CN104865319A - 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, more specifically relate to a kind of ultra high efficiency liquid phase dual wavelength multi objective content assaying method of Liuwei Dihuang preparation.
Background technology
Six drugs containing rehmanniae is as the representative prescription of " nourishing kidney yin ", and its curative effect is indubitable, and the Liuwei Dihuang preparation on market today is numerous, therefore only guarantees its quality, and this classical ancient prescription could be allowed to give full play to its treatment advantage.The assay that 2010 editions Chinese Pharmacopoeias specify: for water-honeyed pill, small honey pill, large these three kinds of formulations of honeyed bolus, containing moutan bark in Paeonol, the every 1g of water-honeyed pill must not be less than 0.90mg; The every lg of small honey pill must not be less than 0.70mg; The every ball of large honeyed bolus must not be less than 6.3mg.Containing wine cornus in loganin, 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 every ball of large honeyed bolus must not be less than 4.5mg.For condensed pill, every lg in loganin, must not be less than 1.4mg containing wine cornus, and every lg in Paeonol, must not be less than 1.8mg containing moutan bark.Capsule every is containing moutan bark in Paeonol, and specification (1) must not be less than 3.0mg; Specification (2) must not be less than 1.5mg, and wine cornus is in ursolic acid, and specification (1) must not be less than 0.60mg; Specification (2) must not be less than 0.30mg.Wherein Paeonol and loganin adopt high performance liquid chromatography to detect, and chromatographic condition is different, determined wavelength is respectively 274nm and 236nm, ursolic acid adopts thin-layered chromatography to carry out assay, complex operation step, consuming time longer, be unfavorable for detection rapidly and efficiently and can not from the overall evaluation and the quality controlling six drugs containing rehmanniae perfect square.
Connect UV-detector about high performance liquid chromatography about the quality controling research report of Liuwei Dihuang preparation is more in the past, but the composition that often can detect is less simultaneously, quality control can not be carried out on the whole, report LC-MS and Micellar Electrokinetic Chromatography is had to carry out multicomponent assay to Liuwei Dihuang Wan, all can detect four kinds of compositions wherein simultaneously, but the universal restriction comparatively rare by instrument of these class methods.Bibliographical information high performance liquid chromatography coupling diode array and evaporative light-scattering detector can detect eight kinds of compositions in Liuwei Dihuang Wan simultaneously in addition, but adopt two kinds of detecting device testing processes comparatively loaded down with trivial details, and time and effort consuming.Ultra Performance Liquid Chromatography (UHPLC) adopts granule filler chromatographic column and extra high voltage system, significantly can improve degree of separation and the detection sensitivity of chromatographic peak, significantly can shorten analytical cycle again, reduce reagent consumption, after being equipped with diode array detector (DAD), sensing range is more widened.Therefore Ultra Performance Liquid Chromatography is adopted, determine the determined wavelength be applicable to through DAD full wavelength scanner, can carry out detecting fast, comprehensively to the principal ingredient in Liuwei Dihuang Wan and capsule, instrument is easy to get, the easy strong operability of method, is expected to the powerful means becoming this compound preparation quality control.
Summary of the invention
The object of the invention is to, for the defect existed in prior art, provide a kind of Liuwei Dihuang preparation multicomponent ultra high efficiency liquid phase dual wavelength content assaying method.Method provided by the invention can realize carrying out detecting fast, comprehensively to the principal ingredient in Liuwei Dihuang preparation, 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 method specifically comprises the following steps:
(1) get Liuwei Dihuang preparation, after pulverizing, precision takes, and is dissolved in methyl alcohol, ultrasonic extraction with concentration 1g/10 ~ 30ml, centrifugal, gets supernatant liquid filtering, obtains Liuwei Dihuang preparation solution, for subsequent use;
(2) precision takes standard items, is dissolved in methyl alcohol, obtains standard solution with concentration 20 ~ 30 μ g/ml, for subsequent use;
(3) adopt ultra-performance liquid chromatography, under identical testing conditions, obtain the chromatogram of Liuwei Dihuang preparation solution and standard solution respectively; Described testing conditions comprises:
Chromatographic column filling agent is octadecylsilane chemically bonded silica;
Adopt gradient elution; Wherein, mobile phase A is the phosphate aqueous solution of concentration 0.08ml/100ml, and Mobile phase B is acetonitrile; Elution program is: 0 ~ 5min, 2 ~ 25% Mobile phase B; 5 ~ 8min, 25 ~ 70% Mobile phase B; 8 ~ 8.1min, 70-95% Mobile phase B; 8.1 ~ 13min, 95% Mobile phase B;
Determined wavelength is 200-220nm and 238-250nm;
(4) according to the peak area of composition corresponding with standard items in chromatogram in peak area in chromatogram of the concentration of standard items, standard items and Liuwei Dihuang preparation, the content of composition corresponding with standard items in Liuwei Dihuang preparation is calculated.
