=0～0.10, X2=0.40～0.70, X3=1.00～1.80, X4=2.50～4.00, X5=10.00～13.00; For satisfying the needs of working curve in the testing, most preferably X1＜0.10, X2=0.50, X3=1.00, X4=3.00, X5=10.00.
Among the present invention; Described uniformity testing is the conventional check sample uniform method of using in this area; Preferably include the following step: each magnitude level is got 7 pig urine samples; Detect Clenbuterol content with reference to GB/T22286-2008 " the mensuration liquid chromatography tandem mass spectrometry of multiple beta-receptor activator residual quantity in the animal derived food ", with the variance test method mixed solution is carried out uniformity testing again.
Among the present invention, the instrument that uses during described packing is the conventional package tools that uses in this area, preferred transfer pipet, bottleneck knockout or liquid-transfering gun, the more preferably liquid-transfering gun of 1mL.
Among the present invention, described lyophilization is the conventional lyophilization in present technique field, preferably includes following steps: will move into then in the freeze drier through the mixed solution of uniformity testing at-60 ℃～-80 ℃ pre-freezes (preferred-70 ℃), freeze-drying gets final product.Preferred 12～36h of the time of described pre-freeze, more preferably 24h.Pollute each other in freeze-drying process for fear of product, the product branch that preferably will fill different magnitude level is placed on and carries out freeze-drying in the different vessels.Described freeze-drying is preferably vacuum freeze-drying.Preferred 12～36h of the time of described vacuum freeze-drying, more preferably 24h.
Preferably, the product after the freeze drying being covered tight exterior and interior cover immediately seals.The product that the tight exterior and interior cover of above-mentioned lid seals preferably is transferred to-17 ℃～-23 ℃ preservations.
The invention still further relates to the Clenbuterol substrate standard substance that the preparation method of aforementioned Clenbuterol substrate standard substance makes.
The described Clenbuterol substrate standard substance that is made by the preparation method of aforementioned Clenbuterol substrate standard substance is preferably for to use and sell or to export by the concentration gradient of X1, X2, X3, X4 and X5 is complete.The concentration (in ng/mL) of the described complete Clenbuterol substrate standard substance of using and selling or exporting preferably is respectively X1=0～0.10, X2=0.40～0.70, X3=1.00～1.80, X4=2.50～4.00, X5=10.00～13.00; More preferably X1＜0.10, X2=0.50, X3=1.00, X4=3.00, X5=10.00.
Agents useful for same of the present invention, raw material be all commercially available to be got or prepares according to the conventional method of this area.Only if definition or explanation are arranged in addition, the same meaning that all technical terms used herein and those skilled in the art are familiar with.
On the basis of this area general knowledge, the optimum condition of each above-mentioned technical characterictic can combination in any obtain preferred embodiments of the present invention among the present invention.
Positive progressive effect of the present invention is:
The present invention discloses the Clenbuterol substrate standard substance first, and has set up the preparation method of Clenbuterol substrate standard substance.For residual of kelengtelu in the daily pig urine detects the magnitude tracing material is provided; Solved the testing result difference problem that matrix difference causes; Accurately guaranteed monitoring, not only protected the people's health, and imported and exported for China live pig the reliable detection substrate standard substance is provided residual of kelengtelu in the live pig product; Guaranteed the technological status in the interests in international trade and China commodity inspection work after the China joined WTO, social benefit has a far reaching influence; Also remedied simultaneously the blank spot of China aspect the Clenbuterol substrate standard substance.
In addition; Clenbuterol substrate standard substance of the present invention provides substrate standard substance for the test experience chamber; Detect the comparability of data between quality that raising detects and laboratory, particularly look into new reaching through Science and Technology of Shanghai Cha Xin referral centre of Chinese Academy of Sciences science and technology and seek advice from, series standard material of the present invention has good novelty and good application market is worth; Through analyzing, complex art of the present invention has reached leading domestic level.
Clenbuterol substrate standard substance of the present invention is investigated through homogeneity and stability, and it is satisfactory to investigate the back, carries out definite value and uncertainty evaluation.Evaluation result shows that the uncertainty that Clenbuterol substrate standard substance of the present invention causes is less, and it is comparatively stable to measure the result, can assess the quality of respectively measuring the Clenbuterol product on the market preferably, guarantees the accuracy of measurement result.
