CN106153811A - Periplaneta americana lyophilized powder method of quality control - Google Patents
Periplaneta americana lyophilized powder method of quality control Download PDFInfo
- Publication number
- CN106153811A CN106153811A CN201610627319.6A CN201610627319A CN106153811A CN 106153811 A CN106153811 A CN 106153811A CN 201610627319 A CN201610627319 A CN 201610627319A CN 106153811 A CN106153811 A CN 106153811A
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- CN
- China
- Prior art keywords
- solution
- lyophilized powder
- reference substance
- ethanol
- periplaneta americana
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 238000000034 method Methods 0.000 title claims abstract description 73
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- 238000003908 quality control method Methods 0.000 title claims abstract description 16
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- 235000001014 amino acid Nutrition 0.000 claims abstract description 38
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Classifications
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- 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
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- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
Landscapes
- Physics & Mathematics (AREA)
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Abstract
The invention discloses a kind of periplaneta americana lyophilized powder method of quality control, carry out aminoacid discriminating including utilizing thin layer chromatography, utilize UV-VIS spectrophotometry aminoacid and polypeptide to carry out assay, utilizes high performance liquid chromatography that uracil, hypoxanthine, creatinine are carried out assay.The periplaneta americana lyophilized powder method of quality control of the present invention, carries out periplaneta americana lyophilized powder quality detecting control in terms of aminoacid qualitative identification, aminoacid, polypeptide, gradient elution assay, amino acid composition analysis, polypeptide molecular weight mensuration etc. are several.The quality of periplaneta americana lyophilized powder is provided strong guarantee, ensures quality and the curative effect of later stage preparation with periplaneta americana lyophilized powder as intermediate.
Description
Technical field
The invention belongs to technical field of Chinese medicines, specifically, relate to a kind of periplaneta americana lyophilized powder method of quality control.
Background technology
Periplaneta americana (Periplaneta americana (L.)) is Insecta Pterigota Blattaria Blattidae Periplaneta
The dry polypide of insecticide, has another name called Blatta seu periplaneta, Shi Jiang, sliding worm, steals oil mother-in-law, tea woman, and the beginning of being used as medicine is loaded in Shennong's Herbal, calls " taste
Salty, cold.Main blood stasis, disease are hard, cold and heat, and removing mass gathers, laryngopharynx numbness, endogenous cold, loss of fecundity ", there is blood circulation promoting and blood stasis dispelling, removing toxic substances disappears infantile malnutrition, diuretic disappears
The effect such as swollen, is used for treating infantile malnutrition, furuncle, carbuncle, throat moth, innominate toxic swelling, syphilis and the disease such as poisonous snake, centipete bite
Sick.Containing plurality of active ingredients such as protein, aminoacid, peptides, saccharides, modern study shows, anticancer, improve immunity,
The aspects such as treatment cardiovascular disease, reparation skin have obvious action.
Cancer is to be only second to the second largest killer of the mankind of cardiovascular disease, serious harm human health, has been increasingly becoming weight
Big social public health problem.Doctor trained in Western medicine is to the treatment of tumor still based on operation, radiation and chemotherapy, and toxic and side effects is serious and wide
General, cause the deterioration of patient's body condition and the decline of quality of life, most of patients is because being impatient at and abandoning cure, the most extremely
In over-treatment.Chinese medicine plays immeasurable effect to the treatment of tumor, and malignant tumor belongs to the traditional Chinese medical science " cancer ", " rock ", " evil
Swollen ", the category such as " accumulation ", treatment by Chinese herbs malignant tumor is based on organic conception, and righting combines with eliminating evil, treating both the principal and secondary aspects of a disease, from
And heighten the effect of a treatment, there is the incomparable unique advantage of doctor trained in Western medicine (the common effect of multipath, Mutiple Targets and too many levels), gradually become
Focus for antitumor research.
Periplaneta americana has the resources advantage of high protein, and its internal gross protein value is between 20%-70%, the highest
In meats such as Carnis Sus domestica, beef, Carnis Gallus domesticus.Periplaneta americana also rich in amino acid/11 more than 8 kinds, including human body semi-dispensable amino acid 2 kinds and
Essential amino acid 7 kinds.Document reports that isolation identification goes out more than 50 kind of neuropeptide, such as neuropeptide proctodeum from periplaneta americana
Peptide, insect antimicrobial peptide etc..Wherein insect antimicrobial peptide is to have biological activity short chain polypeptides, and tool is to spies such as thermally-stabilised, broad-spectrum antiseptics
Point, can suppress kinds of tumor cells and the growth of animal carcasses tumor, and will not be to normal cell damage.Liao Ji is " cockroach
Oils and fats is in health food and application pharmaceutically and preparation method " (CN1500495A) points out: " cockroach contains oleic acid, linoleic acid,
The unsaturated fatty acids such as linolenic acid, the effects such as its content is up to 71.903%, has antibacterial and repairs wound, enhancing immunity ".
Covering pine year, burnt spring fragrant etc. uses GC-MS to be analyzed the extract of periplaneta americana, find containing more enol, olefin(e) acid class and
Alkane, polyhydric alcohol, macrolide.Additionally, possibly together with pheromone, saccharide (such as mucopolysaccharide etc.) in periplaneta americana body, enzyme,
Esterase, coenzyme A and abundant mineral and trace element.
Summary of the invention
In view of this, the technical problem to be solved is to provide a kind of periplaneta americana lyophilized powder quality control side
Method.
The invention discloses a kind of periplaneta americana lyophilized powder method of quality control, carry out amino including utilizing thin layer chromatography
Acid differentiates, utilizes ultraviolet visible spectrophotometry to carry out aminoacid and polypeptide assay, utilize high performance liquid chromatography pair
Uracil, hypoxanthine, creatinine carry out assay.
