CN106872602A - Calm the nerves the multicomponent content assaying method of precious particle - Google Patents

Abstract

The invention discloses a kind of multicomponent content assaying method of precious particle of calming the nerves, belong to the technical field of Chinese medicine preparation quality control.The present invention uses high performance liquid chromatography, while Saponin A, scopolactone, three kinds of compounds of Quercetin carry out assay in precious particle of calming the nerves under same chromatographic condition.The present invention is determined simultaneously using many contents, with globality, characteristic, stability, can combined reaction calm the nerves the inherent quality of precious granular preparation, be the quality control level for further improving precious particle of calming the nerves, ensure product quality, can ensure that the stability between precious granules batch of calming the nerves.

Description

Calm the nerves the multicomponent content assaying method of precious particle

Technical field

The invention belongs to Chinese medicine preparation field of quality control, it is related to a kind of multicomponent assay side of precious particle of calming the nerves Method.

Background technology

Calm the nerves precious particle tonifying kidney and benefiting sperm, mental-tranquilization.For insomnia forgetfulness, dizziness and tinnitus, soreness and weakness of waist and knees.It is for treating The herbaceous plant prescription Chinese patent drug of insomnia forgetfulness, formulation is granule.The spina date seed of the selected Xingtai area of this product is closed with matrimony vine, north Vigorously, scientific formula, compatibility are reasonable.The existing detection method of this preparation is extracted for precious preparation of calming the nerves using thin layer discriminating, n-butanol Thing carries out quality control, has not met the development of drug inspection technology at this stage.With China's pharmaceutical production quality control The raising of level, State Food and Drug Administration proposes " national drug standards raising action plan ", with General Promotion medicine The quality control level of product.Therefore, a kind of method of the precious granular mass that can fast, accurately and comprehensively control to calm the nerves is set up, it is right to realize Comprehensive control of its material group, has great importance.

The main chemical compositions of matrimony vine have LBP-X (LBP), matrimony vine flavones, glycine betaine (betaine), carotenoid And carotenoid ester, vitamin C, scopoletin (scopoletin), several amino acids and elements K, Na, Ca, Mg, Cu, The compositions such as Fe, Mn, Zn, P [11].Additionally, still from matrimony vine isolated cyclic peptide (Cyclicpeptides) and matrimony vine element A-D [12], cerebroside [13] etc..Wherein, LBP-X is matrimony vine main active, with enhancing immunization；Glycine betaine, Zeaxanthine and the palmitic acid of zeaxanthine two, cerebroside have protective effect to the hepatic lesion that carbon tetrachloride causes；Matrimony vine Plain A and B have the activity [1 for suppressing angiotensin converting enzyme.

Spina date seed contains alkaloid：Spina date seed alkali (sanjoinine) A, B, D, E, F, G1, G2, Ia, Ib, K, also containing spina date seed Cyclic peptide (sanjoinenine).Also contain triterpenes：Betulic acid (betulinicacid), betulin (betulin), America Boheic acid (ceanothic acid), alphitolic acid (alphitolicacid), jujuboside (jujuboside) A, B, and recklessly Glucoraphenin (daucosterol).Contain flavonoids again：Spinosin (spinosin), wild jujube flavine (zivulgarin), 6 " '-mustard Sub- acyl spinosin (6 " '-sinapoylspinosin), 6 " '-asafoetide acyl spinosin (6 " '-feruloylspinosin), 6 " '-to coumaric acyl spinosin (6 " '-p-coumaroylspinosin), swertisin (swer-tisin), 6,8- bis--C- Portugals Grape glycosyl apiolin (vicenin II), apiolin -6-C- [(6-O- para hydroxybenzenes formyl)-β-D- glucopyranosyls (1 → 2)]-β-D- glucopyanosides [apigenin-6-C- [(6-O-p-hydroxybenzoyl)-β-D-glucopyranosyl (1 → 2)]-β-D-glucopyranoside] etc..Also (threonine) containing threonine, valine (valine), methionine (methionine), leucine (leucine), isoleucine (isoleucine), lysine (lysine), phenylalanine (phenylalanine) Determination of multiple metal elements such as 17 kinds of amino acid and potassium, sodium, calcium, zinc, iron, copper, manganese such as.Contain forulic acid again (ferulic acid), vitamin C and phytosterol, CAMP (cyclic adenosine 3', 5'- Monophosphate) etc..

