CN1773277A - Method for discriminating donkey-hide gelatin utilizing liquid-liquid-liquid three phase static extracting efficient liquid-phase chromatograph - Google Patents
Method for discriminating donkey-hide gelatin utilizing liquid-liquid-liquid three phase static extracting efficient liquid-phase chromatograph Download PDFInfo
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Abstract
The present invention discloses a method adopting liquid-liquid-liquid three-phase static extraction high performance liquid chromatography to identify ass-hide glue. Said invention adopts three solvents with different polarities and different specific weights, uses non-polar solvent as light phase, the polar solvent is intermediate phase (the ass-hide glue can be previously dissolved in said phase). After the phases are separated, three polar extract test solutions are obtained, and said three test solutions are undergone the process of gradient separation by using different mobile phases, then said invention utilizes high-performance chromatography to make determination, records chromatogram and three-dimensional spectrum so as to implement identification of ass-hide glue.
Description
Technical field
The present invention relates to a kind of Chinese medicine analysis field.---liquid---liquid three phase static extracting efficient liquid-phase chromatograph is differentiated the method for donkey-hide gelatin to be specifically related to a kind of liquid.
Background technology
Donkey-hide gelatin is famous Chinese medicine, is the skin by the animal donkey, the jelly made from auxiliary material after decocting.Donkey-hide gelatin mainly is made up of collagen and catabolite thereof, and as polypeptide and each seed amino acid (aliphatics, alkalescence, acidity, aromatic series, heterocyclic amino acid), its range of molecular weight distributions is wide, and the molecular polarity scope is big; Also contain numerous other compositions such as sterols in addition, be difficult to carry out qualitative analysis and quality control.Mule hide, the skin of ox-hide and other animal also can be used for boiling glue class preparation, and its appearance character and chemical constitution and donkey-hide gelatin are closely similar, are difficult to they are differentiated with conventional method.Especially donkey-hide gelatin and mule and horse hide glue derive from the skin of equal genus (equine Equidae Equus Eqnns) animal, and the composition between them is similar, differentiates difficulty more.Therefore, the real and fake discrimination of donkey-hide gelatin fails effectively to be solved always for many years.
At present, the real and fake discrimination of donkey-hide gelatin mainly adopts classic method and experience to differentiate, adopts thin-layer method to differentiate donkey-hide gelatin as version pharmacopeia in 2005, only detects glycocoll wherein, and specificity is not strong, can't monitor its inherent quality.The modern study of donkey-hide gelatin discrimination method comprises that mainly spectrum differentiates (infrared, ultraviolet, circular dichroism spectra etc.), and chromatogram is differentiated (PC, TLC, HPLC, electrophoresis etc.) etc.Spectroscopic methodology is not because of possessing separation function, and the spectral mixture of mensuration is stack spectrum, and composition is many more, complicated more, and the spectral information that obtains is simple more, is used for the discriminating of glue classes such as donkey-hide gelatin, and diagnostic characteristics is not obvious.Though chromatography has separation function, the composition system at the donkey-hide gelatin complexity adopts existing sample pretreating method and chromatographic condition, can not reach desirable separating effect.Separating effect is bad, and the characteristic chemical constituent of negligible amounts will be covered by numerous non-characteristic chemical constituents, and the chromatogram of the various glue of surveying will be very similar, and the true and false of donkey-hide gelatin will be difficult to judge.
Given this, for donkey-hide gelatin is effectively differentiated and Control of Internal Quality, be necessary very much to set up a kind of method of analyzing with overall quality that donkey-hide gelatin is clearly differentiated.
Summary of the invention
At the deficiencies in the prior art, the purpose of this invention is to provide the method that a kind of employing liquid---liquid---liquid three phase static extracting efficient liquid-phase chromatograph is differentiated donkey-hide gelatin, and with the donkey-hide gelatin high performance liquid chromatography as one of index of donkey-hide gelatin real and fake discrimination and quality control.
The employing three phase static extracting efficient liquid-phase chromatograph that the present invention relates to is differentiated the method for donkey-hide gelatin, and key step is as follows:
1, the preparation of donkey-hide gelatin sample solution:
Method a, precision took by weighing the donkey-hide gelatin sample powder of No. 4 sieves, put in the glass container, phase solvent in the middle of adding, and jolting 1～3 minute is put in the ultrasonic cleaning machine and to be made it dissolving in ultrasonic 15～20 minutes, makes the solution of 20mg/ml; Then phase solution in the middle of described is slowly added along wall in the container that fills the heavy phase solvent, slowly add light phase solvent along wall again, make light phase: middle phase: the volume ratio of heavy phase is: 1: 1: 1 or 1: 1: 2 or 2: 1: 1 or 1: 2: 1 or 2: 1: 2; Seal, leave standstill extraction 6 hours～60 hours; Carry out three phase separation after the extraction, light, heavy phase extract are put evaporate to dryness in the water-bath respectively, residue adds anhydrous alcohol solution and constant volume respectively,, heavy phase test liquid light as sample; Will be in the middle of light, heavy two-phase solvent be stripped phase solution, with 3000～4000 rev/mins centrifugal 5～10 minutes, get supernatant after the separation as phase test liquid in the middle of the sample;
Or method b, precision took by weighing the donkey-hide gelatin sample powder of No. 4 sieves, put in the glass container, and phase solvent in the middle of adding, jolting 1～3 minute is put in the ultrasonic cleaning machine and to be made it dissolving in ultrasonic 15～20 minutes, makes the solution of 20mg/ml; Light phase solvent is slowly added in the container that fills middle phase solution along wall, and make light phase: the volume ratio of middle phase is: 1: 1 or 2: 1 or 1: 2; Seal, leave standstill extraction 6 hours～60 hours; Carry out two-phase after the extraction and separate, light phase extract is put evaporate to dryness in the water-bath, and residue adds anhydrous alcohol solution and constant volume, as the light phase test liquid of sample; To slowly be added in the container that fills the heavy phase solvent along wall by the stripped middle phase solution of light phase solvent, phase solution in the middle of making: the volume ratio of heavy phase is: 1: 1 or 1: 2 or 2: 1; Seal, leave standstill extraction 6 hours～60 hours; Carry out two-phase after the extraction and separate, the heavy phase extraction solution is put evaporate to dryness in the water-bath, and residue adds anhydrous alcohol solution and constant volume, as the heavy phase test liquid of sample; With successively respectively by middle phase solution light, the heavy phase solvent extraction, with 3000～4000 rev/mins centrifugal 5～10 minutes, get supernatant after the separation as the middle phase test liquid of sample;
2, the preparation of reference substance solution:
Get DL-β-phenylalanine and DL-tyrosine, add the solution that absolute ethyl alcohol or water are mixed with 5 μ g/ml respectively, as the reference substance solution of middle phase test liquid chromatogram;
Get cholerythrin and add the solution that absolute ethyl alcohol is made 1 μ g/ml, as the reference substance solution of light phase test liquid chromatogram;
Get Quercetin and add the solution that absolute ethyl alcohol is made 5 μ g/ml, as the reference substance solution of heavy phase test liquid chromatogram;
3, the preparation of oxhide gelatin and assorted hide glue sample solution:
Preparation with the donkey-hide gelatin sample solution.