Description of drawings
Fig. 1 is the Clenbuterol substrate standard substance preparation method process flow diagram of embodiment 1.
Fig. 2 is a gold test strip bar testing result; A-E is that the pig of variable concentrations urinates Clenbuterol substrate standard substance in the freeze-dried powder, A:＜0.10ng/mL, B:0.50ng/mL, C:1.00ng/mL, D:3.00ng/mL, E:10.0ng/mL; F-G is WS Quality Control, F: negative control, G: positive control.
Embodiment
Mode through embodiment further specifies the present invention below, but does not therefore limit the present invention among the described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example according to conventional method and condition, or is selected according to catalogue.
Among the following embodiment, used major equipment and reagent:
Magnetic stirring apparatus (IKA); Freezing freeze dryer (LABCONCO); AP I 4000 liquid chromatography-mass spectrographies/mass spectrometer (American AB company); MS2 type vortex vortex mixer (IKA); 24 solid-phase extraction devices (Supelco); MCX mixed type cation exchange pillar (60mg/3mL) (CNW); Water-bath nitrogen dries up appearance (Zymark); PH meter (PHS-3C type, last Nereid section) KQ-600B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); (Milli-Q ultrapure water production system (Millipore, the U.S.).
Clenbuterol (BW3815); (purity is greater than 98%, Dr.Ehrenstorfer for Clenbuterol-D9; Methyl alcohol (chromatographically pure, German MERCK); Acetonitrile (chromatographically pure, German MERCK); Ammonium acetate (chemical pure, Dihua worker company limited is won in Tianjin); Glacial acetic acid (analyze pure, the cruel clever Fine Chemical Co., Ltd in Shanghai); Ammoniacal liquor (chemical pure, Shanghai development chemical industry two factories); Formic acid (analyze pure, Shanghai Ling Feng chemical reagent company limited); Perchloric acid (analyze pure, Shanghai gold deer chemical industry company limited); Clenbuterol (Clenbuterol) ELISA detection kit (Shanghai Jie Menbaolinmai bioengineering company limited).
Fresh healthy swine urine is provided by the supervision of Nanhui, Shanghai City animal health, and through the LC/MS/MS check, Clenbuterol content is less than 0.10ng/mL.
The development of Clenbuterol substrate standard substance in the embodiment 1 pig urine freeze-dried powder
The first step, the freeze-dried powder preparation
Preparation flow is referring to accompanying drawing 1.
1, material is prepared
The about 2L of fresh pig urine samples of the animal health supervision health pig of gathering in Nanhui, Shanghai City is put in and is transported to the laboratory in the ice chest at once.Utilize quantitative test filter paper room temperature (20 ℃) to filter the pig urine and remove solid matter, collect the pig urine samples after filtering in the 5L beaker, and to wherein adding a clean magneton, stirring 30min makes its abundant mixing under the fast 500rpm stirring with magnetic stirring apparatus.
Adopt LC/MS/MS and ELISA method that the pig urine samples of above-mentioned mixing is tested, Clenbuterol content is less than 0.10ng/mL, and as the matrix urine sample, it is subsequent use to be kept at 4 ℃ of refrigerators.
The Clenbuterol standard solution: adopt 100,000/ electronic balance take by weighing Clenbuterol standard substance 0.02000g and place beaker; Fully dissolve with ultrapure water; Be transferred in the 1L volumetric flask, be 20 μ g/mL Clenbuterol standard solution with ultrapure water constant volume mixing.Pipette 20 μ g/mL Clenbuterol standard solution 1mL with the transfer pipet of 1mL again and be transferred in the 10mL volumetric flask, be 2 μ g/mL Clenbuterol standard solution with ultrapure water constant volume mixing, it is subsequent use to be kept at 4 ℃ of refrigerators.