Cany silktree, mental-tranquilization；And blood and relieving pain.Main palpitation and insomnia；Forgetful dreaminess；Toothache；Muscle and bone ache；Waist-leg is numb；Fall Injure bitterly.Main chemical compositions are flavone compound, such as Quercetin, rutin.

The selection of drug inspection method, should be according to the principle of " accurate, sensitive, easy, quick ", it is however emphasized that method is applicable Property, and note absorbing Domestic Scientific Research achievement and foreign advanced experience；Current country's physical condition should be considered, new skill is reacted again The application development of art, further improves and improves detection level.

The content of the invention

The present invention is to provide for a kind of multicomponent content assaying method of precious particle of calming the nerves.

The present invention does not carry out content control for the existing quality standard of precious particle of calming the nerves, it is proposed that precious particle of calming the nerves is while enter The multicomponent assay of row, will have the main component of effect of calming the nerves in spina date seed：Saponin A, Spine Date Seed jujubosideB, There is the composition of definite calmness effect in matrimony vine：Scopolactone；Cany silktree main component Quercetin as assay parameter, Make to calm the nerves precious particle quality standard with more globality, characteristic and stability, and to instrument, chromatographic column, detector, Detection wavelength, chromatogram flow phase, column temperature study repeatedly, optimized, by the quantitative control of multi-target ingredient, can be comprehensive Reflect the inherent quality of precious particle of calming the nerves, lift the quality control level of preparation, it is ensured that the steady quality of the multiple batches of preparation of preparation Uniformity.

Multicomponent content assaying method of the present invention is under same chromatographic condition, while to institute in precious particle of calming the nerves The Saponin A that contains, scopolactone, three kinds of chemical compositions of Quercetin carry out liquid chromatogram assay.

Multicomponent content assaying method of the present invention is comprised the steps of：

First, chromatographic condition and system suitability test：

Chromatographic column：Agilent Zorbox SB C18 4.6mm × 150mm, 5 μ

Column temperature：35 DEG C of column temperature

Detection：EISD

Sample size：10μl

Eluent gradient elutes table：Mobile phase：A is methyl alcohol, and B is 0.5% glacial acetic acid

2nd, the preparation of reference substance solution：Precision weighs Saponin A, scopolactone, Quercetin reference substance and fits respectively Amount, is respectively placed in 10mL volumetric flasks, plus methyl alcohol dissolves and is diluted to scale, shakes up, and obtains final product reference substance storing solution；It is accurate respectively Above-mentioned reference substance storing solution 1.0ml is measured to be placed in same 20ml volumetric flasks, plus methanol constant volume is to scale, shakes up, as right According to product solution.

3rd, the preparation of need testing solution：This product powder (cross No. four sieve) about 1g is taken, it is accurately weighed, in putting conical flask with cover, Precision plus the ethanol 50ml of people 70%, weighed weight is heated to reflux 6 hours, lets cool, then weighed weight, is supplied with 70% ethanol and subtracted The weight of mistake, shakes up, filtration.Precision measures subsequent filtrate 25ml, puts in flask, is concentrated into about l ml, plus 3mol/L hydrochloric acid solutions L0ml, heats hydrolysis 2 hours in water-bath, cool down immediately, moves in people's separatory funnel, with water 10ml gradation washing containers, is incorporated to point In liquid funnel, plus sodium chloride 2g, being extracted 5 times with chloroform strength shaking, each 15m1 merges chloroform liquid, plus anhydrous Sodium sulphate 2g, stirring, filtration, container wash with a small amount of chloroform, filters, and filtrate merges, and less than 70 DEG C are concentrated into and closely do, vertical I.e. plus methyl alcohol makes dissolving, it is transferred in 10ml measuring bottles, and is diluted to scale, shake up, obtains final product need testing solution.

4th, determination method：Determined according to step (1) chromatographic condition, obtained final product.

Multicomponent content assaying method research of the present invention is the preferred plan obtained by a large amount of screening tests, with Lower experimental study is preferred process of the invention, chromatographic condition and system suitability test：

Precious granular recipe of calming the nerves be spina date seed, matrimony vine, cany silktree, wherein, in spina date seed main component be jujuboside A, Spine Date Seed jujubosideB, matrimony vine main component are glycine betaine, scopolactone, and cany silktree main component is flavones ingredient, quercitrin Element, Kaempferol etc., in order to reduce test operation, improve the efficiency of detection, and we are studied for main component, try hard to same When determine above-mentioned main component.