Further, described aminoacid discrimination method for weighing 0.5g periplaneta americana lyophilized powder, the ethanol 10ml adding 70%,
Ultrasonic 10min, filters, and collects filtrate, and water-bath volatilizes, and the ethanol 5ml that residue adds 70% dissolves, as need testing solution；
Take alanine, phenylalanine, glycine reference substance respectively, accurately weighed, put in brown measuring bottle, the ethanol adding 70%
Make the reference substance solution of 1mg/ml；
Test according to thin layer chromatography, draw each 1 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with body
Long-pending than be the n-butyl alcohol-formic acid-aqueous solution of 75:15:10 be developing solvent, launch after saturated 30min, take out, dry, spray with
0.2% ethanol solution of ninhydrin, 105 DEG C are heated to clear spot；
Observe silica gel g thin-layer plate, in test sample chromatograph, on position corresponding with reference substance chromatograph, show phase in the sunlight
Speckle with color.
Further, described method for determining content of peptides is:
Precision weighs bovine serum albumin reference substance (BSA) 51.80mg, puts in 100ml volumetric flask, adds distilled water and is dissolved to
Scale, shakes up, and is made into every 1ml reference substance solution containing 0.5180mg bovine serum albumin；
Precision weighs lyophilized powder 0.2g, puts in volumetric flask, adds the ethanol 2.0ml of 70%, accurately weighed weight, ultrasonic carries
Take 10min, let cool, supply the weight of less loss with 70% ethanol, shake up, take supernatant 1ml 70% ethanol constant volume and hold in 100ml
In measuring bottle, shake up, as need testing solution；
Take need testing solution and each 1ml of reference substance solution, add alkaline copper reagent 5.00ml respectively, mixing, rapidly join phenol
Reagent 0.5ml, quickly shakes up, 55 DEG C of water-bath 15min, takes out and is cooled to room temperature, after colour developing, according to spectrophotography, at 740nm
Measure absorbance, calculate and get final product.
Further, described Contents of Amino Acids method is:
Precision weighs 16.08mg alanine reference substance, puts in 100ml volumetric flask, and the isopropanol adding 10% dissolves, and dilutes
To scale, as mother solution；Precision measures described mother solution 4ml, to 50ml volumetric flask, is diluted with water to scale, is configured to every 1ml
Reference substance solution containing 12.86 μ g alanine；
Precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, adds the ethanol 10ml of 70%, accurately weighed weight, surpass
Sound extracts 15min, lets cool, supplies the weight of less loss with 70% ethanol, shakes all, filters, take subsequent filtrate 1ml 70% ethanol constant volume
In 100ml volumetric flask, shake up, as need testing solution；
Take in need testing solution and reference substance solution each 2ml to 10ml tool plug scale test tube, be separately added into distilled water extremely
2ml, adds 0.1ml freshly prepared 1%Vc solution, 3ml 1,2,3-indantrione monohydrate alcoholic solution, mixing, heats 15min, take in 100 DEG C of water-baths
Go out and be cooled to rapidly room temperature, add 60% ethanol to scale, at 570nm, measure absorbance, calculate and get final product.
Further, described uracil, hypoxanthine, creatinine carry out content assaying method and are
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；Methanol is flowing phase
A, 0.7% glacial acetic acid solution is that Mobile phase B carries out gradient elution；Detection wavelength is 254nm, column temperature 30 DEG C, flow velocity 0.7ml/
min；
Gradient is:
0.7% glacial acetic acid solution of 0.7% glacial acetic acid solution-99% of 0-15min 100%,
0.7% glacial acetic acid solution of 0.7% glacial acetic acid solution-92% of 15-40min 99%,
0.7% glacial acetic acid solution of 0.7% glacial acetic acid solution-45% of 40-50min 92%；
The preparation of reference substance solution: take uracil, hypoxanthine, inosine reference substance, accurately weighed, put in measuring bottle, add super
Pure water is made containing uracil 5.02 μ g/ml, hypoxanthine 25.2 μ g/ml, the mixing reference substance solution of inosine 127.5 μ g/ml；
The preparation of need testing solution: precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, distilled water 10ml, accurate title
Determining weight, supersound extraction 10min, let cool, supply the weight of less loss with distilled water, shake all, filter, precision measures subsequent filtrate 5ml,
Being evaporated, residue adds water and is settled in 10ml measuring bottle, and 0.45 μm membrane filtration takes subsequent filtrate as need testing solution；
Assay method: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, surveys
Fixed, calculate and get final product, lyophilized powder every gram must not be less than 0.22% containing uracil, hypoxanthine must not be less than 0.77%, creatinine must not
Less than 4.62%.
Compared with prior art, the present invention can obtain and include techniques below effect:
1) the periplaneta americana lyophilized powder method of quality control of the present invention, from aminoacid qualitative identification, aminoacid, polypeptide, core
Several aspects such as methods of glycosides assay, amino acid composition analysis, polypeptide molecular weight mensuration are to periplaneta americana lyophilized powder quality
Carry out detecting control.The quality of periplaneta americana lyophilized powder is provided strong guarantee, has ensured with periplaneta americana lyophilized powder and be
The quality of the later stage preparation of intermediate and curative effect.
2) the periplaneta americana lyophilized powder method of quality control of the present invention, improve further periplaneta americana decoction pieces, extract and
The quality standard of its preparation, has certain reference value to periplaneta americana decoction pieces, extract and preparation thereof.
Certainly, the arbitrary product implementing the present invention must be not necessarily required to reach all the above technique effect simultaneously.
Accompanying drawing explanation
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this
Bright schematic description and description is used for explaining the present invention, is not intended that inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 show the embodiment of the present invention 1 Central America big Lian lyophilized powder thin-layer chromatogram；
Fig. 2 show the embodiment of the present invention 2 Central America big Lian lyophilized powder UV scanning figure (reference substance)；
Fig. 3 show the embodiment of the present invention 2 Central America big Lian lyophilized powder UV scanning figure (test sample)；
Fig. 4 show bovine serum albumin canonical plotting in the embodiment of the present invention 2；
Fig. 5 show the embodiment of the present invention 3 Central America big Lian lyophilized powder UV scanning figure (reference substance)；
Fig. 6 show the embodiment of the present invention 3 Central America big Lian lyophilized powder UV scanning figure (test sample)；
Fig. 7 show alanine reference substance canonical plotting in the embodiment of the present invention 3；
Fig. 8 show in the embodiment of the present invention 4 mixing reference substance high-efficient liquid phase chromatogram；
Fig. 9 show the embodiment of the present invention 4 Central America big Lian lyophilized powder high-efficient liquid phase chromatogram；
Figure 10 show uracil canonical plotting in the embodiment of the present invention 4；
Figure 11 show hypoxanthine canonical plotting in the embodiment of the present invention 4；
Figure 12 show creatinine canonical plotting in the embodiment of the present invention 4.