2, preparation contains the pig urine samples of Clenbuterol
According to selected magnitude level (X1＜0.10ng/mL, X2=0.50ng/mL, X3=1.00ng/mL, X4=3.00ng/mL, X5=10.00ng/mL); Adopt 2 μ g/mL Clenbuterol standard solution of preparation that the matrix urine is added, with the abundant stirring and evenly mixing of magnetic stirring apparatus.Choose 7 pig urine samples of each magnitude level; Detect Clenbuterol content with reference to GB/T22286-2008 " the mensuration liquid chromatography tandem mass spectrometry of multiple beta-receptor activator residual quantity in the animal derived food ", the pig urine samples that adds behind the mixing is carried out uniformity testing with the variance test method.After uniformity testing is qualified, utilize range to be distributed into the 1mL/ bottle for the liquid-transfering gun of 1mL.
The preparation of the pig urine samples of magnitude level X1＜0.10ng/mL Clenbuterol: above-mentioned matrix urine sample promptly is.
The preparation of the pig urine samples of magnitude level X2=0.50ng/mL Clenbuterol: utilizing range is that the liquid-transfering gun of 100 μ L accurately pipettes the above-mentioned 2 μ g/mL Clenbuterol standard solution of 50 μ L; Be transferred in the 200mL volumetric flask; Be placed in the beaker with matrix urine sample constant volume, stir 30min and make its abundant mixing stirring under the fast 500rpm with magnetic stirring apparatus.
The preparation of the pig urine samples of magnitude level X3=1.00ng/mL Clenbuterol: utilizing range is that the liquid-transfering gun of 100 μ L accurately pipettes the above-mentioned 2 μ g/mL Clenbuterol standard solution of 100 μ L; Be transferred in the 200mL volumetric flask; Be placed in the beaker with matrix urine sample constant volume, stir 30min and make its abundant mixing stirring under the fast 500rpm with magnetic stirring apparatus.
The preparation of the pig urine samples of magnitude level X4=3.00ng/mL Clenbuterol: utilizing range is that the liquid-transfering gun of 100 μ L accurately pipettes the above-mentioned 2 μ g/mL Clenbuterol standard solution of 150 μ L; Be transferred in the 200mL volumetric flask; Be placed in the beaker with matrix urine sample constant volume, stir 30min and make its abundant mixing stirring under the fast 500rpm with magnetic stirring apparatus.
The preparation of the pig urine samples of magnitude level X5=10.00ng/mL Clenbuterol: utilize range accurately to pipette above-mentioned 1mL 2 μ g/mL Clenbuterol standard solution for the liquid-transfering gun of 1mL; Be transferred in the 200mL volumetric flask; Be placed in the beaker with matrix urine sample constant volume, stir 30min and make its abundant mixing stirring under the fast 500rpm with magnetic stirring apparatus.
3, freeze-drying
The sample that branch is installed is placed on-70 ℃ of pre-freeze 24h, and the sample branch that will contain different magnitude level then is placed on and is transferred in the different vessels in the freeze drier, carries out vacuum freeze-drying 24h.Sample after the freeze-drying covers tight exterior and interior cover immediately and seals, and is transferred to-20 ℃ of preservations.The sample that is prepared into is Clenbuterol substrate standard substance in the pig urine freeze-dried powder.
Second step, the detection definite value of Clenbuterol content in the freeze-dried powder
1 experimental implementation part
1.1, the configuration of storing solution
Adopt 100,000/ electronic balance take by weighing an amount of Clenbuterol standard items, use methyl alcohol to be mixed with the standard inventory solution of mass concentration as 100ng/mL ,-20 ℃ of refrigerators are preserved subsequent use.
Adopt 100,000/ electronic balance take by weighing an amount of Clenbuterol-D9, use methyl alcohol to be mixed with in the Clenbuterol of mass concentration as 100ng/mL and mark working fluid ,-20 ℃ of refrigerators are preserved subsequent use.
1.2, the configuration of standard operation solution
Draw a certain amount of Clenbuterol storing solution and Clenbuterol-D9 storing solution; Dilute with water is configured to the standard operation solution that concentration is 0ng/mL, 0.1ng/mL, 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, and every milliliter of this standard operation solution contains mark 5.0ng in Clenbuterol-D9 isotope.
