CN108535386A - The method for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography - Google Patents
The method for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a kind of methods measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography, are related to analytical chemistry field.This method carries out gradient elution with the mobile phase being made of mobile phase A and Mobile phase B；Mobile phase A is the aqueous solution containing hexane sulfonate, wherein hexane sulfonate concentration is 0.05% 0.1%, pH is 2.5 3.0；Mobile phase B is the mixed solution of methanol and acetonitrile, and the volume ratio of methanol and acetonitrile in Mobile phase B is 4.5 5.5:1；Can 5 kinds of components of vitamin B1, vitamin B6, Hydrochioro, dihydralazine sulfate, promethazine hydrochloride in FUFANG LIXUEPING PIAN effectively be detached into appearance, theoretical cam curve and separating degree meet the requirements, separating effect is apparent, and the dissolution rate of 5 kinds of components in FUFANG LIXUEPING PIAN can be measured by external standard method；This method has the characteristics that simple, quick, high sensitivity, selectivity are good.
Description
Technical field
The present invention relates to analytical chemistry fields, and compound profit is measured with ultra-performance liquid chromatography in particular to a kind of
The method of 5 kinds of component dissolution rates in blood plain film.
Background technology
FUFANG LIXUEPING PIAN is the multicomponent designed by former Shanghai Research Institute of Hypertension chief Kuang An Kun professors, low dose
Compound hypertension medicine is a landmark traditional immobilised compound preparation in China's hypertension prevention and control history.This product is recorded in 2015
Version Chinese Pharmacopoeia, is suitable for early and middle portion high blood pressure.
Due to only having recorded the dissolution of two components (Hydrochioro and promethazine hydrochloride) in 2015 editions Chinese Pharmacopoeia standards,
Limit is to be not less than 70% in 45 minutes；Method is slurry processes, 50 turns；Medium is 0.1mol/L hydrochloric acid solutions 900mL.Dissolve out measurement side
Method is traditional liquid-phase chromatography method, and the run time of each sample is 25 minutes.But no other 4 components (reserpine, dimension lifes
Plain B1, vitamin B6, dihydralazine sulfate) dissolution, because content of the reserpine in FUFANG LIXUEPING PIAN is atomic, every 32 μ
G, dissolution medium volume are 900mL, and the theory of reserpine dissolves out a concentration of 0.036 μ g/mL, and concentration is too low, therefore now measures 5 groups
The dissolution rate (vitamin B1, vitamin B6, dihydralazine sulfate, Hydrochioro and promethazine hydrochloride) divided.And use UPLC methods
Measure five kinds of ingredients in FUFANG LIXUEPING PIAN simultaneously：Hydrochioro, dihydralazine sulfate, promethazine hydrochloride, vitamin B1 and dimension
Raw element B6, there is not yet report.Variation tendency because measuring FUFANG LIXUEPING PIAN 5 kinds of main component concentration in process in leaching exists
Sample size is big, the longer disadvantage of official method sample run time, therefore, a kind of rapid assay methods of dissolution is developed, to medicine
The quality evaluation of product has a very important significance.
In consideration of it, special propose the present invention.
Invention content
The purpose of the present invention is to provide 5 kinds of components in a kind of measurement FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography are molten
The method of out-degree, using this method can effectively by FUFANG LIXUEPING PIAN vitamin B1, vitamin B6, Hydrochioro,
5 kinds of dihydralazine sulfate, promethazine hydrochloride components detach appearance, and separating effect is apparent, in conjunction with reference substance, that is, vitamin B6, dimension life
The concentration and peak area of plain B1, dihydralazine sulfate, Hydrochioro and promethazine hydrochloride can measure compound profit by external standard method
The dissolution rate of 5 kinds of components in blood plain film, and then stripping curve is obtained, in addition, this method has simple, quick, high sensitivity, choosing
The advantages such as selecting property is good.