Liuwei Dihuang preparation of the present invention is LIUWEIDIHUANG SHUIMIWAN, large honeyed bolus, condensed pill or capsule.
Step of the present invention (1) is optimized the extraction of Liuwei Dihuang preparation and the compound method of solution, thus adds the accuracy of detection.Specifically, the ultrasonic frequency of described ultrasonic extraction is preferably 35000 ~ 45000Hz, and extraction time is preferably 20 ~ 40min under said extracted condition, and centrifugal speed is preferably 5000 ~ 20000 revs/min, and centrifugation time is 5 ~ 15min.After centrifugal, the membrane filtration in 0.2 ~ 0.3 μm, aperture can be used to filter supernatant.Preferred by above-mentioned condition, can improve the accuracy detected in subsequent step.
As a kind of preferred version, step of the present invention (1) is specially: get Liuwei Dihuang preparation, after pulverizing, precision takes, be dissolved in methyl alcohol with concentration 1g/10ml, extract 30min with the ultrasound wave of frequency 40000Hz, with the centrifugation 10min of 10000 revs/min, get supernatant, with the membrane filtration in 0.22 μm, aperture, obtain Liuwei Dihuang preparation solution.
Step of the present invention (2) described standard items point to be chosen according to being detected as of the effective constituent in Liuwei Dihuang preparation and States Pharmacopoeia specifications, comprise gallic acid, protocatechuic acid, 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin.In the present invention, the concentration of described standard items refers to that often kind of standard items divide other concentration, when practical operation, by after various standard items respectively precision weighing, can merge, be dissolved in the methyl alcohol of specified quantitative, and mix, obtain hybrid standard product solution.The concentration of described standard items is preferably: gallic acid, protocatechuic acid, 1,2, the concentration of 3,4,6-Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin is 25 μ g/ml.
Because heterogeneity goes out peak situation difference at different wavelengths, therefore, the present invention preferably detects gallic acid, protocatechuic acid, 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose, Paeonol under the condition being 200-220nm at determined wavelength; Be under the condition of 238-250nm at determined wavelength, detect 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid, benzoylpaeoniflorin.
The wavelength of step of the present invention (3) described detection is preferably 210nm and 238nm.Adopt these two wavelength to detect mentioned component, best analytical effect can be realized.
The implication of the described phosphate aqueous solution concentration 0.08ml/100ml of step of the present invention (3) is: containing 0.08ml phosphoric acid in every 100ml solution.In described gradient elution program, the number percent of Mobile phase B refers to that Mobile phase B accounts for the percent by volume of two-phase volume sum.
Step of the present invention (3) can also comprise following testing conditions: the specification of chromatographic column is 100 ~ 250mm × 2.1 ~ 4.6mm, is preferably 100mm × 2.1mm.The particle diameter of chromatographic column filling agent is 1.8 ~ 5.0 μm, is preferably 1.8 ~ 2.0 μm, more preferably 1.8 μm.Described filling agent can select the Agilent SB C purchased from Agilent Technologies
18.The column temperature of chromatographic column is 30 ~ 40 DEG C, is preferably 35 DEG C.Flow rate of mobile phase is 0.1 ~ 1.0ml/min, is preferably 0.4ml/min.Sample size is 1 ~ 5 μ l, is preferably 1 ~ 2 μ l, more preferably 1 μ l.
Because standard solution is identical with the testing conditions of Liuwei Dihuang preparation solution, in chromatogram, in Liuwei Dihuang preparation one-tenth corresponding with standard items separate the retention time at peak should be substantially identical with standard items with peak shape, therefore, composition corresponding in two width chromatograms can be judged by identical retention time, and confirm by the shape at peak is auxiliary.Step of the present invention (4) is by the peak area of corresponding composition in standard of comparison product solution chromatogram and Liuwei Dihuang preparation solution chromatogram, according to the concentration known of standard items, calculate the concentration of composition corresponding with standard items in Liuwei Dihuang preparation solution, calculate the content of composition corresponding with standard items in Liuwei Dihuang preparation further.
