CN104865320B - HPLC dual-wavelength fingerprint determination method for multiple components of six-ingredient glutinous rehmannia preparation - Google Patents
HPLC dual-wavelength fingerprint determination method for multiple components of six-ingredient glutinous rehmannia preparation Download PDFInfo
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Abstract
The invention discloses an HPLC dual-wavelength fingerprint determination method for multiple components of a six-ingredient glutinous rehmannia preparation. The method comprises the following steps: (1) taking the six-ingredient glutinous rehmannia preparation and dissolving the six-ingredient glutinous rehmannia preparation in methanol so as to obtain a six-ingredient glutinous rehmannia preparation solution with a concentration of 1 g/10-30 ml; (2) dissolving a standard substance in methanol so as to obtain a standard substance solution with a concentration of 20-30 [mu]g/ml; (3) respectively acquiring the chromatograms of the six-ingredient glutinous rehmannia preparation solution and the standard substance solution under same detection conditions by using HPLC, wherein the detection conditions are that octadecyl silane bonded silica glue is used as a filler for a chromatographic column, gradient elution is carried out, a mobile phase A is an aqueous phosphoric acid solution with a concentration of 0.08 ml/100 ml, a mobile phase B is acetonitrile, and detection wavelengths are in a range of 200 to 220 nm and a range of 238 to 250 nm, respectively; and (4) calculating the contents of components in the six-ingredient glutinous rehmannia preparation corresponding to components in the standard substance according to the concentration and chromatogram of the standard substance. The method provided by the invention can realize rapid and comprehensive detection of main components in the six-ingredient glutinous rehmannia preparation and has the advantages of easy availability of instruments, simplicity and good operationality.
Description
Technical field
The present invention relates to Chinese medicine technical field of measurement and test, it is more particularly to a kind of Liuwei Dihuang preparation multicomponent hplc double wave
Long finger print measuring method.
Background technology
, as the representative prescription of " nourishing kidney yin ", its curative effect is indubitable for six drugs containing rehmanniae, the six drugs containing rehmanniae on market today
Preparation is numerous, therefore only guarantees its quality, and this classical ancient prescription could be allowed to give full play to its treatment advantage.2010 editions Chinese Pharmacopoeias
The assay of regulation: for water-honeyed pill, small honey pill, these three formulations of big honeyed bolus, containing moutan bark in terms of Paeonol, water-honeyed pill is every
1g must not be less than 0.90mg；The every lg of small honey pill must not be less than 0.70mg；The big every ball of honeyed bolus must not be less than 6.3mg.Containing wine-prepared fructus corni with
Loganin meter, the every lg of water-honeyed pill must not be less than 0.70mg；The every lg of small honey pill must not be less than 0.50mg；The big every ball of honeyed bolus must not be less than
4.5mg.For condensed pill, every lg contains wine-prepared fructus corni in terms of loganin, must not be less than 1.4mg, and every lg contains moutan bark in terms of Paeonol,
1.8mg must not be less than.Capsule every contains moutan bark in terms of Paeonol, and specification (1) must not be less than 3.0mg；Specification (2) must not be lacked
In 1.5mg, in terms of ursolic acid, specification (1) must not be less than 0.60mg to wine-prepared fructus corni；Specification (2) must not be less than 0.30mg.The wherein root bark of tree peony
Phenol and loganin adopt high performance liquid chromatography detection, and chromatographic condition is different, and Detection wavelength is respectively 274nm and 236nm, black bearberry
Acid carries out assay using thin-layered chromatography, and quality standard is simple, coarse, not science.Generally use single component or several one-tenth
The quality divided marking and control compound preparation, certain composition of the simply medicine that it controls, rather than medicine itself.If
In drug production process, quality of medicinal material does not meet medicinal requirements, when meeting content requirement specified in patent medicine quality standard again,
Arise that the phenomenon adding chemical standard product or other compositions in the product.The interpolation of chemical standard product changes original
Composition and effectiveness, its curative effect also just changes, and gives people medication and brings dangerous or unavailability factor.