1.3, the pre-treatment of Clenbuterol substrate standard substance in the pig urine freeze-dried powder
(1) redissolves
The magnitude level that embodiment 1 is made is that the Clenbuterol substrate standard substance is placed equilibrium at room temperature 30min in the pig urine freeze-dried powder of X1～X5, beats the Packaging Bottle bottom then gently, a bottle interior lyophilized products is concentrated on bottle as far as possible at the bottom of.Take off bottle stopper lentamente, accurately add the 1.000mL ultrapure water, build bottle cap, jiggle bottle two to three times; Place 10min, slowly rotation is shaken bottle a bottle interior material is fully mixed again, placement 30min; Rotation is shaken again, places 10min again, and mixing at last for several times turns upside down.
(2) extract and purify
Precision is measured above-mentioned 1.000mL pig urine freeze-dried powder and is redissolved thing in the 50mL centrifuge tube; Add the 15mLpH=5.2 ammonium acetate; Abundant mixing, the interior mark of the Clenbuterol working fluid that adds 50 μ L 100ng/mL is added a cover concussion 15min in testing sample; Add 0.1mol/L perchloric acid solution 10mL again, fully mixing.
MCX mixed type cation exchange pillar (60mg/3mL) is connected on the solid-phase extraction device, uses 3mL methyl alcohol, 3mL water activation pillar successively.All be transferred to sample solution in the pillar then; Also thoroughly drain with 1mL methyl alcohol and 1mL 2% methanol aqueous solution washing pillar successively, use 7mL 5% ammoniacal liquor methanol solution wash-out at last, eluent is after 38 ℃ of water-baths dry up solvent with nitrogen; Accurately add 1.000mL acetonitrile solution (2:3); The whirlpool mixing through the organic filtering with microporous membrane of 0.22 μ m, supplies HPLC/MS/MS to measure.
1.4, instrument parameter and condition determination
(1) chromatographic condition
Chromatographic column: Atlantics dC18 (2.1*150mm, 5 μ m, Waters); Pre-column: Atlantics C18 (2.1*10mm, 5 μ m, Waters);
Column temperature: 30 ℃;
Moving phase: A phase: 0.1% aqueous formic acid, B phase: 0.1% formic acid acetonitrile solution
20%A+80%B; Flow velocity: 0.2mL/min;
Sampling volume: 20 μ L.
(2) mass spectrum condition
Molecular structure according to Clenbuterol; Select ESI (+) as the ionization pattern; Clenbuterol standard operation solution is mixed with the acetonitrile solution that concentration is 100ng/mL; Respectively conditions such as ionization voltage, taper hole voltage, source temperature, desolventizing temperature are optimized, adopted the full scan mode, its mass spectrum parameter is: electron spray ESI positron ionization pattern; Capillary voltage: 3.44kv; Taper hole voltage: 25v; RF lens: 0.2v; Ion source temperature: 100 ℃; Desolventizing temperature degree: 350 ℃; Taper hole air blowing flow: 20L/hr; Desolventizing airshed: 600L/hr; The monitoring ion pair is seen table 1:
Table 1 monitoring ion pair
| Measured object | Parent ion (m/z) | Daughter ion (m/z) | Quantitative daughter ion (m/z) |
| Clenbuterol | 277 | 203、259 | 203 |
| Clenbuterol-D9 | 286 | 204 | 204 |
After Clenbuterol substrate standard substance preparation is accomplished, can according to above-mentioned experiment condition to the homogeneity of Clenbuterol substrate standard substance test, stability is investigated, definite value and carry out probabilistic detection.
1.5 uniformity testing
According to JJG 1006-1994 " primary standard material technical manual " homogeneity draw samples requirement, Clenbuterol substrate standard substance (RM from the pig urine freeze-dried powder of 4 magnitude level of this batch
20.5ng/mL, RM
31ng/mL, RM
43ng/mL, RM
59ng/mL removes the blank RM of matrix
1Do not detect) randomly draw 15 bottles respectively, to get each sample replication 3 times.
1.6 study on the stability
Stable is the key link of biological species standard substance development, keeps the measure of sample stability must run through the overall process that standard substance is developed.Because standard substance in use unavoidably will relate to factors such as transportation, therefore, we have carried out long-time stability respectively to this cover standard substance and have investigated and the short-term stability investigation.
