CN104316621B - Method for measuring total nucleotide in protein products - Google Patents
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Abstract
The invention relates to a method for measuring the total nucleotide in protein products. The method includes the following steps: (1) basic group mixed standard liquid is prepared; (2) a solution of a to-be-measured sample is prepared, the basic group mixed standard liquid in the step (1) serves as reference liquid, a content value of free basic groups in the sample is obtained through peak area comparison with the high performance liquid chromatography, and the total content of the free basic groups is obtained; (3) complete hydrolysis processing is carried out on the to-be-measured sample, and the completely-hydrolyzed total basic group content is measured with the high performance liquid chromatography; (4) the content of the nucleotide is obtained through molecular weight conversion after the content, obtained in the step (2), of the free basic groups is subtracted from the total basic group content obtained in the step (3). The method is accurate in measurement, the content of the nucleotide in the single-cell protein products can be accurately reflected, and the nutritive value and the quality of the products are accordingly measured. Except for single-cell proteins, the method is further suitable for raw materials such as dairy products, feed and algae which are rich in nucleotide.
Description
Technical field
The present invention relates to nucleotide determination techniques field.
Background technology
Single cell protein (Single cell protein is called for short SCP), also known as microprotein or tropina, is to utilize
Industrial wastewater, waste gas, natural gas, petroleum alkane class, agricultural and sideline converted products and organic waste etc., as culture medium, cultivate ferment
The unicellular microorganism such as pathogenic antibacterial female, non-, miniature bacterium, fungus, are then passed through making after purge drying processes, are food works
Industry and the important protein source of feed industry.
A single cell protein big advantage compared with other albumen is exactly its less cell incubation time, and microorganism is organic
Other traditional protein productivity of the productivity ratio of body protein product wants height.Single cell protein (SCP) has compared with traditional protein and is not subject to
The impact of natural environment and climate, raw material be easy to get, can produce continuously, easily controllable, pollute the advantages such as little.
Single cell protein product contains rich in protein, amino acid whose A wide selection of colours and designs, and ratio is suitable.Single cell protein is also
Containing abundant fat, carbohydrate, nucleic acid, vitamin and inorganic salt, and containing ore deposits such as calcium, phosphorus, potassium, ferrum, magnesium, sodium, manganese
Material element, possibly together with multiple enzyme.
Nucleotide is that cell forms main component, is composition DNA and RNA monomer, at cellularity, metabolism, energy and tune
The aspects such as joint function play an important role, and its biochemical functions has: as the raw material of RNA and DNA synthesis；Regulation as metabolism
Material；As the composition of coenzyme, such as coenzyme A；Activate glycogen and the important mediator of glycoprotein synthesis, also in phospholipid synthesis
Jie's thing, activates intermediate product；Participation hereditary information is transmitted；ATP is the direct energy of life process；The contraction of AMP regulation smooth muscle
Property scalable blood flow；Nucleotide is also used as the donor etc. of methyl.Therefore extraneous nucleotide has important biomolecule work
With.
In feed industry, single cell protein is inexpensively and the most reliably originating of extraneous nucleotide.Accordingly, it is determined that raw material
In the content of nucleotide accurately be exactly a very important job.
At present the assay method of nucleic acid can be divided into two big classes: the quantitative analysis of nucleic acid and the mensuration of nucleic acid hydrolysis thing.
The quantitative approach of nucleic acid mainly has: fixing phosphorus method, ultraviolet method, orcin method.The mensuration of nucleic acid hydrolysis thing comprises mensuration
Nucleotide, nucleoside and various base, the method for these hydrolysate comparative maturities uses high performance liquid chromatograph to carry out, some
There are the application of maturation, such as, the mensuration of free nucleotide in Milk Powder Formula For Infants.But, said method is all respectively arranged with feature
And shortcoming, there is its specific scope of application, a lot of problem can be produced when being applied to the mensuration measuring single cell protein nucleotide,
Thus cause measurement result that mistake occurs.
Fixing phosphorus method measuring principle is that in structural nucleic acid molecule, containing a certain proportion of phosphorus, (RNA phosphorus content is about 8.5%~
9.0%), measure its phosphorus content and can converse the amount of nucleic acid.But method cannot be distinguished by nucleic acid phosphorus and other organophosphors, this meeting
Cause the nucleic acid phosphorus in protein raw materials and other organophosphors to be all calculated as the phosphorous of nucleic acid, thus cause measurement result the most inclined
High.