Content of the invention
It is an object of the invention to, for defect present in prior art, provide a kind of Liuwei Dihuang preparation multicomponent
High performance liquid chromatography (hplc) dual wavelength finger print measuring method.The method that the present invention provides can be realized to six drugs containing rehmanniae
Main component in preparation carries out quick, comprehensive detection, and instrument is easy to get, and method is easy, strong operability.
The invention provides a kind of multicomponent hplc dual wavelength finger print measuring method of Liuwei Dihuang preparation, described side
Method comprises the following steps:
(1) take Liuwei Dihuang preparation, after pulverizing, precision weighs, be dissolved in methyl alcohol with concentration 1g/10～30ml, ultrasonic carry
Take, centrifugation, take supernatant liquid filtering, obtain final product Liuwei Dihuang preparation solution, standby；
(2) precision weighs standard items, is dissolved in methyl alcohol with concentration 20～30 μ g/ml, obtains final product standard solution, standby；
(3) adopt high performance liquid chromatography, obtain Liuwei Dihuang preparation solution and mark under identical testing conditions respectively
The chromatogram of quasi- product solution；Described testing conditions include:
Chromatogram column packing is octadecylsilane chemically bonded silica；
Using gradient elution；Wherein, mobile phase a is the phosphate aqueous solution of concentration 0.08ml/100ml, and mobile phase b is second
Nitrile；Elution program is: 0～15min, 2～15% mobile phases b；15～25min, 15～25% mobile phases b；25～40min, 25
～70% mobile phase b；
Detection wavelength is 200-220nm and 238-250nm；
(4) according to the concentration of standard items, standard items in the peak area in chromatogram and Liuwei Dihuang preparation with standard
Peak area in chromatogram for the condition tie element, calculates the content of composition corresponding with standard items in Liuwei Dihuang preparation.
Liuwei Dihuang preparation of the present invention is LIUWEIDIHUANG SHUIMIWAN, big honeyed bolus, condensed pill or capsule.
Step (1) of the present invention is optimized to the extraction of Liuwei Dihuang preparation and the compound method of solution, thus increasing
The degree of accuracy of detection.Specifically, the ultrasonic frequency of described ultrasonic extraction is preferably 35000～45000hz, extraction time
It is preferably 20～40min under the conditions of said extracted, the speed of centrifugation is preferably 5000～20000 revs/min, centrifugation time is 5
～15min.After centrifugation, using the membrane filtration in 0.2～0.3 μm of aperture, supernatant can be filtered.By to above-mentioned condition
Preferred, the degree of accuracy of detection can be improved in subsequent step.
As a kind of preferred version, step (1) of the present invention particularly as follows: taking Liuwei Dihuang preparation, after pulverizing, precision weighs, with
Concentration 1g/10ml is dissolved in methyl alcohol, with ultrasonic wave extraction 30min of frequency 40000hz, with 10000 revs/min of centrifugation
10min, takes supernatant, with 0.22 μm of the membrane filtration in aperture, obtains final product Liuwei Dihuang preparation solution.
The described standard items of step (2) of the present invention become with the detection of States Pharmacopoeia specifications according to the active ingredient in Liuwei Dihuang preparation
Divide and chosen, including gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, Paeonol, 5- methylol chaff
Aldehyde, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin.In the present invention, described standard items
Concentration refers to every kind of standard items concentration respectively, in practical operation, can merge after various standard items respectively precision weighing,
It is dissolved in the methyl alcohol of specified quantitative, and mixes, obtain hybrid standard product solution.The concentration of described standard items is preferably: does not have
Gallate-based, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin,
The concentration of sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin is 25 μ g/ml.
Due to heterogeneity, appearance situation is different at different wavelengths, and therefore, the present invention in Detection wavelength is being preferably
Detection gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, Paeonol under conditions of 200-220nm；In inspection
Survey wavelength for, under conditions of 238-250nm, detecting 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzene first
Acid, benzoylpaeoniflorin.
The wavelength of the described detection of step (3) of the present invention is preferably 210nm and 238nm.Using this two wavelength to above-mentioned one-tenth
Divide and detected, it is possible to achieve optimal analytical effect.
The implication of described phosphate aqueous solution concentration 0.08ml/100ml of step (3) of the present invention is: contains in every 100ml solution
0.08ml phosphoric acid.In described gradient elution program, the percentage of mobile phase b refers to that mobile phase b accounts for the volume of two-phase volume sum
Percentage.
