CN106442359A - Method for detecting total amino acid content in periplaneta americana solution - Google Patents
Method for detecting total amino acid content in periplaneta americana solution Download PDFInfo
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- CN106442359A CN106442359A CN201610887165.4A CN201610887165A CN106442359A CN 106442359 A CN106442359 A CN 106442359A CN 201610887165 A CN201610887165 A CN 201610887165A CN 106442359 A CN106442359 A CN 106442359A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N2021/3185—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry typically monochromatic or band-limited
Abstract
The invention provides a method for detecting total amino acid content in a periplaneta americana solution, which comprises the following steps: providing an alanine reference solution with a concentration of 0.014-0.018 mg/mL; diluting a to-be-tested periplaneta americana solution with water, reacting with a ninhydrin solution to obtain a pretreated to-be-tested product, mixing with an ethanol solution, detecting by an ultraviolet spectrophotometric method, and measuring the absorbance of the to-be-test product at a 570 nm wavelength; operating the alanine reference solution at the same time and following the same method as the diluted to-be-tested product, and measuring the absorbance of the reference; and calculating the total amino acid content in the periplaneta americana solution with the following formula: the total amino acid content in the to-be-tested periplaneta americana solution=concentration of the alanine reference solution/absorbance of the reference * absorbance of the to-be-tested product * dilution ratio of the to-be-tested product. The detection method provided by the invention is simple and greatly reduces the detection time, and the precision and repeatability meet the requirements of production on the total amino acid content.
Description
Technical field
The present invention relates to amino acid detection techniques field, total amino acidss content in more particularly, to a kind of periplaneta americana solution
Detection method.
Background technology
Kangfuxin Liquid is a kind of surgery common medicine, and its raw material is periplaneta americana.Kangfuxin Liquid is clinically widely used
In burn, scald, war wound, traumatic infection and intractable ulcer, decubital ulcer etc., simple for use, preferably, this single variety produces curative effect
Product market sales volume is fine.Kangfuxin Liquid product is rich in 18 kinds of aminoacid such as alanine, leucine and L-Valine, therefore total amino acidss
Content is one of main evaluation index of product quality.The raw extract of Kangfuxin Liquid be periplaneta americana ethanol extraction, half
Finished product is that Kangfuxin Liquid treats that liquid filling and finished product Kangfuxin Liquid are both needed to carry out quality control to total amino acidss content, due to therein third
Histidine content proportion is maximum, therefore using alanine as the evaluation criteria of total amino acidss content.
In existing Kangfuxin Liquid, the detection method of total amino acidss content comprises the following steps：The preparation of reference substance solution：
Precision weighs the alanine reference substance 50mg in 105 DEG C of dryings to constant weight, is placed in 100mL measuring bottle, is diluted with water to scale, shakes
Even；Precision measures the solution after 5mL dilution shakes up, and is placed in 100mL measuring bottle, is diluted with water to scale, shake up, obtains final product.Standard
The foundation of curve：Precision measure described reference substance solution 0.0,0.2,0.4,0.6,0.8,1.0mL, be respectively placed in 10mL tool plug examination
Guan Zhong, adds water to 1mL；Then 0.2mol/L citrate buffer (pH5.0) 1.0mL, 1% ascorbic acid solution are sequentially added
0.1mL, 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution 3.0mL, shake up, and are placed in boiling water bath and heat 15 minutes, take out, let cool；Add
80% ethanol 3.0mL, shakes up, and using ultraviolet spectrophotometry test, measures trap at 570nm wavelength respectively, to absorb
Spend for vertical coordinate, concentration is abscissa, draws standard curve.The mensure of product to be tested：Precision measures Kangfuxin Liquid product to be tested 2mL,
It is placed in 100mL measuring bottle, is diluted with water to scale, shake up；Precision measures the solution after 1mL dilution shakes up, and is placed in 10mL tool plug
In test tube, then sequentially add 0.2mol/L citrate buffer (pH5.0) 1.0mL, 1% ascorbic acid solution 0.1mL, indenes three
Ketone ethylene glycol monomethyl ether test solution 3.0mL, shakes up, and is placed in boiling water bath and heats 15 minutes, takes out, lets cool；Add 80% ethanol
3.0mL, shakes up, and measures ultraviolet absorptivity at 570nm wavelength, reads aminoacid in product to be tested solution from described standard curve
Content, calculate, obtain final product.National Specification：The every 1mL of Kangfuxin Liquid finished product contains total amino acidss with alanine (C3H7NO2) meter,
0.72mg must not be less than.