The inventive method adopts ultra high efficiency liquid phase dual wavelength multi objective to carry out chromatogram detection, only need can detect at least 11 kinds of principal ingredients in Liuwei Dihuang Wan and capsule less than 10 minutes, and carry out quantitatively it.Compared with traditional high performance liquid chromatography, analysis time can be saved more than 3/4, meanwhile, save reagent and expend.Method provided by the invention adopts double UV check, thus covers the composition that in Liuwei Dihuang preparation, most of content is higher, and detection method accurately, reliably.
Ultra high efficiency liquid phase dual wavelength multi objective content assaying method provided by the invention, can accurate, easy, detect Liuwei Dihuang preparation fast, and be conducive to the quality evaluating Liuwei Dihuang preparation on the whole, guarantee its clinical efficacy.
Accompanying drawing explanation
Fig. 1 is hybrid standard product under 210nm wavelength and six drugs containing rehmanniae condensed pill sample (No. 34 samples) chromatogram; Mark in figure: a, hybrid standard product, b, six drugs containing rehmanniae condensed pill sample; 1, gallic acid, 2, protocatechuic acid, 3,1,2,3,4,6-Penta-O-galloyl-D-glucopyranose, 4, Paeonol.
Fig. 2 is hybrid standard product under 238nm wavelength and six drugs containing rehmanniae condensed pill sample (No. 34 samples) chromatogram; Mark in figure: c, hybrid standard product, d, six drugs containing rehmanniae condensed pill sample; 5,5 hydroxymethyl furfural, 6, morroniside, 7, loganin, 8, sweroside, 9, Paeoniflorin, 10, benzoic acid, 11, benzoylpaeoniflorin.
Embodiment
Below in conjunction with drawings and Examples, embodiments of the present invention are described in further detail.Following examples for illustration of the present invention, but can not be used for limiting the scope of the invention.
The instrument that the embodiment of the present invention adopts and reagent comprise: Agilent 1290 Ultra Performance Liquid Chromatography instrument, preparation G4204A quaternary pump, G4226A automatic sampler, G1316C temperature control box, G4212A diode array detector, ten thousand/electronic balance (ME204E, Mettler-Toledo), ultrapure water (Milli-Q, Millipore), acetonitrile (chromatographically pure, Merk), phosphoric acid (chromatographically pure, DikmaPure).Standard items: (hundred careless company limiteds are matched in Beijing to gallic acid, 130718), (hundred careless company limiteds are matched in Beijing to protocatechuic acid, 130106), 1, 2, 3, 4, (hundred careless company limiteds are matched in Beijing to 6-Penta-O-galloyl-D-glucopyranose, 130508), Paeonol (Chinese food Drug Administration, 110708-200506), (hundred careless company limiteds are matched in Beijing to 5 hydroxymethyl furfural, 131124), (hundred careless company limiteds are matched in Beijing to morroniside, 130912), (hundred careless company limiteds are matched in Beijing to loganin, 1201204), (hundred careless company limiteds are matched in Beijing to sweroside, 140425), (hundred careless company limiteds are matched in Beijing to Paeoniflorin, 130726), benzoic acid (hundred careless company limiteds are matched in Beijing), (hundred careless company limiteds are matched in Beijing to benzoylpaeoniflorin, 131108).
Embodiment 1
According to following steps, Liuwei Dihuang preparation is detected:
(1) Liuwei Dihuang preparation is got, after pulverizing, precision takes, be dissolved in methyl alcohol with concentration 1g/10ml, 30min is extracted with the ultrasound wave of frequency 40000Hz, with the centrifugation 10min of 10000 revs/min, get supernatant, with the membrane filtration in 0.22 μm, aperture, obtain Liuwei Dihuang preparation solution, for subsequent use;
(2) precision takes gallic acid, protocatechuic acid, 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin, be solvent with methyl alcohol, be mixed with the standard solution that concentration is respectively 25 μ g/mL, for subsequent use;
(3) adopt ultra-performance liquid chromatography, under identical testing conditions, obtain the chromatogram of six drugs containing rehmanniae condensed pill solution and standard solution respectively; Described testing conditions comprises:
Chromatographic column filling agent is octadecylsilane chemically bonded silica Agilent SB C
18, particle diameter is 1.8 μm; Chromatographic column specification is 100mm × 2.1mm;
Adopt gradient elution; Wherein, mobile phase A is the phosphate aqueous solution of concentration 0.08ml/100ml, and Mobile phase B is acetonitrile; Elution program is: 0 ~ 5min, 2 ~ 25% Mobile phase B; 5 ~ 8min, 25 ~ 70% Mobile phase B; 8 ~ 8.1min, 70 ~ 95% Mobile phase B; 8.1 ~ 13min, 95% Mobile phase B;
Determined 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) according to the peak area of composition corresponding with standard items in chromatogram in peak area in chromatogram of the concentration of standard items, standard items and Liuwei Dihuang preparation, the content of composition corresponding with standard items in Liuwei Dihuang preparation is calculated.