Absorbance method measures the 260nm ultraviolet absorptivity that the principle of nucleic acid is the conjugated double bond utilizing base in nucleic acid to contain
Feature be measured.The shortcoming of the method is: 1) the most quantitative is the content of conjugated double bond；2) containing conjugated double bond
Material includes: nucleic acid and the non-core acids things such as nucleic acid, oligonucleotide, nucleotide, nucleoside, base, 1,3 butadiene, cyclohexene
Matter；4) the content size of conjugated double bond is successively: base > nucleoside > nucleotide；6) indicate on molecular biology that ultraviolet method measures
It is only applicable to pure nucleic acid samples, is not suitable for the sample containing multiple nucleic acids hydrolysate.Yeast, enzymolysis it are not suitable for the most yet
The single cell protein raw materials such as yeast, because they are except there being nucleic acid also to contain the hydrolysate of a lot of nucleic acid.
Orcin method measures the principle of nucleic acid: after an acidic hydrolysis, the ribose that degraded is formed is changed into furfural, the latter to nucleic acid
Generate green compounds with orcin (3,5-vesorcinol) effect, at 670nm, have absorption maximum.Its shortcoming is: orcin
Atopic is poor, and all pentoses all have this to react, and nucleic acid hydrolysis thing and glucide are bigger on the impact of its measurement result.Therefore
The products measure result meeting error of single cell protein class complicated and diversified for chemical constituent is the biggest.
Nucleic acid hydrolysis thing includes nucleotide, nucleoside, base.Nucleotide include guanyl, adenylic acid, uridylic acid, cytidylic acid,
Inosinic acid.Nucleoside includes guanosine, adenosine, uridnine, inosine, cytidine.The base of ribonucleic acid has four kinds of adenine, guanine, born of the same parents
Pyrimidine, uracil, DNA (deoxyribonucleic acid) also has thymus pyrimidine.These hydrolysates existing document report can use high-efficient liquid phase color
Spectrum measures its content.
Bulletin grant number is the patent of invention<milk powder nucleotide high-performance liquid chromatogram determination method>of CN102680601B
Disclose a kind of method measuring nucleotide in milk powder, locate before disclosing the use liquid-phase condition of liquid chromatograph, standard substance, sample
Reason methods etc., well solve the mensuration of milk powder nucleotide.But similar with the method that other liquid phases measure nucleotide, answering
Inapplicable situation is shown when the nucleotide of single cell protein measures.Inapplicable reason is: 1) method mostly is free
The nucleotide of state；2) only determining nucleotide and do not measure nucleoside, for Animal nutrition angle, nucleic acid hydrolysis thing is except base
The function of nutrition and immunity can be played in addition；3) endonuclear non-free state nucleotide can not measure；4) at actual list
The form of diverse of cell protein raw material nucleotide, has the acid of nucleoside form, nucleotide form, nucleoside diphosphate, nucleoside triphosphate
Acid, oligonucleotide, these all do not have to have embodied in assay method.
Summary of the invention
Nucleotide on nutrition significance not only includes the nucleotide of monomer, also include other forms such as oligonucleoside
Acid, nucleotide, nucleoside.Base then cannot provide nutrition and immunologic function.Have research display animal take in external source base (purine and
Pyrimidine) have problems with: 1) purine of food source and pyrimidine seldom utilize by body；2) purine and pyrimidine meeting are directly taken in
Increase the weight of the burden of Liver and kidney metabolism；3) the direct absorption of purine can cause uric acid higher；4) adenine itself is poisonous, can cause male not
Educate and chronic kidney hypofunction, also can affect growth of animal.Therefore, it is thus achieved that all ucleotides materials in addition to base are on threpsology
Have great importance.Correspondingly, the content of ucleotides material in detection food just has important medical science, food nutrition
And commercial value.This is also the technical problem to be solved in the present invention.
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that one can measure in single cell protein product contained
The method of total nucleotide content.
To achieve these goals, present invention employs techniques below scheme: the total nucleotide assay method of protein product,
It is characterized in that use following steps:
(1) preparation base hybrid standard liquid；
(2) prepare the solution of testing sample, using the base hybrid standard liquid described in step 1 as tested liquid, use efficiently
Liquid chromatography relatively obtains the content value of free base in sample by going out peak area ratio, and obtains the total content of free base；
(3) testing sample carries out complete hydrolysis process, with the total alkali after quantitative determination of high-performance liquid complete hydrolysis
Base content；(4) convert through molecular weight deduct the free base contents of step 2 gained with total base contents of step 3 gained after
Content to nucleotide.