The invention is realized in this way：
A method of measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN, 5 kinds of components point with ultra-performance liquid chromatography
It is not：Vitamin B1, vitamin B6, Hydrochioro, dihydralazine sulfate, promethazine hydrochloride；
Described method includes following steps：
FUFANG LIXUEPING PIAN is taken to dissolve out prepare liquid, carrying out gradient with the mobile phase being made of mobile phase A and Mobile phase B washes
It is de-；
Mobile phase A is the aqueous solution containing hexane sulfonate, wherein hexane sulfonate concentration is 0.05%-0.1%, pH
For 2.5-3.0；
Mobile phase B is the mixed solution of methanol and acetonitrile, and the volume ratio of methanol and acetonitrile in Mobile phase B is 4.5-5.5:
1。
Preferably, in some embodiments of the present invention, the hexane sulfonate concentration of mobile phase A is 0.07%, pH is
2.75。
Preferably, in some embodiments of the present invention, the volume ratio of the methanol in Mobile phase B and acetonitrile is 5:1.
Further, in some embodiments of the present invention, the program of gradient elution is as follows：
| Time (min) | Mobile phase A (%V/V) | Mobile phase B (%V/V) |
| 0-0.3 | 98-90 | 2-10 |
| 0.3-3 | 94-40 | 6-60 |
| 3-4.7 | 40 | 60 |
| 4.7-5.0 | 40-94 | 60-6 |
| 5.0-6.8 | 90-98 | 10-2 |
Table Program is illustrated, in the time range of 0-0.3min, is carried out in a manner of isocratic elution, mobile phase
The volume ratio of A is 98%-90%, and the volume ratio of Mobile phase B is 2%-10%, the volume of volume+Mobile phase B of mobile phase A=
100%；It in the time range of 0.3-3min, is carried out with gradient elution mode, the volume ratio of mobile phase A is by 0.3min's
94% becomes the 40% of 3min, and the volume ratio of Mobile phase B is from becoming the 60% of 3min the 6% of 0.3min；
It in the time range of 3-4.7min, is carried out in a manner of isocratic elution, the volume ratio of mobile phase A is 40%, the volume ratio of Mobile phase B
It is 60%；It in the time range of 4.7-5.0min, is carried out with gradient elution mode, the volume ratio of mobile phase A is 4.7min
40% become the 94% of 5min, the volume ratio of Mobile phase B is to become the 6% of 5min from the 60% of 4.7min；
It in the time range of 5.0-6.8min, is carried out in a manner of isocratic elution, the volume ratio of mobile phase A is 90%-98%, Mobile phase B
Volume ratio be 10%-2%, volume=100% of volume+Mobile phase B of mobile phase A.
Further, in some embodiments of the present invention, the program of gradient elution is as follows：
Table Program is illustrated, in the time range of 0-0.3min, is carried out in a manner of isocratic elution, mobile phase
The volume ratio of A is 94%, and the volume ratio of Mobile phase B is 6；In the time range of 0.3-3min, carried out with gradient elution mode,
The volume ratio of mobile phase A is from becoming the 40% of 3min the 94% of 0.3min, and the volume ratio of Mobile phase B is by the
The 6% of 0.3min becomes the 60% of 3min；It in the time range of 3-4.7min, is carried out, is flowed in a manner of isocratic elution
The volume ratio of phase A is 40%, and the volume ratio of Mobile phase B is 60%；In the time range of 4.7-5.0min, with gradient elution side
Formula carries out, and the volume ratio of mobile phase A is the 40% of 4.7min to become the 94% of 5min, and the volume ratio of Mobile phase B is by the
The 60% of 4.7min becomes the 6% of 5min；It in the time range of 5.0-6.8min, is carried out, is flowed in a manner of isocratic elution
The volume ratio of dynamic phase A is 94%, and the volume ratio of Mobile phase B is 6%.
Preferably, in some embodiments of the present invention, the program of gradient elution is：
It is 94% in the volume ratio of 0min, mobile phase A, the volume ratio of Mobile phase B is 6%；
It is 94% in the volume ratio of 0.3min, mobile phase A, the volume ratio of Mobile phase B is 6%；
It is 40% in the volume ratio of 3min, mobile phase A, the volume ratio of Mobile phase B is 60%；
It is 40% in the volume ratio of 4.7min, mobile phase A, the volume ratio of Mobile phase B is 60%；
It is 94% in the volume ratio of 5min, mobile phase A, the volume ratio of Mobile phase B is 6%；
It is 94% in the volume ratio of 6.8min, mobile phase A, the volume ratio of Mobile phase B is 6%.
Sample minute is 6.8min, improves the efficiency of sample measurement.