Long-time stability are investigated and are adopted classical stability study, and promptly As time goes on the sample of preparation is measured under the same conditions simultaneously.Respectively after the same day, 1 month are accomplished in specimen preparation, after 2 months, after 3 months, after 5 months, after 6 months, after 8 months, after 12 months to the standard substance (RM of 5 magnitude level of preparation
1, RM
2, RM
3, RM
4, RM
5) randomly draw 3 bottles, each sample replication 3 times is positioned under 4 ℃ the temperature and preserves, and carries out long-time stability and investigates.
Short-term stability is investigated and is adopted the synchronism stability Journal of Sex Research, even the measurement of all stability studies can be carried out under repeated condition, one-shot measurement is done primary calibration.The condition that the experimental simulation sample possibly run in transit is with the standard substance (RM of 5 magnitude level that prepare
1, RM
2, RM
3, RM
4, RM
5) respectively get 9 bottles, respectively be divided into 3 groups, 3 bags every group, be placed on respectively and test the investigation short-term stability simultaneously, each sample replication 3 times after-20 ℃, 4 ℃ and room temperature condition preserved for two weeks down.
1.7 standard substance definite value
Requirement according to ISO guide rule 35 " rule and the statistical method of standard substance/standard model definite value "; This batch of standard substance adopts the method for Duo Jia laboratory cooperation definite value; Invited 10 families such as Shanghai Entry-Exit Inspection and Quarantine bureau, Shanghai City food and medicine quality supervision institute, Shanghai City Center for Disease Control to have the common definite value of mechanism of Clenbuterol detectability, 5 magnitude level standard substance (RM that each unit randomly draws respectively
1, RM
2, RM
3, RM
4, RM
5) each 1 bottle, the definite value analysis is carried out in replicate determination 10 times.
2 investigate and the definite value result
2.1 uniformity testing result
Adopt method of analysis of variance (specifically with reference to ISO guide rule 35 " rule and the statistical method of standard substance/standard model definite value ") that this cover standard substance is carried out uniformity testing, the result sees table 2, and under 95% confidence level, the F value is all less than F
0.05(14,30) meet the primary standard material development requirement that evenly is up to the standards, and prove that this cover standard substance all is uniform.
Homogeneity statistics between the bottle of Clenbuterol substrate standard substance (RM) in the table 2 pig urine freeze-dried powder
2.2 study on the stability result
Clenbuterol substrate standard substance study on the stability result sees table 3, table 4 in the pig urine freeze-dried powder.
Short-term stability investigates the result and the homogeneity experimental result adopts the t check to compare, on 95% confidence level, and the two no significant difference, Clenbuterol refers under traffic condition to be stable in 14 days in the standard substance, satisfies the actual shipment requirement.
Clenbuterol substrate standard substance (RM) short-term stability assay in the table 3 pig urine freeze-dried powder
Clenbuterol substrate standard substance (RM) long-time stability assay in the table 4 pig urine freeze-dried powder
| The investigation time | RM 1(ng/mL) | RM 2(ng/mL) | RM 3(ng/mL) | RM 4(ng/mL) | RM 5(ng/mL) |
| The same day | Do not detect | 0.63 | 1.35 | 3.75 | 11.47 |
| One month | Do not detect | 0.58 | 1.28 | 3.69 | 11.11 |
| Two months | Do not detect | 0.60 | 1.09 | 3.43 | 10.10 |
| Three months | Do not detect | 0.60 | 1.21 | 3.79 | 11.44 |
| Five months | Do not detect | 0.64 | 1.26 | 3.78 | 11.09 |
| Six months | Do not detect | 0.55 | 1.35 | 3.74 | 10.58 |
| Eight months | Do not detect | 0.52 | 1.18 | 3.65 | 10.57 |
| 12 months | Do not detect | 0.59 | 1.26 | 3.94 | 11.66 |
Because do not have physics or chemical model can describe the mechanism of degradation of Clenbuterol in the pig urine freeze-dried powder truly, so select the empirical model of linear model as this standard substance for use, The statistical testing results is seen table 5.In fact the characteristic value (Clenbuterol concentration) in this model (pig urine freeze-dried powder), people expect that the intercept (in range of uncertainty) of straight line equals the definite value result of this standard substance; Simultaneously, the slope of straight line levels off to zero.Can know long-time stability by table 5 | β
1|＜t
0.95,6* s (β
1), slope is not remarkable, shows that Clenbuterol does not all have significantly rising or downtrending at the appointed time in this cover; And variation range is all in characteristic value and range of uncertainty thereof; Therefore, under 4 ℃ of conditions, preserve, can judge in 12 months it is stable.