4, the high-performance liquid chromatogram determination of donkey-hide gelatin sample three-phase test liquid
(1), chromatographic condition:
Chromatographic column filler is an octadecylsilane chemically bonded silica; Moving phase is acetonitrile-water, and (gradient that middle phase, light phase, heavy phase test liquid adopt is respectively gradient elution: 0min → 20min → 100min, acetonitrile (V/V): 1% → 1% → 10%; 0min → 5min → 10min → 20min → 40min → 60min → 110min, acetonitrile (V/V): 50% → 50% → 60% → 70% → 85% → 95% → 95%; 0min → 10min → 20min → 30min → 40min → 100min → 120min, acetonitrile (V/V): 30% → 30% → 35% → 35% → 40% → 90% → 90%); Column temperature: 30 ℃; The detection wavelength is 205nm;
(2), chromatographic determination:
Accurate respectively light phase, middle phase, heavy phase test liquid and the reference substance solution thereof drawn with different eluent gradients, according to high effective liquid chromatography for measuring, writes down 100 or 120 minutes chromatogram and three-dimensional spectrum.
5, oxhide gelatin and assorted hide glue sample three-phase test liquid high-performance liquid chromatogram determination:
With donkey-hide gelatin sample three-phase test liquid high-performance liquid chromatogram determination.
6, the HPLC chromatogram is resolved:
HPLC collection of illustrative plates to three kinds of glue is analyzed, and gets the chromatogram of cophase supply test solution, observes chromatogram, three-dimensional plot global feature; Find out the chromatogram peak number of every kind of glue in certain retention time scope, list the three-dimensional spectrum data of every kind of glue in certain retention time scope the chromatographic peak of the terminal absorption spectrum of ultraviolet (only have disregard).
In the middle of donkey-hide gelatin, oxhide gelatin, the assorted hide glue in the HPLC collection of illustrative plates of phase test liquid, in 10～100 minutes retention times, the chromatogram peak number is respectively: 42,50,54.Three-dimensional plot spectrum color spectrum peak number in 25～95 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is respectively: 13,7,18, and the wherein retention time (min) and the ultraviolet maximum absorption wavelength (λ thereof at the three-dimensional collection of illustrative plates of donkey-hide gelatin peak
Max) and minimal absorption wavelength (λ
Min) data are: 26.71min, λ
Max274nm, λ
Min238nm; 37.97min, λ
Max270nm, λ
Min250nm; 38.74
Min, λ
Max274nm, 222nm, λ
Min258nm, 214nm; 44.20min, λ
Max250nm, λ
Min222nm; 54.27min, λ
Max262nm, λ
Min234nm; 57.06min, λ
Max274nm, λ
Min242nm 69.51min, λ
Max246nm, λ
Min234nm; 71.33min, λ
Max254nm, λ
Min226nm; 75.85min, λ
Max254nm, λ
Min226nm; 76.86min, λ
Max274nm, λ
Min242nm; 81.86min, λ
Max270nm, λ
Min242nm; 89.05min, λ
Max262nm, λ
Min234nm; 92.28min, λ
Max262nm, λ
Min242nm;
In the HPLC collection of illustrative plates of donkey-hide gelatin, oxhide gelatin, the assorted light phase test liquid of hide glue, in 10～110 minutes retention times, the chromatogram peak number is respectively: 51,51,56; Three-dimensional plot spectrum color spectrum peak number in 50～65 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is respectively: 19,11,18, and the wherein retention time (min) and the ultraviolet maximum absorption wavelength (λ thereof of donkey-hide gelatin three-dimensional plot spectrum chromatographic peak
Max) and minimal absorption wavelength (λ
Min) data are: 50.39min, λ
Max236nm, λ
Min212nm; 51.19min, λ
Max232nm, λ
Min208nm; 51.94min, λ
Max232nm, λ
Min204nm; 52.73min, λ
Max232nm, λ
Min204nm; 54.13min, λ
Max276nm, 232nm, λ
Min272nm, 212nm; 54.37min, λ
Max268nm, 232nm, λ
Min264nm, 212nm; 54.69min, λ
Max232nm, λ
Min204nm; 55.06min, λ
Max280nm, 228nm, λ
Min256nm, 216nm; 56.53min, λ
Max232nm; 57.77min, λ
Max308nm, 272nm, 230nm, λ
Min284nm, 216nm; 58.63min, λ
Min232nm, λ
Min204nm; 59.15min, λ
Max276nm, λ
Min232nm; 59.77min, λ
Max276nm, λ
Min224nm; 60.43min, λ
Max276nm, λ
Min232nm; 61.56min, λ
MaxBe 276nm, λ
Min228nm; 62,09min, λ
Max222nm, λ
Min216nm; 62.57min, λ
Max276nm, 224nm, λ
Min264nm, 216nm; 63.87min, λ
Max276nm, 220nm, λ
Min256nm, 216nm; 64.67min, λ
Max224nm, λ
Min212nm;
In the HPLC collection of illustrative plates of donkey-hide gelatin, oxhide gelatin, assorted hide glue heavy phase test liquid, in 25～110 minutes retention times, the chromatogram peak number is respectively: 48,44,48.Three-dimensional plot spectrum color spectrum peak number in 25～100 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is respectively: 12,15,24, and the wherein retention time (min) and the ultraviolet maximum absorption wavelength (λ thereof of donkey-hide gelatin three-dimensional plot spectrum chromatographic peak
Max) and minimal absorption wavelength (λ
Min) data are: 25.98min, λ
Max252nm, λ
Min240nm; 61.72min, λ
Max324nm, 228nm, λ
Min268nm, 216nm; 79.12min, λ
Max224nm, λ
Min220nm; 86.12min, λ
Max284nm, 240nm, λ
Min272nm, 212nm; 90.04min, λ
Max276nm, 224nm, λ
Min260nm, 216nm; 90.44min, λ
Max276nm, 224nm, λ
Min260nm, 216nm; 91.58min, λ
Max216nm, λ
Min212nm; 92.24min, λ
Max212nm, λ
Min208nm; 93.66min, λ
Max212nm, λ
Min208nm; 94.51min, λ
Max256nm, 212nm, λ
Min248nm, 208nm; 95.91min, λ
Max212nm, λ
Min208nm; 97.11min, λ
Max272nm, 220nm, λ
Min248nm, 212nm;
As mentioned above, utilize the collection of illustrative plates of three-phase test liquid and data or collection of illustrative plates and data or any a collection of illustrative plates and the data of test liquid mutually of two-phase test liquid arbitrarily, all can make clear and definite judgement the true and false of donkey-hide gelatin.