Further, in some embodiments of the present invention, flow rate of mobile phase 0.46-0.60mL/min.
Preferably, in some embodiments of the present invention, flow rate of mobile phase 0.53mL/min.
Further, in some embodiments of the present invention, sampling volume is 1-3 μ L.
Preferably, in some embodiments of the present invention, sampling volume is 2 μ L.
Further, in some embodiments of the present invention, Detection wavelength 215-225nm.
Preferably, in some embodiments of the present invention, Detection wavelength 220nm.
Further, in some embodiments of the present invention, using cyano column as chromatographic column.
Preferably, in some embodiments of the present invention, chromatographic column is Waters Acquity H-Class CYANO
Chromatographic column：1.8μm 3.0*100mm.
Further, in some embodiments of the present invention, column temperature is 34-36 DEG C.
Preferably, in some embodiments of the present invention, column temperature is 35 DEG C.
Further, in some embodiments of the present invention, FUFANG LIXUEPING PIAN dissolves out prepare liquid by the following method
It obtains：
FUFANG LIXUEPING PIAN sample is dissolved with dissolution medium, obtains FUFANG LIXUEPING PIAN dissolution prepare liquid；
Wherein, the dissolution medium is water, hydrochloric acid solution, acetate buffer solution or phosphate buffer solution.
Further, in some embodiments of the present invention, in 7 Smart digestion instruments of SOTAX AT, dissolution is added
Medium and FUFANG LIXUEPING PIAN, control rotating speed are 50 revs/min, take solution 10mL to filter respectively at different time points, and in time
Identical medium 10mL is supplemented in process container, takes subsequent filtrate, and prepare liquid is dissolved out as FUFANG LIXUEPING PIAN.
The setting of different time points can be selected according to actual conditions, such as can be 5,10,15,20,30,45min etc..
Further, in some embodiments of the present invention, hydrochloric acid solution pH is 1.9-2.1, acetate buffer solution
PH is 4.4-4.6, and phosphate buffer solution pH is 6.7-6.9.
Preferably, in some embodiments of the present invention, hydrochloric acid solution pH is 2.0, and acetate buffer solution pH is
4.5, phosphate buffer solution pH are 6.8.
In short, provided by the invention measure 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
Method for example carries out gradient elution by using suitable chromatographic condition with the mobile phase being made of mobile phase A and Mobile phase B；
Mobile phase A is the aqueous solution containing hexane sulfonate, wherein hexane sulfonate concentration is 0.05%-0.1%, pH 2.5-
3.0；Mobile phase B is the mixed solution of methanol and acetonitrile, and the volume ratio of methanol and acetonitrile in Mobile phase B is 4.5-5.5:1；It can
With effectively by vitamin B1, vitamin B6, Hydrochioro, dihydralazine sulfate, the promethazine hydrochloride 5 in FUFANG LIXUEPING PIAN
Kind component detaches appearance, and theoretical cam curve and separating degree meet the requirements, and separating effect is apparent；In conjunction with reference substance, that is, vitamin
The concentration and peak area of B6, vitamin B1, dihydralazine sulfate, Hydrochioro and promethazine hydrochloride, can be surveyed by external standard method
The dissolution rate of 5 kinds of components in FUFANG LIXUEPING PIAN is obtained, and then obtains stripping curve, in addition, this method has simple, quick, spirit
The advantages such as sensitivity is high, selectivity is good.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 be medium be pH2.0 hydrochloric acid solution when FUFANG LIXUEPING PIAN dissolution prepare liquid dissolution curve.
Fig. 2 be medium be pH2.0 hydrochloric acid solution when FUFANG LIXUEPING PIAN dissolution prepare liquid dissolution sample chromatogram.
Fig. 3 be medium be pH4.5 acetate buffer solution when FUFANG LIXUEPING PIAN dissolution prepare liquid dissolution curve.
Fig. 4 be medium be pH4.5 acetate buffer solution when FUFANG LIXUEPING PIAN dissolution prepare liquid dissolution sample color
Spectrogram.
Fig. 5 be medium be pH6.8 phosphate buffer solution when FUFANG LIXUEPING PIAN dissolution prepare liquid dissolution curve.
Fig. 6 be medium be pH6.8 phosphate buffer solution when FUFANG LIXUEPING PIAN dissolution prepare liquid dissolution sample color
Spectrogram.