In described step 3, testing sample carries out complete hydrolysis process by following operation realization:
Weigh testing sample, be placed in pressure digestive tube；
In pressure digestive tube, add Perchloric Acid Digestion liquid, make testing sample hydrolyze；
Put into baking oven 100～110 DEG C of digestion 50～70min；
Use 10～20% potassium hydroxide solution adjust pH to 6.5～7.5, standby.
In described Perchloric Acid Digestion liquid, the mass percent concentration of perchloric acid is 70%.
Described step 2 measures and free base contents and step 3 measure the chromatographic condition of total base contents is: flowing phase
A uses 0.1mol/L potassium dihydrogen phosphate, pH=4.9～5.1；Mobile phase B uses 25% methanol solution.
Measure total base contents use following gradient elution program:
The mensuration free base contents following gradient elution program of employing:
Ribonucleic acid produces oligonucleotide, nucleotide, nucleoside, base, phosphoric acid and sugar, complete in the case of not exclusively hydrolysis
Purine bases, pyrimidine bases, phosphoric acid and sugar it is decomposed in the case of fully hydrolyzed.Use base after HPLC quantitative determination complete hydrolysis
The content of (cytosine Cytosine, uracil Uracil, guanine Guanine, adenine Adenine) is total base and contains
Amount, then measure the base of free state in sample, total base deducts free base and is the base deriving from nucleotide, then basis
Molecular weight conversion obtains the content of nucleotide.Method used in the present invention is measured accurately, can produce by accurate response single cell protein
The content of product nucleotide, thus weigh nutritive value and the quality good or not of this product.In addition to single cell protein, the present invention
Method apply also for the raw material rich in nucleotide such as milk product, feedstuff, algae.
Accompanying drawing explanation
Fig. 1 is the eluting result figure using the HPLC method total base of examination criteria product.
Fig. 2 is to use HPLC method examination criteria product to dissociate the eluting result figure of base.
Fig. 3 is to use the eluting result figure of free base contents in HPLC method detection sample.
Fig. 4 is to use the eluting result figure of the content of total base in HPLC method detection sample.
Detailed description of the invention
It is below the enforcement step of the present invention:
1 reagent and solution
1.1 except as otherwise noted, and this law agents useful for same is analytical pure, and water is deionized water, meets GB/T 6,682 2 grades
The regulation of water.
1.2 potassium dihydrogen phosphates: analytical pure.
1.3 potassium hydroxide: analytical pure.
1.4 methanol: chromatographically pure.
1.5 acetonitriles: chromatographically pure.
1.6 perchloric acid: analytical pure.
1.720%w/v potassium hydroxide solution: weigh potassium hydroxide 100 grams, be dissolved in 500ml water, be stored in volumetric flask
In.
1.8 mobile phase A: 0.1M potassium dihydrogen phosphate, weigh potassium dihydrogen phosphate 27.2 grams, are dissolved in 1000ml water,
Add 20% potassium hydroxide solution 1.0ml, add water and be settled to 2 liters, pH=4.97.
1.9 Mobile phase B: 25% methanol solution, measure methanol 250ml and are dissolved in 750ml water, 0.45um after stirring and evenly mixing
After organic system membrane filtration standby.
1.10 cytosine Cytosine:sigma#V900462.
1.11 uracil Uracil:sigma#V900439.
1.12 guanine Guanine:sigma#V900473.
1.13 thymus pyrimidine Thymine:sigma#V900437.
1.14 adenine Adenine:sigma#V900471.
1.15 cytosine standard reserving solutions: weigh cytosine 1.0044 grams, add water and be settled to 250ml, 70 DEG C of hot baths add
Heat of solution.
1.16 uracil standard reserving solutions: weigh uracil 0.9976 gram, add water and be settled to 250ml, 70 DEG C of hot baths add
Heat of solution.
1.17 guanine standard reserving solutions: weigh guanine 0.1000 gram, add 0.1M sodium hydroxide solution 150～180ml,
Add water after all dissolving and be settled to 250ml.