Detailed description of the invention
Describe embodiments of the present invention in detail below in conjunction with drawings and Examples, thereby how the present invention is applied
Technological means solves technical problem and reaches the process that realizes of technology effect and can fully understand and implement according to this.
Embodiment 1 aminoacid differentiates
Weigh 0.5g periplaneta americana lyophilized powder, the ethanol 10ml adding 70%, ultrasonic 10min, filter, collect filtrate, water-bath
Volatilizing, the ethanol 5ml that residue adds 70% dissolves, as need testing solution.
Take alanine, phenylalanine, glycine reference substance respectively, accurately weighed, put in brown measuring bottle, the ethanol adding 70%
Make the reference substance solution of 1mg/ml.
Test according to thin layer chromatography, draw each 1 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with just
Butanol-formic acid-water (75:15:10) is developing solvent, launches after saturated 30min, takes out, dries, and spray is with 0.2% 1,2,3-indantrione monohydrate ethanol
Solution, 105 DEG C are heated to clear spot, inspect in the sunlight.
Result as it is shown in figure 1, figure 1 is alanine, 3 be phenylalanine, 5 for glycine；2,4,6 it is periplaneta americana and freezes
Dry powder.In figure in test sample chromatograph, on position corresponding with control medicinal material chromatograph, show the speckle of same color in the sunlight.
Embodiment 2 determining content of peptides
In periplaneta americana lyophilized powder, principle active component was component polypeptides, according to version " Chinese Pharmacopoeia " annex in 2010
Pertinent regulations under VA spectrophotometry item, reference literature data measures total polypeptide with bovine serum albumin for reference substance
Content.
Prepared by 2.1 reference substance solution
Precision weighs bovine serum albumin reference substance (BSA) 51.80mg, puts in 100ml volumetric flask, adds distilled water and is dissolved to
Scale, shakes up, and is made into every 1ml reference substance solution containing 0.5180mg bovine serum albumin.
The selection of 2.2 detection wavelength
Precision measures reference substance solution, and each 1ml of sample solution, in 10ml tool plug scale test tube, adds alkaline copper reagent
5.0ml, mixing, rapidly join phenol reagent 0.5ml, quickly shake up, 55 DEG C of water-bath 15min, take out and be cooled to room temperature, after colour developing,
It is scanned in the range of 400-800nm, See Figure 2 and 3.
From Fig. 2 and 3, reference substance solution, need testing solution all have absorption maximum at 740nm, therefore it is beautiful for selecting 740nm
The mensuration wavelength of continent big Lian polypeptide.
2.3 ranges of linearity are investigated
Accurate BSA reference substance solution 0 (blank) of drawing, 0.2,0.4,0.6,0.8,1.0ml, add distilled water and complement to
1.0ml.Add alkaline copper reagent 5.00ml, mixing, rapidly join phenol reagent 0.5ml, quickly shake up, 55 DEG C of water-bath 15min, take out
It is cooled to room temperature, after colour developing, according to spectrophotography (pharmacopeia version I portion in 2010 of middle Chinese republic, annex VA), at 740nm
Measuring absorbance, with absorbency Y as vertical coordinate, peptide concentration X is abscissa, draws standard curve, and result is shown in Fig. 4.Straight line returns
Return the equation to be: Y=0.0071X+0.087 (r=0.9993), show that bovine serum albumin is at 15.938 μ g/ml～79.692 μ g/
In ml, sample concentration is good with absorbance linear relationship.
Prepared by 2.4 test sample solution
2.4.1 the selection of extracting method
Precision weighs lyophilized powder 0.02g, puts in volumetric flask, adds the ethanol 2.0ml of 70%, and accurately weighed weight, with not
Same mode extracts 15min, lets cool, supplies the weight of less loss with 70% ethanol, shake up, and takes supernatant 1ml 70% ethanol fixed
It is dissolved in 100ml volumetric flask, shakes up, according to the method under 2.3, from " adding alkaline copper reagent 5.00ml ", measure extinction in accordance with the law
Degree, calculates, the results are shown in Table 1.
The comparison of table 1 extracting method
| Extracting method | Shaking | Ultrasonic |
| Content of peptides (%) | 65.25 | 67.89 |
As shown in Table 1, ultrasonic method is better than shaking, therefore selects supersound extraction.
2.4.2 the investigation of Extraction solvent
Precision weighs lyophilized powder 0.02g, adds water respectively, 70% ethanol, the 90% each 2.0m1 of ethanol, accurately weighed weight, super
Sound 15min, lets cool, and supplies the weight of less loss with corresponding solvent, with 2.3 lower methods, from " adding alkaline copper reagent 5.00ml "
Rise, measure absorbance in accordance with the law, calculate, the results are shown in Table 2.
The comparison of table 2 Extraction solvent
| Extraction solvent | Distilled water | 70% ethanol | 90% ethanol |
| Content of peptides (%) | 66.78 | 68.35 | 67.55 |
As shown in Table 2,70% ethanol extraction is more more than distilled water and 90% ethanol, therefore selects 70% ethanol as extraction
Solvent.
2.4.3 the investigation of Extraction solvent consumption
Precision weighs lyophilized powder 0.02g, is separately added into 70% ethanol of 2ml, 3ml, 4ml, and accurately weighed weight is ultrasonic
15min, lets cool, and supplies the weight of less loss with 70% ethanol, with 2.3 times methods, measures absorbance in accordance with the law, calculates content of peptides,
The results are shown in Table 3.
The investigation of table 3 Extraction solvent consumption
| Solvent load (ml) | 2 | 3 | 4 |
| Content of peptides (%) | 68.31 | 68.46 | 68.24 |
As shown in Table 3, the extraction of polypeptide is affected little by solvent load, therefore determines that solvent load is 2ml.
