CN104865319B - Ultra performance liquid chromatographic dual-wavelength multi-index content determination method for six-ingredient glutinous rehmannia preparation - Google Patents
Ultra performance liquid chromatographic dual-wavelength multi-index content determination method for six-ingredient glutinous rehmannia preparation Download PDFInfo
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Abstract
The invention discloses an ultra performance liquid chromatographic dual-wavelength multi-index content determination method for 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/10-30 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 ultra performance liquid chromatography, 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, is more particularly to a kind of ultra high efficiency liquid phase double wave of Liuwei Dihuang preparation
Long multi objective content assaying method.
Background technology
Representative prescription of the six drugs containing rehmanniae as " nourishing kidney yin ", its curative effect are indubitable, 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, big honeyed pills these three dosage forms, containing Cortex Moutan in terms of paeonol, water-honeyed pill is every
1g must not be less than 0.90mg；Small honey pill must not be less than 0.70mg per lg；Big honeyed pills must not be less than 6.3mg per ball.Containing wine-prepared fructus corni with
Loganin meter, water-honeyed pill must not be less than 0.70mg per lg；Small honey pill must not be less than 0.50mg per lg；Big honeyed pills must not be less than per ball
4.5mg.For concentrated pill, per lg containing wine-prepared fructus corni in terms of loganin, 1.4mg must not be less than, per lg containing Cortex Moutan in terms of paeonol,
1.8mg must not be less than.Containing Cortex Moutan in terms of paeonol, specification (1) must not be less than 3.0mg to capsule per；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.Wherein Cortex Moutan
Phenol and loganin adopt high performance liquid chromatography detection, and chromatographic condition is different, and Detection wavelength is respectively 274nm and 236nm, Folium Vaccinii vitis-idaeae
Acid carries out assay using thin layer chromatography, complex operation step, takes longer, is unfavorable for detection rapidly and efficiently and can not
From the overall evaluation and the quality of control six drugs containing rehmanniae perfect square.
What the conventional quality controling research report about Liuwei Dihuang preparation was more is purple with regard to high performance liquid chromatography connection
External detector, but tend to while the composition for detecting is less, quality control can not be carried out on the whole, have been reported that LC-MS
Multicomponent assay is carried out to LIUWEI DIHUANG WAN with Micellar Electrokinetic Chromatography, can detect simultaneously four kinds therein into
Point, but the popularization of such method limited by instrument is more rare.Also document report high performance liquid chromatography is combined diode battle array
Row and evaporative light scattering detector can detect eight kinds of compositions in LIUWEI DIHUANG WAN simultaneously, but adopt two kinds of detector detection process
It is relatively complicated, and time and effort consuming.Ultra Performance Liquid Chromatography (UHPLC) adopts little particle filler chromatographic column and extra high voltage system, can
To significantly improve the separating degree and detection sensitivity of chromatographic peak, can significantly shorten analytical cycle again, reduce reagent consumption, be equipped with
After diode array detector (DAD), detection range is more widened.Therefore Ultra Performance Liquid Chromatography is adopted, through DAD all-wave lengths
Scanning determines the Detection wavelength being adapted to, and can carry out quick, comprehensive detection, instrument to the main component in LIUWEI DIHUANG WAN and capsule
Device is easy to get, method simplicity strong operability, is expected to become the powerful means of the compound preparation quality control.
The content of the invention
It is an object of the present invention to be directed to defect present in prior art, there is provided a kind of Liuwei Dihuang preparation multicomponent
Ultra high efficiency liquid phase dual wavelength content assaying method.The present invention provide method can realize to Liuwei Dihuang preparation in it is main
Composition carries out quick, comprehensive detection, and instrument is easy to get, and method is easy, strong operability.