Chemical research and the assay report of jujuboside constituents are more, and being summed up main analysis method has Colorimetric method, thin layer chromatography scanning and high performance liquid chromatography, wherein it is more accurate with high effective liquid chromatography for measuring, but due to wild jujube The UV absorption of benevolence saponin A and B is very weak, therefore, though having document report with conventional HPLC-UV methods, detection sensitivity is very It is low.RPLC --- evaporative light-scattering detector (RP-HPLC-ELSD) is conventional in recent years to be suitable for not having The detection method of UV absorption composition, can equally be applicable simultaneously for the composition for having UV absorption.

Beet alkali content is determined AAS, thin layer chromatography scanning, non-aqueous titration and high performance liquid chromatography etc..It is high Effect liquid phase chromatogram method is gradually applied to separation analysis and the content of chemical composition of Chinese materia medica as a kind of modern separate analytical technique Determine, the application in terms of plant beet alkali content measure is also increasingly extensive, but due in betaine structure without conjugated system, only Can be detected with refractometer or be determined in low UV absorption wavelength (190~220nm), condition requirement is higher, and sensitivity or selection Property is not ideal enough.Therefore, the general high performance liquid chromatography for using is surveyed after column front derivation is carried out to beet alkali extract Fixed, this method destroys the primitive component of beet alkali extract.Young etc. [31] reports can be direct without derivative using NH2 posts The content of northern Lycium chinense glycine betaine is determined, method is quick, easy

Scopolactone is water insoluble, dissolves in methyl alcohol.Universal method is to extract scopolin with ethanol, using acid Hydrolyze, then extract scopolactone and detected, general Detection wavelength is 298nm.

The Detection wavelength of Quercetin is extracted using methyl alcohol substantially in 256nm, generally and detected.

1st, Detection wavelength is determined

The ultraviolet detection wavelength of comprehensive several main components is different, and ultraviolet detection ambient interferences are than larger More preferably while being applicable the detection of multiple compositions, we are not had using EISD (HPLC-ELSD) to UV absorption Require, it is adaptable to most composition detection.

2nd, the research of mobile phase

The preparation of reference substance solution：Precision weighs Saponin A, Spine Date Seed jujubosideB, scopolactone, beet respectively Alkali, Quercetin reference substance are appropriate, plus methanol solution is configured to the solution of 1.0mg/mL respectively, used as reference substance solution.Adopt first Preliminary experiment, chromatographic column are carried out with following mobile phase：AgilentZorbox SB C18 4.6mm × 150mm, 5 μ, 35 DEG C of column temperature, Flow velocity 1.0ml/min, EISD detection is as a result as follows：

1. mobile phase is：0.5% glacial acetic acid-methyl alcohol (32：68), difference sample introduction Saponin A, Spine Date Seed jujubosideB, east Henbane lactone, glycine betaine, Quercetin reference substance reference substance solution, in detection Saponin A, Spine Date Seed jujubosideB, Anisodus luridus Ester, Quercetin, scopolactone and Quercetin appearance time cannot be separated too early.

2. mobile phase is：Water-methanol-acetic acid (45:50:5), difference sample introduction Saponin A, Spine Date Seed jujubosideB, east Liang Henbane lactone, glycine betaine, Quercetin reference substance reference substance solution, detection Saponin A, Spine Date Seed jujubosideB, scopolactone, Quercetin, Quercetin appearance time cannot be separated too early, and jujube kernel saponin A, Spine Date Seed jujubosideB appearance time are long, and peak shape is not inconsistent Close and require.

3. mobile phase is：Methanol-water-glacial acetic acid (32:68:0.16), difference sample introduction Saponin A, jujuboside B, scopolactone, glycine betaine, Quercetin reference substance reference substance solution, detection Quercetin, other peak shapes are undesirable.

According to above result of the test, while detecting that multicomponent needs to carry out gradient elution, each constituents are separated.

3rd, eluent gradient wash-out research

It is prepared by reference substance solution：Respectively precision weigh Saponin A, Spine Date Seed jujubosideB, scopolactone, glycine betaine, Quercetin reference substance is appropriate, is respectively placed in 10mL volumetric flasks, plus methyl alcohol dissolves and is diluted to scale, shakes up, and obtains final product reference substance Storing solution；Precision measures above-mentioned reference substance storing solution 1.0ml and is placed in same 20ml volumetric flasks respectively, plus methanol constant volume is extremely carved Degree, shakes up, as reference substance solution.