2.4.4 the investigation of extraction time
Precision weighs lyophilized powder 0.02g, adds 70% ethanol 2.0ml, accurately weighed weight, respectively ultrasonic 10min, 15min,
20min, lets cool, and supplies the weight of less loss with 70% ethanol, with 2.3 times methods, measures absorbance in accordance with the law, calculates, the results are shown in Table
4。
The investigation of table 4 extraction time
| Extraction time (min) | 10 | 15 | 20 |
| Content of peptides (%) | 68.33 | 68.37 | 67.30 |
As shown in Table 4, during ultrasonic 10min, polypeptide proposes the most substantially, therefore ultrasonic time is set to 10min.
2.5 precision test
Take same reference substance solution, replication 6 times, calculate RSD value, the results are shown in Table 5.
Table 5 precision test
As shown in Table 5, the absorbance that 6 times measure reference substance solution, its RSD ＜ 3%, show that instrument precision is good.
2.6 stability test
Take need testing solution to measure absorbance respectively at 0h, 0.5h, 1h, 1.5h, 2h, calculate its RSD, the results are shown in Table 6.
Table 6 stability test
As shown in Table 6: the RSD ＜ 3% of need testing solution absorbance, test sample liquid is stable in 2h.
2.7 replica test
Precision weighs with a collection of test sample 6 parts, prepares sample test liquid by the preparation method of need testing solution, carries out respectively
Measure, calculate content, the results are shown in Table 7.
Table 7 replica test
As shown in Table 7: RSD < 3%, this method has good repeatability.
2.8 average recovery tests
Precision takes the appropriate 0.01g of test sample of known content, add a certain amount of bovine serum albumin reference substance (concentration:
6.52mg/ml), prepare new need testing solution, measure absorbance according to said determination method, the results are shown in Table 8.
Table 8 is loaded recovery test result
As shown in Table 8, average recovery between 95%～105%, RSD < 3%, show that this method measures content of peptides accurate
The most feasible.
2.9 method for determining content of peptides finally determined
Precision weighs bovine serum albumin reference substance (BSA) 51.80mg, puts in 100ml volumetric flask, adds distilled water and is dissolved to
Scale, shakes up, and is made into every 1ml reference substance solution containing 0.5180mg bovine serum albumin.
Precision weighs lyophilized powder 0.2g, puts in volumetric flask, adds the ethanol 2.0ml of 70%, accurately weighed weight, ultrasonic carries
Take 10min, let cool, supply the weight of less loss with 70% ethanol, shake up, take supernatant 1ml 70% ethanol constant volume and hold in 100ml
In measuring bottle, shake up, as need testing solution.
Take need testing solution and each 1ml of reference substance solution, add alkaline copper reagent 5.00ml respectively, mixing, rapidly join phenol
Reagent 0.5ml, quickly shakes up, 55 DEG C of water-bath 15min, takes out and is cooled to room temperature, after colour developing, according to spectrophotography, at 740nm
Measure absorbance, calculate and get final product.
Measure three batches of lyophilized powder content of peptides as stated above, the results are shown in Table 9.
The mensuration of table 9 lyophilized powder content of peptides
As shown in Table 9, in lyophilized powder, average polypeptide content is 67.43%, it is contemplated that the source of decoction pieces, processing, and preparation is raw
The factors such as product, tentative lyophilized powder every gram must not be less than 53.94% in terms of bovine serum albumin containing polypeptide.
Embodiment 3 Contents of Amino Acids
In periplaneta americana lyophilized powder, aminoacid was one of effective ingredient, according to version " Chinese Pharmacopoeia " annex VA in 2010
Pertinent regulations under spectrophotometry item, measure total amino acids content with alanine for reference substance.
3.1 the preparation of reference substance solution
Precision weighs 16.08mg alanine reference substance, puts in 100ml volumetric flask, and the isopropanol adding 10% dissolves, and dilutes
To scale, as mother solution.Precision measures 4ml, to 50ml volumetric flask, with water to scale, is configured to every 1ml containing 12.86 μ g third
The reference substance solution of propylhomoserin.
The selection of 3.2 detection wavelength
Precision measures reference substance solution, sample solution is filled in scale test tube in 10ml tool, adds 0.lml freshly prepared
1%Vc solution, 3m1 1,2,3-indantrione monohydrate alcoholic solution, mixing, 100 DEG C of water-baths are heated 15min, takes out and be cooled to rapidly room temperature, add
60% ethanol, to scale, is scanned in the range of 400-700nm, sees Fig. 5 and 6.
From Fig. 5 and 6, at 570nm, reference substance solution and test sample all have absorption maximum, therefore selecting 570nm is detection
Wavelength.
3.3 ranges of linearity are investigated
Accurate draw alanine reference substance solution (12.86 μ g/ml) 0 (blank), 0.4,0.8,1.2,1.6,2ml to 10ml
In tool plug scale test tube, being separately added into distilled water to 2ml, add 0.lml freshly prepared 1%Vc solution, 3m1 1,2,3-indantrione monohydrate alcohol is molten
Liquid, mixing, 100 DEG C of water-baths are heated 15min, takes out and be cooled to rapidly room temperature, add 60% ethanol to scale, at 570nm
Measuring absorbance, with concentration X as abscissa, absorbency Y is vertical coordinate, draws standard curve, and result is shown in Fig. 7.Rectilinear regression side
Cheng Wei: Y=0.249X+0.1137 (r=0.9996), show alanine in 0.5146 μ g/ml～2.5728 μ g/ml, sample
Concentration is good with absorbance linear relationship.
The preparation of 3.4 need testing solutions
3.4.1 the selection of extracting method
Precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, adds the ethanol 10ml of 70%, accurately weighed weight, use
Different modes extracts 15min, lets cool, and supplies the weight of less loss with 70% ethanol, shakes all, filters, take subsequent filtrate 1ml with 70%
Ethanol constant volume, in 100ml volumetric flask, shakes up, and according to the method under 3.3, measures absorbance in accordance with the law, calculate, knot at 570nm
Fruit is shown in Table 10.