Existing standard curve method can detect that the content of total amino acidss in Kangfuxin Liquid, but detection method operation is multiple
Miscellaneous, detection takes longer (typically in more than 2h), affects the production cycle.
Content of the invention
In view of this, the application provides a kind of detection method of total amino acidss content in periplaneta americana solution, and the application carries
For detection method easy and simple to handle, detection duration shorter, the production cycle is shorter.
The application provides a kind of detection method of total amino acidss content in periplaneta americana solution, comprises the following steps：
The alanine reference substance solution that concentration is 0.014mg/mL～0.018mg/mL is provided；
By periplaneta americana solution product to be tested dilute with water, the product to be tested after dilution and ninhydrin solution are reacted, and obtain
The product to be tested of pretreatment；Described alanine reference substance solution and described ninhydrin solution are equally reacted simultaneously, obtained pre-
The reference substance processing；
After the reference substance of the product to be tested of described pretreatment, pretreatment is mixed with ethanol solution respectively, using ultraviolet spectrometry
Photometry detects, respectively obtains the product to be tested trap at 570nm wavelength and reference substance trap；
Calculate according to formula shown in formula 1, obtain total amino acidss content in periplaneta americana solution；
Periplaneta americana solution product to be tested total amino acidss content=alanine reference substance solution concentration/reference substance trap * is treated
Survey product trap * product to be tested extension rate formula 1.
Preferably, described periplaneta americana solution is selected from Kangfuxin Liquid or Kangfuxin Liquid treats liquid filling；
Described ninhydrin solution is 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution.
Preferably, the product to be tested after described dilution and the reaction of ninhydrin solution exist in buffer and ascorbic acid solution
Under conditions of carry out；
Described buffer does not contain the group with ninhydrin reaction, and the pH value of described buffer is 5～7.
Preferably, described buffer is selected from phosphate buffer, phthalate buffer or citron acids buffer.
Preferably, described product to be tested extension rate is 50 times, and the consumption of the product to be tested after dilution is 1mL；
The pH value of described buffer is 5, and concentration is 0.2mol/L, and consumption is 1mL；
The mass concentration of described ascorbic acid solution is 1%, and consumption is 0.1mL；
The consumption of described ninhydrin solution is 3mL.
Preferably, the mass concentration of described ethanol solution is 80%, and consumption is 3mL.
Preferably, heating realization in boiling water bath is passed through in the product to be tested after described dilution and the reaction of ninhydrin solution.
Preferably, the time of described heating is 15min.
Compared with prior art, the application provide periplaneta americana solution in total amino acidss content detection method include with
Lower step：The alanine reference substance solution that concentration is 0.014mg/mL～0.018mg/mL is provided；Will be to be measured for periplaneta americana solution
Product dilute with water, the product to be tested after being diluted；Product to be tested after described dilution is reacted with ninhydrin solution, obtains pre- place
The product to be tested of reason, then by itself and ethanol solution mixing, according to ultraviolet spectrophotometry detection, record to be measured at 570nm wavelength
Product trap；Described alanine reference substance solution is operated with method with the product to be tested after dilution simultaneously, records reference substance trap；
Calculate according to formula shown in formula 1, obtain final product.The application utilizes the chromogenic reaction of total amino acidss and 1,2,3-indantrione monohydrate in periplaneta americana solution,
And amino acid concentration has good linear relationship, using single-point reference substance relative method with the ultraviolet absorptivity of 570nm wavelength
Detection obtains total amino acidss content in periplaneta americana solution, takes within 30min.Therefore, the detection method that the application provides
Easy and simple to handle, detection time can be greatly reduced, its precision and repeatability etc. all meet production and total amino acidss content is examined simultaneously
The requirement surveyed.