1. preliminary experiment：

Mobile phase：A is methyl alcohol, and B is 0.5% glacial acetic acid

Time	Mobile phase A %	Mobile phase B %
			0	2	98
7	4	96
			12	18	82
15	20	80
			22	23	77
32	32	68
			42	35	62
50	90	10

Because mobile phase ratio has an aritical ratio in change procedure during the presence of gradient elution peak, that ratio is reached The absorption value of mobile phase just occurs larger change when example, and the detection of disturbed specimen, the appearance of several reference substances is produced Raw interference.We attempt changing gradient slope allowing it to change position and peak type.

2. gradient system is adjusted

Gradient system is changed to gradual change system, reduces interference of the suddenly change of mobile phase absorption value to reference substance appearance.

Time	Mobile phase A %	Mobile phase B %
			0-5	2-10	98-90
5-15	10	90
			15-25	10-20	90-80
25-35	20	80
			35-45	20-30	80-70
45-50	30-35	70-65
			50-70	90	10

Peak shape improves, and wherein Saponin A, Quercetin peak shape are good, Spine Date Seed jujubosideB, scopolactone, glycine betaine Appearance time is in eluent gradient and changes interval, does not reach baseline separation.Continue to adjust each interval gradient of mobile phase.Fig. 2

3. gradient system is determined

Time	Mobile phase A %	Mobile phase B %
			0-5	2-10	98-90
5-15	10	90
			15-30	10-30	90-70
30-40	30	70
			40-50	30-45	70-55
50-60	45	55
			60-80	90	10

Peak shape improves, and peak shape improves, and Saponin A, Spine Date Seed jujubosideB, scopolactone, glycine betaine, Quercetin go out Peak time is in proportion of mobile phase stable region, reaches baseline separation.Fig. 3

4th, need testing solution preparation research

Gradient system is studied with reference substance, and because impurity is numerous in sample, sample refinement method determines that impurity is It is no that interference is produced to composition to be measured, We conducted the research of need testing solution process for purification.

1. during precision steelyard takes the volumetric flask of sample 2g to 10ml, ultrasonic extraction 20min is put to room temperature, with methanol constant volume, is filled Divide and shake up, 0.45 μm of miillpore filter is crossed, as need testing solution 1.

2. precision steelyard takes sample 2g in apparatus,Soxhlet's, plus 50ml petroleum ethers, and heating water bath is extracted 2 hours, abandons or adopts stone Oily ether, then with 50ml methyl alcohol surname extraction 3 hours, methanol extract liquid is collected, reclaim methyl alcohol and be quantitatively transferred to the capacity of 10ml In bottle, with methanol constant volume, fully shake up, 0.45 μm of miillpore filter is crossed, as need testing solution 2.

3. this product powder (crossing No. four sieves) about 1g is taken, it is accurately weighed, to put in conical flask with cover, precision adds the ethanol of people 70% 50ml, weighed weight is heated to reflux 6 hours, lets cool, then weighed weight, and the weight of less loss is supplied with 70% ethanol, is shaken up, filter Cross.Precision measures subsequent filtrate 25ml, puts in flask, is concentrated into about l ml, plus 3mol/L hydrochloric acid solution l0ml, and water is heated in water-bath Solution 2 hours, cools down immediately, moves in people's separatory funnel, with water 10ml gradation washing containers, is incorporated in separatory funnel, plus sodium chloride 2g, is extracted 5 times with chloroform strength shaking, and each 15m1 merges chloroform liquid, plus anhydrous sodium sulfate 2g, stirring, filter Cross, container is washed with a small amount of chloroform, filter, filtrate merges, less than 70 DEG C be concentrated into it is near dry, immediately plus methyl alcohol makes dissolving, It is transferred in 10ml measuring bottles, and is diluted to scale, shake up, obtains final product need testing solution 3.

Chromatographic condition：

Mobile phase：Mobile phase：A is methyl alcohol, and B is 0.5% glacial acetic acid, and flow velocity 1ml/min, EISD enters The μ l of sample amount 10.