1.18 thymus pyrimidine standard reserving solutions: weigh thymus pyrimidine 1.0133 grams, add water and be settled to 250ml, 70 DEG C of hot water
Bath heating for dissolving.
1.19 adenine standard reserving solutions: weigh adenine 1.0171 grams, add water and be settled to 250ml, 70 DEG C of hot baths add
Heat of solution.
1.20 base hybrid standard liquid: measure cytosine, uracil, guanine, thymus pyrimidine, the storage of adenine standard successively
In standby liquid 1.0ml, 1.0ml, 10.0ml, 1.0ml, 1.0ml to 250ml volumetric flask, add water and be settled to 250ml, hybrid standard liquid
Cytosine, uracil, guanine, thymus pyrimidine, the concentration of adenine is respectively 16.07,15.99,19.55,16.21,
16.27ug/ml.Subpackage in 0.5ml cryopreservation tube ,-20 DEG C of preservations, be valid for one year.
2 instrument and equipments
2.1 Shimadzu LC-20AD high performance liquid chromatograph systems (gradient elution, ultraviolet detection, controlled column temperature).
2.2 chromatographic columns: Athena C18,4.6 × 150mm.
2.3pH meter.
2.4 analytical balance.
2.5 ultrasonic cleaner.
2.61mL disposable syringe.
2.70.45um disposable aqueous phase filter.
2.80.5ml centrifuge tube.
2.9100ml glass beaker.
2.10250ml volumetric flask.
2.1116ml pressure blind nut vial.
2.1240ml blind nut glass tubing
2.131000ul pipettor.
2.145000ul pipettor.
3 determination steps
3.1 total base hydrolysis
Accurately weigh 0.1000 gram of sample, be placed in 16ml pressure blind nut vial.(each testing sample repeated sampling is extremely
Few two groups.)
Add 70% Perchloric Acid Digestion liquid 10ml.(being divided into twice addition, add 5ml for the first time, the concussion of slight whirlpool makes
Sample is not adhere to bottom digestive tube, whirlpool concussion time the page can not be more than the 10ml page at, add after vortex residue 5ml
Perchloric acid.
Put into 105 DEG C of baking oven digestion 60min.(after digestion 10～15min, again taking out whirlpool concussion)
Take out digestive tube after digestion, rapidly Digestive system is all pipetted to 100ml beaker.
20% potassium hydroxide is used to adjust pH to 6.5～7.5.
The Digestive system of modulated pH is all pipetted to 250ml volumetric flask, pipettes 2ml Digestive system after constant volume to 5ml plastics
Pipe is interior, preserves, after adding one times of mixing of 2ml water dilution, the above machine that filters standby.
Use glass bomb with cover as hydrolysis pipe substitute boiling water bath advantage be to avoid uncovered boiling water bath time test solution boiling
Rise and spill, it is ensured that stablizing of hydrolyzed solution, and be avoided that the further oxidation of hydrolysate.
The advantage using 70% perchloric acid: replace other digestion solutions such as mixed acid of trifluoroacetic acid formic acid, sulphuric acid, hydrochloric acid,
Perchloric acid uses potassium hydroxide to carry out acid-base neutralization after clearing up end can generate insoluble potassium hyperchlorate white precipitate, thus not
The salt ion in hydrolyzed solution can be increased, thus farthest protect the chromatographic column of liquid chromatogram measuring, extend making of chromatographic column
Use the life-span.
3.2 dissociate base
Accurately weigh 0.1000 gram of sample as in 40ml glass tubing.(each testing sample repeated sampling at least two group.)
Add water 15ml, and whirlpool shakes 3 times, each 1min, every minor tick 20min.
Ultrasonic 20min after concussion.Whirlpool concussion again.
Take in glass tubing in test solution 3ml to 5ml centrifuge tube.12000g high speed centrifugation 15min.
Taking in supernatant 2ml to 5ml centrifuge tube, add water 2ml, dilutes one times.The above machine that filters is preserved standby after mixing.
3.3 mensuration that by high performance liquid chromatography, sample is carried out total base contents, total alkali primary colours spectral condition
Ambient temperature: 25 DEG C～30 DEG C, cleannes require: no-sundries, abnormal flavour etc.
Chromatographic column: Athena C18,4.6 × 150mm.
Detection wavelength: 260nm.
Column temperature: 30 DEG C.
Mobile phase A: 0.1mol/L potassium dihydrogen phosphate.