According to method described in the present embodiment, detect commercially available 81 kinds of Liuwei Dihuang preparations (comprising 34 kinds of six drugs containing rehmanniae condensed pills, 17 kinds of LIUWEIDIHUANG SHUIMIWANs, 11 kinds of large honeyed bolus of six drugs containing rehmanniae and 10 kinds of LIUWEIDIHUANG JIAONANG agent), testing result is as shown in table 1.
Wherein, the chromatogram of No. 34th, sample (i.e. No. 34th, six drugs containing rehmanniae condensed pill) under 210nm wavelength is see Fig. 1, and the chromatogram under 238nm wavelength is see 2.
Table 1: Liuwei Dihuang preparation Contents of Main Components (ND=does not detect)
Embodiment 2
According to following steps, Liuwei Dihuang preparation is detected:
(1) Liuwei Dihuang preparation is got, after pulverizing, precision takes, be dissolved in methyl alcohol with concentration 1g/15ml, 40min is extracted with the ultrasound wave of frequency 35000Hz, with the centrifugation 15min of 5000 revs/min, get supernatant, with the membrane filtration in 0.2 μm, aperture, obtain Liuwei Dihuang preparation solution, for subsequent use;
(2) precision takes gallic acid, protocatechuic acid, 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin, be solvent with methyl alcohol, be mixed with the standard solution that concentration is respectively 25 μ g/mL, for subsequent use;
(3) adopt ultra-performance liquid chromatography, under identical testing conditions, obtain the chromatogram of six drugs containing rehmanniae condensed pill solution and standard solution respectively; Described testing conditions comprises:
Chromatographic column filling agent is octadecylsilane chemically bonded silica Agilent SB C
18, particle diameter is 2.0 μm; Chromatographic column specification is 100mm × 4.6mm;
Adopt gradient elution; Wherein, mobile phase A is the phosphate aqueous solution of concentration 0.08ml/100ml, and Mobile phase B is acetonitrile; Elution program is: 0 ~ 5min, 2 ~ 25% Mobile phase B; 5 ~ 8min, 25 ~ 70% Mobile phase B; 8 ~ 8.1min, 70 ~ 95% Mobile phase B; 8.1 ~ 13min, 95% Mobile phase B;
Determined 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) according to the peak area of composition corresponding with standard items in chromatogram in peak area in chromatogram of the concentration of standard items, standard items and Liuwei Dihuang preparation, the content of composition corresponding with standard items in Liuwei Dihuang preparation is calculated.
Embodiment 3
According to following steps, Liuwei Dihuang preparation is detected:
(1) Liuwei Dihuang preparation is got, after pulverizing, precision takes, be dissolved in methyl alcohol with concentration 1g/10ml, 20min is extracted with the ultrasound wave of frequency 45000Hz, with the centrifugation 5min of 20000 revs/min, get supernatant, with the membrane filtration in 0.3 μm, aperture, obtain Liuwei Dihuang preparation solution, for subsequent use;
(2) precision takes gallic acid, protocatechuic acid, 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin, be solvent with methyl alcohol, be mixed with the standard solution that concentration is respectively 25 μ g/mL, for subsequent use;
(3) adopt ultra-performance liquid chromatography, under identical testing conditions, obtain the chromatogram of six drugs containing rehmanniae condensed pill solution and standard solution respectively; Described testing conditions comprises:
Chromatographic column filling agent is octadecylsilane chemically bonded silica Agilent SB C
18, particle diameter is 5 μm; Chromatographic column specification is 250mm × 4.6mm;
Adopt gradient elution; Wherein, mobile phase A is the phosphate aqueous solution of concentration 0.08ml/100ml, and Mobile phase B is acetonitrile; Elution program is: 0 ~ 5min, 2 ~ 25% Mobile phase B; 5 ~ 8min, 25 ~ 70% Mobile phase B; 8 ~ 8.1min, 70 ~ 95% Mobile phase B; 8.1 ~ 13min, 95% Mobile phase B;
Determined 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) according to the peak area of composition corresponding with standard items in chromatogram in peak area in chromatogram of the concentration of standard items, standard items and Liuwei Dihuang preparation, the content of composition corresponding with standard items in Liuwei Dihuang preparation is calculated.