Gradient elution：

Time	Mobile phase A %	Mobile phase B %
			0-5	2-10	98-90
5-15	10	90
			15-30	10-30	90-70
30-40	30	70
			40-50	30-45	70-55
50-60	45	55
			60-80	90	10

As a result：

Need testing solution 1 is because without removal step, impurity peaks produce severe jamming to peak to be measured.

Not exclusively, impurity peaks still have interference to the removal of impurities of need testing solution 2.

, due to adding hydrolysis removal step, glycine betaine is without appearance, Quercetin, Saponin A, east Liang for need testing solution 3 Henbane lactone appearance is good, and noiseless, Spine Date Seed jujubosideB appearance partly overlaps with impurity peaks, does not reach separation.

Adjustment gradient system, increases the washing time of 30%A mobile phases.

Result is as above：, without appearance, Quercetin, Saponin A, scopolactone appearance are good, noiseless, acid for glycine betaine Jujube kernel saponin(e B appearances partly overlap with impurity peaks, do not reach separation.

In sum, precious particle multicomponent detection method of calming the nerves is defined as：

First, chromatographic condition and system suitability test：

Chromatographic column：Agilent Zorbox SB C18 4.6mm × 150mm, 5 μ

Column temperature：35 DEG C of column temperature

Detection：EISD

Sample size：10μ

Eluent gradient elutes table：Mobile phase：A is methyl alcohol, and B is 0.5% glacial acetic acid

Time	Mobile phase A %	Mobile phase B %
			0-5	2-10	98-90
5-15	10	90
			15-30	10-30	90-70
30-40	30	70
			40-55	30-45	70-55
55-65	45	55
			65-80	90	10

2nd, the preparation of reference substance solution：Respectively precision weigh Saponin A, scopolactone,

Quercetin reference substance is appropriate, is respectively placed in 10mL volumetric flasks, plus methyl alcohol dissolves and is diluted to scale, shakes up, i.e., Obtain reference substance storing solution；Precision measures above-mentioned reference substance storing solution 1.0ml and is placed in same 20ml volumetric flasks respectively, plus methyl alcohol Scale is settled to, is shaken up, as reference substance solution.

3rd, the preparation of need testing solution：This product powder (cross No. four sieve) about 1g is taken, it is accurately weighed, in putting conical flask with cover, Precision plus the ethanol 50ml of people 70%, weighed weight is heated to reflux 6 hours, lets cool, then weighed weight, is supplied with 70% ethanol and subtracted The weight of mistake, shakes up, filtration.Precision measures subsequent filtrate 25ml, puts in flask, is concentrated into about l ml, plus 3mol/L hydrochloric acid solutions L0ml, heats hydrolysis 2 hours in water-bath, cool down immediately, in moving into separatory funnel, with water 10ml gradation washing containers, is incorporated to point In liquid funnel, plus sodium chloride 2g, being extracted 5 times with chloroform strength shaking, each 15m1 merges chloroform liquid, plus anhydrous Sodium sulphate 2g, stirring, filtration, container wash with a small amount of chloroform, filters, and filtrate merges, and less than 70 DEG C are concentrated into and closely do, vertical I.e. plus methyl alcohol makes dissolving, it is transferred in 10ml measuring bottles, and is diluted to scale, shake up, obtains final product need testing solution.

4th, determination method：Determined according to step (1) chromatographic condition, obtained final product.Fig. 4

Brief description of the drawings

Fig. 1：The liquid chromatogram of preliminary experiment one

Fig. 2：The liquid chromatogram of preliminary experiment two

Fig. 3：The liquid chromatogram of preliminary experiment three

Fig. 4：Determine experiment condition liquid chromatogram

Specific embodiment

Be set forth below embodiment further describe the present invention, the embodiment be merely to illustrate the present invention and to the present invention Without limitation.

Embodiment 1：Calm the nerves contained Saponin A, scopolactone, three kinds of height of compound of Quercetin in precious particle Effect liquid phase chromatogram assay.