The invention provides a kind of ultra high efficiency liquid phase dual wavelength multi objective content assaying method of Liuwei Dihuang preparation, the party
Method specifically includes following steps：
(1) Liuwei Dihuang preparation is taken, precision is weighed after crushing, is dissolved in methanol with concentration 1g/10～30ml, ultrasound is carried
Take, be centrifuged, take supernatant liquid filtering, obtain final product Liuwei Dihuang preparation solution, it is standby；
(2) precision weighs standard substance, is dissolved in methanol with 20～30 μ g/ml of concentration, obtains final product standard solution, standby；
(3) adopt ultra-performance liquid chromatography, obtained under identical testing conditions respectively Liuwei Dihuang preparation solution and
The chromatogram of standard solution；The testing conditions include：
Chromatograph column packing is octadecylsilane chemically bonded silica；
Using gradient elution；Wherein, phosphate aqueous solution of the mobile phase A for concentration 0.08ml/100ml, Mobile phase B is second
Nitrile；Elution program is：0～5min, 2～25% Mobile phase Bs；5～8min, 25～70% Mobile phase Bs；8～8.1min, 70-
95% Mobile phase B；8.1～13min, 95% Mobile phase B；
Detection wavelength is 200-220nm and 238-250nm；
(4) with standard in peak area and Liuwei Dihuang preparation of the concentration, standard substance according to standard substance in chromatogram
Peak area of the condition tie element in chromatogram, calculates the content of composition corresponding with standard substance in Liuwei Dihuang preparation.
Liuwei Dihuang preparation of the present invention is LIUWEIDIHUANG SHUIMIWAN, big honeyed pills, concentrated pill or capsule.
Step (1) of the present invention is optimized to the extraction of Liuwei Dihuang preparation and the compound method of solution, so as to increase
The accuracy of detection.Specifically, the ultrasonic frequency of the supersound extraction is preferably 35000～45000Hz, extraction time
Under the conditions of said extracted, the speed of centrifugation is preferably 5000～20000 revs/min to preferably 20～40min, and centrifugation time is 5
～15min.After centrifugation, supernatant can be filtered using the membrane filtration in 0.2～0.3 μm of aperture.By to above-mentioned condition
Preferred, the accuracy detected during subsequent step can be improved.
Used as a kind of preferred version, step (1) of the present invention is specially：Liuwei Dihuang preparation is taken, precision is weighed after crushing, with
Concentration 1g/10ml is dissolved in methanol, with ultrasonic 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.
Step (2) of the present invention described standard substance according to the detection of the effective ingredient in Liuwei Dihuang preparation and States Pharmacopoeia specifications into
Divide and chosen, including gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol, 5- methylol brans
Aldehyde, morroniside, loganin, sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin.In the present invention, the standard substance
Concentration refers to every kind of standard substance concentration respectively, in practical operation, can distinguish various standard substance after precision weighing, merge,
It is dissolved in the methanol of specified quantitative, and mix homogeneously, obtain hybrid standard product solution.The concentration of the standard substance is preferably：Not yet
Gallate-based, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol, 5 hydroxymethyl furfural, morroniside, loganin,
The concentration of sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin is 25 μ g/ml.
Due to heterogeneity, appearance situation is different at different wavelengths, therefore, the present invention preferably in Detection wavelength is being
Gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol is detected under conditions of 200-220nm；In inspection
Wavelength is surveyed for, under conditions of 238-250nm, detecting 5 hydroxymethyl furfural, morroniside, loganin, sweroside, peoniflorin, benzene first
Acid, benzoylpaeoniflorin.
The wavelength of step (3) of the present invention described detection is preferably 210nm and 238nm.Using the two wavelength to it is above-mentioned into
Divide and detected, it is possible to achieve optimal analytical effect.
The implication of step (3) of the present invention described phosphate aqueous solution concentration 0.08ml/100ml is：Contain in per 100ml solution
0.08ml phosphoric acid.In the gradient elution program, the percentage ratio of Mobile phase B refers to that Mobile phase B accounts for the volume of biphase volume sum
Percentage ratio.
Step (3) of the present invention can also include following testing conditions：The specification of chromatographic column be 100～250mm × 2.1～
4.6mm, preferably 100mm × 2.1mm.The particle diameter of chromatograph column packing is 1.8～5.0 μm, preferably 1.8～2.0 μm, enters one
Step is preferably 1.8 μm.The filler can select the Agilent SB C purchased from Agilent Technologies18.The column temperature of chromatographic column
For 30～40 DEG C, preferably 35 DEG C.Flow rate of mobile phase is 0.1～1.0ml/min, preferably 0.4ml/min.Sample size is 1～5
μ l, preferably 1～2 μ l, more preferably 1 μ l.