Differentiate that at above-mentioned employing three phase static extracting efficient liquid-phase chromatograph described donkey-hide gelatin is a said donkey-hide gelatin formulation on any pharmacy in the method for donkey-hide gelatin.
Differentiate in the method for donkey-hide gelatin at above-mentioned employing three phase static extracting efficient liquid-phase chromatograph, the preparation of described donkey-hide gelatin need testing solution also comprise with in the middle of the two-phase static extracting system preparation of middle and light phase composition mutually, light phase test liquid and two-phase static extracting system middle and the heavy phase composition the middle phase, the heavy phase test liquid that prepare.
Differentiate that at above-mentioned employing three phase static extracting efficient liquid-phase chromatograph described light phase solvent is normal hexane or cyclohexane or pentane or heptane or sherwood oil or ether in the method for donkey-hide gelatin; Middle phase solvent is that water or percent by volume are 1%～50% ethanol or 1%～50% methyl alcohol or 1%～50% acetonitrile; The heavy phase solvent is chloroform or methylene chloride or isoamylol.
Wherein, preferably normal hexane or cyclohexane of described light phase solvent; Middle phase solvent is water preferably; The heavy phase solvent is chloroform preferably.
Differentiate that at above-mentioned employing three phase static extracting efficient liquid-phase chromatograph described light phase: middle phase: the volume ratio of heavy phase is preferably: 1: 1: 1 or 1: 1: 2 in the method for donkey-hide gelatin.Described leaving standstill preferably 20 hours～30 hours extraction time.Described stripped middle phase solution preferably separates with 3000 rev/mins of centrifugal conditions of 6 minutes.
Differentiate in the method for donkey-hide gelatin at above-mentioned application three phase static extracting efficient liquid-phase chromatograph, criterion as a result is example with donkey-hide gelatin, oxhide gelatin, assorted hide glue, in the HPLC collection of illustrative plates of its middle phase test liquid, in 10～100 minutes retention times, the chromatogram peak number is respectively: 42,50,54; Three-dimensional plot spectrum color spectrum peak number in 25～95 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is respectively: 13,7,18; In the HPLC collection of illustrative plates of its light phase test liquid, in 10～110 minutes retention times, the chromatogram peak number is respectively: 51,51,56; Three-dimensional plot spectrum color spectrum peak number in 50～65 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is respectively: 19,11,18; In the HPLC collection of illustrative plates of its heavy phase test liquid, in 25～110 minutes retention times, the chromatogram peak number is respectively: 48,44,48; Three-dimensional plot spectrum color spectrum peak number in 25～100 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is respectively: 12,15,24.
Principle of the present invention is in view of the diversity of donkey-hide gelatin composition and complicacy, adopt that common liquid---liquid-liquid extraction method can produce serious emulsion, be difficult to layering and be separated, cause chromatographic resolution degree and poor reproducibility easily, cause the situation that discriminating work can't finish and a kind of sample pretreating method---liquid---liquid---the liquid three phase static extracting method that adopts.According to similar mix principle and molecular diffusion effect, sample is dissolved in middle phase solvent, form the three phase extraction system by middle phase solution and heavy phase with light phase solvent, under static state, the molecule of opposed polarity is extracted by the solvent of corresponding polarity.Suppose to look three kinds of solvent extracts impurity each other, so by extraction, can obtain three kinds of " pure " components that polarity is different, so not only ingeniously solve the refining and issues of purification of sample, also effectively eliminated the serious emulsification phenomenon that common extracting process produces.HPLC chromatographic determination to the three phase extraction thing has been equivalent to prolong effective retention time, has improved detection sensitivity, has improved the degree of separation and the symmetry of chromatographic peak, characteristic peak is fully separated, to reach the purpose that collection of illustrative plates is attractive in appearance, diagnostic characteristics is obvious.
Advantage of the present invention is as follows:
1, the high performance liquid chromatography of the light phase of donkey-hide gelatin sample, middle phase, heavy phase extract has been represented the donkey-hide gelatin main active.The three-phase solvent extract is carried out chromatographic determination, just can be implemented in wideer polarity scope and the longer interior characteristic chemical constituent of finding donkey-hide gelatin of seeking of retention time, with three kinds of chromatograms of three-phase solvent extract, can carry out real and fake discrimination and express its inherent quality comprehensively donkey-hide gelatin exactly.Therefore, adopt liquid---liquid---liquid three phase static extracting efficient liquid-phase chromatograph to differentiate donkey-hide gelatin.
2, adopt the three phase static extracting legal system to be equipped with sample solution, be based on the chemical composition of donkey-hide gelatin complexity, the separation of before analytical test, the opposed polarity composition being taked, enrichment method.In middle phase, by centre and heavy phase and light interface mutually, the non-polar molecule in the donkey-hide gelatin is to gently diffusion mutually with the donkey-hide gelatin sample dissolution, and the low pole molecule spreads to heavy phase, phase in the middle of polar molecule is then stayed.The three-phase solvent extract of supposing donkey-hide gelatin is impurity each other, that is all can regard any two-phase extraction thing wherein as the interfering material of another phase extract chromatographic determination, pass through three phase extraction so, each that promptly is equivalent in the three-phase solvent extract is all made with extra care mutually, therefore the very big interfering material of amplitude minimizing chromatographic determination.Chromatographic determination by to the three phase extraction thing of sample can make the component in each phase extract fully be separated in chromatogram.Like this, help setting up desirable chromatographic condition, improve detection sensitivity, the degree of separation of improving chromatographic peak and symmetry, reduction baseline wander and reduce the quantity at irregular peak, obtain significant, the whole looks of diagnostic characteristics chromatogram attractive in appearance.
Description of drawings
Phase extract HPLC spectrogram (15～100 minutes) in the middle of Fig. 1 .1 donkey-hide gelatin sample
Phase extract HPLC spectrogram (15～100 minutes) in the middle of Fig. 1 .2 oxhide gelatin sample
Phase extract HPLC spectrogram (15～100 minutes) in the middle of the assorted hide glue sample of Fig. 1 .3
Fig. 1 .4 reference substance HPLC spectrogram.A left side: DL-tyrosine; Right: DL-β-phenylalanine (object of reference)
Phase extract HPLC three-dimensional spectrum (15～95 minutes) in the middle of Fig. 1 .5 donkey-hide gelatin sample
Phase extract HPLC three-dimensional spectrum (15～95 minutes) in the middle of Fig. 1 .6 oxhide gelatin sample
Phase extract HPLC three-dimensional spectrum (15～95 minutes) in the middle of the assorted hide glue sample of Fig. 1 .7
The light phase extract of Fig. 2 .1 donkey-hide gelatin sample HPLC three-dimensional spectrum (50～65 minutes)
The light phase extract of Fig. 2 .2 oxhide gelatin sample HPLC three-dimensional spectrum (50～65 minutes)
Fig. 2 .3 light phase extract of hide glue sample HPLC three-dimensional spectrum (50～65 minutes) of mixing
Fig. 3 .1 donkey-hide gelatin sample heavy phase extract HPLC three-dimensional spectrum (25～100 minutes)
Fig. 3 .2 oxhide gelatin sample heavy phase extract HPLC three-dimensional spectrum (25～100 minutes)
Fig. 3 .3 hide glue sample heavy phase extract HPLC three-dimensional spectrum (25～100 minutes) of mixing
Embodiment
The invention will be further described below in conjunction with embodiment, and following embodiment only is used to the present invention is described and is not limitation of the present invention.