Clenbuterol substrate standard substance (RM) long-time stability statistics in the table 5 pig urine freeze-dried powder
2.3 standard substance definite value and uncertain assessment
2.3.1 the definite value of standard substance
The Clenbuterol substrate standard substance adopts 8 tame laboratories to work in coordination with definite value in the pig urine freeze-dried powder; Testing result is through Ke Kelun check and the check of lattice cloth Lars; The data of all unit all can keep and participate in final definite value, and the mean value and the population mean of each laboratory detection result are seen table 6.
Clenbuterol substrate standard substance definite value result in the table 6 pig urine freeze-dried powder
2.3.2 the standard substance definite value is uncertainty evaluation as a result
The uncertainty source of Clenbuterol substrate standard substance is made up of four parts in the pig urine freeze-dried powder: the uncertainty that pig urine freeze-dried powder redissolution process causes; The uncertainty that the unevenness of standard substance causes; The uncertainty that standard substance mobility before the deadline causes; The uncertainty that the definite value process of standard substance is brought comprises the uncertainty of measuring process introducing and the uncertainty that the measurement data statistics is introduced.Statistics is seen table 7.
Clenbuterol substrate standard substance uncertainty tabulation in the table 7 pig urine freeze-dried powder
Combined standard uncertainty multiply by comprise factor k=2 (fiducial interval P=0.95), uncertainty is expanded.
According to the relevant rules regulation of standard substance development, we are through aforementioned a series of investigation and definite value, obtain finally that Clenbuterol substrate standard substance definite value result and uncertainty see table 8 in the pig urine freeze-dried powder.
Clenbuterol substrate standard substance definite value result in the table 8 pig urine freeze-dried powder
| Clenbuterol base in the pig urine freeze-dried powder | Definite value result (ng/mL) | Standard uncertainty (ng/mL) | Expanded uncertainty |
| The body standard substance | U(k＝2)(％) | ||
| RM 1 | Do not detect | / | / |
| RM 2 | 0.65 | 0.072 | 0.15 |
| RM 3 | 1.28 | 0.22 | 0.44 |
| RM 4 | 3.78 | 0.23 | 0.46 |
| RM 5 | 11.48 | 0.86 | 1.72 |
Effect embodiment 1 enzyme linked immunological kit detects Clenbuterol substrate standard substance in the pig urine freeze-dried powder
1, sample preparation
(1) redissolves
The magnitude level that before using embodiment 1 is made is placed equilibrium at room temperature 30min as Clenbuterol substrate standard substance in the pig urine freeze-dried powder of X1～X5, beats the Packaging Bottle bottom then gently, a bottle interior lyophilized products is concentrated on bottle as far as possible at the bottom of.Take off bottle stopper lentamente, accurately add the 1.000mL ultrapure water, build bottle cap, jiggle bottle two to three times; Place 10min, slowly rotation is shaken bottle a bottle interior material is fully mixed again, placement 30min; Rotation is shaken again, places 10min again, and mixing at last for several times turns upside down.
(2) centrifugal
With 4 ℃ of centrifugal 5min of above-mentioned pig urine freeze-dried powder redissolution thing 5000rpm, get supernatant and detect.
2, mensuration program (operating under 20 ℃～25 ℃ conditions)
(1), prepares the washing working fluid of suitable concn according to the enzyme labeling solution of kit instructions preparation suitable concn.