Brief description
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Have technology description in required use accompanying drawing be briefly described it should be apparent that, drawings in the following description be only this
Inventive embodiment, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing providing obtains other accompanying drawings.
The precision test broken line graph of the detection method that Fig. 1 provides for the embodiment of the present application.
Specific embodiment
Below the embodiment it is clear that described is clearly and completely described to the technical scheme in the embodiment of the present application
It is only a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the application, the common skill in this area
The every other embodiment that art personnel are obtained under the premise of not making creative work, broadly falls into the model of the application protection
Enclose.
This application provides in a kind of periplaneta americana solution total amino acidss content detection method, comprise the following steps：
The alanine reference substance solution that concentration is 0.014mg/mL～0.018mg/mL is provided；
By periplaneta americana solution product to be tested dilute with water, the product to be tested after dilution and ninhydrin solution are reacted, and obtain
The product to be tested of pretreatment；Described alanine reference substance solution and described ninhydrin solution are equally reacted simultaneously, obtained pre-
The reference substance processing；
After the reference substance of the product to be tested of described pretreatment, pretreatment is mixed with ethanol solution respectively, using ultraviolet spectrometry
Photometry detects, respectively obtains the product to be tested trap at 570nm wavelength and reference substance trap；
Calculate according to formula shown in formula 1, obtain total amino acidss content in periplaneta americana solution；
Periplaneta americana solution product to be tested total amino acidss content=alanine reference substance solution concentration/reference substance trap * is treated
Survey product trap * product to be tested extension rate formula 1.
This application provides a kind of quick detection skill of total amino acidss content (Kangfuxin Liquid and periplaneta americana extracting solution etc.)
Art, efficiency high.
The embodiment of the present application is prepared into the alanine reference substance solution that concentration is 0.014mg/mL～0.018mg/mL, concentration
Optimal in 0.016mg/mL.The source of described alanine reference substance can be Chinese pharmaceutical biological product identification institute (140680-
201002), content is 100.0%.
The embodiment of the present application precision measures periplaneta americana solution product to be tested, is placed in measuring bottle and is diluted with water to scale, shakes up,
Product to be tested after being diluted.
In this application, described periplaneta americana solution includes periplaneta americana extracting solution such as periplaneta americana ethanol extract or dilute
American-cockroach-extract, Kangfuxin Liquid (finished product) and the Kangfuxin Liquid of releasing dissolving treat liquid filling (Kangfuxin Liquid semi-finished product, total amino acidss
Requirement controls in more than 0.77mg/mL) etc..Periplaneta americana solution described in the embodiment of the present application is selected from Kangfuxin Liquid or rehabilitation is new
Liquid treats liquid filling, and preferably Kangfuxin Liquid treats liquid filling.
In this application, described product to be tested extension rate is preferably 50 times, and such as precision measures product to be tested 2mL, is placed in 100mL
In measuring bottle, it is diluted with water to scale, shake up, the product to be tested after being diluted.The embodiment of the present application precision measures treating after dilution
Survey product 1mL, is placed in 10mL tool plug test tube.
After product to be tested after being diluted, the embodiment of the present application adds ninhydrin solution to be reacted, and obtains pretreatment
Product to be tested；Described alanine reference substance solution and described ninhydrin solution are equally reacted simultaneously, obtained the right of pretreatment
According to product.