Due to standard solution it is identical with the testing conditions of Liuwei Dihuang preparation solution, in chromatogram, six drugs containing rehmanniae system
In agent, the retention time and peak shape of composition appearance corresponding with standard substance should be essentially identical with standard substance, therefore, it can by phase
With retention time judge corresponding composition in two width chromatograms, and the shape auxiliary confirmation by 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 substance in Liuwei Dihuang preparation solution, further calculates six
The content of composition corresponding with standard substance in taste rehmannia preparation.
The inventive method carries out chromatograph detection using ultra high efficiency liquid phase dual wavelength multi objective, it is only necessary to can inspection less than 10 minutes
At least 11 kinds main components in LIUWEI DIHUANG WAN and capsule are surveyed, and which is carried out quantitatively.With traditional high performance liquid chromatography phase
Than, analysis time can be saved more than 3/4, meanwhile, save reagent consuming.The method that the present invention is provided is examined using dual wavelength
Survey, so as to cover the higher composition of most of content in Liuwei Dihuang preparation, and detection method is accurate, reliable.
The present invention provide ultra high efficiency liquid phase dual wavelength multi objective content assaying method, can accurately, simplicity, quick detection six
Taste rehmannia preparation, and be conducive to evaluating the quality of Liuwei Dihuang preparation on the whole, it is ensured that its clinical efficacy.
Description of the drawings
Fig. 1 is hybrid standard product and six drugs containing rehmanniae concentrated pill sample (No. 34 samples) chromatogram under 210nm wavelength；In figure
Labelling：A, hybrid standard product, b, six drugs containing rehmanniae concentrated pill sample；1st, gallic acid, 2, protocatechuic acid, 3,1,2,3,4,6- five do not have
Infanticide acyl glucose, 4, paeonol.
Fig. 2 is hybrid standard product and six drugs containing rehmanniae concentrated pill sample (No. 34 samples) chromatogram under 238nm wavelength；In figure
Labelling：C, hybrid standard product, d, six drugs containing rehmanniae concentrated pill sample；5th, 5 hydroxymethyl furfural, 6, morroniside, 7, loganin, 8, river deer
Tooth dish glycosides, 9, peoniflorin, 10, benzoic acid, 11, benzoylpaeoniflorin.
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 illustrated, but can not be used for limiting the scope of the present invention.
The instrument that the embodiment of the present invention is adopted is included with reagent：1290 Ultra Performance Liquid Chromatography instrument of Agilent, prepares
G4204A quaternary pump, G4226A automatic samplers, G1316C temperature control boxs, G4212A diode array detector, a ten thousandth electricity
Sub- balance (ME204E, Mettler-Toledo), ultra-pure water (Milli-Q, Millipore), acetonitrile (chromatographically pure, Merk), phosphorus
Sour (chromatographically pure, DikmaPure).Standard substance：Gallic acid (Beijing match BAICAO company limited, 130718), protocatechuic acid (Beijing
Match BAICAO company limited, 130106), 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose (Beijing match BAICAO company limited, 130508),
Paeonol (Chinese food Drug Administration, 110708-200506), 5 hydroxymethyl furfural (Beijing match BAICAO company limited,
131124), morroniside (Beijing match BAICAO company limited, 130912), loganin (Beijing match BAICAO company limited, 1201204),
Sweroside (Beijing match BAICAO company limited, 140425), peoniflorin (Beijing match BAICAO company limited, 130726), benzoic acid
(Beijing match BAICAO company limited), and benzoylpaeoniflorin (Beijing match BAICAO company limited, 131108).
Embodiment 1
Liuwei Dihuang preparation is detected according to following steps：
(1) Liuwei Dihuang preparation is taken, precision is weighed after crushing, is dissolved in methanol with concentration 1g/10ml, uses frequency
Ultrasonic 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-glucopyranoses, paeonol, 5- hydroxyl first
Base furfural, morroniside, loganin, sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin, with methanol as solvent, are configured to
Concentration is respectively the standard solution of 25 μ g/mL, standby；
(3) ultra-performance liquid chromatography is adopted, six drugs containing rehmanniae concentrated pill solution is obtained under identical testing conditions respectively
With the chromatogram of standard solution；The testing conditions include：
Chromatograph column packing is octadecylsilane chemically bonded silica Agilent SB C18, particle diameter is 1.8 μm；Chromatographic column is advised
Lattice are 100mm × 2.1mm；
Using gradient elution；Wherein, phosphate aqueous solution of the mobile phase A for concentration 0.08ml/100ml, Mobile phase B is second
Nitrile；Elution program is：0～5min, 2～25% Mobile phase Bs；5～8min, 25～70% Mobile phase Bs；8～8.1min, 70～
95% Mobile phase B；8.1～13min, 95% Mobile phase B；
Detection wavelength is 210nm and 238nm；
Column temperature is 35 DEG C；
Flow rate of mobile phase is 0.4mL/min；
Sample size is 1 μ L；
(4) with standard in peak area and Liuwei Dihuang preparation of the concentration, standard substance according to standard substance in chromatogram
Peak area of the condition tie element in chromatogram, calculates the content of composition corresponding with standard substance in Liuwei Dihuang preparation.