(2) take out the microwell plate that has fixed antibody and at room temperature placed 20 minutes, equilibrium temperature is to room temperature;
(3) add supernatant or the detected sample that the centrifugal pig that obtains of 50 μ L urinates Clenbuterol substrate standard substance in the freeze-dried powder in each reacting hole; Add enzyme labeling Clenbuterol working fluid subsidiary in the 100 μ L kits then, ELISA Plate is moved in a circle with abundant mixing in the plane.Wherein two nanopores only add 150 μ L water, as blank; With the shrouding film microwell plate is sealed up, hatched 30 minutes in 20 ℃～25 ℃;
(4) pour out liquid in the micropore, and do to guarantee the removing liquid in the micropore fully at the thieving paper arsis, charge in the hole with 250 μ L washing lotions, outwell liquid in the micropore once more, repetitive operation twice again, and is dried at the thieving paper arsis at last;
(5) add substrate solution subsidiary in the 100 μ L kits in every hole, microwell plate is sealed up, hatched 20 minutes for 20 ℃～25 ℃ with the shrouding film;
(6) add reaction terminating liquid subsidiary in the 100 μ L kits in every hole, fully mixing with ELIASA reading under 450nm, is deducted blank and drawing standard curve.
3, result and discussion
All are detected the absorbance of the signal deduction blank in hole; Utilize the data of gained to set up working curve then; As shown in table 9, the logarithm of absorbance A and Clenbuterol concentration C is good linear relationship: A=0.553-0.512lgC, correlation coefficient r=0.996.
Table 9 working curve data
| CLB concentration (ng/mL) | 0 | 0.1 | 0.3 | 0.9 | 2.7 | 8.1 |
| Absorbance (O.D. value) | 1.083 | 1.046 | 0.873 | 0.546 | 0.309 | 0.107 |
With the working curve of above-mentioned foundation, calculate the mensuration result of Clenbuterol substrate standard substance in the pig urine freeze-dried powder, as shown in table 10:
Clenbuterol substrate standard substance (RM) is measured the result in the table 10 pig urine freeze-dried powder
Testing result and standard substance value relatively can be found; There is some difference for result that the kit detection obtains and standard substance value; Between 85%～125%, reference reagent box quality standard, this association can be used as the significant data of assessment kit quality through the statistics error of indication.Therefore, the Clenbuterol substrate standard substance can be used to guarantee the accuracy of measurement result in the pig urine freeze-dried powder of our development.
Effect embodiment 2 gold mark detection test paper methods detect Clenbuterol substrate standard substance in the pig urine freeze-dried powder
1, sample preparation is with effect embodiment 1
2, mensuration program (operating under 20～25 ℃ of conditions of room temperature)
Mainly operate based on product description:
(1) test card is taken out from packaging bag, lie against experiment table.
(2) get about 100 μ L samples with transfer pipet, be added drop-wise to the well place of test card.React and read the result in 8～10 minutes.
3, result and discussion
We detect Clenbuterol substrate standard substance appearance in the pig urine freeze-dried powder of five kinds of magnitude level; For testing result is proved conclusively; We have detected one group of Quality Control appearance simultaneously, comprise negative control (blank water sample) and positive control 10ng/mL Clenbuterol water sample.
Testing result is as shown in Figure 1: each detects on the test paper has two lines, a nature controlling line (C), and a response line (T), the C line all demonstrates redness on all test paper, represents gold size that normal immune response takes place, and explains that test paper is effectively available.Concrete testing result is divided into two groups: A-E represents series concentration pig urine examination result, from A to E, represents the CLB concentration that contains in the pig urine sample to be successively:＜0.10ng/mL, 0.50ng/mL, 1.00ng/mL; 3.00ng/mL 10.00ng/mL is based on competition immune response principle; CLB is many more in the pig urine sample, and the color of T line is shallow more, when CLB concentration reaches 1.00ng/mL; The T line color obviously shoals, when reaching 3.00ng/mL, and the complete obiteration of T line; F represents blank water sample negative control, and the T line has tangible redness, and G represents 10.00ng/mL WS matter positive control, the complete obiteration of T line.
Concrete gold size test strips testing result is seen Figure of description 2.
Clenbuterol substrate standard substance phenomenon clearly can detect the standard substance of Clenbuterol content more than or equal to 3.00ng/mL in this gold test strip bar detection pig urine freeze-dried powder.With reference to relevant criterion (YY/T1164-2009 human chorionic gonadotrophin (HCG) detects test paper (colloidal gold immunity chromatography)), this result can be used for the quality evaluation of gold size test strips.
