CN105929083A - Method for simultaneously determining five active components in liver smoothing and stomach strengthening pill - Google Patents
Method for simultaneously determining five active components in liver smoothing and stomach strengthening pill Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
A method for simultaneously determining five active components in a liver smoothing and stomach strengthening pill comprises the following steps: 1, preparing a reference solution: respectively preparing a 241[mu]g.mL<-1> paeoniflorin solution, a 1420[mu]g.mL<-1> hesperidin solution, a 208[mu]g.mL<-1> naringin solution, a 438[mu]g.mL<-1> honokiol solution and a 458[mu]g.mL<-1> magnolol solution, placing all the solutions in a 5mL volumetric flask, and adding methanol to the volumetric flask to a certain volume in order to obtain a mixed reference solution; 2, preparing a sample solution; 3, setting chromatographic conditions as follows: a TC-C18 chromatographic column is adopted; an acetonitrile (A)-water (B) is adopted as a mobile phase, and gradient elution is set according to 0-15min, 22%-22% acetonitrile; 15-25min, 22%-72% acetonitrile; 25-35min, 72%-72% acetonitrile; 35-40min, 72%-90% acetonitrile; 40-50min, 90%-90% acetonitrile and 50-55min, 90%-22% acetonitrile; the detection wavelength is 0-10min, 230nm; 10-20nm, 280nm; and 20-55min, 294nm; the flow velocity is 1.0mL.min<-1>; a VWD detector is adopted as a detector; the column temperature is 25DEG C; and the sample size is 10[mu]L; and 3, determining. The method is simple and accurate, has good reappearance and stability, and can be used comprehensively evaluate and control the quality of the liver smoothing and stomach strengthening pill.
Description
Technical field
The present invention relates to Chinese patent medicine detection technique field, be specifically related to the detection side of a kind of Chinese patent medicine Shugan Jianpi Pills
Method.
Background technology
Shugan Jianpi Pills have soothing the liver dissipating depression of QI, intestinal stasis relieving and in, stomach function regulating analgesic efficacy, especially for incoordination between the liver and stomach
The epigastric distending pain, the fullness in the chest and hypochondrium that cause are vexed, vomit acid regurgitation and abdominal distention constipation.
Shugan Jianpi Pills formula and preparation method: Cortex Magnoliae Officinalis (processed with Rhizoma Zingiberis Recens) 300g, Rhizoma Cyperi (vinegar system) 400g, the Radix Paeoniae Alba (bran
Fry) 500g, Radix Bupleuri (vinegar system) 300g, Pericarpium Citri Reticulatae Viride (vinegar-fried) 200g, Fructus Citri 300g, Pericarpium Citri Reticulatae 500g, Lignum Santali Albi 300g,
Fructus Amomi Rotundus 300g, Fructus Aurantii 300g, Endothelium Corneum Gigeriae Galli (stir-fry) 500g, Semen Arecae 500g, Rhizoma Corydalis (vinegar-fried) 300g, five
Oletum Trogopterori (vinegar system) 300g, Semen Pharbitidis (stir-fry) 300g, weigh above ten five kinds of Chinese medicine on request, be ground into fine powder, mistake
Sieve, mixing, with water pill, it is dried；Every 500g dry ball 90g Pulvis Talci, 9g Resina persicae coating, polishing,
Obtain.This kind is recorded in the Sanitation Ministry medicine standard Traditional Chinese medicine historical preparation the 5th page 182.Initial quality control
Method processed is difficult to product is carried out effective quality control.Therefore, it is necessary to set up a kind of fast, accurate and comprehensive
Separate, detect the method for various active composition in Shugan Jianpi Pills, in order to preferably this product is carried out quality
Monitoring.
Summary of the invention
It is an object of the invention to provide one measure simultaneously peoniflorin in Shugan Jianpi Pills, Hesperidin, naringin,
Honokiol, the method for five kinds of active component of magnolol, solve existing Shugan Jianpi Pills quality control difficult, it is impossible to
Enough difficult problems the most accurately controlling drug quality.