The application adds ninhydrin solution, and the chromogenic reaction using total amino acidss in periplaneta americana solution and 1,2,3-indantrione monohydrate is carried out
The detection of total amino acidss content.In embodiments herein, described ninhydrin solution is 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution.This
Application is not particularly limited to the source of described ninhydrin solution, specifically, the preparation of described 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution
Method includes：Take 2.5g 1,2,3-indantrione monohydrate, add 50mL ethylene glycol monomethyl ether to make dissolving, the potassium cyanide separately taking concentration to be 0.01mol/L is molten
Liquid 5mL, spent glycol methyl ether is diluted to 250mL, will be molten to above-mentioned 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether solution and potassium cyanide ethylene glycol monomethyl ether
Liquid both solution mixes, and places and obtains final product test solution in 24 hours, and described test solution preserves in dark place at 2 DEG C～8 DEG C, and storage is treated for one week
With；The consumption of described 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution can be 3mL.
In a preferred embodiment of the invention, the reaction of product to be tested and the ninhydrin solution after described dilution in buffer and
Ascorbic acid solution is carried out under conditions of existing, and obtains the product to be tested of pretreatment.Wherein, the quality of described ascorbic acid solution is dense
Degree is preferably 1%, and consumption is preferably 0.1mL.
Described buffer is general not to be contained with the group such as amido of ninhydrin reaction etc., and the pH value of described buffer is preferably
5～7；Described buffer preferably is selected from phosphate buffer, phthalate buffer or citron acids buffer such as citric acid
Buffer.In the preferred embodiment of the application, the pH value of described buffer is 5, and concentration can be 0.2mol/L, and consumption can be
1mL.
In an embodiment of the application, described buffer is potassium dihydrogen phosphate-sodium hydroxide solution, and pH value is 6.8,
Compound method can be：The potassium dihydrogen phosphate of 25mL 0.2mol/L is mixed homogeneously with 23.6mL 0.1mol/L sodium hydroxide, plus
Water is diluted to 100mL.In an embodiment of the application, the phosphate buffer that described buffer is 5 for pH value, preparation side
Method can be：Take 0.2mol/L sodium dihydrogen phosphate a certain amount of, adjust pH value to 5 with sodium hydroxide test solution, obtain final product.In the application
An embodiment in, the phthalate buffer that described buffer is 5.6 for pH value, compound method can be：Take adjacent benzene
Diformazan potassium hydrogen phthalate 10g, add water 900mL, is stirred to dissolve, with sodium hydroxide test solution (if necessary use dilute hydrochloric acid) adjust pH value to
5.6, it is diluted with water to 1000mL, mix, obtain final product.In an embodiment of the application, described buffer is 5.8 for pH value
Phosphate buffer, compound method can be：Take potassium dihydrogen phosphate 8.34g and dipotassium hydrogen phosphate 0.87g, add water and make to be dissolved into
1000mL, obtains final product.In an embodiment of the application, the citrate buffer that described buffer is 5 for pH value, concentration is
0.2mol/L, consumption is 1mL.In an embodiment of the application, the citrate buffering that described buffer is 6.2 for pH value
Liquid, compound method can be：Take citric acid 4.2g, plus 20% ethanol hydrogen production sodium hydroxide solution 40mL of 1mol/L makes dissolving, then use
20% ethanol dilution, to 100mL, obtains final product；Or, take 2.1% aqueous solution of citric acid, adjust pH value with 50% sodium hydroxide solution
To 6.2, obtain final product.In an embodiment of the application, the phosphate buffer that described buffer is 6.5 for pH value, preparation side
Method can be：Take potassium dihydrogen phosphate 0.68g, plus 0.1mol/L sodium hydroxide solution 15.2mL, it is diluted with water to 100mL, obtain final product.?
In one embodiment of the application, the phosphate buffer that described buffer is 6.6 for pH value, compound method can be：Take phosphoric acid
Sodium dihydrogen 1.74g, disodium hydrogen phosphate 2.7g and sodium chloride 1.7g, add water and make to be dissolved into 400mL, obtain final product.
In a preferred embodiment of the invention, the product to be tested after described dilution and the reaction of ninhydrin solution are passed through in boiling water
In bath, heating is realized, and takes out reaction vessel, let cool, obtain the product to be tested of pretreatment after heating certain time.Described boiling water bath is
Well-known to those skilled in the art, temperature is 98 DEG C～100 DEG C；The time of described heating is preferably 15min.