Step (3) of the present invention can also include following testing conditions: the specification of chromatographic column is 250mm × 4.6mm.Chromatographic column
The particle diameter of filler is 5 μm.Described filler can be selected for the agilent c purchased from Agilent Technologies18.The column temperature of chromatographic column
For 25～35 DEG C, preferably 30 DEG C.Flow rate of mobile phase is 0.5～1.5ml/min, preferably 1.0ml/min.Sample size be 10～
30 μ l, preferably 20 μ l.
Because standard solution is identical with the testing conditions of Liuwei Dihuang preparation solution, in chromatogram, six drugs containing rehmanniae system
In agent, the retention time of composition appearance corresponding with standard items and peak shape should be essentially identical with standard items, therefore, it can by phase
Same retention time judges corresponding composition in two width chromatograms, and is confirmed by the shape auxiliary at peak.Step (4) of the present invention
By the peak area of corresponding composition in standard of comparison product solution chromatogram and Liuwei Dihuang preparation solution chromatogram, according to standard
The concentration known of product, calculates the concentration of composition corresponding with standard items in Liuwei Dihuang preparation solution, calculates six further
The content of composition corresponding with standard items in taste rehmannia preparation.
The present invention selectes multiple compositions that two wavelength detect in Liuwei Dihuang preparation simultaneously, comprehensively quantitative six drugs containing rehmanniae
Ball and capsule preparations, are conducive to evaluating the quality of this Liuwei Dihuang preparation on the whole it is ensured that its clinical efficacy.
Brief description
Fig. 1 is first six drugs containing rehmanniae condensed pill sample and contrast under 210nm wavelength for the hybrid standard product in embodiment 1
Figure；In figure marks: a, six drugs containing rehmanniae sample, b, hybrid standard product, and 1, gallic acid, 2, protocatechuic acid, 3,1,2,3,4,6- five
Galloyl glucose, 4, Paeonol.
Fig. 2 is first six drugs containing rehmanniae condensed pill sample and contrast under 238nm wavelength for the hybrid standard product in embodiment 1
Figure；In figure marks: a, six drugs containing rehmanniae sample, b, hybrid standard product；5th, 5 hydroxymethyl furfural, 6, morroniside, 7, loganin, 8, river deer
Tooth dish glycosides, 9, Paeoniflorin, 10, benzoic acid, 11, benzoylpaeoniflorin.
Fig. 3 be other five different batches in embodiment 1 six drugs containing rehmanniae concentrated pill sample dual wavelength (210nm and
238nm) the chromatogram under pattern；In figure marks: a, Detection wavelength are 210nm, and b, Detection wavelength are 238nm；1st, second batch, 2,
3rd batch, the 3, the 4th batch, the 4, the 5th batch, the 5, the 6th batch.
Fig. 4 is the Liuwei Dihuang preparation sample of four kinds of different dosage forms in embodiment 1 in dual wavelength (210nm and 238nm) mould
Chromatogram under formula；In figure marks: a, Detection wavelength are 210nm, and b, Detection wavelength are 238nm；1st, six drugs containing rehmanniae condensed pill, 2,
LIUWEIDIHUANG SHUIMIWAN, 3, the big honeyed bolus of six drugs containing rehmanniae, 4, LIUWEIDIHUANG JIAONANG.
Specific embodiment
With reference to the accompanying drawings and examples embodiments of the present invention are described in further detail.Following examples are used for
The present invention is described, but can not be used for limiting the scope of the present invention.
The instrument that the embodiment of the present invention adopts is included with reagent: Agilent 1260 high performance liquid chromatograph, diode array
Detector, ten thousand/electronic balance (me204e, mettler-toledo), ultra-pure water (milli-q, millipore), acetonitrile
(chromatographically pure, merk), phosphoric acid (chromatographically pure, dikmapure).Standard items: gallic acid (the careless Co., Ltd of Beijing match hundred,
130718), protocatechuic acid (the careless Co., Ltd of Beijing match hundred, 130106), 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose (match by Beijing
Hundred careless Co., Ltds, 130508), Paeonol (Chinese food Drug Administration, 110708-200506), 5- methylol chaff
Aldehyde (the careless Co., Ltd of Beijing match hundred, 131124), morroniside (the careless Co., Ltd of Beijing match hundred, 130912), loganin (match by Beijing
Hundred careless Co., Ltds, 1201204), sweroside (the careless Co., Ltd of Beijing match hundred, 140425), (hundred grass are matched in Beijing to be had Paeoniflorin
Limit company, 130726), benzoic acid (the careless Co., Ltd of Beijing match hundred), benzoylpaeoniflorin (the careless Co., Ltd of Beijing match hundred,
131108).