Through comparing, in three embodiments provided by the invention, the accuracy resultant effect of embodiment 1 is optimum.
Experimental example: methodological study
1, linear relationship is investigated: get standard items, and prepare the standard solution of five variable concentrations from high to low, take standard concentration as horizontal ordinate, chromatographic peak area is that ordinate drawing standard curve obtains regression equation.Result shows, 11 kinds of standard items are all good in above-mentioned concentration lower linear relation, and the range of linearity of concrete each standard items equation of linear regression and concentration is in table 2.Accepted standard product concentration of the present invention is in the range of linearity.
Table 2: linear relationship investigates result
| Compound | Determined wavelength | Typical curve | Related 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 |
| Paeoniflorin | 238nm | y＝2528.373x+6.6397 | 0.9991 | 20.00-141.40 |
| 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose | 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 |
2, precision test: the method that getting six drugs containing rehmanniae condensed pill sample (No. 34, sample) provides according to embodiment 1 prepares 1 part of Liuwei Dihuang preparation solution, and according to the method that embodiment 1 provides, 6 detections are carried out respectively to this solution.According to the result that 6 times are detected, relative standard deviation (RSD) value of 11 compositions is all not more than 1.6% (as shown in table 3), illustrates that the precision of instrument is good.
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 |
| Paeoniflorin | 0.33 |
| 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose | 0.17 |
| Benzoic acid | 0.20 |
| Benzoylpaeoniflorin | 1.60 |
| Paeonol | 0.28 |
3, repeated experiment: the method that getting six drugs containing rehmanniae condensed pill sample (No. 34, sample) provides according to embodiment 1 prepares 6 parts of Liuwei Dihuang preparation solution respectively, and detects respectively 6 parts of solution according to the method that embodiment 1 provides.According to testing result, gallic acid, protocatechuic acid, 1,2,3, the RSD value of 4,6-Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin is all less than 1.75%, illustrates that the method is reproducible.
4, stability test: the method that getting six drugs containing rehmanniae condensed pill sample (No. 34, sample) provides according to embodiment 1 prepares 1 part of Liuwei Dihuang preparation solution, got equivalent solution respectively at the 0th, 2,4,6,8,12 and 24 hour, the method provided according to embodiment 1 detects.According to testing result, gallic acid, protocatechuic acid, 1,2,3,4, the RSD value of 6-Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin is all less than 1.99%, proves in this sample 24 hours stable.
5, accuracy test: the six drugs containing rehmanniae condensed pill sample getting known each component content, add the standard items of composition equivalent corresponding to sample, then detect the content of each composition in sample after application of sample, often kind of standard items application of sample 6 times respectively, the method provided according to embodiment 1 detects.Application of sample reclaims and the results are shown in Table 4, and the average recovery of each standard items, all between 95.25 ~ 104.64%, shows that this content assaying method accurately, reliably.
Table 4: application of sample recovery test result
Above experimental result display, method repeatability provided by the invention, stability, precision, accuracy are all good, and it is feasible for adopting ultra high efficiency liquid phase dual wavelength multi objective content assaying method provided by the invention to carry out quality control to six drugs containing rehmanniae pill.
Above embodiment is only for illustration of the present invention, but not limitation of the present invention.Although with reference to embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, various combination, amendment or equivalent replacement are carried out to technical scheme of the present invention, do not depart from the spirit and scope of technical solution of the present invention, all should be encompassed in the middle of right of the present invention.