The comparison of table 10 extracting method
| Extracting method | Shaking | Ultrasonic |
| Aminoacid (%) | 5.58 | 5.32 |
As shown in Table 10, supersound extraction can extract more aminoacid than shaking, and ultrasonic method is simpler, therefore selects ultrasonic
Process.
3.4.2 the investigation of Extraction solvent
Precision weighs lyophilized powder 0.2g, adds water respectively, 70% ethanol, the 90% each 10m1 of ethanol, and accurately weighed weight is ultrasonic
15min, lets cool, and supplies the weight of less loss respectively with corresponding solvent, with 3.3 lower methods, measures absorbance in accordance with the law, calculates,
The results are shown in Table 11.
The comparison of table 11 Extraction solvent
| Extraction solvent | 90% ethanol | 70% ethanol | Distilled water |
| Aminoacid (%) | 5.61 | 5.66 | 5.43 |
As shown in Table 11,70% ethanol and 90% ethanol extraction effect are more or less the same, and 70% ethanol is the most more, select
70% ethanol is as Extraction solvent.
3.4.3 the investigation of Extraction solvent consumption
Precision weighs lyophilized powder 0.2g, is separately added into 70% ethanol of 5ml, 10ml, 15ml, and accurately weighed weight is ultrasonic
15min, supplies the weight of less loss with 70% ethanol, with 3.3 lower methods, measures absorbance in accordance with the law, calculates, the results are shown in Table 12.
The investigation of table 12 Extraction solvent consumption
| Solvent load (ml) | 5 | 10 | 15 |
| Aminoacid (%) | 5.34 | 5.63 | 5.67 |
As shown in Table 12, when adding 15ml, amino acid whose content is maximum, but does not has much difference with 10ml ethanol,
10ml ethanol also can extract completely, therefore determines that solvent load is 10ml.
3.4.4 the investigation of extraction time
Precision weighs lyophilized powder 0.2g, the ethanol 10ml adding 70%, accurately weighed weight, difference supersound extraction 10min,
15min, 20min, supply the weight of less loss with 70% ethanol, with 3.3 lower methods, measures absorbance in accordance with the law, calculates, and result is shown in
Table 13.
The investigation of table 13 extraction time
| Extraction time (min) | 10 | 15 | 20 |
| Aminoacid (%) | 5.58 | 5.71 | 5.64 |
As shown in Table 13, during supersound process 15min, the aminoacid in lyophilized powder can be proposed completely, therefore when determining ultrasonic
Between be 15min.
3.5 precision test
Take same reference substance solution, replication 6 times, calculate RSD value, the results are shown in Table 14.
Table 14 Precision test result
As shown in Table 14, the absorbance that 6 times measure reference substance solution, its RSD ＜ 3%, show that instrument precision is good.
3.6 stability test
Take need testing solution to measure absorbance respectively at 0h, 0.5h, 1h, 1.5h, 2h, calculate its RSD, the results are shown in Table 15.
Table 15 stability test result
As shown in Table 15: the RSD ＜ 3% of need testing solution absorbance, test sample liquid is stable in 2h.
3.7 replica test
Precision weighs with a collection of test sample 6 parts, prepares sample test liquid by the preparation method of need testing solution, carries out respectively
Measure, calculate content, the results are shown in Table 16.
Table 16 replica test result
As shown in Table 16: RSD < 3%, this method has good repeatability.
3.8 average recovery tests
Precision takes the test sample 0.1g nine parts of known content, and precision adds alanine reference substance (concentration: 5.082mg/ respectively
0.3 ml), 0.4,0.5ml, prepare new need testing solution, measure absorbance according to said determination method, the results are shown in Table 17.
Table 17 average recovery is tested
As shown in Table 17, average recovery between 95%～105%, RSD < 3%, show this method measure amino acid content
The most feasible.
The Contents of Amino Acids method that 3.9 finally determine
Precision weighs 16.08mg alanine reference substance, puts in 100ml volumetric flask, and the isopropanol adding 10% dissolves, and dilutes
To scale, as mother solution；Precision measures described mother solution 4ml, to 50ml volumetric flask, is diluted with water to scale, is configured to every 1ml
Reference substance solution containing 12.86 μ g alanine；
Precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, adds the ethanol 10ml of 70%, accurately weighed weight, surpass
Sound extracts 15min, lets cool, supplies the weight of less loss with 70% ethanol, shakes all, filters, take subsequent filtrate 1ml 70% ethanol constant volume
In 100ml volumetric flask, shake up, as need testing solution；
Take in need testing solution and reference substance solution each 2ml to 10ml tool plug scale test tube, be separately added into distilled water extremely
2ml, adds 0.1ml freshly prepared 1%Vc solution, 3ml 1,2,3-indantrione monohydrate alcoholic solution, mixing, heats 15min, take in 100 DEG C of water-baths
Go out and be cooled to rapidly room temperature, add 60% ethanol to scale, at 570nm, measure absorbance, calculate and get final product.
Measure three batches of lyophilized powder amino acid contents as stated above, the results are shown in Table 18.
The mensuration of table 18 lyophilized powder amino acid content
As shown in Table 18, in lyophilized powder, amino acid content is 5.64%, it is contemplated that the provenance of decoction pieces, preparation production etc.
Factor, tentative lyophilized powder every gram amino acid content must not be less than 4.51% in terms of alanine.
Embodiment 4 gradient elution assay
Containing more gradient elution in periplaneta americana, it is the intermediate of biological oxidation, antitumor drug, has important
Biological activity, report with reference to relevant document, use HPLC-UV method to measure main gradient elution urine in periplaneta americana lyophilized powder
The content of pyrimidine, hypoxanthine and inosine.
4.1 the selection of chromatographic condition
Chromatographic column: Sepax HP-C18 post (250 × 4.6mm, 5 μm)
Flowing phase: chromatographic condition: flowing phase: methanol (A)-0.7% glacial acetic acid solution (B) gradient elution: 0～15min,
0.7% glacial acetic acid solution of 100%～0.7% glacial acetic acid solution of 99%；15～40min, 0.7% glacial acetic acid solution of 99%
～0.7% glacial acetic acid solution of 92%；40～50min, 0.7% glacial acetic acid solution of 92%～0.7% glacial acetic acid of 45% are molten
Liquid；Detection wavelength 254nm；Sample size 10 μ l；Temperature 30 DEG C, volume flow 0.7ml/min.