The mensuration of embodiment 1 donkey-hide gelatin HPLC collection of illustrative plates
1 instrument and reagent
1.1 instrument
Agilent 1100 high performance liquid chromatographs; Agilent DAD diode array UV-detector; The Agilent chem workstation.
1.2 reagent
DL-β-phenylalanine, DL-tyrosine, cholerythrin reference substance, chromatography is pure, available from Shandong Province pharmaceuticals; The Quercetin standard items are available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute; Acetonitrile (import), chromatographically pure; High purity water; Chloroform, normal hexane, absolute ethyl alcohol, it is pure to be analysis; Donkey-hide gelatin, oxhide gelatin and assorted hide glue are that Shandong Province Liaocheng City medicine inspecting institute provides.
2 chromatographic conditions
Chromatographic column filler is an octadecylsilane chemically bonded silica; Moving phase is acetonitrile-water, and (gradient that middle phase, light phase, heavy phase test liquid adopt is respectively gradient elution: 0min → 20min → 100min, acetonitrile (V/V): 1% → 1% → 10%; 0min → 5min → 10min → 20min → 40min → 60min → 110min, acetonitrile (V/V): 50% → 50% → 60% → 70% → 85% → 95% → 95%; 0min → 10min → 20min → 30min → 40min → 100min → 120min, acetonitrile (V/V): 30% → 30% → 35% → 35% → 40% → 90% → 90%); Column temperature: 30 ℃; The detection wavelength is 205nm; Theoretical cam curve is calculated with DL-β-phenylalanine peak, should be not less than 2500.
3 donkey-hide gelatin chromatographic determinations:
3.1 the preparation of donkey-hide gelatin sample solution:
3.1.1 sample dissolution
Got the donkey-hide gelatin powder 200mg of No. 4 sieves, accurate claimed surely, put in the 10ml measuring bottle, added water (middle phase solvent), and put in the ultrasonic cleaning instrument and to make dissolving in ultrasonic 15 minutes to scale.
3.1.2 extraction
Precision is measured chloroform (heavy phase solvent) 20ml, puts in the 50ml taper separating funnel, and Xian Yanbi slowly adds the good donkey-hide gelatin aqueous solution of above-mentioned dissolving, slowly adds 20ml normal hexane (light phase solvent) along wall again, leaves standstill extraction 30 hours.
3.1.3 sample solution preparation
After leaving standstill extraction, be separated, the heavy phase extraction solution is put evaporate to dryness in the water-bath respectively with light extraction solution mutually, residue adds anhydrous alcohol solution respectively and is settled to 1ml, promptly gets the heavy phase and light test liquid mutually of sample; Middle phase solution is put in the centrifuge tube, and 3000 left the heart 6 minutes, got supernatant as middle phase test liquid.
3.2 the preparation of oxhide gelatin and assorted hide glue sample solution
Preparation with above-mentioned donkey-hide gelatin sample solution.
3.3 the preparation of reference substance solution
3.3.1 measuring, middle phase test liquid uses reference substance solution
Each is an amount of to get DL-β-phenylalanine and DL-tyrosine, adds water respectively and makes the solution that contains 5.0mg among every 1ml.
3.3.2 light phase test liquid is measured and is used reference substance solution
It is an amount of to get cholerythrin, adds absolute ethyl alcohol and makes the solution that contains 1.0mg among every 1ml.
3.3.3 measuring, the heavy phase test liquid uses reference substance solution
It is an amount of to get Quercetin, adds absolute ethyl alcohol and makes the solution that contains 5.0mg among every 1ml.
3.4 donkey-hide gelatin chromatographic determination
3.4.1 the mensuration of middle phase test liquid, accurate middle phase test liquid of donkey-hide gelatin sample and the reference substance solution drawn, according to high effective liquid chromatography for measuring (it is 1: 99 and 90: 10 that the eluent gradient initial sum stops volume ratio), write down 100 minutes chromatogram and three-dimensional spectrum.Selecting DL-β-phenylalanine for use is object of reference (S).With the relative retention time of the chromatographic peak (S peak) of DL-β-phenylalanine is 1 to calculate the relative retention time of other chromatographic peak.
3.4.2 the mensuration of light phase test liquid, accurate light phase test liquid of donkey-hide gelatin sample and the reference substance solution drawn according to high effective liquid chromatography for measuring, writes down 120 minutes chromatogram and three-dimensional spectrum.Selecting cholerythrin for use is object of reference (S).Relative retention time with bilirubinic chromatographic peak (S peak) is the relative retention time of 1 other chromatographic peak of calculating.
3.4.3 the mensuration of heavy phase test liquid, accurate donkey-hide gelatin sample heavy phase test liquid and the reference substance solution drawn according to high effective liquid chromatography for measuring, writes down 120 minutes chromatogram and three-dimensional spectrum.Selecting Quercetin for use is object of reference (S).With the relative retention time of the chromatographic peak (S peak) of Quercetin is 1 to calculate the relative retention time of other chromatographic peak.
4 oxhide gelatin and assorted hide glue chromatographic determination
With the donkey-hide gelatin chromatographic determination.
5 donkey-hide gelatin chromatograms are resolved:
By the mensuration of donkey-hide gelatin, oxhide gelatin, assorted hide glue chromatogram, relatively its chromatogram is listed chromatogram and three-dimensional spectrum data, sees Table 1～table 3.