According to the present embodiment methods described, to commercially available 81 kinds of Liuwei Dihuang preparations (including 34 kinds of six drugs containing rehmanniae concentrated pills,
17 kinds of LIUWEIDIHUANG SHUIMIWAN, 11 kinds of six drugs containing rehmanniae big honeyed pills and 10 kinds of LIUWEIDIHUANG JIAONANG agent) detected, testing result is such as
Shown in table 1.
Wherein, chromatogram of the sample the 34th (i.e. six drugs containing rehmanniae concentrated pill the 34th) under 210nm wavelength be referring to Fig. 1,
Chromatogram under 238nm wavelength is referring to 2.
Table 1：Liuwei Dihuang preparation Contents of Main Components (ND=is not detected by)
Embodiment 2
Liuwei Dihuang preparation is detected according to following steps：
(1) Liuwei Dihuang preparation is taken, precision is weighed after crushing, is dissolved in methanol with concentration 1g/15ml, uses frequency
Ultrasonic 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-glucopyranoses, paeonol, 5- hydroxyl first
Base furfural, morroniside, loganin, sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin, with methanol as solvent, are configured to
Concentration is respectively the standard solution of 25 μ g/mL, standby；
(3) ultra-performance liquid chromatography is adopted, six drugs containing rehmanniae concentrated pill solution is obtained under identical testing conditions respectively
With the chromatogram of standard solution；The testing conditions include：
Chromatograph column packing is octadecylsilane chemically bonded silica Agilent SB C18, particle diameter is 2.0 μm；Chromatographic column is advised
Lattice are 100mm × 4.6mm；
Using gradient elution；Wherein, phosphate aqueous solution of the mobile phase A for concentration 0.08ml/100ml, Mobile phase B is second
Nitrile；Elution program is：0～5min, 2～25% Mobile phase Bs；5～8min, 25～70% Mobile phase Bs；8～8.1min, 70～
95% Mobile phase B；8.1～13min, 95% Mobile phase B；
Detection wavelength is 200nm and 238nm；
Column temperature is 30 DEG C；
Flow rate of mobile phase is 0.1mL/min；
Sample size is 2 μ L；
(4) with standard in peak area and Liuwei Dihuang preparation of the concentration, standard substance according to standard substance in chromatogram
Peak area of the condition tie element in chromatogram, calculates the content of composition corresponding with standard substance in Liuwei Dihuang preparation.
Embodiment 3
Liuwei Dihuang preparation is detected according to following steps：
(1) Liuwei Dihuang preparation is taken, precision is weighed after crushing, is dissolved in methanol with concentration 1g/10ml, uses frequency
Ultrasonic 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-glucopyranoses, paeonol, 5- hydroxyl first
Base furfural, morroniside, loganin, sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin, with methanol as solvent, are configured to
Concentration is respectively the standard solution of 25 μ g/mL, standby；
(3) ultra-performance liquid chromatography is adopted, six drugs containing rehmanniae concentrated pill solution is obtained under identical testing conditions respectively
With the chromatogram of standard solution；The testing conditions include：
Chromatograph column packing is octadecylsilane chemically bonded silica Agilent SB C18, particle diameter is 5 μm；Chromatographic column specification
For 250mm × 4.6mm；
Using gradient elution；Wherein, phosphate aqueous solution of the mobile phase A for concentration 0.08ml/100ml, Mobile phase B is second
Nitrile；Elution program is：0～5min, 2～25% Mobile phase Bs；5～8min, 25～70% Mobile phase Bs；8～8.1min, 70～
95% Mobile phase B；8.1～13min, 95% Mobile phase B；
Detection wavelength is 220nm and 250nm；
Column temperature is 40 DEG C；
Flow rate of mobile phase is 1mL/min；
Sample size is 5 μ L；
(4) with standard in peak area and Liuwei Dihuang preparation of the concentration, standard substance according to standard substance in chromatogram
Peak area of the condition tie element in chromatogram, calculates the content of composition corresponding with standard substance in Liuwei Dihuang preparation.