For achieving the above object, to be achieved in that the method specifically includes following for technical scheme of the present invention
Step:
(1) preparation of reference substance solution
Precision weigh peoniflorin 1.25mg, Hesperidin 7.45mg, naringin 1.10mg, honokiol 2.21mg,
Magnolol standard substance 2.32mg, is dissolved separately in 5mL volumetric flask by methanol constant volume to graduation mark position,
Being settled to graduation mark position after supersound process 10min, methanol is supplied；Prepare concentration 241 μ g mL respectively-1、
1420μg·mL-1、208μg·mL-1、438μg·mL-1With 458 μ g mL-1Single reference substance solution；
Respectively precision draws five kinds of single reference substance solution again, is positioned in the volumetric flask of 5mL, by methanol constant volume,
I.e. prepare the mixing reference substance solution of known each concentration；
(2) preparation of need testing solution
Taking Shugan Jianpi Pills some, uncoating is the most finely ground with glass mortar, and precision weighs 0.2000g, puts
In 5mL volumetric flask, add 5mL methanol, ultrasonic dissolution 30min, high speed centrifugation 20min, take upper strata
Clear liquid is as need testing solution, for test；
(3) chromatographic condition
Use TC-C18Chromatographic column (4.6mm × 250mm, 5 μm)；Acetonitrile (A)-water (B) is as flowing
Phase, gradient elution: 0-15min, 22%-22% acetonitrile (percent by volume)；15-25min, 22%-72%
Acetonitrile (percent by volume)；25-35min, 72%-72% acetonitrile (percent by volume)；35-40min, 72%-90%
Acetonitrile (percent by volume)；40-50min, 90%-90% acetonitrile (percent by volume)；50-55min, 90%-22%
Acetonitrile；Detection wavelength: 0-10min, 230nm；10-20min, 280nm；20-55min, 294nm；
Flow velocity 1.0mL min-1；Detector: VWD detector；Column temperature: 25 DEG C；Sample size: 10 μ L；
(4) measure: precision draws the reference substance solution and each 10 μ l of need testing solution prepared, note respectively
Enter liquid chromatograph, measure, the chromatogram of record 60min, to obtain final product.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of peoniflorin of the present invention.
Fig. 2 is the high-efficient liquid phase chromatogram of Hesperidin of the present invention.
Fig. 3 is the high-efficient liquid phase chromatogram of naringin of the present invention.
The high-efficient liquid phase chromatogram of Fig. 4 honokiol of the present invention.
Fig. 5 is the high-efficient liquid phase chromatogram of magnolol of the present invention.
Fig. 6 is the high-efficient liquid phase chromatogram that the present invention mixes reference substance solution.
Fig. 7 is the high-efficient liquid phase chromatogram of inventive samples.
Wherein: A, peoniflorin；B, Hesperidin；C, naringin；D, honokiol；E, magnolol.
Advantages of the present invention and good effect:
(1) the method for the invention simple, accurately, repeatability and good stability, can be thoroughly evaluating and control
Shugan Jianpi Pills quality processed is provided fundamental basis and scientific basis.
(2) the inventive method uses high performance liquid chromatography, utilizes gradient elution mode, it is achieved that survey simultaneously
Determine peoniflorin in Shugan Jianpi Pills, Hesperidin, naringin, honokiol, magnolol 5 kinds of effective ingredient
Content, chromatographic peak peak shape is good, and separating degree is higher, and detection sensitivity is high, can comprehensively, more effectively control soothing liver-QI
The quality of JIANWEI WAN.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is made with further elucidated above, in order to the skill of this area
Art personnel know more about the present invention, but do not limit the present invention with this.
Instrument and reagent
Instrument: Agilent 1100 high performance liquid chromatograph (Co., Ltd in U.S.'s Agilent Technologies)；
GWA-UN4 Superpure water machine (Beijing Puxi General Instrument Co., Ltd)；FA1104N ten thousand/analysis sky
Flat (manufacture of balance equipment factory of Shanghai Precision Scientific Apparatus Co., Ltd)；SK5200H ultrasonic cleaner
(200W59HZ Shanghai Ke Dao Instrument Ltd.).
Reagent: peoniflorin reference substance (lot number: 110736-201438；Content is 96.4%；Source: China
Food and medicine calibrating academy)；Hesperidin reference substance (lot number: 110721-201316；Content is 95.3%；
Source: National Institute for Food and Drugs Control)；Naringin reference substance (lot number: 1110722-201312；Contain
Amount is 94.7%；Source: National Institute for Food and Drugs Control)；Honokiol reference substance (lot number:
110730-201313；Content is 99.1%；Source: National Institute for Food and Drugs Control)；Magnolol compares
Product (lot number: 110729-201513；Content is 98.8%；Source: National Institute for Food and Drugs Control)；
Shugan Jianpi Pills (Shanxi Wang Long legendary god of farming pharmaceutcal corporation, Ltd；Lot number: 20131002, according to Ministry of Public Health medicine mark
Formulation and technology in quasi-Traditional Chinese medicine historical preparation makes two batches by oneself, and lot number is 20151011,20151221)；Methanol (color
Compose pure)；Water (secondary deionized water).