Embodiment 1
According to following steps, Liuwei Dihuang preparation is detected:
(1) take Liuwei Dihuang preparation, after pulverizing, precision weighs, be dissolved in methyl alcohol with concentration 1g/10ml, use frequency
Ultrasonic wave extraction 30min of 40000hz, with 10000 revs/min of centrifugation 10min, takes supernatant, with 0.22 μm of aperture
Membrane filtration, obtains final product Liuwei Dihuang preparation solution, standby；
(2) precision weighs gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, Paeonol, 5- hydroxyl first
Base furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin, with methyl alcohol as solvent, are configured to
Concentration is respectively the standard solution of 25 μ g/ml, standby；
(3) adopt high performance liquid chromatography, obtain Liuwei Dihuang preparation solution and mark under identical testing conditions respectively
The chromatogram of quasi- product solution；Described testing conditions include:
Chromatogram column packing is octadecylsilane chemically bonded silica agilent c18, particle diameter be 5 μm；Chromatographic column specification is
250mm×4.6mm；
Using gradient elution；Wherein, mobile phase a is the phosphate aqueous solution of concentration 0.08ml/100ml, and mobile phase b is second
Nitrile；Elution program is: 0～15min, 2～15% mobile phases b；15～25min, 15～25% mobile phases b；25～40min, 25
～70% mobile phase b；
Detection wavelength is 210nm and 238nm；
Column temperature is 30 DEG C；
Flow rate of mobile phase is 1.0ml/min；
Sample size is 20 μ l；
(4) according to the concentration of standard items, standard items in the peak area in chromatogram and Liuwei Dihuang preparation with standard
Peak area in chromatogram for the condition tie element, calculates the content of composition corresponding with standard items in Liuwei Dihuang preparation.
Using the method for the present embodiment, to commercially available six drugs containing rehmanniae condensed pill, LIUWEIDIHUANG SHUIMIWAN, the big honey of six drugs containing rehmanniae
Each six batches of totally four kinds of formulations are detected for ball, LIUWEIDIHUANG JIAONANG.Wherein, first six drugs containing rehmanniae condensed pill is in 210nm
Chromatogram under wavelength is referring to Fig. 1, the chromatogram under 238nm wavelength referring to Fig. 2, the in addition six drugs containing rehmanniae concentration of five batches
Chromatogram under 210nm and 238nm wavelength for the ball is referring to Fig. 3；Six drugs containing rehmanniae condensed pill, LIUWEIDIHUANG SHUIMIWAN, six drugs containing rehmanniae
Big honeyed bolus, LIUWEIDIHUANG JIAONANG each batch of totally four kinds of formulations in the chromatogram under 210nm and 238nm wavelength referring to Fig. 4.
Embodiment 2
According to following steps, Liuwei Dihuang preparation is detected:
(1) take Liuwei Dihuang preparation, after pulverizing, precision weighs, be dissolved in methyl alcohol with concentration 1g/15ml, use frequency
Ultrasonic wave extraction 40min of 35000hz, with 5000 revs/min of centrifugation 15min, takes supernatant, with 0.2 μm of the filter in aperture
Membrane filtration, obtains final product Liuwei Dihuang preparation solution, standby；
(2) precision weighs gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, Paeonol, 5- hydroxyl first
Base furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin, with methyl alcohol as solvent, are configured to
Concentration is respectively the standard solution of 25 μ g/ml, standby；
(3) adopt ultra-performance liquid chromatography, obtain six drugs containing rehmanniae condensed pill solution under identical testing conditions respectively
Chromatogram with standard solution；Described testing conditions include:
Chromatogram column packing is octadecylsilane chemically bonded silica agilent c18, particle diameter be 5 μm；Chromatographic column specification is
250mm×4.6mm；
Using gradient elution；Wherein, mobile phase a is the phosphate aqueous solution of concentration 0.08ml/100ml, and mobile phase b is second
Nitrile；Elution program is: 0～15min, 2～15% mobile phases b；15～25min, 15～25% mobile phases b；25～40min, 25
～70% mobile phase b；
Detection wavelength is 200nm and 238nm；
Column temperature is 25 DEG C；
Flow rate of mobile phase is 0.5ml/min；
Sample size is 10 μ l；
(4) according to the concentration of standard items, standard items in the peak area in chromatogram and Liuwei Dihuang preparation with standard
Peak area in chromatogram for the condition tie element, calculates the content of composition corresponding with standard items in Liuwei Dihuang preparation.