First, chromatographic condition and system suitability test：

Chromatographic column：Agilent Zorbox SB C18 4.6mm × 150mm, 5 μ

Column temperature：35 DEG C of column temperature

Detection：EISD

Sample size：10μ

Eluent gradient elutes table：Mobile phase：A is methyl alcohol, and B is 0.5% glacial acetic acid

Time	Mobile phase A %	Mobile phase B %
			0-5	2-10	98-90
5-15	10	90
			15-30	10-30	90-70
30-40	30	70
			40-55	30-45	70-55
55-65	45	55
			65-80	90	10

2nd, the preparation of reference substance solution：Precision weighs Saponin A, scopolactone, Quercetin reference substance and fits respectively Amount, is respectively placed in 10mL volumetric flasks, plus methyl alcohol dissolves and is diluted to scale, shakes up, and obtains final product reference substance storing solution；It is accurate respectively Above-mentioned reference substance storing solution 1.0ml is measured to be placed in same 20ml volumetric flasks, plus methanol constant volume is to scale, shakes up, as right According to product solution.

3rd, the preparation of need testing solution：This product powder (cross No. four sieve) about 1g is taken, it is accurately weighed, in putting conical flask with cover, Precision plus the ethanol 50ml of people 70%, weighed weight is heated to reflux 6 hours, lets cool, then weighed weight, is supplied with 70% ethanol and subtracted The weight of mistake, shakes up, filtration.Precision measures subsequent filtrate 25ml, puts in flask, is concentrated into about l ml, plus 3mol/L hydrochloric acid solutions L0ml, heats hydrolysis 2 hours in water-bath, cool down immediately, in moving into separatory funnel, with water 10ml gradation washing containers, is incorporated to point In liquid funnel, plus sodium chloride 2g, being extracted 5 times with chloroform strength shaking, each 15m1 merges chloroform liquid, plus anhydrous Sodium sulphate 2g, stirring, filtration, container wash with a small amount of chloroform, filters, and filtrate merges, and less than 70 DEG C are concentrated into and closely do, vertical I.e. plus methyl alcohol makes dissolving, it is transferred in 10ml measuring bottles, and is diluted to scale, shake up, obtains final product need testing solution.

4th, determination method：Determined according to step (1) chromatographic condition, obtained final product.

Claims (1)

1. calm the nerves the multicomponent content assaying method of precious particle, it is characterised in that：Described multicomponent content assaying method be Under same chromatographic condition, while to Saponin A contained in precious particle of calming the nerves, scopolactone, three kinds of chemistry of Quercetin Composition carries out liquid chromatogram assay：

The multicomponent content assaying method is carried out in the steps below：

First, chromatographic condition and system suitability test：

Chromatographic column：Agilent Zorbox SB C18 4.6mm × 150mm, 5 μ

Column temperature：35 DEG C of column temperature

Detection：EISD

Sample size：10μ

Eluent gradient elutes table：Mobile phase：A is methyl alcohol, and B is 0.5% glacial acetic acid

Time Mobile phase A % Mobile phase B % 0-5 2-10 98-90 5-15 10 90 15-30 10-30 90-70 30-40 30 70 40-55 30-45 70-55 55-65 45 55 65-80 90 10

2nd, the preparation of reference substance solution：Precision weighs Saponin A, scopolactone, Quercetin reference substance in right amount respectively, It is respectively placed in 10mL volumetric flasks, plus methyl alcohol dissolves and is diluted to scale, shakes up, and obtains final product reference substance storing solution；It is accurate respectively to measure Above-mentioned reference substance storing solution 1.0ml is taken to be placed in same 20ml volumetric flasks, plus methanol constant volume is to scale, shakes up, as control Product solution.

3rd, the preparation of need testing solution：This product powder (cross No. four sieve) about 1g is taken, it is accurately weighed, it is accurate in putting conical flask with cover Plus the ethanol 50ml of people 70%, weighed weight is heated to reflux 6 hours, lets cool, then weighed weight, and less loss is supplied with 70% ethanol Weight, shakes up, filtration.Precision measures subsequent filtrate 25ml, puts in flask, is concentrated into about lml, plus 3mol/L hydrochloric acid solution l0ml, Hydrolysis 2 hours is heated in water-bath, is cooled down immediately, moved in people's separatory funnel, with water 10ml gradation washing containers, be incorporated to separatory funnel In, plus sodium chloride 2g, being extracted 5 times with chloroform strength shaking, each 15m1 merges chloroform liquid, plus anhydrous sodium sulfate 2g, stirring, filtration, container wash with a small amount of chloroform, filters, and filtrate merges, and less than 70 DEG C are concentrated into and closely do, and first is added immediately Alcohol makes dissolving, is transferred in 10ml measuring bottles, and is diluted to scale, shakes up, and obtains final product need testing solution.

4th, determination method：Determined according to step (1) chromatographic condition, obtained final product.