The embodiment of the present application precision measures described alanine reference substance solution, grasps with method with the product to be tested after dilution simultaneously
Make, as equally reacted described alanine reference substance solution and described ninhydrin solution simultaneously, obtain the comparison of pretreatment
Product.Here, not repeating to the process of the reference substance obtaining pretreatment.
After obtaining the product to be tested of pretreatment and the reference substance of pretreatment, the embodiment of the present application adds ethanol solution respectively,
Shake up, according to ultraviolet spectrophotometry detection, respectively obtain the product to be tested trap at 570nm wavelength and reference substance trap；
Finally calculate according to formula shown in formula 1, obtain final product total amino acidss content in periplaneta americana solution.
In an embodiment of the application, the mass concentration of described ethanol solution is 80%, and consumption can be 3mL.Ultraviolet
(visible ray) spectrophotography, is that the absorption of the electromagnetic wave for this scope of 200nm～760nm to wavelength is special according to material molecule
One kind that property is set up is qualitative, quantitation and structure analysis method.The application adopts ultraviolet spectrophotometry, using aminoacid
Concentration has good linear relationship with the ultraviolet absorptivity of 570nm wavelength, detects that America is big by single-point reference substance relative method
Total amino acidss content in Lian solution.The application is not particularly limited to the detection of described ultraviolet spectrophotometry, at 570nm wavelength
Record the trap of product to be tested and reference substance with method simultaneously.
Obtain the trap of product to be tested and reference substance, the application calculates according to formula shown in formula 1, obtains periplaneta americana solution
Middle total amino acidss content；
Periplaneta americana solution product to be tested total amino acidss content=alanine reference substance solution concentration/reference substance trap * is treated
Survey product trap * product to be tested extension rate formula 1.In formula 1, " * " represents multiplication sign.
The detection method that the application provides is applicable to test in laboratory specific total amino acidss content, easy and simple to handle, energy
Greatly reduce detection time, its precision and repeatability etc. all meet the requirement producing to total amino acidss content detection simultaneously.
For a further understanding of the application, with reference to amino total in the periplaneta americana solution that embodiment provides to the application
The detection method of acid content is specifically described.
The source of alanine reference substance used by following examples is Chinese pharmaceutical biological product identification institute (140680-
201002), content is 100.0%；Kangfuxin Liquid and Kangfuxin Liquid semi-finished product are Hunan Cologne Pharmaceutical Co., Ltd and produce；Purple
Outer spectrophotometer model TU-1950 (Beijing Pu Xi company)；The compound method of 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution used
Including：Take 2.5g 1,2,3-indantrione monohydrate, add 50mL ethylene glycol monomethyl ether to make dissolving, separately take the potassium cyanide solution that concentration is 0.01mol/L
5mL, spent glycol methyl ether is diluted to 250mL, by above-mentioned 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether solution and potassium cyanide ethylene glycol monomethyl ether solution
Both solution mix, place obtain final product test solution within 24 hours, described test solution 2 DEG C～8 DEG C in dark place preserve, storage one week stand-by.
Embodiment 1 precision test
Precision weighs alanine reference substance 16mg, is placed in 100mL measuring bottle, is diluted with water to scale, shakes up；Accurate amount again
Take the reference substance 5mL after dilution, be placed in 100mL measuring bottle, be diluted with water to scale, shake up, obtain final product that concentration is 16 μ g/mL third
Propylhomoserin reference substance solution.
Precision measures Kangfuxin Liquid (M140941) product to be tested 2mL, takes 10 parts (1 part is done 1 test), is respectively placed in 100mL
In measuring bottle, it is diluted with water to scale, shake up；Precision measures the product to be tested 1mL after dilution respectively, is placed in 10mL tool plug test tube,
Sequentially add 0.2mol/L citrate buffer (pH5.0) 1.0mL, 1% ascorbic acid solution 0.1mL, 1,2,3-indantrione monohydrate ethylene glycol first
Ether test solution 3.0mL, shakes up, and is placed in boiling water bath and heats 15 minutes, takes out, lets cool；Add 80% ethanol 3.0mL, shake up, press
According to ultraviolet spectrophotometry test, measure trap at 570nm wavelength.