Above-mentioned two effect embodiment utilize Clenbuterol substrate standard substance of the present invention respectively, and two kinds of detection methods have been carried out quality evaluation, and testing result shows that two kinds of methods can reach consistance preferably, meets request for utilization.
Claims (11)
1. the preparation method of a Clenbuterol substrate standard substance; It is characterized in that: it comprises the steps: the fresh pig urine of removing behind the solid matter is mixed; Add the Clenbuterol standard substance to magnitude level, mix again mixed solution, with this mixed solution behind uniformity testing; Packing, freeze drying gets final product again.
2. preparation method as claimed in claim 1 is characterized in that: described fresh pig urine is the pig urine of Clenbuterol residual volume＜0.10ng/mL.
3. preparation method as claimed in claim 1 is characterized in that: described method of removing solid matter is centrifugal separation or quantitative test filter paper filtering method.
4. preparation method as claimed in claim 1 is characterized in that: described mixing adopted the magnetic stirrer mixing.
5. preparation method as claimed in claim 4 is characterized in that: the stir speed (S.S.) of described magnetic stirring apparatus is 200～1000rpm; Mixing time is 10-40min.
6. preparation method as claimed in claim 1; It is characterized in that: the form of described Clenbuterol standard substance is the solid form or the solution form of Clenbuterol standard substance; Preferred Clenbuterol standard substance solution more preferably adds the WS of Clenbuterol standard substance.
7. preparation method as claimed in claim 1; It is characterized in that: described magnitude level is the concentration of Clenbuterol solution; With the ng/mL score be not: X1, X2, X3, X4, X5; Wherein X1=0～0.30, X2=0.30～0.80, X3=0.50～2.00, X4=1.50～8.00, X5=5.00～15.00, and meet the following conditions: X1＜X2＜X3＜X4＜X5; Preferred X1=0～0.10, X2=0.40～0.70, X3=1.00～1.80, X4=2.50～4.00, X5=10.00～13.00, more preferably X1＜0.10, X2=0.50, X3=1.00, X4=3.00, X5=10.00.
8. preparation method as claimed in claim 1 is characterized in that: the instrument that uses during packing is transfer pipet, bottleneck knockout or liquid-transfering gun; Described lyophilization comprise the steps: with through the mixed solution of uniformity testing-60 ℃～-80 ℃ pre-freezes, move into then in the freeze drier, freeze-drying gets final product; Described freeze-drying is carried out freeze-drying for the product branch that will contain different magnitude level is placed in the different vessels; The time of described pre-freeze is 12～36h.
9. preparation method as claimed in claim 1 is characterized in that: behind the described lyophilization, the sample after the freeze-drying covers tight exterior and interior cover immediately and seals, and is transferred to-17～-23 ℃ of preservations.
10. like the prepared Clenbuterol substrate standard substance of the preparation method of each described Clenbuterol substrate standard substance of claim 1～9.
11. Clenbuterol substrate standard substance as claimed in claim 10 is characterized in that: described Clenbuterol substrate standard substance is used and sold or is exported by the concentration gradient of X1, X2, X3, X4 and X5 is complete; The concentration of described Clenbuterol substrate standard substance is respectively X1=0～0.10, X2=0.40～0.70, X3=1.00～1.80, X4=2.50～4.00, X5=10.00～13.00, in ng/mL.
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| CN106908537B (en) * | 2016-04-06 | 2020-01-21 | 中国计量科学研究院 | Preparation method of honey standard substance containing chloramphenicol |
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| CN112379005A (en) * | 2020-09-11 | 2021-02-19 | 司法鉴定科学研究院 | Freeze-dried quality control product for analyzing organic poison in human blood/urine and preparation method thereof |
| CN112903874A (en) * | 2021-01-25 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Free and conjugated clenbuterol standard substance contained in pig urine and preparation method thereof |
| CN112903874B (en) * | 2021-01-25 | 2023-02-17 | 中国农业科学院农产品加工研究所 | Free and conjugated clenbuterol standard substance contained in pig urine and preparation method thereof |
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