Jing compares, and in three embodiments that the present invention is provided, the accuracy resultant effect of embodiment 1 is optimum.
Experimental example：Methodological study
1st, linear relationship is investigated：Standard substance are taken, the standard solution of five variable concentrations is prepared from high to low, with standard substance
Concentration is abscissa, and chromatographic peak area is that vertical coordinate drafting standard curve obtains regression equation.As a result show, 11 kinds of standard substance are upper
State that the offline sexual intercourse of concentration is good, the range of linearity of concrete each standard substance equation of linear regression and concentration is shown in Table 2.This
Bright accepted standard product concentration is in linear scope.
Table 2：Linear relationship investigates result
| Compound | Detection wavelength | Standard curve | Correlation coefficient (r) | The range of linearity (μ g/mL) |
| Gallic acid | 210nm | Y=20730.713x-11.3787 | 0.9994 | 9.70-203.70 |
| 5 hydroxymethyl furfural | 238nm | Y=1927.059x+22.9385 | 0.9997 | 19.62-1418.00 |
| Protocatechuic acid | 210nm | Y=15846.456x+0.6091 | 0.9998 | 2.52-25.25 |
| Morroniside | 238nm | Y=3743.123x-7.5733 | 0.9992 | 20.00-120.00 |
| Loganin | 238nm | Y=4151.934x-5.1161 | 1.0000 | 15.15-303.00 |
| Sweroside | 238nm | Y=1033.950x-0.4331 | 0.9998 | 5.05-171.70 |
| Peoniflorin | 238nm | Y=2528.373x+6.6397 | 0.9991 | 20.00-141.40 |
| 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses | 210nm | Y=11472.582x+4.2163 | 0.9999 | 4.95-123.70 |
| Benzoic acid | 238nm | Y=8079.100x+0.4757 | 0.9998 | 5.00-25.00 |
| Benzoylpaeoniflorin | 238nm | Y=3760.100x+2.2759 | 0.9999 | 9.90-128.70 |
| Paeonol | 210nm | Y=11710.942x+35.3310 | 0.9994 | 18.25-351.70 |
2nd, precision test：Take six drugs containing rehmanniae concentrated pill sample (sample 34) 1 is prepared according to the method that embodiment 1 is provided
Part Liuwei Dihuang preparation solution, and the method provided according to embodiment 1 carries out 6 detections respectively to the solution.Detected according to 6 times
Result, relative standard deviation (RSD) value of 11 compositions illustrates the precision of instrument no more than 1.6% (as shown in table 3)
Well.
Table 3：Precision testing result
| Compound | RSD (%) |
| Gallic acid | 1.07 |
| 5 hydroxymethyl furfural | 0.27 |
| Protocatechuic acid | 0.45 |
| Morroniside | 0.91 |
| Loganin | 0.42 |
| Sweroside | 1.36 |
| Peoniflorin | 0.33 |
| 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses | 0.17 |
| Benzoic acid | 0.20 |
| Benzoylpaeoniflorin | 1.60 |
| Paeonol | 0.28 |
3rd, repeated experiment：Take six drugs containing rehmanniae concentrated pill sample (sample 34) to distinguish according to the method that embodiment 1 is provided
6 parts of Liuwei Dihuang preparation solution are prepared, and the method provided according to embodiment 1 is detected respectively to 6 parts of solution.According to detection
As a result, gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol, 5 hydroxymethyl furfural, morroniside,
The RSD values of loganin, sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin are respectively less than 1.75%, illustrate that the method repeats
Property is good.