Mobile phase B: 25% methanol solution.
Sample size: 20ul
Flow velocity: 0.5mL/min
Elution program is as shown in table 1 below:
Table 1
The flowing of low concentration can reduce the infringement of chromatographic column mutually, extends the service life of chromatographic column.
The pH value 4.97 of mobile phase A, the flowing of low pH is relatively big, in balanced-out peak separating degree and chromatograph relative to chromatographic column damage
The angle of post protection, pH=4.9～5.1 is ideal range.
The advantage of eluting degree is the batch operation that can carry out Multi-example mensuration, ensures that the performance of chromatographic column is steady simultaneously
Fixed.
3.4 carry out the mensuration of free base contents, free alkali primary colours spectral condition by high performance liquid chromatography to sample
Ambient temperature: 25 DEG C～30 DEG C, cleannes require: no-sundries, abnormal flavour etc.
Chromatographic column: Athena C18,4.6 × 150mm.
Detection wavelength: 260nm.
Column temperature: 30 DEG C.
Mobile phase A: 0.1mol/L potassium dihydrogen phosphate.
Mobile phase B: 25% methanol solution.
Sample size: 20ul
Flow velocity: 0.5mL/min
Elution program is as shown in table 2 below:
Table 2
Use the content of base in external standard method sample.As Fig. 4 reflects the eluting result of total base, as shown in Figure 3
Reflect the eluting result of free base.When measuring free base, free nucleotide, free nucleoside in product all can go out
Peak, the advantage of elution program is 5 kinds of nucleotide, 5 kinds of nucleoside, 5 kinds of bases can be separated, and will not be overlapped mutually, thus
Guarantee to obtain the result of base of dissociating accurately.
4 results calculate
Base contents ω in sample, identifies with mass fraction, and numerical value represents with %, calculates by formula:
Δ Wx=WT-WF························(3)
W=Δ ωA×1.49+ΔωU×2.89+ΔωC×2.91+ΔωG×2.40·····(4)
Measure the free base of a certain yeast product and always base and free base are respectively as follows:
| Project | Total base % | Free base % |
| Cytosine | 0.39 | 0.01 |
| Uracil | 0.56 | 0.12 |
| Guanine | 0.62 | 0.12 |
| Thymus pyrimidine | 0.03 | 0.03 |
| Adenine | 1.10 | 0.40 |
| Add up to | 2.70 | 0.68 |
With base calculate nucleotide content for 2.02%, be scaled total nucleotide according to the molecular weight of each nucleotide and contain
Amount is
| Project | The nucleotide of base meter | Molecular weight multiple | Nucleotide |
| Cytosine | 0.38 | 2.91 | 1.11 |
| Uracil | 0.44 | 2.89 | 1.27 |
| Guanine | 0.50 | 2.4 | 1.20 |
| Thymus pyrimidine | 0.00 | / | 0.00 |
| Adenine | 0.70 | 1.49 | 1.04 |
| 2.02 | 4.62 |
The total nucleotide result obtained is 4.62%
In formula:
The chromatographic peak area of A sample oligonucleotide, nucleoside or base
AstThe chromatographic peak area of standard substance
CstThe concentration of standard substance
X represents four kinds of bases: A, U, C, G；
C base concentration ug/ml；
V volume of dissolution ml；
M example weight (g)；
WXBase contents %；
WTTotal base contents；
WF dissociates base contents；
△ W nucleotide in terms of base；
W total nucleotide content
The arithmetic mean of instantaneous value of measurement result parallel assay represents, retains 2 position effective digitals.
5 precision
The absolute difference of the twice independent measurement result obtained under the conditions of repeatability must not exceed arithmetic mean of instantaneous value
10%.