Precision measures described alanine reference substance solution 1mL, is placed in 10mL tool plug test tube, same with the product to be tested after dilution
Shi Tongfa operates, and measures trap.
Calculate according to formula shown in formula 1, obtain total amino acidss (in terms of alanine) content in described Kangfuxin Liquid, result is joined
It is shown in Table 1 and Fig. 1, the Precision test result of the detection method that table 1 provides for the embodiment of the present application, Fig. 1 is the embodiment of the present application
The precision test broken line graph of the detection method providing.
The Precision test result of the detection method that table 1 the embodiment of the present application provides
With this method of croup, data in table 1 and Fig. 1 is accepted or rejected, measuring meansigma methodss for ten times is 0.81992mg/mL, pole
Difference is 0.0171, and standard deviation is 0.00601, and relative standard deviation is 0.73%.
Inspection maximum：T=(0.8277-0.81992)/0.00601=1.29；
Inspection minima：T=(0.81992-0.8106)/0.00601=1.55；
Table look-up and understand, T0.99,10=3.25；
Because the T of maximum, minima all meets T ＜ T0.99,10, therefore maximum, minima should retain, and carry out statistical computation.Meter
Calculate result to include：
The precision of detection method provided in an embodiment of the present invention is：RSD=0.73%；
According to Precision Experiment result, look into mensure frequency n=10；
Level of significance α=0.01 (confidence level is 99%)；
Confidence coefficient T=3.17；
The fiducial limit of single measurements is：X ± TS=0.81992 ± 0.01905；
According to single measurements fiducial range, determine that relative error standard is：
TS/X=± 0.01905/0.81992*100%=± 2.3%.
It follows that the application has 99% assurance, the relative error of the data of parallel assay is less than twice
2.3%.
Embodiment 2 recovery test
Precision measures Kangfuxin Liquid (M140516) product to be tested 2mL, is placed in 100mL measuring bottle, is diluted with water to scale, shakes
Even；Precision measures the product to be tested 1mL after dilution, is placed in 10mL tool plug test tube, sequentially adds 0.2mol/L citrate buffer
(pH5.0) 1.0mL, 1% ascorbic acid solution 0.1mL, 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution 3.0mL, shake up, are placed in boiling water bath
Heating 15 minutes, takes out, lets cool；Add 80% ethanol 3.0mL, shake up, according to ultraviolet spectrophotometry test, in 570nm
Trap is measured at wavelength.
Precision measures alanine reference substance solution 1mL that concentration is 16 μ g/mL, is placed in 10mL tool plug test tube, with dilution
Product to be tested afterwards operates with method simultaneously, measures trap.Calculate according to formula shown in formula 1, obtain total ammonia in described Kangfuxin Liquid
Base acid (in terms of alanine) content.
The Kangfuxin Liquid M140604 (content is 0.80mg/mL) recording total amino acidss content is made solvent, takes 9 parts of (numberings
For 1 to 9), every part of 100mL, dissolving, the alanine reference substance of dilution different quality respectively, then adopt above-mentioned uv-spectrophotometric
Method measures and extrapolates the actual measurement quality of reference substance.The detection method that result provides for the embodiment of the present application referring to table 2, table 2
Recovery test result.
The recovery test result of the detection method that table 2 the embodiment of the present application provides
Actual measured amount refers to the standard substance actual measured amount after removing sample measurement value, and described sample measurement value is 0.80mg/mL.
Statistical analysiss are carried out to the experimental result of table 2 accuracy, analysis result includes：Average recovery rate is 100.58%,
Relative standard deviation is 0.94%, reaches the required precision producing to inspection；The response rate is between 98.0%～102.0%.By
This understands, the detection method accuracy that the embodiment of the present application provides is good.