Embodiment 3
According to following steps, Liuwei Dihuang preparation is detected:
(1) take Liuwei Dihuang preparation, after pulverizing, precision weighs, be dissolved in methyl alcohol with concentration 1g/10ml, use frequency
Ultrasonic wave extraction 20min of 45000hz, with 20000 revs/min of centrifugation 5min, takes supernatant, with 0.3 μm of the filter in aperture
Membrane filtration, obtains final product Liuwei Dihuang preparation solution, standby；
(2) precision weighs gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, Paeonol, 5- hydroxyl first
Base furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin, with methyl alcohol as solvent, are configured to
Concentration is respectively the standard solution of 25 μ g/ml, standby；
(3) adopt ultra-performance liquid chromatography, obtain six drugs containing rehmanniae condensed pill solution under identical testing conditions respectively
Chromatogram with standard solution；Described testing conditions include:
Chromatogram column packing is octadecylsilane chemically bonded silica agilent c18, particle diameter be 5 μm；Chromatographic column specification is
250mm×4.6mm；
Using gradient elution；Wherein, mobile phase a is the phosphate aqueous solution of concentration 0.08ml/100ml, and mobile phase b is second
Nitrile；Elution program is: 0～15min, 2～15% mobile phases b；15～25min, 15～25% mobile phases b；25～40min, 25
～70% mobile phase b；
Detection wavelength is 220nm and 250nm；
Column temperature is 35 DEG C；
Flow rate of mobile phase is 1.5ml/min；
Sample size is 30 μ l；
(4) according to the concentration of standard items, standard items in the peak area in chromatogram and Liuwei Dihuang preparation with standard
Peak area in chromatogram for the condition tie element, calculates the content of composition corresponding with standard items in Liuwei Dihuang preparation.
Through comparing, in three embodiments that the present invention provides, the accuracy resultant effect of embodiment 1 is optimum.
Experimental example: Method validation
1st, linear relationship is investigated: takes standard items, prepares the standard solution of five variable concentrations from high to low, with standard items
Concentration is abscissa, and chromatographic peak area is drawn calibration curve for ordinate and obtained regression equation.Result shows, gallic acid, former youngster
Boheic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Chinese herbaceous peony
Medicine glycosides, benzoic acid and benzoylpaeoniflorin are all good in the offline sexual intercourse of above-mentioned concentration, the standard concentration 20 that the present invention provides
～30 μ g/ml are in linear scope.
2nd, precision test: take first six drugs containing rehmanniae condensed pill sample, prepare 1 part according to the method that embodiment 1 provides
Liuwei Dihuang preparation solution, and according to the method that embodiment 1 provides, this solution is carried out respectively with 6 detections.According to 6 detections
As a result, relative standard deviation (rsd) value of 11 compositions, no more than 2%, illustrates that the precision of instrument is good.
3rd, repeated experiment: take first six drugs containing rehmanniae condensed pill sample, prepare respectively according to the method that embodiment 1 provides
6 parts of Liuwei Dihuang preparation solution, and according to the method that embodiment 1 provides, 6 parts of solution are detected respectively.According to detection knot
Really, gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, Paeonol, 5 hydroxymethyl furfural, morroniside, horse
The rsd value of money glycosides, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin is respectively less than 2%, illustrates that the method is reproducible.