Claims (2)
1. the total nucleotide assay method of protein product, it is characterised in that employing following steps:
(1) preparation base hybrid standard liquid:
Preparation cytosine standard reserving solution: weighing cytosine 1.0044 grams, add water and be settled to 250ml, 70 DEG C of hot baths add thermosol
Solve；
Preparation uracil standard reserving solution: weighing uracil 0.9976 gram, add water and be settled to 250ml, 70 DEG C of hot baths add thermosol
Solve；
Preparation guanine standard reserving solution: weigh guanine 0.1000 gram, add 0.1M sodium hydroxide solution 150～180ml, all
Add water after dissolving and be settled to 250ml；
Preparation thymus pyrimidine standard reserving solution: weighing thymus pyrimidine 1.0133 grams, add water and be settled to 250ml, 70 DEG C of hot baths add
Heat of solution；
Preparation adenine standard reserving solution: weighing adenine 1.0171 grams, add water and be settled to 250ml, 70 DEG C of hot baths add thermosol
Solve；
Measure cytosine standard reserving solution 1.0ml, uracil standard reserving solution 1.0ml, guanine standard reserving solution successively
In 10.0ml, thymus pyrimidine standard reserving solution 1.0ml, adenine standard reserving solution 1.0ml to 250ml volumetric flask, add water constant volume
To 250ml, hybrid standard vacuole's pyrimidine, uracil, guanine, thymus pyrimidine, the concentration of adenine is respectively 16.07,
15.99,19.55,16.21,16.27ug/ml, in subpackage to 0.5ml cryopreservation tube ,-20 DEG C of preservations, thus join and to obtain base mixing mark
Quasi-liquid；
(2) prepare the solution of testing sample, using the base hybrid standard liquid described in step 1 as tested liquid, use efficient liquid phase
Chromatography relatively obtains in sample the content value of free base by going out peak area ratio, and obtains the total content of free base,
(3) testing sample carries out complete hydrolysis process, contains by the total base after quantitative determination of high-performance liquid complete hydrolysis
Amount, complete hydrolysis is processed and is realized by following operation:
Accurately weigh 0.1000 gram of sample, be placed in 16ml pressure blind nut vial,
Adding 70% Perchloric Acid Digestion liquid 10ml, be divided into twice addition, add 5ml for the first time, the concussion of slight whirlpool makes sample
Be not adhere to bottom digestive tube, whirlpool concussion time the page can not be more than the 10ml page at, add after vortex residue 5ml；
Put into 105 DEG C of baking oven digestion 60min, wherein after digestion 10～15min, again take out whirlpool concussion,
Take out digestive tube after digestion, Digestive system all pipetted to 100ml beaker rapidly,
20% potassium hydroxide is used to adjust pH to 6.5～7.5, standby；
(4) core is obtained through molecular weight conversion deduct the free base contents of step 2 gained with total base contents of step 3 gained after
The content of thuja acid,
Wherein in step (2) and step (3), wherein total alkali primary colours spectral condition is:
Chromatographic column: Athena C18,4.6 × 150mm,
Detection wavelength: 260nm,
Column temperature: 30 DEG C,
Mobile phase A: 0.1mol/L potassium dihydrogen phosphate,
Mobile phase B: 25% methanol solution,
Sample size: 20ul,
Flow velocity: 0.5mL/min
Elution program is as follows:
Free alkali primary colours spectral condition is:
Chromatographic column: Athena C18,4.6 × 150mm,
Detection wavelength: 260nm,
Column temperature: 30 DEG C,
Mobile phase A: 0.1mol/L potassium dihydrogen phosphate,
Mobile phase B: 25% methanol solution,
Sample size: 20ul
Flow velocity: 0.5mL/min
Elution program is as follows:
Use the content of base in external standard method sample.
The total nucleotide assay method of protein product the most according to claim 1, it is characterised in that: described step 2 is surveyed
Surely the chromatographic condition measuring total base contents in base contents and the step 3 of dissociating is: mobile phase A uses 0.1mol/L biphosphate
Potassium solution, pH=4.9～5.1.
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| CN110763792A (en) * | 2019-10-24 | 2020-02-07 | 湖北省宏源药业科技股份有限公司 | Method for detecting guanine-related substances |
| CN111077206A (en) * | 2019-12-30 | 2020-04-28 | 成都市海通药业有限公司 | Method for detecting content of polyinosinic cells in polyinosinic cell injection |
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| US3730844A (en) * | 1971-08-27 | 1973-05-01 | Purdue Research Foundation | Polynucleotide analysis |
| CN103884676A (en) * | 2014-02-27 | 2014-06-25 | 浙江大学 | Rapid detection method for contents of multi-index ingredients in traditional Chinese medicinal materials |
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| US3730844A (en) * | 1971-08-27 | 1973-05-01 | Purdue Research Foundation | Polynucleotide analysis |
| CN103884676A (en) * | 2014-02-27 | 2014-06-25 | 浙江大学 | Rapid detection method for contents of multi-index ingredients in traditional Chinese medicinal materials |
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