Embodiment 3
Precision weighs alanine reference substance 16mg, is placed in 100mL measuring bottle, is diluted with water to scale, shakes up；Accurate amount again
Take the reference substance 5mL after dilution, be placed in 100mL measuring bottle, be diluted with water to scale, shake up, obtain final product that concentration is 16 μ g/mL third
Propylhomoserin reference substance solution.
Precision measures Kangfuxin Liquid (M140516) product to be tested 2mL, is placed in 100mL measuring bottle, is diluted with water to scale, shakes
Even；Precision measures the product to be tested 1mL after dilution, is placed in 10mL tool plug test tube, sequentially adds 0.2mol/L citrate buffer
(pH5.0) 1.0mL, 1% ascorbic acid solution 0.1mL, 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution 3.0mL, shake up, are placed in boiling water bath
Heating 15 minutes, takes out, lets cool；Add 80% ethanol 3.0mL, shake up, according to ultraviolet spectrophotometry test, in 570nm
Trap is measured at wavelength.
Precision measures described alanine reference substance solution 1mL, is placed in 10mL tool plug test tube, same with the product to be tested after dilution
Shi Tongfa operates, and measures trap.Calculate according to formula shown in formula 1, obtain in described Kangfuxin Liquid total amino acidss (with alanine
Meter) content.
According to the method described above, check above-mentioned Kangfuxin Liquid product to be tested respectively by two coroners, check 10 times, result referring to
Table 3, table 3 is the testing result to Kangfuxin Liquid M140516 and M140627 for the embodiment of the present invention.
Embodiment 4
By the detection method of embodiment 3, check Kangfuxin Liquid (M140627) product to be tested respectively by described two coroners,
Inspection 10 times, result is referring to table 3.
The testing result to Kangfuxin Liquid M140516 and M140627 for table 3 embodiment of the present invention
Statistical analysiss are carried out to table 3 assay, referring to table 4, table 4 is the embodiment of the present invention to Kangfuxin Liquid to analysis result
The statistic analysis result of M140516 and M140627 testing result.
The statistic analysis result to Kangfuxin Liquid M140516 and M140627 testing result for table 4 embodiment of the present invention
Find out from above-mentioned data：The embodiment of the present application detection data all can meet≤and 2.3% relative error requires, RSD
All meet the precision requirement of production, and be 25min through each sample determination time of statistical average.
Embodiment 5
Precision measures the Kangfuxin Liquid semi-finished product product to be tested 2mL that lot number is M140604, is placed in 100mL measuring bottle, dilute with water
Release to scale, shake up；Precision measures the product to be tested 1mL after dilution, is placed in 10mL tool plug test tube, sequentially adds 0.2mol/L Chinese holly
Rafter acid buffer (pH5.0) 1.0mL, 1% ascorbic acid solution 0.1mL, 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution 3.0mL, shake up, put
Heat 15 minutes in boiling water bath, take out, let cool；Add 80% ethanol 3.0mL, shake up, according to ultraviolet spectrophotometry examination
Test, measure trap at 570nm wavelength, its result is 0.4755.
Precision measures alanine reference substance solution 1mL that concentration is 0.016mg/mL, is placed in 10mL tool plug test tube, and dilute
Product to be tested after releasing operates with method simultaneously, measures trap, and its result is 0.4730.
Calculate according to formula shown in formula 1：
Semi-finished product product to be tested total amino acidss content=0.016/0.4730*0.4755*50=0.80mg/mL；
In described Kangfuxin Liquid semi-finished product, total amino acidss (being counted with alanine) content, as 0.80mg/mL, is met and is no less than
The regulation of 0.77mg/mL.Described semi-finished product detection level is qualified, can fill finished product.