The preparation of 4.2 reference substance solution
Take uracil, hypoxanthine, inosine reference substance in right amount, accurately weighed, put in measuring bottle, add ultra-pure water and make containing urine phonetic
Pyridine 5.02 μ g/ml, hypoxanthine 25.2 μ g/ml, the mixing reference substance solution of inosine 127.5 μ g/ml, to obtain final product.
4.3 ranges of linearity are investigated
Above-mentioned mixing reference substance solution 4,6,8,10,12, the 14 μ l of accurate absorption enters successively according to above-mentioned chromatographic condition respectively
Sample, measures peak area, and with peak area as vertical coordinate, sample size is abscissa, draws standard curve, the results are shown in Table 19, and it is right to mix
Seeing Fig. 8 and 9 respectively according to product reference substance, periplaneta americana lyophilized powder high-efficient liquid phase chromatogram, standard curve is shown in Figure 10-12.
The investigation of table 19 range of linearity
As shown in figs. 10-12, uracil linear regression equation is: Y=59.2789X+10.7480 (r=0.9997)；Secondary
Xanthine linear regression equation is: Y=53.0102X-24.1614 (r=0.9999)；Inosine linear regression equation is: Y=
25.1879X-29.6832 (r=0.9998), shows that uracil is 2.008 × 10-2～7.028 × 10-2μg；In hypoxanthine
10.08 × 10-2～35.28 × 10-2μg；Inosine is 51.00 × 10-2～178.50 × 10-2μ g, in good linear relationship.
4.4. the preparation of need testing solution
4.4.1 the selection of extracting method
Precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, adds distilled water 10ml, and accurately weighed weight, by difference
Mode extract 10min, let cool, supply the weight of less loss with distilled water, shake up, filter, precision measures subsequent filtrate 5ml, is evaporated,
Residue adds water and is settled in 10ml measuring bottle, and 0.45 μm membrane filtration takes subsequent filtrate and is measured by the method under 4.1, result
It is shown in Table 20.
The comparison of table 20 extracting method
| Extracting method | Shaking | Ultrasonic |
| Uracil (%) | 0.26 | 0.28 |
| Hypoxanthine (%) | 0.95 | 0.97 |
| Creatinine (%) | 5.63 | 5.75 |
As shown in Table 20, ultrasonic method is better than shaking, and the simplest, therefore selects supersound extraction.
4.4.2 the investigation of Extraction solvent
Precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, adds distilled water 10ml, the 70% each 10m1 of ethanol, accurate
Weigh, supersound extraction 10min, let cool, supply the weight of less loss by same solvent, prepare sample and 4.1 according to method below 4.4.1
Under method be measured, the results are shown in Table 21.
The investigation of table 21 Extraction solvent
| Extraction solvent | Distilled water | 70% ethanol |
| Uracil (%) | 0.28 | 0.27 |
| Hypoxanthine (%) | 0.97 | 0.94 |
| Creatinine (%) | 5.75 | 5.58 |
As shown in Table 21, distilled water extraction effect is better than 70% ethanol, therefore selects distilled water as Extraction solvent.
4.4.3 the investigation of Extraction solvent consumption
Precision weighs lyophilized powder 0.2g, is separately added into 5ml, 10ml, 15ml distilled water, precise weighing, ultrasonic 15min, shines
4.4.1 the method that lower section method is prepared under sample and 4.1 is measured, and the results are shown in Table 22.
The investigation of table 22 Extraction solvent consumption
| Solvent load (ml) | 5 | 10 | 15 |
| Uracil (%) | 0.27 | 0.28 | 0.27 |
| Hypoxanthine (%) | 0.88 | 0.97 | 0.96 |
| Creatinine (%) | 5.63 | 5.75 | 5.77 |
As shown in Table 22, solvent load 10ml and 15ml is little on the impact of gradient elution content, therefore determines solvent load
For 10ml.
4.4.4 the investigation of extraction time
Precision weighs lyophilized powder 0.2g, adds distilled water 10ml, precise weighing, respectively ultrasonic 5min, 10min, 15min, shines
4.4.1 the method that lower section method is prepared under sample and 4.1 is measured, and the results are shown in Table 23.
The investigation of table 23 extraction time
| Extraction time (min) | 5 | 10 | 15 |
| Uracil (%) | 0.26 | 0.28 | 0.27 |
| Hypoxanthine (%) | 0.93 | 0.97 | 0.95 |
| Creatinine (%) | 5.57 | 5.75 | 5.59 |
As shown in Table 23, during ultrasonic 10min, the most substantially can propose gradient elution, therefore determine that ultrasonic time is 10min.
4.4.5 test is verified
Summary result of the test, prepares 3 batches of test samples, and its method is as follows: precision weighs lyophilized powder 0.2g, puts tool plug cone
In shape bottle, add distilled water 10ml, precise weighing, supersound extraction 10min, let cool, supply the weight of less loss with distilled water, shake
Even, filter, precision measures subsequent filtrate 5ml, is evaporated, and residue adds water and is settled in 10ml measuring bottle, 0.45 μm membrane filtration, takes continuous filter
Liquid is measured by the method under 4.1, the results are shown in Table 24.
Result of the test verified by table 24
| Test sequence number | 1 | 2 | 3 |
| Uracil (%) | 0.28 | 0.29 | 0.28 |
| Hypoxanthine (%) | 0.97 | 0.96 | 0.95 |
| Creatinine (%) | 5.75 | 5.79 | 5.71 |
As shown in Table 24, the preparation method reasonable of this test sample.
4.5 precision test
Take same mixing reference substance solution, replication 6 times, calculate RSD value, the results are shown in Table 25.