The three-dimensional spectrum data (25～95 minutes) of phase extract in the middle of table 1 donkey-hide gelatin, oxhide gelatin, the assorted hide glue
| Sample | Peak number | Retention time (min) | λ max(intensity) | Spectroscopic data | ||
| λ min(intensity) | λ max(intensity) | λ min(intensity) | ||||
| Ah | 1 2 3 4 5 6 7 | 26.71 37.97 38.74 44.20 54.27 57.06 69.51 | 274(10.31) 270(3.21) 274(1.50) 250(9.50) 262(4.99) 274(7.50) 246(1.75) | 238(0.70) 250(2.53) 258(0.67) 222(4.00) 234(1.96) 242(0.55) 234(1.13) | 222(7.99) | 214(7.39) |
| Glue | 8 9 10 11 12 13 | 71.33 75.85 76.86 81.86 89.05 92.28 | 254(9.88) 254(1.52) 274(4.64) 270(3.07) 262(4.42) 262(2.32) | 226(3.48) 226(0.90) 242(0.72) 242(1.07) 234(1.94) 242(1.717) | ||
| Oxhide gelatin | 1 2 3 4 5 6 7 | 38.13 43.36 53.46 70.89 75.35 81.61 92.17 | 276(2.05) 252(5.04) 268(2.53) 256(1.26) 256(2.27) 268(2.85) 260(1.98) | 248(0.57) 224(2.01) 240(0.97) 236(0.12) 236(0.52) 236(0.00) 236(0.00) | 224(11.80) | 216(11.04) |
| Assorted hide glue | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 | 26.5 28.69 37.12 38.06 43.13 52.28 52.76 56.23 59.07 65.13 68.33 70.15 72.42 74.71 76.24 81.31 88.42 91.67 | 272(1.97) 252(2.27) 272(4.75) 276(1.27) 248(12.30) 272(1.92) 264(6.17) 272(2.51) 268(1.57) 264(1.59) 248(3.59) 262(14.37) 248(1.92) 252(3.12) 272(1.73) 268(3.35) 260(3.95) 260(2.95) | 240(0.64) 232(1.41) 256(3.74) 260(0.96) 224(6.06) 244(1.26) 232(3.06) 244(0.64) 244(1.03) 240(0.97) 228(2.32) 224(4.99) 240(1.46) 232(1.66) 232(0.64) 240(1.06) 232(1.16) 232(0.91) | 220(6.98) 236(1.66) | 216(6.84) 232(1.35) |
Table 2 donkey-hide gelatin, oxhide gelatin, the three-dimensional spectrum data (50～65 minutes) of the light phase extract of assorted hide glue
| Sample | Peak number | Relative retention time (min) | Spectroscopic data | |||
| λ max(intensity) | λ min(intensity) | λ max(intensity) | λ min(intensity) | |||
| Donkey-hide gelatin | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 | 50.39 51.19 51.94 52.73 54.13 54.37 54.69 55.06 56.53 57.77 58.63 59.15 59.77 60.43 61.56 62.09 62.57 63.87 64.67 | 236(19.64) 232(21.92) 232(79.26) 232(83.06) 276(8.72) 268(9.64) 232(28.80) 280(8.74) 232(153.31) 308(84.11) 220(114.88) 232(70.69) 276(49.71) 276(69.77) 276(42.68) 276(64.99) 220(18.60) 276(14.89) 276(4.71) 224(13.81) | 212(12.76) 208(13.27) 204(26.86) 204(25.45) 272(8.66) 264(9.32) 204(19.45) 256(6.75) 284(15.32) 204(30.06) 232(11.50) 224(11.06) 232(9.19) 228(10.02) 216(18.52) 264(12.78) 256(3.68) 212(11.82) | 232(31.38) 232(19.38) 228(15.14) 272(30.32) 224(88.86) 220(8.50) | 212(21.58) 212(15.41) 216(28.22) 212(12.55) 216(82.48) 216(8.34) |
| Yellow | 1 2 3 | 51.82 53.02 54.44 | 222(7.23) 298(5.45) 274(3.52) | 218(7.04) 262(2.53) 262(3.02) | ||
| Gelatin | 4 5 6 7 8 9 10 11 | 54.98 55.67 56.34 57.60 59.60 61.32 62.38 64.49 | 274(3.93) 290(2.50) 230(17.26) 306(22.64) 242(22.85) 274(6.43) 270(6.98) 274(11.88) 214(8.61) | 262(3.33) 266(2.37) 206(10.64) 282(14.61) 234(22.26) 242(3.97) 234(4.33) 262(10.34) 210(8.40) | 214(8.60) 214(9.43) 270(28.72) 218(52.52) 226(72.55) | 210(8.49) 210(9.32) 246(22.59) 218(69.02) |
| Assorted hide glue | 1 2 3 4 5 6 7 8 9 10 11 12 l3 14 15 16 17 l8 | 50.29 51.87 52.67 53.17 54.03 54.59 55.05 55.68 56.45 57.63 58.53 59.12 59.68 60.39 61.43 62.43 63.06 64.50 | 234(16.98) 234(62.06) 234(63.12) 298(5.65) 290(2.75) 282(4.06) 282(5.20) 218(10.82) 230(31.35) 350(4.80) 270(32.06) 218(61.01) 222(9.75) 278(9.04) 270(18.07) 282(7.27) 270(17.42) 274(14.42) 222(9.58) 222(21.02) | 210(11.91) 262(2.81) 274(2.64) 274(3.56) 258(4.08) 210(10.22) 206(14.06) 334(4.76) 250(26.07) 214(8.95) 242(5.04) 238(6.39) 242(3.99) 238(6.09) 262(12.38) 214(9.07) 206(13.00) | 218(8.06) 230(14.33) 226(12.68) 218(10.68) 306(25.27) 242(27.00) 218(9.00) 226(92.97) | 214(7.81) 214(12.40) 214(11.42) 214(10.23) 286(16.30) 238(26.67) 214(8.62) 218(88.44) |
Table 3 donkey-hide gelatin, oxhide gelatin, the three-dimensional spectrum data of assorted hide glue heavy phase extract
| Sample | Peak number | Relative retention time (min) | Spectroscopic data | |||
| λ max(intensity) | λ min(intensity) | λ max(intensity) | λ min(intensity) | |||
| Donkey-hide gelatin | 1 2 3 4 5 6 7 8 9 10 11 12 | 25.98 61.72 79.12 86.12 90.04 90.44 91.58 92.24 93.66 94.51 95.91 97.11 | 252(3.18) 324(213.37) 224(8.86) 284(3.60) 276(5.22) 276(8.88) 216(7.54) 212(10.40) 212(11.15) 256(2.80) 212(10.83) 272(38.44) | 240(2.56) 268(19.65) 220(8.65) 272(3.20) 260(4.26) 264(7.46) 212(7.41) 208(10.13) 208(11.08) 248(2.38) 208(10.62) 248(26.58) | 228(143.74) 240(38.58) 224(28.638) 224(59.28) 212(7.65) 220(79.59) | 216(117.61) 212(12.48) 216(27.86) 216(55.75) 208(7.62) 212(75.32) |
| Oxhide gelatin | 1 2 3 4 5 6 7 8 9 10 11 12 13 | 50.22 81.46 82.07 82.66 80.06 89.14 90.01 90.41 92.46 93.58 95.67 95.88 97.03 | 214(3.52) 214(4.24) 210(5.76) 214(7.82) 290(2.45) 290(1.36) 274(4.23) 274(8.84) 222(4.91) 222(8.02) 218(4.47) 218(4.87) 222(4.64) | 206(2.01) 206(4.72) 282(2.25) 278(1.27) 266(3.64) 262(7.41) 206(0.40) 206(3.74) 206(2.44) 206(1.65) 210(3.75 | 238(23.71) 250(3.39) 226(24.96) 226(57.39) | 210(3.12) 230(2.25) 214(22.67) 218(53.38) |
| 14 15 | 98.64) 99.55 | 226(4.60) 218(3.82) | 206(1.58) | |||