Detailed description of the invention
Embodiment 1
(1) preparation of reference substance solution: precision weighs peoniflorin 1.25mg, Hesperidin 7.45mg, naringin
1.10mg, honokiol 2.21mg, magnolol standard substance 2.32mg, be dissolved separately in 5mL volumetric flask use
Methanol constant volume, to graduation mark position, is settled to graduation mark position after supersound process 10min, methanol is supplied；
Prepare concentration 241 μ g mL respectively-1、1420μg·mL-1、208μg·mL-1、438μg·mL-1With
458μg·mL-1Single reference substance solution；Precision draws five kinds of single reference substance solution respectively again, is positioned over
In the volumetric flask of 5mL, by methanol constant volume, i.e. prepare the mixing reference substance solution of known each concentration.
(2) preparation of need testing solution: take Shugan Jianpi Pills some, uncoating is the most finely ground with glass mortar,
Precision weighs 0.2000g, is placed in 5mL volumetric flask addition 5mL methanol, ultrasonic dissolution 30min, height
The centrifugal 20min of speed, takes the supernatant as need testing solution, for test.
(3) chromatographic condition: use TC-C18Chromatographic column (4.6mm × 250mm, 5 μm)；Acetonitrile (A)-
Water (B) is as flowing phase, gradient elution: 0-15min, 22%-22% acetonitrile (percent by volume)；15-25
Min, 22%-72% acetonitrile (percent by volume)；25-35min, 72%-72% acetonitrile (percent by volume)；35-40
Min, 72%-90% acetonitrile (percent by volume)；40-50min, 90%-90% acetonitrile (percent by volume)；50-55
Min, 90%-22% acetonitrile (percent by volume)；Detection wavelength: 0-10min, 230nm；10-20min,
280nm；20-55min, 294nm；Flow velocity 1.0mL min-1；Detector: VWD detector；Column temperature:
25℃；Sample size: 10 μ L.
(4) measure: the above-mentioned reference substance solution prepared of accurate absorption and each 10 μ l of need testing solution respectively,
Inject liquid chromatograph, measure, the chromatogram of record 60min, to obtain final product.
Under this chromatographic condition peoniflorin, Hesperidin, naringin, honokiol, the appearance time of magnolol and
Specifically go out peak situation and see Fig. 1 to Fig. 7.
Methodology is examined or check
(1) linear relationship: accurate draw the five kinds of single reference substance solution prepared, sample size is respectively 0.1,
0.5,1,2,2.5,5,7.5,8,9,10 μ L, is measured under the chromatographic condition described in step (3),
With chromatographic peak area as vertical coordinate, concentration is abscissa, makees linear relationship curve, and concentration is made linear by peak area
Equation.Obtain linear equation and the results are shown in Table 1.
Table 1 linear relationship equation
(2) Precision Experiment: the mixing reference substance solution that accurate absorption prepares, in the color described in step (3)
Continuous sample introduction 6 times under spectral condition, record the peak of peoniflorin, naringin, Hesperidin, honokiol, magnolol
Area, calculates relative standard deviation and is respectively 1.71%, 0.61%, 0.32%, 0.62% and 0.71% (n=6),
Result shows that instrument precision is good, is shown in Table 2.
Table 2 precision test experiments data
(3) repeatability experiment: take the identical Shugan Jianpi Pills of lot number 6 parts, according to step (2) described test sample
The preparation method of solution prepares need testing solution, and is measured under the chromatographic condition described in step (3), root
According to calculated by peak area peoniflorin, naringin, Hesperidin, honokiol, the relative standard deviation of Determination of Magnolol
It is respectively 0.59%, 0.79%, 1.17%, 2.00% and 1.30%, is shown in Table 3, show that method repeatability is good.