Embodiment 6
Precision measure Kangfuxin Liquid (concentration known the be 0.81mg/mL) 0.5mL that lot number is M140516,1mL, 2mL,
4mL, 6mL, are respectively placed in 100mL measuring bottle, are diluted with water to scale, shake up, be respectively prepared testing sample solution 1.～5.；Point
Precision does not measure the product to be tested 1mL after dilution, is placed in 10mL tool plug test tube, sequentially adds 0.2mol/L citrate buffer
(pH5.0) 1.0mL, 1% ascorbic acid solution 0.1mL, 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution 3.0mL, shake up, are placed in boiling water bath
Heating 15 minutes, takes out, lets cool；Add 80% ethanol 3.0mL, shake up, according to ultraviolet spectrophotometry test, in 570nm
Trap is measured at wavelength.
It is 0.004mg/mL, 0.008mg/mL, 0.016mg/mL, 0.032mg/mL, 0.048mg/mL that precision measures concentration
Alanine reference substance solution 1mL, be placed in 10mL tool plug test tube in, with dilution after product to be tested simultaneously with method operate, measure inhale
Receipts degree, and calculate product to be tested content, result such as table 5 according to formula shown in formula 1：
Table 5 embodiment of the present invention adopts the statistic analysis result of variable concentrations reference substance solution testing result
Find out from above-mentioned data：When reference substance concentration is 0.016mg/mL, detected value is connect the most with sample concentration known
Closely.
As seen from the above embodiment, the application obtains periplaneta americana solution (as health using the detection of single-point reference substance relative method
Multiple new liquid finished product and semi-finished product) in total amino acidss content, take within 30min.The detection method operation letter that the application provides
Just, detection time can be greatly reduced, its precision and repeatability etc. all meet production and total amino acidss content detection is wanted simultaneously
Ask.
Claims (8)
1. in a kind of periplaneta americana solution total amino acidss content detection method, comprise the following steps：
The alanine reference substance solution that concentration is 0.014mg/mL～0.018mg/mL is provided；
By periplaneta americana solution product to be tested dilute with water, the product to be tested after dilution and ninhydrin solution are reacted, and obtain pre- place
The product to be tested of reason；Described alanine reference substance solution and described ninhydrin solution are equally reacted simultaneously, obtained pretreatment
Reference substance；
After the reference substance of the product to be tested of described pretreatment, pretreatment is mixed with ethanol solution respectively, using uv-spectrophotometric
Method detects, respectively obtains the product to be tested trap at 570nm wavelength and reference substance trap；
Calculate according to formula shown in formula 1, obtain total amino acidss content in periplaneta americana solution；
Periplaneta americana solution product to be tested total amino acidss content=alanine reference substance solution concentration/reference substance trap * product to be tested
Trap * product to be tested extension rate formula 1.
2. detection method according to claim 1 is it is characterised in that described periplaneta americana solution is selected from Kangfuxin Liquid or health
Multiple new liquid treats liquid filling；
Described ninhydrin solution is 1,2,3-indantrione monohydrate ethylene glycol monomethyl ether test solution.
3. detection method according to claim 1 is it is characterised in that product to be tested after described dilution and ninhydrin solution
Reaction is carried out under conditions of buffer and ascorbic acid solution exist；
Described buffer does not contain the group with ninhydrin reaction, and the pH value of described buffer is 5～7.
4. detection method according to claim 3 is it is characterised in that described buffer is selected from phosphate buffer, adjacent benzene
Diformate buffer or citron acids buffer.
5. detection method according to claim 3 is it is characterised in that described product to be tested extension rate is 50 times, after dilution
Product to be tested consumption be 1mL；
The pH value of described buffer is 5, and concentration is 0.2mol/L, and consumption is 1mL；
The mass concentration of described ascorbic acid solution is 1%, and consumption is 0.1mL；
The consumption of described ninhydrin solution is 3mL.
6. detection method according to claim 5, it is characterised in that the mass concentration of described ethanol solution is 80%, is used
Measure as 3mL.
7. detection method according to claim 3 is it is characterised in that product to be tested after described dilution and ninhydrin solution
Reaction is realized by heating in boiling water bath.
8. detection method according to claim 7 is it is characterised in that the time of described heating is 15min.
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