| Assorted hide glue | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 | 53.38 61.62 68.60 68.71 70.95 72.10 72.82 81.40 82.23 82.67 83.23 83.92 84.99 85.12 86.00 87.94 88.92 89.27 90.00 90.40 91.52 92.19 93.66 97.07 | 286(1.56) 322(75.04) 206(61.50) 270(3.41) 274(3.80) 258(4.78) 282(4.22) 274(5.00) 218(12.79) 286(3.36) 210(12.49) 222(8.06) 286(4.59) 226(8.39) 282(4.09) 286(3.55) 282(5.18) 266(4.43) 282(3.83) 266(5.04) 274(5.90) 274(14.18) 222(8.75) 274(17.03) 224(12.31) 274(177.51) | 266(0.99) 270(7.62) 258(3.29) 250(3.44) 242(4.63) 258(4.06) 270(4.92) 206(9.01) 270(2.89) 206(11.86) 214(6.84) 270(3.60) 214(7.34) 270(3.49) 270(2.92) 278(4.29) 250(3.68) 254(3.76) 238(3.97) 262(5.32) 262(11.72) 206(6.20) 254(13.44) 212(10.52) 250(117.37) | 210(17.25) 230(45.25) 222(8.99) 214(9.95) 242(7.43) 222(11.88) 226(11.65) 238(43.90) 222(6.21) 222(8.50) 218(5.87) 226(30.88) 226(77.20) 222(41.78) 222(346.42) | 218(39.48) 218(8.68) 206(9.07) 238(7.07) 214(9.89) 214(10.73) 206(4.82) 214(7.45) 214(5.20) 214(28.66) 214(71.71) 214(39.35) 210(315.04) |
The result judges: in the middle of the donkey-hide gelatin in the HPLC collection of illustrative plates of phase test liquid, in 10～100 minutes retention times, the chromatogram peak number is 42.Three-dimensional plot spectrum color spectrum peak number in 25～95 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is 13, retention time of its chromatographic peak (min) and ultraviolet maximum absorption wavelength (λ thereof
Max) and minimal absorption wavelength (λ
Min) data are: 26.71min, λ
Max274nm, λ
Min238nm; 37.97min, λ
Max270nm, λ
Min250nm; 38.74min, λ
Max274nm, 222nm, λ
Min258nm, 214nm; 44.20
Min, λ
Max250nm, λ
Min222nm; 54.27min, λ
Max262nm, λ
Min234nm; 57.06
Min, λ
Max274nm, λ
Min242nm; 69.51min, λ
Max246nm, λ
Min234nm; 71.33min, λ
Max254nm, λ
Min226nm; 75.85min, λ
Max254nm, λ
Min226nm; 76.86min, λ
Max274nm, λ
Min242nm; 81.86min, λ
Max270nm, λ
Min242nm; 89.05min, λ
Max262nm, λ
Min234nm; 92.28min, λ
Max262nm, λ
Min242nm;
In the HPLC collection of illustrative plates of the light phase test liquid of donkey-hide gelatin, in 10～110 minutes retention times, the chromatogram peak number is 51; Three-dimensional plot spectrum color spectrum peak number in 50～65 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is 19, the retention time (min) and the ultraviolet maximum absorption wavelength (λ thereof of its three-dimensional plot spectrum chromatographic peak
Max) and minimal absorption wavelength (λ
Min) data are: 50.39min, λ
Max236nm, λ
Min212nm; 51.19min, λ
Max232nm, λ
Min208nm; 51.94min, λ
Max232nm, λ
Min204nm; 52.73min, λ
Max232nm, λ
Min204nm; 54.13min, λ
Max276nm, 232nm, λ
Min272nm, 212nm; 54.37min, λ
Max268nm, 232nm, λ
Min264nm, 212nm; 54.69min, λ
Max232nm, λ
Min204nm; 55.06min, λ
Max280nm, 228nm, λ
Min256nm, 216nm; 56.53min, λ
Max232nm; 57.77min, λ
Max308nm, 272nm, 230nm, λ
Min284nm, 216nm; 58.63min, λ
Max232nm, λ
Min204nm; 59.15min, λ
Max276nm, λ
Min232nm; 59.77min, λ
Max276nm, λ
Min224nm; 60.43min, λ
Max276nm, λ
Min232nm; 61.56min, λ
MaxBe 276nm, λ
Min228nm; 62,09min, λ
Max222nm, λ
Min216nm; 62.57min, λ
Max276nm, 224nm, λ
Min264nm, 216nm; 63.87min, λ
Max276nm, 220nm, λ
Min256nm, 216nm; 64.67min, λ
Max224nm, λ
Min212nm;
In the HPLC collection of illustrative plates of donkey-hide gelatin heavy phase test liquid, in 25～110 minutes retention times, the chromatogram peak number is 48.Three-dimensional plot spectrum color spectrum peak number in 25～100 minutes retention times peak of the terminal absorption spectrum of ultraviolet (only have disregard) is: 12, and the retention time (min) and the ultraviolet maximum absorption wavelength (λ thereof of its three-dimensional plot spectrum chromatographic peak
Max) and minimal absorption wavelength (λ
Min) data are: 25.98min, λ
Max252nm, λ
Min240nm; 61.72min, λ
Max324nm, 228nm, λ
Min268nm, 216nm; 79.12min, λ
Max224nm, λ
Min220nm; 86.12min, λ
Max284nm, 240nm, λ
Min272nm, 212nm; 90.04min, λ
Max276nm, 224nm, λ
Min260nm, 216nm; 90.44min, λ
Max276nm, 224nm, λ
Min260nm, 216nm; 91.58min, λ
Max216nm, λ
Min212nm; 92.24min, λ
Max212nm, λ
Min208nm; 93.66min, λ
Max212nm, λ
Min208nm; 94.51min, λ
Max256nm, 212nm, λ
Min248nm, 208nm; 95.91min, λ
Max212nm, λ
Min208nm; 97.11min, λ
Max272nm, 220nm, λ
Min248nm, 212nm;
The experiment of 6 chromatogram reappearances
With the donkey-hide gelatin is test sample (N=5), operate with reference to finger-print reappearance prescriptive procedure, to total peak relative retention time and there is the relative peak area at the total peak (having the total peak of regulation promptly to surpass the fingerprint peaks of total peak area 10%) of regulation to add up, RSD% is no more than 3% respectively.The results are shown in Table 2
The experiment of 7 chromatogram precision
Operate with reference to finger-print reappearance prescriptive procedure, with the donkey-hide gelatin is test sample, get 1 part, continuous sample introduction 5 times, to total peak relative retention time and there is the relative peak area at the total peak (having the total peak of regulation promptly to surpass the fingerprint peaks of total peak area 10%) of regulation to add up, RSD% is no more than 3% respectively.