Table 3 repeated experiment data
(4) stability test: take same need testing solution, surveys under the chromatographic condition described in step (3) respectively
Fixed 0,4,8,12,16, the chromatogram of 24h, measure its stability.According to peoniflorin, naringin, orange
Skin glycosides, honokiol, the peak area of magnolol, calculate relative standard deviation and be respectively 1.54%, and 1.43%,
1.19%, 1.18% and 1.88%, be shown in Table 3, by experimental data illustrate need testing solution in 24h basicly stable.
Table 4 stability experiment data
(5) recovery test: precision weighs Shugan Jianpi Pills 6 parts and is respectively placed in volumetric flask, and according to five
Plant the content of active component, every part of sample all adds suitable peoniflorin, naringin, Hesperidin and thickness
Piao phenol, magnolol reference substance, preparation method and step (3) according to step (2) described need testing solution are described
Chromatographic condition be measured.The response rate of each component is shown in Table 5.Test result indicate that the method response rate is preferable,
Accuracy rate is higher.
The recovery of standard addition test data of the various composition of table 5
Sample size measures: according to preparation method and step (3) the described color of step (2) described need testing solution
Spectral condition measures three batches of need testing solutions, the results are shown in Table 6.
Table 6 sample size measurement result
In Shugan Jianpi Pills, active component is more, and contains for active component multiple in Shugan Jianpi Pills simultaneously
Amount method for measuring is the most visible.Easy according to operability, the feature of favorable reproducibility, for improve determination efficiency and
Saving solvent, this experiment uses high performance liquid chromatography to be measured.Have easy and simple to handle, precision good, inspection
Survey highly sensitive feature, can comprehensively, more effectively control the quality of this medicine.
Claims (2)
1. one kind measures the method for five kinds of active component in Shugan Jianpi Pills simultaneously, it is characterised in that comprise the following steps:
(1) preparation of reference substance solution
Precision weighs peoniflorin, Hesperidin, naringin, honokiol, magnolol standard substance, is dissolved separately in volumetric flask, by methanol constant volume to graduation mark position, prepares concentration 241 g mL respectively-1、 1420 µg·mL-1、 208 µg·mL-1、 438 µg·mL-1With 458 g mL-1Single reference substance solution；Precision draws five kinds of single reference substance solution respectively again, is positioned in the volumetric flask of 5 mL, by methanol constant volume, i.e. prepares the mixing reference substance solution of known each concentration；
(2) preparation of need testing solution
Taking Shugan Jianpi Pills some, uncoating is the most finely ground with glass mortar, and precision weighs 0.2000 g, is placed in 5 mL volumetric flasks and adds 5 mL methanol, ultrasonic dissolution 30 min, high speed centrifugation 20 min, takes the supernatant as need testing solution, for test；
(3) chromatographic condition
Use TC-C18Chromatographic column；Acetonitrile-water is as flowing phase, and gradient elution: 0-15 min, volume ratio is the acetonitrile of 22%-22%；15-25 min, volume ratio is the acetonitrile of 22%-72%；25-35 min, volume ratio is the acetonitrile of 72%-72%；35-40 min, volume ratio is the acetonitrile of 72%-90%；40-50 min, volume ratio is the acetonitrile of 90%-90%；50-55 min, volume ratio is the acetonitrile of 90%-22%；Detection wavelength: 0-10min, 230nm；10-20 min, 280nm；20-55 min, 294
nm；Flow velocity 1.0 mL min-1；Detector: VWD detector；Column temperature: 25 DEG C；Sample size: 10 L；
(4) measure: the accurate reference substance solution and each 10 μ l of need testing solution prepared of drawing, injection liquid chromatograph respectively, measure, the chromatogram of record 60min, to obtain final product.
The method of five kinds of active component in a kind of Shugan Jianpi Pills of mensuration simultaneously the most according to claim 1, it is characterized in that, the concrete operation step of preparing of step (1) reference substance solution is: precision weighs peoniflorin 1.25mg, Hesperidin 7.45mg, naringin 1.10mg, honokiol 2.21mg, magnolol standard substance 2.32mg, it is dissolved separately in 5 mL volumetric flasks by methanol constant volume to graduation mark position, being settled to graduation mark position after supersound process 10 min, methanol is supplied；Prepare concentration 241 g mL respectively-1、 1420 µg·mL-1、 208 µg·mL-1、 438 µg·mL-1With 458 g mL-1Single reference substance solution.
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Application publication date: 20160907 |