The dissolution curve for FUFANG LIXUEPING PIAN dissolution prepare liquid that Fig. 7 is medium when being water.
The dissolution sample chromatogram figure for FUFANG LIXUEPING PIAN dissolution prepare liquid that Fig. 8 is medium when being water.
Fig. 9 is Detection wavelength when being 215nm in FUFANG LIXUEPING PIAN dissolution prepare liquid vitamin B1, vitamin B6, hydrogen chlorine
The chromatographic fractionation figure of thiazine, dihydralazine sulfate, promethazine hydrochloride.
Figure 10 is vitamin B1, vitamin B6, Hydrochioro, sulphur in 6 FUFANG LIXUEPING PIAN of embodiment dissolution prepare liquid
The chromatographic fractionation figure of sour Dihydralazine, promethazine hydrochloride.
Vitamin B1, vitamin B6, the hydrogen that Figure 11 is Detection wavelength when being 225nm in FUFANG LIXUEPING PIAN dissolution prepare liquid
The chromatographic fractionation figure of chlorothiazide, dihydralazine sulfate, promethazine hydrochloride.
Vitamin B1, vitamin B6, the esodrix that Figure 12 is chromatographic column when being C18 in FUFANG LIXUEPING PIAN dissolution prepare liquid
The chromatographic fractionation figure of piperazine, dihydralazine sulfate, promethazine hydrochloride.
Figure 13 is flow velocity when being 0.60mL/min in FUFANG LIXUEPING PIAN dissolution prepare liquid vitamin B1, vitamin B6,
The chromatographic fractionation figure of Hydrochioro, dihydralazine sulfate, promethazine hydrochloride.
Figure 14 is flow velocity when being 0.46mL/min in FUFANG LIXUEPING PIAN dissolution prepare liquid vitamin B1, vitamin B6,
The chromatographic fractionation figure of Hydrochioro, dihydralazine sulfate, promethazine hydrochloride.
Vitamin B1, vitamin B6, the esodrix that Figure 15 is sample size when being 1 μ L in FUFANG LIXUEPING PIAN dissolution prepare liquid
The chromatographic fractionation figure of piperazine, dihydralazine sulfate, promethazine hydrochloride.
Vitamin B1, vitamin B6, the esodrix that Figure 16 is sample size when being 3 μ L in FUFANG LIXUEPING PIAN dissolution prepare liquid
The chromatographic fractionation figure of piperazine, dihydralazine sulfate, promethazine hydrochloride.
Vitamin B1, the vitamin that Figure 17 is Mobile phase B initial proportion when being 2% in FUFANG LIXUEPING PIAN dissolution prepare liquid
The chromatographic fractionation figure of B6, Hydrochioro, dihydralazine sulfate, promethazine hydrochloride.
Figure 18 is Mobile phase B initial proportion when being 10% in FUFANG LIXUEPING PIAN dissolution prepare liquid vitamin B1, dimension life
The chromatographic fractionation figure of plain B6, Hydrochioro, dihydralazine sulfate, promethazine hydrochloride.
Figure 19 be when being eluted using isocratic elution mode FUFANG LIXUEPING PIAN dissolution prepare liquid in vitamin B1, dimension
The chromatographic fractionation figure of raw element B6, Hydrochioro, dihydralazine sulfate, promethazine hydrochloride.
Figure 20 be the vitamin B1 of reference substance solution in embodiment 1, vitamin B6, Hydrochioro, dihydralazine sulfate,
The chromatographic fractionation figure of promethazine hydrochloride.
Specific implementation mode
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The side provided in this embodiment that 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN are measured with ultra-performance liquid chromatography
Method is as follows：
(1) precision weighs vitamin B1 reference substance, vitamin B6 reference substance, Hydrochioro reference substance, promethazine hydrochloride pair
Appropriate according to product, dihydralazine sulfate reference substance, it is respectively vitamin B1 pair to ultimately join corresponding dissolution medium and be diluted to concentration
According to 1 μ g/mL of product, 1 μ g/mL of vitamin B6 reference substance, 3.1 μ g/mL of Hydrochioro reference substance, 2.1 μ g/ of promethazine hydrochloride reference substance
The mixed solution of mL, 4.2 μ g/mL of dihydralazine sulfate reference substance, product solution, is examined with Ultra Performance Liquid Chromatography instrument as a contrast
It surveys, Figure 20 is as a result seen, the results show that the retention time of VB6 is 2.027, the retention time of VB1 is 2.412, dihydralazine sulfate
Retention time be 2.645, the retention time of Hydrochioro is 3.242, the retention time of promethazine hydrochloride is 4.803.