4th, stability test：Take six drugs containing rehmanniae concentrated pill sample (sample 34) 1 is prepared according to the method that embodiment 1 is provided
Part Liuwei Dihuang preparation solution, took equivalent solution respectively at the 0th, 2,4,6,8,12 and 24 hours, according to the side that embodiment 1 is provided
Method is detected.According to testing result, gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol,
The RSD values of 5 hydroxymethyl furfural, morroniside, loganin, sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin are respectively less than
1.99%, it was demonstrated that the sample was stablized in 24 hours.
5th, accuracy test：Take the six drugs containing rehmanniae concentrated pill sample of known each component content, add it is corresponding to sample into
The standard substance of the amount of grading, then detect the content of each composition in sample after being loaded, and every kind of standard substance are loaded 6 times respectively, according to reality
The method for applying the offer of example 1 is detected.Sample-adding recovery the results are shown in Table 4, the average recovery of each standard substance 95.25～
Between 104.64%, show that the content assaying method is accurate, reliable.
Table 4：Sample-adding recovery test result
Above experimental result shows that the method repeatability of present invention offer, stability, precision, accuracy are good, adopt
The ultra high efficiency liquid phase dual wavelength multi objective content assaying method provided with the present invention carries out quality control to six drugs containing rehmanniae pill
It is feasible.
Embodiment of above is merely to illustrate the present invention, rather than limitation of the present invention.Although with reference to embodiment to this
It is bright to 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, all should cover will in right of the invention
Ask in the middle of scope.
Claims (6)
1. a kind of ultra high efficiency liquid phase dual wavelength multi objective content assaying method of Liuwei Dihuang preparation, it is characterised in that the method
Including step：
(1) Liuwei Dihuang preparation is taken, precision is weighed after crushing, is dissolved in methanol with concentration 1g/10～30ml, supersound extraction,
Centrifugation, takes supernatant liquid filtering, obtains final product Liuwei Dihuang preparation solution, standby；
(2) precision weighs standard substance, is dissolved in methanol with 20～30 μ g/ml of concentration, obtains final product standard solution, standby；It is described
Standard substance include gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol, 5 hydroxymethyl furfural,
Morroniside, loganin, sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin；
(3) ultra-performance liquid chromatography is adopted, Liuwei Dihuang preparation solution and standard is obtained under identical testing conditions respectively
The chromatogram of product solution；The testing conditions include：
Chromatograph column packing is octadecylsilane chemically bonded silica；The specification of the chromatographic column be 100～250mm × 2.1～
4.6mm；The particle diameter of filler is 1.8～5 μm；
Using gradient elution；Wherein, phosphate aqueous solution of the mobile phase A for concentration 0.08ml/100ml, Mobile phase B is acetonitrile；Wash
De- program is：0～5min, 2～25% Mobile phase Bs；5～8min, 25～70% Mobile phase Bs；8～8.1min, 70～95% streams
Dynamic phase B；8.1～13min, 95% Mobile phase B；
Detection wavelength is 200-220nm and 238-250nm；Detection wavelength be 200-220nm under conditions of detect gallic acid,
Protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol；Under conditions of Detection wavelength is 238-250nm, detection
5 hydroxymethyl furfural, morroniside, loganin, sweroside, peoniflorin, benzoic acid, benzoylpaeoniflorin；
The testing conditions also include：The column temperature of chromatographic column is 30～40 DEG C；Flow rate of mobile phase is 0.1～1.0ml/min；Sample introduction
Measure as 1～5 μ l；
(4) with standard condition in peak area and Liuwei Dihuang preparation of the concentration, standard substance according to standard substance in chromatogram
Peak area of the tie element in chromatogram, calculates the content of composition corresponding with standard substance in Liuwei Dihuang preparation.
2. method according to claim 1, it is characterised in that the Detection wavelength described in step (3) is 210nm and 238nm.
3. method according to claim 1 and 2, it is characterised in that in step (1) supersound extraction, ultrasonic frequency
For 35000～45000Hz, extraction time is 20～40min.
4. method according to claim 3, it is characterised in that in step (1) centrifugation, the speed of centrifugation is 5000～
20000 revs/min, centrifugation time is 5～15min.
5. method according to claim 4, it is characterised in that step (1) is described to be filtered using 0.2～0.3 μm of aperture
Membrane filtration.
6. method according to claim 1 and 2, it is characterised in that the dosage form of the Liuwei Dihuang preparation is：Water-honeyed pill,
Big honeyed pills, concentrated pill or capsule.
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