Table 25 Precision test result
| Test sequence number | Uracil (mAU) | Hypoxanthine (mAU) | Creatinine (mAU) |
| 1 | 312.35 | 1323.08 | 3209.46 |
| 2 | 309.01 | 1306.34 | 3173.34 |
| 3 | 315.48 | 1328.43 | 3211.53 |
| 4 | 314.98 | 1327.01 | 3216.83 |
| 5 | 310.86 | 1302.45 | 3168.58 |
| 6 | 310.04 | 1316.36 | 3194.76 |
| Average peak area | 312.12 | 1317.28 | 3195.75 |
| RSD (%) | 0.85 | 0.83 | 0.64 |
As shown in Table 25, the absorbance that 6 times measure reference substance solution, its RSD ＜ 3%, show that instrument precision is good.
4.6 stability test
Take with a collection of test sample, prepare sample test liquid by the preparation method of need testing solution, respectively at 0h, 2h, 4h,
8h, 16h, 24h sample introduction, measures peak area according to 4.1, investigates stability, calculates its RSD, the results are shown in Table 26.
Table 26 stability test result
| Minute (h) | Uracil (mAU) | Hypoxanthine (mAU) | Creatinine (mAU) |
| 0 | 338.81 | 1010.4 | 2901.08 |
| 2 | 340.11 | 1023.09 | 2910.66 |
| 4 | 332.34 | 1015.38 | 2894.09 |
| 8 | 329.13 | 1008.73 | 2881.53 |
| 16 | 325.43 | 991.31 | 2873.53 |
| 24 | 320.85 | 983.52 | 2863.76 |
| Average peak area | 331.11 | 1005.41 | 2887.44 |
| RSD (%) | 2.27 | 1.49 | 0.61 |
As shown in Table 26: the RSD ＜ 3% of need testing solution absorbance, test sample liquid is stable in 2h.
4.7 replica test
Precision weighs with a collection of test sample 6 parts, prepares sample test liquid by the preparation method of need testing solution, according to 4.1
It is measured, calculates content, the results are shown in Table 27.
Table 27 replica test result
As shown in Table 27: RSD < 3%, this method has good repeatability.
4.8 average recovery tests
Precision takes the appropriate 0.1g of test sample of known content, adds a certain amount of uracil (concentration: 0.2813mg/ respectively
Ml), hypoxanthine (concentration: 1.05mg/ml), inosine (concentration: 5.51mg/ml) reference substance, by the preparation side of need testing solution
Method prepares new sample test liquid, is measured according to 4.1, calculates content, the results are shown in Table 28.
Table 28 is loaded recovery test result
As shown in Table 28, average recovery between 95%～105%, RSD < 3%, show that this method is the most feasible.
The content assaying method of 4.9 gradient elutions finally determined
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；Methanol is flowing phase
A, 0.7% glacial acetic acid solution is that Mobile phase B carries out gradient elution；Detection wavelength is 254nm, column temperature 30 DEG C, flow velocity 0.7ml/
min。
Gradient is:
The preparation of reference substance solution: take uracil, hypoxanthine, inosine reference substance, accurately weighed, put in measuring bottle, add super
Pure water is made containing uracil 5.02 μ g/ml, hypoxanthine 25.2 μ g/ml, the mixing reference substance solution of inosine 127.5 μ g/ml.
The preparation of need testing solution: precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, distilled water 10ml, accurate title
Determining weight, supersound extraction 10min, let cool, supply the weight of less loss with distilled water, shake all, filter, precision measures subsequent filtrate 5ml,
Being evaporated, residue adds water and is settled in 10ml measuring bottle, and 0.45 μm membrane filtration takes subsequent filtrate as need testing solution.
Assay method: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, surveys
Fixed, calculate and get final product, lyophilized powder every gram must not be less than 0.22% containing uracil, hypoxanthine must not be less than 0.77%, creatinine must not
Less than 4.62%.
Measure three batches of lyophilized powder content of peptides as stated above, the results are shown in Table 29.
The assay of table 29 lyophilized powder gradient elution
| Tested number | Uracil (%) | Hypoxanthine (%) | Creatinine (%) |
| 1 | 0.28 | 0.96 | 5.79 |
| 2 | 0.27 | 0.95 | 5.74 |
| 3 | 0.28 | 0.97 | 5.81 |
| Average content (%) | 0.28 | 0.96 | 5.78 |
| RSD (%) | 2.09 | 1.04 | 0.62 |
As shown in Table 29, in lyophilized powder mean urinary pyrimidine content be 0.28%, average hypoxanthine content be 0.96%, flat
The factors such as all Inosine Contents are 5.78%, it is contemplated that the source of decoction pieces, processing, preparation production, tentative lyophilized powder every gram is phonetic containing urine
Pyridine must not be less than 0.22%, hypoxanthine must not be less than 0.77%, creatinine must not be less than 4.62%.
The periplaneta americana lyophilized powder method of quality control of the present invention, from aminoacid qualitative identification, aminoacid, polypeptide, nucleoside
Periplaneta americana lyophilized powder quality is entered by several aspects such as constituents assay, amino acid composition analysis, polypeptide molecular weight mensuration
Row detection controls.The quality of periplaneta americana lyophilized powder is provided strong guarantee, in having ensured and with periplaneta americana lyophilized powder being
The quality of the later stage preparation of mesosome and curative effect.The periplaneta americana lyophilized powder method of quality control of the present invention, improves America further
Big Lian decoction pieces, extract and the quality standard of preparation thereof, have certain reference price to periplaneta americana decoction pieces, extract and preparation thereof
Value.
As employed some vocabulary in the middle of description and claim to censure special component or method.Art technology
Personnel are it is to be appreciated that same composition may be called with different nouns in different regions.This specification and claims are not
In the way of the difference of title is used as distinguishing composition." comprising " as mentioned by the middle of description and claim in the whole text is
One open language, therefore " comprise but be not limited to " should be construed to." substantially " refer in receivable range of error, this area
Technical staff can solve described technical problem in the range of certain error, basically reaches described technique effect.Description is follow-up
It is described as implementing the better embodiment of the present invention, for the purpose of right described description is the rule so that the present invention to be described, not
In order to limit the scope of the present invention.Protection scope of the present invention is when being as the criterion depending on the defined person of claims.