8 chromatogram stability experiments
Operate with reference to finger-print reappearance prescriptive procedure, with the donkey-hide gelatin is test sample, investigate 24 hours solution-stabilized, to total peak relative retention time and there is the relative peak area at the total peak (having the total peak of regulation promptly to surpass the fingerprint peaks of total peak area 10%) of regulation to add up, RSD% is no more than 3% respectively.Test sample was stablized in 24 hours.
The mensuration of embodiment 2 donkey-hide gelatin HPLC collection of illustrative plates
Except that different with 3.1.2 extraction among the embodiment 1, all the other are all identical.
The extracting process of embodiment 2 is: will dissolve good middle phase solution, and put in the 50ml taper separating funnel, and slowly add the 20ml cyclohexane along wall, and seal, leave standstill extraction 50 hours.After extraction is finished, be separated, light phase extract is put evaporate to dryness in the water-bath, residue adds anhydrous alcohol solution and is settled to concentration is 1g/ml, as the light phase test liquid of sample; To slowly be added in another 50ml taper separating funnel that fills the 20ml methylene chloride along wall by the stripped middle phase solution of light phase solvent, seal, leave standstill extraction 55 hours.Be separated after the extraction, the heavy phase extraction solution is put evaporate to dryness in the water-bath, and residue adds anhydrous alcohol solution and is settled to concentration is 1g/ml, as the heavy phase test liquid of sample; To be put in the centrifuge tube by middle phase solution light, the heavy phase solvent extraction, 3500 left the heart 8 minutes, got supernatant as middle phase test liquid.
Claims (7)
1. method that adopts liquid---liquid---liquid three phase static extracting efficient liquid-phase chromatograph to differentiate donkey-hide gelatin is characterized in that key step is as follows:
The first step, donkey-hide gelatin HPLC collection of illustrative plates is measured:
(1) preparation of donkey-hide gelatin need testing solution:
Method a, precision took by weighing the donkey-hide gelatin sample powder of No. 4 sieves, put in the glass container, phase solvent in the middle of adding, and jolting 1～3 minute is put in the ultrasonic cleaning machine and to be made it dissolving in ultrasonic 15～20 minutes, makes the solution of 20mg/ml; Then phase solution in the middle of described is slowly added along wall in the container that fills the heavy phase solvent, slowly add light phase solvent along wall again, make light phase: middle phase: the volume ratio of heavy phase is: 1: 1: 1 or 1: 1: 2 or 2: 1: 1 or 1: 2: 1 or 2: 1: 2; Seal, leave standstill extraction 6 hours～60 hours; Carry out three phase separation after the extraction, light, heavy phase extract are put evaporate to dryness in the water-bath respectively, residue adds anhydrous alcohol solution and constant volume respectively,, heavy phase test liquid light as sample; Will be in the middle of light, heavy two-phase solvent be stripped phase solution, with 3000～4000 rev/mins centrifugal 5～10 minutes, get supernatant after the separation as phase test liquid in the middle of the sample;
Or method b, precision took by weighing the donkey-hide gelatin sample powder of No. 4 sieves, put in the glass container, and phase solvent in the middle of adding, jolting 1～3 minute is put in the ultrasonic cleaning machine and to be made it dissolving in ultrasonic 15～20 minutes, makes the solution of 20mg/ml; Light phase solvent is slowly added in the container that fills middle phase solution along wall, and make light phase: the volume ratio of middle phase is: 1: 1 or 2: 1 or 1: 2; Seal, leave standstill extraction 6 hours～60 hours; Carry out two-phase after the extraction and separate, light phase extract is put evaporate to dryness in the water-bath, and residue adds anhydrous alcohol solution and constant volume, as the light phase test liquid of sample; To slowly be added in the container that fills the heavy phase solvent along wall by the stripped middle phase solution of light phase solvent, phase solution in the middle of making: the volume ratio of heavy phase is: 1: 1 or 1: 2 or 2: 1; Seal, leave standstill extraction 6 hours～60 hours; Carry out two-phase after the extraction and separate, the heavy phase extraction solution is put evaporate to dryness in the water-bath, and residue adds anhydrous alcohol solution and constant volume, as the heavy phase test liquid of sample; With successively respectively by middle phase solution light, the heavy phase solvent extraction, with 3000～4000 rev/mins centrifugal 5～10 minutes, get supernatant after the separation as the middle phase test liquid of sample;
(2) preparation of reference substance solution:
Get DL-β-phenylalanine and DL-tyrosine, add the solution that absolute ethyl alcohol or water are mixed with 5 μ g/ml respectively, as the reference substance solution of middle phase test liquid chromatogram;
Get cholerythrin and add the solution that absolute ethyl alcohol is made 1 μ g/ml, as the reference substance solution of light phase test liquid chromatogram;
Get Quercetin and add the solution that absolute ethyl alcohol is made 5 μ g/ml, as the reference substance solution of heavy phase test liquid chromatogram;
(3) chromatographic condition:
Chromatographic column filler is an octadecylsilane chemically bonded silica; Moving phase is acetonitrile-water, gradient elution, and the gradient that middle phase, light phase, heavy phase test liquid adopt is respectively: 0min → 20min → 100min, acetonitrile (V/V): 1% → 1% → 10%; 0min → 5min → 10min → 20min → 40min → 60min → 110min, acetonitrile (V/V): 50% → 50% → 60% → 70% → 85% → 95% → 95%; 0min → 10min → 20min → 30min → 40min → 100min → 120min, acetonitrile (V/V): 30% → 30% → 35% → 35% → 40% → 90% → 90%; Column temperature: 30 ℃; The detection wavelength is 205nm;
(4) measure:
Phase, light phase, heavy phase test liquid and corresponding with it reference substance solution according to high effective liquid chromatography for measuring, write down 100 or 120 minutes chromatogram and three-dimensional spectrum in the middle of accurate respectively the absorption;
Second step is to measure the HPLC collection of illustrative plates that identical method is measured oxhide gelatin and assorted hide glue respectively with above-mentioned donkey-hide gelatin HPLC collection of illustrative plates;
The 3rd step was analyzed the HPLC collection of illustrative plates of the cophase supply test solution of donkey-hide gelatin, oxhide gelatin, assorted hide glue, observed the difference of chromatogram, three-dimensional plot global shape; List the three-dimensional spectrum data of every kind of glue in certain retention time, wherein, the ultra-violet absorption spectrum of chromatographic peak only has terminal disregarding of absorbing.
2. donkey-hide gelatin discrimination method as claimed in claim 1 is characterized in that: described donkey-hide gelatin is the formulation of said donkey-hide gelatin on any pharmacy.
3. donkey-hide gelatin discrimination method as claimed in claim 1 is characterized in that: the preparation of described donkey-hide gelatin need testing solution also comprise adopt in the middle of with the two-phase static extracting system preparation of light phase composition in the middle of mutually, light phase test liquid and adopt in the middle of and the two-phase static extracting system of heavy phase the composition middle phase, the heavy phase test liquid that prepare.