4th, stability test: take first six drugs containing rehmanniae condensed pill sample, prepare 1 part according to the method that embodiment 1 provides
Liuwei Dihuang preparation solution, takes equivalent solution respectively in the 0th, 2,4,6,8,12 and 24 hours after the production, provides according to embodiment 1
Method detected.According to testing result, gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, the root bark of tree peony
The rsd value of phenol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin is all little
In 2.5% it was demonstrated that this sample was stablized in 24 hours.
5th, accuracy test: take the six drugs containing rehmanniae condensed pill sample of known each component content, add corresponding to sample become
The standard items of the amount of grading, the content of each composition in sample after then detection is loaded, every kind of standard items are loaded 6 times respectively, according to reality
The method applying example 1 offer is detected.The average recovery of each standard items, all between 96.5-103.8%, shows that this contains measurement
Method of determining is accurate, reliable.
Above experimental result shows, the method repeatability of present invention offer, stability, precision, the degree of accuracy are all good, adopt
It is feasible for carrying out quality control with the hplc dual wavelength multi objective content assaying method that the present invention provides to six drugs containing rehmanniae pill
's.
Embodiment of above is merely to illustrate the present invention, rather than limitation of the present invention.Although with reference to embodiment to this
Bright be described in detail, it will be understood by those within the art that, technical scheme is carried out various combinations,
Modification or equivalent, without departure from the spirit and scope of technical solution of the present invention, the right that all should cover in the present invention will
Ask in the middle of scope.
Claims (7)
1. a kind of multicomponent hplc dual wavelength finger print measuring method of Liuwei Dihuang preparation is it is characterised in that methods described
Comprise the following steps:
(1) take Liuwei Dihuang preparation, after pulverizing, precision weighs, be dissolved in methyl alcohol with concentration 1g/10～30ml, ultrasonic extraction,
Centrifugation, takes supernatant liquid filtering, obtains final product Liuwei Dihuang preparation solution, standby；
(2) precision weighs standard items, is dissolved in methyl alcohol with concentration 20～30 μ g/ml, obtains final product standard solution, standby；
Described standard items include gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, Paeonol, 5- hydroxyl
Methyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin；
(3) adopt high performance liquid chromatography, obtain Liuwei Dihuang preparation solution and standard items under identical testing conditions respectively
The chromatogram of solution；Described testing conditions include:
Chromatogram column packing is octadecylsilane chemically bonded silica；The specification of described chromatographic column is 250mm × 4.6mm；Filler
Particle diameter be 5 μm；
Using gradient elution；Wherein, mobile phase a is the phosphate aqueous solution of concentration 0.08ml/100ml, and mobile phase b is acetonitrile；Wash
De- program is: 0～15min, 2～15% mobile phases b；15～25min, 15～25% mobile phases b；25～40min, 25～70%
Mobile phase b；
Detection wavelength is 200-220nm and 238-250nm；Under conditions of Detection wavelength is for 200-220nm detection gallic acid,
Protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, Paeonol；Under conditions of Detection wavelength is for 238-250nm, detection
5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid, benzoylpaeoniflorin；
(4) according to the concentration of standard items, standard items in the peak area in chromatogram and Liuwei Dihuang preparation with standard condition
Peak area in chromatogram for the tie element, calculates the content of composition corresponding with standard items in Liuwei Dihuang preparation.
2. method according to claim 1 is it is characterised in that the described testing conditions of step (3) also include: the post of chromatographic column
Temperature is 25～35 DEG C；Flow rate of mobile phase is 0.5～1.5ml/min；Sample size is 10～30 μ l.
3. method according to claim 1 and 2 it is characterised in that Detection wavelength described in step (3) be 210nm and
238nm.
4. it is characterised in that the described ultrasonic extraction of step (1), ultrasonic frequency is method according to claim 1 and 2
35000～45000hz, extraction time is 20～40min.
5. method according to claim 4 is it is characterised in that the described centrifugation of step (1), and the speed of centrifugation is 5000～
20000 revs/min, centrifugation time is 5～15min.
6. method according to claim 5 is it is characterised in that the described filtration of step (1), using 0.2～0.3 μm of aperture
Membrane filtration.
7. method according to claim 1 is it is characterised in that the formulation of described Liuwei Dihuang preparation is: water-honeyed pill, greatly honey
Ball, condensed pill or capsule.
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