Claims (10)
1. a ultra high efficiency liquid phase dual wavelength multi objective content assaying method for Liuwei Dihuang preparation, it is characterized in that, the method comprising the steps of:
(1) get Liuwei Dihuang preparation, after pulverizing, precision takes, and is dissolved in methyl alcohol, ultrasonic extraction with concentration 1g/10 ~ 30ml, centrifugal, gets supernatant liquid filtering, obtains Liuwei Dihuang preparation solution, for subsequent use;
(2) precision takes standard items, is dissolved in methyl alcohol, obtains standard solution with concentration 20 ~ 30 μ g/ml, for subsequent use;
(3) adopt ultra-performance liquid chromatography, under identical testing conditions, obtain the chromatogram of Liuwei Dihuang preparation solution and standard solution respectively; Described testing conditions comprises:
Chromatographic column filling agent is octadecylsilane chemically bonded silica;
Adopt gradient elution; Wherein, mobile phase A is the phosphate aqueous solution of concentration 0.08ml/100ml, and Mobile phase B is acetonitrile; Elution program is: 0 ~ 5min, 2 ~ 25% Mobile phase B; 5 ~ 8min, 25 ~ 70% Mobile phase B; 8 ~ 8.1min, 70 ~ 95% Mobile phase B; 8.1 ~ 13min, 95% Mobile phase B;
Determined wavelength is 200-220nm and 238-250nm;
(4) according to the peak area of composition corresponding with standard items in chromatogram in peak area in chromatogram of the concentration of standard items, standard items and Liuwei Dihuang preparation, the content of composition corresponding with standard items in Liuwei Dihuang preparation is calculated.
2. method according to claim 1, it is characterized in that, standard items described in step (2) comprise gallic acid, protocatechuic acid, 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose, Paeonol, 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid and benzoylpaeoniflorin.
3. method according to claim 2, is characterized in that, is to detect gallic acid, protocatechuic acid, 1,2,3,4,6-Penta-O-galloyl-D-glucopyranose, Paeonol under the condition of 200-220nm at determined wavelength; Be under the condition of 238-250nm at determined wavelength, detect 5 hydroxymethyl furfural, morroniside, loganin, sweroside, Paeoniflorin, benzoic acid, benzoylpaeoniflorin.
4. the method according to claims 1 to 3 any one, is characterized in that, the specification of step (3) described chromatographic column is 100 ~ 250mm × 2.1 ~ 4.6mm; The particle diameter of filling agent is 1.8 ~ 5 μm.
5. the method according to any one of Claims 1 to 4, is characterized in that, step (3) described testing conditions also comprises: the column temperature of chromatographic column is 30 ~ 40 DEG C; Flow rate of mobile phase is 0.1 ~ 1.0ml/min; Sample size is 1 ~ 5 μ l.
6. the method according to any one of Claims 1 to 5, is characterized in that, the determined wavelength described in step (3) is 210nm and 238nm.
7. the method according to claim 1 ~ 6 any one, is characterized in that, in the described ultrasonic extraction of step (1), ultrasonic frequency is 35000 ~ 45000Hz, and extraction time is 20 ~ 40min.
8. method according to claim 7, is characterized in that, step (1) described centrifugal in, centrifugal speed is 5000 ~ 20000 revs/min, and centrifugation time is 5 ~ 15min.
9. method according to claim 8, is characterized in that, step (1) is described filters the membrane filtration adopting 0.2 ~ 0.3 μm, aperture.
10. the method according to claim 1 ~ 9 any one, is characterized in that, the formulation of described Liuwei Dihuang preparation is: water-honeyed pill, large honeyed bolus, condensed pill or capsule.
Priority Applications (1)
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| CN108267524A (en) * | 2018-03-26 | 2018-07-10 | 马海周 | A kind of method for detecting burnt rehmanin B in burnt rehmanin A |
| CN113820403A (en) * | 2020-06-19 | 2021-12-21 | 九芝堂股份有限公司 | Method for detecting content of compound medicine dregs |
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| CN108267524A (en) * | 2018-03-26 | 2018-07-10 | 马海周 | A kind of method for detecting burnt rehmanin B in burnt rehmanin A |
| CN108267524B (en) * | 2018-03-26 | 2019-03-08 | 马海周 | A method of detecting burnt rehmanin B in burnt rehmanin A |
| CN113820403A (en) * | 2020-06-19 | 2021-12-21 | 九芝堂股份有限公司 | Method for detecting content of compound medicine dregs |
| CN113820403B (en) * | 2020-06-19 | 2023-11-07 | 九芝堂股份有限公司 | Method for detecting content of compound medicine residues |
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