4. as the described donkey-hide gelatin discrimination method of one of claim 1～3, it is characterized in that: described light phase extraction solvent is hexane or cyclohexane or pentane or heptane or sherwood oil or ether; Middle phase extraction solvent is water or 1%～50% ethanol or 1%～50% methyl alcohol or 1%～50% acetonitrile; The heavy phase extraction solvent is chloroform or methylene chloride or isoamylol.
5. as claim 1 or 3 described donkey-hide gelatin discrimination methods, it is characterized in that: it is light phase that described three-phase solvent extraction is compared: middle phase: the ratio of heavy phase volume is: 1: 1: 1 or 1: 1: 2.
6. donkey-hide gelatin discrimination method as claimed in claim 1 is characterized in that: describedly leave standstill extraction, the extraction time is 20 hours～30 hours.
7. donkey-hide gelatin discrimination method as claimed in claim 1 is characterized in that: in the middle of described donkey-hide gelatin, oxhide gelatin, the assorted hide glue in the HPLC collection of illustrative plates of phase test liquid, in 10～100 minutes retention times, the chromatogram peak number is respectively: 42,50,54; Three-dimensional plot spectrum color spectrum peak number in 25～95 minutes retention times wherein only has a peak of the terminal absorption spectrum of ultraviolet to disregard, and is respectively: 13,7,18, and the wherein retention time (min) and the ultraviolet maximum absorption wavelength (λ thereof at the three-dimensional collection of illustrative plates of donkey-hide gelatin peak
Max) and minimal absorption wavelength (λ
Min) data are: 26.71min, λ
Max274nm, λ
Min238nm; 37.97min, λ
Max270nm, λ
Min250nm; 38.74min, λ
Max274nm, 222nm, λ
Min258nm, 214nm; 44.20min, λ
Max250nm, λ
Min222nm; 54.27min, λ
Max262nm, λ
Min234nm; 57.06min, λ
Max274nm, λ
Min242nm; 69.51min, λ
Max246nm, λ
Min234nm; 71.33min, λ
Max254nm, λ
Min226nm; 75.85min, λ
Max254nm, λ
Min226nm; 76.86min, λ
Max274nm, λ
Min242nm; 81.86min, λ
Max270nm, λ
Min242nm; 89.05min, λ
Max262nm, λ
Min234nm; 92.28min, λ
Max262nm, λ
Min242nm;
In the HPLC collection of illustrative plates of described donkey-hide gelatin, oxhide gelatin, the assorted light phase test liquid of hide glue, in 10～110 minutes retention times, the chromatogram peak number is respectively: 51,51,56; Three-dimensional plot spectrum color spectrum peak number in 50～65 minutes retention times wherein only has a peak of the terminal absorption spectrum of ultraviolet to disregard, and is respectively: 19,11,18, and the wherein retention time (min) and the ultraviolet maximum absorption wavelength (λ thereof of donkey-hide gelatin three-dimensional plot spectrum chromatographic peak
Max) and minimal absorption wavelength (λ
Min) data are: 50.39min, λ
Max236nm, λ
Min212nm; 51.19min, λ
Max232nm, λ
Min208nm; 51.94min, λ
Max232nm, λ
Min204nm; 52.73min, λ
Max232nm, λ
Min204nm; 54.13min, λ
Max276nm, 232nm, λ
Min272nm, 212nm; 54.37min, λ
Max268nm, 232nm, λ
Min264nm, 212nm; 54.69min, λ
Max232nm, λ
Min204nm; 55.06min, λ
Max280nm, 228nm, λ
Min256nm, 216nm; 56.53min, λ
Max232nm; 57.77min, λ
Max308nm, 272nm, 230nm, λ
Min284nm, 216nm; 58.63min, λ
Max232nm, λ
Min204nm; 59.15min, λ
Max276nm, λ
Min232nm; 59.77min, λ
Max276nm, λ
Min224nm; 60.43min, λ
Max276nm, λ
Min232nm; 61.56min, λ
MaxBe 276nm, λ
Min228nm; 62,09min, λ
Max222nm, λ
Min216nm; 62.57min, λ
Max276nm, 224nm, λ
Min264nm, 216nm; 63.87min, λ
Max276nm, 220nm, λ
Min256nm, 216nm; 64.67min, λ
Max224nm, λ
Min212nm;
In the HPLC collection of illustrative plates of described donkey-hide gelatin, oxhide gelatin, assorted hide glue heavy phase test liquid, in 25～110 minutes retention times, the chromatogram peak number is respectively: 48,44,48; Three-dimensional plot spectrum color spectrum peak number in 25～100 minutes retention times wherein only has a peak of the terminal absorption spectrum of ultraviolet to disregard, and is respectively: 12,15,24, and the wherein retention time (min) and the ultraviolet maximum absorption wavelength (λ thereof of donkey-hide gelatin three-dimensional plot spectrum chromatographic peak
Max) and minimal absorption wavelength (λ
Min) data are: 25.98min, λ
Max252nm, λ
Min240nm; 61.72min, λ
Max324nm, 228nm, λ
Min268nm, 216nm; 79.12min, λ
Max224nm, λ
Min220nm; 86.12min, λ
Max284nm, 240nm, λ
Min272nm, 212nm; 90.04min, λ
Max276nm, 224nm, λ
Min260nm, 216nm; 90.44min, λ
Max276nm, 224nm, λ
Min260nm, 216nm; 91.58min, λ
Max216nm, λ
Min212nm; 92.24min, λ
Max212nm, λ
Min208nm; 93.66min, λ
Max212nm, λ
Min208nm; 94.51min, λ
Max256nm, 212nm, λ
Min248nm, 208nm; 95.91min, λ
Max212nm, λ
Min208nm; 97.11min, λ
Max272nm, 220nm, λ
Min248nm, 212nm.
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| CN102406749A (en) * | 2010-11-22 | 2012-04-11 | 九芝堂股份有限公司 | HPLC (high-performance liquid chromatography) detection method capable of simultaneously detecting contents of six amino acids in donkey-hide glue blood-supplementing preparation |
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| CN102406749A (en) * | 2010-11-22 | 2012-04-11 | 九芝堂股份有限公司 | HPLC (high-performance liquid chromatography) detection method capable of simultaneously detecting contents of six amino acids in donkey-hide glue blood-supplementing preparation |
| CN102406749B (en) * | 2010-11-22 | 2013-08-28 | 九芝堂股份有限公司 | HPLC (high-performance liquid chromatography) detection method capable of simultaneously detecting contents of six amino acids in donkey-hide glue blood-supplementing preparation |
| CN103630621A (en) * | 2013-10-28 | 2014-03-12 | 山东东阿阿胶股份有限公司 | Method for detecting sheep-derived ingredients in glue type traditional Chinese medicines and products thereof |
| CN105203696A (en) * | 2015-10-27 | 2015-12-30 | 河北中医学院 | Multi-developing solvent rapid thin-layer chromatographic method for medicinal material donkey-hide gelatin and water extract thereof |
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