Also, it should be noted term " includes ", " comprising " or its any other variant are intended to nonexcludability
Comprise, so that include that the commodity of a series of key element or system not only include those key elements, but also include the most clearly
Other key elements listed, or also include the key element intrinsic for this commodity or system.In the feelings not having more restriction
Under condition, statement " including ... " key element limited, it is not excluded that in the commodity including described key element or system also
There is other identical element.
Described above illustrate and describes some preferred embodiments of the present invention, but as previously mentioned, it should be understood that the present invention
Be not limited to form disclosed herein, be not to be taken as the eliminating to other embodiments, and can be used for other combinations various,
Amendment and environment, and can be in invention contemplated scope described herein, by above-mentioned teaching or the technology of association area or knowledge
It is modified.And the change that those skilled in the art are carried out and change are without departing from the spirit and scope of the present invention, the most all should be at this
In the protection domain of bright claims.
Claims (5)
1. periplaneta americana lyophilized powder method of quality control, it is characterised in that include utilizing thin layer chromatography carry out aminoacid discriminating,
Utilize ultraviolet visible spectrophotometry aminoacid and polypeptide are carried out assay, utilize high performance liquid chromatography to uracil,
Hypoxanthine, creatinine carry out assay.
2. periplaneta americana lyophilized powder method of quality control as claimed in claim 1, it is characterised in that described aminoacid discriminating side
Method for weighing 0.5g periplaneta americana lyophilized powder, the ethanol 10ml adding 70%, ultrasonic 10min, filter, collect filtrate, water-bath volatilizes,
The ethanol 5ml that residue adds 70% dissolves, as need testing solution；
Take alanine, phenylalanine, glycine reference substance respectively, accurately weighed, to put in brown measuring bottle, the ethanol adding 70% is made
The reference substance solution of 1mg/ml；
Test according to thin layer chromatography, draw each 1 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio
N-butyl alcohol-formic acid-aqueous solution for 75:15:10 is developing solvent, launches after saturated 30min, takes out, dries, and spray is with 0.2% indenes
Triketone ethanol solution, 105 DEG C are heated to clear spot；
Observe silica gel g thin-layer plate, in test sample chromatograph, on position corresponding with reference substance chromatograph, show identical face in the sunlight
The speckle of color.
3. periplaneta americana lyophilized powder method of quality control as claimed in claim 1, it is characterised in that described determining content of peptides
Method is:
Precision weighs bovine serum albumin reference substance 51.80mg, puts in 100ml volumetric flask, adds distilled water and is dissolved to scale, shakes
Even, it is made into every 1ml reference substance solution containing 0.5180mg bovine serum albumin；
Precision weighs lyophilized powder 0.2g, puts in volumetric flask, adds the ethanol 2.0ml of 70%, accurately weighed weight, supersound extraction
10min, lets cool, and supplies the weight of less loss with 70% ethanol, shakes up, and takes supernatant 1ml 70% ethanol constant volume in 100ml capacity
In Ping, shake up, as need testing solution；
Take need testing solution and each 1ml of reference substance solution, add alkaline copper reagent 5.00ml respectively, mixing, rapidly join phenol reagent
0.5ml, quickly shakes up, 55 DEG C of water-bath 15min, takes out and is cooled to room temperature, after colour developing, according to spectrophotography, measures at 740nm
Absorbance, calculates and get final product.
4. periplaneta americana lyophilized powder method of quality control as claimed in claim 1, it is characterised in that described amino acid content is surveyed
The method of determining is:
Precision weighs 16.08mg alanine reference substance, puts in 100ml volumetric flask, and the isopropanol adding 10% dissolves, and is diluted to carve
Degree, as mother solution；Precision measures described mother solution 4ml, to 50ml volumetric flask, is diluted with water to scale, is configured to every 1ml and contains
The reference substance solution of 12.86 μ g alanine；
Precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, adds the ethanol 10ml of 70%, accurately weighed weight, ultrasonic carry
Take 15min, let cool, supply the weight of less loss with 70% ethanol, shake all, filter, take subsequent filtrate 1ml 70% ethanol constant volume in
In 100ml volumetric flask, shake up, as need testing solution；
Take in need testing solution and reference substance solution each 2ml to 10ml tool plug scale test tube, be separately added into distilled water to 2ml, add
0.1ml freshly prepared 1%Vc solution, 3ml 1,2,3-indantrione monohydrate alcoholic solution, mixing, 100 DEG C of water-baths are heated 15min, takes out rapidly
It is cooled to room temperature, adds 60% ethanol to scale, at 570nm, measure absorbance, calculate and get final product.
5. periplaneta americana lyophilized powder method of quality control as claimed in claim 1, it is characterised in that described uracil, secondary Huang
Purine, creatinine carry out content assaying method
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；Methanol is mobile phase A,
0.7% glacial acetic acid solution is that Mobile phase B carries out gradient elution；Detection wavelength is 254nm, column temperature 30 DEG C, flow velocity 0.7ml/min；
Gradient is:
0.7% glacial acetic acid solution of 0.7% glacial acetic acid solution-99% of 0-15min 100%,
0.7% glacial acetic acid solution of 0.7% glacial acetic acid solution-92% of 15-40min 99%,
0.7% glacial acetic acid solution of 0.7% glacial acetic acid solution-45% of 40-50min 92%；
The preparation of reference substance solution: take uracil, hypoxanthine, inosine reference substance, accurately weighed, put in measuring bottle, add ultra-pure water
Make containing uracil 5.02 μ g/ml, hypoxanthine 25.2 μ g/ml, the mixing reference substance solution of inosine 127.5 μ g/ml；
The preparation of need testing solution: precision weighs lyophilized powder 0.2g, puts in tool plug conical flask, and distilled water 10ml is accurately weighed heavy
Amount, supersound extraction 10min, let cool, supply the weight of less loss with distilled water, shake all, filter, precision measures subsequent filtrate 5ml, steams
Dry, residue adds water and is settled in 10ml measuring bottle, and 0.45 μm membrane filtration takes subsequent filtrate as need testing solution；
Assay method: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, meter
Calculate and get final product, lyophilized powder every gram containing uracil must not less than 0.22%, hypoxanthine must not less than 0.77%, creatinine must not be less than
4.62%.
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