(2) compound profit Blood piece sample (manufacturer is taken：Changzhou Pharmaceutical Factory Co., Ltd, every weight 129mg, every contains：
0.032mg reserpines, 3.1mg Hydrochioros, 4.2mg dihydralazine sulfates, 2.1mg promethazine hydrochlorides, 1.0mg vitamin B1s,
1.0mg vitamin B6s), using the 7 Smart digestion instruments of SOTAX AT of SOTAX group companies of Switzerland, dissolution medium is added
Hydrochloric acid solution (0.01mol/L) 900ml of pH2.0, dissolves out 37 DEG C of cup temperature, 50 revs/min of rotating speed, respectively at 5,10,15,
It takes within 20,30,45 minutes solution 10mL to filter respectively, and supplements identical dissolution medium 10mL in process container in time, take continuous
Filtrate is detected as test sample with Ultra Performance Liquid Chromatography instrument.The dissolution rate at sample each time point is measured with external standard method, and is painted
Dissolution profiles processed.Parallel determination 12 times, results are averaged.
Wherein, the chromatographic condition detected with Ultra Performance Liquid Chromatography instrument is as follows：
Chromatographic column：Waters Acquity H-Class CYANO：1.8μm 3.0*100mm.
Column temperature：35℃.
Detection wavelength：220nm.
Sample size：2μL.
Mobile phase：Mobile phase A：0.07% sodium hexanesulfonate (with phosphorus acid for adjusting pH value to 2.75)；Mobile phase B：Methanol：
Acetonitrile (5:1).
Flow velocity：0.53mL/min.
Type of elution：Gradient elution.
Gradient elution program：
| Time (min) | Mobile phase A (%V/V) | Mobile phase B (%V/V) |
| 0-0.3 | 94 | 6 |
| 0.3-3 | 94-40 | 6-60 |
| 3-4.7 | 40 | 60 |
| 4.7-5.0 | 40-94 | 60-6 |
| 5.0-6.8 | 94 | 6 |
I.e.：
It is 94% in the volume ratio of 0min, mobile phase A, the volume ratio of Mobile phase B is 6%；
It is 94% in the volume ratio of 0.3min, mobile phase A, the volume ratio of Mobile phase B is 6%；
It is 40% in the volume ratio of 3min, mobile phase A, the volume ratio of Mobile phase B is 60%；
It is 40% in the volume ratio of 4.7min, mobile phase A, the volume ratio of Mobile phase B is 60%；
It is 94% in the volume ratio of 5min, mobile phase A, the volume ratio of Mobile phase B is 6%；
It is 94% in the volume ratio of 6.8min, mobile phase A, the volume ratio of Mobile phase B is 6%.
The result is shown in Figure 1 and Fig. 2.
In the dissolution medium of the hydrochloric acid solution of pH2.0, each active component (vitamin B1, vitamin B6, Hydrochioro,
Dihydralazine sulfate, promethazine hydrochloride) peak shape it is good (see Fig. 2), can accurate quantitative analysis, each active component after 45min
Dissolution rate in 85% or more (Fig. 1).
Embodiment 2
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, the difference is that, the sodium acetate buffer that the dissolution medium used in the present embodiment is pH4.5.
As a result see Fig. 3 and Fig. 4.
In the dissolution medium of the sodium acetate buffer of pH4.5, each active component (vitamin B1, vitamin B6, hydrogen chlorine
Thiazine, dihydralazine sulfate, promethazine hydrochloride) peak shape it is good (Fig. 4), can accurate quantitative analysis, each activearm after 45min
The dissolution rate divided is in 85% or more (Fig. 3).
Embodiment 3
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, the difference is that, the buffer solution of sodium phosphate that the dissolution medium used in the present embodiment is pH6.8.
As a result see Fig. 5 and Fig. 6.
In the dissolution medium of the buffer solution of sodium phosphate of pH6.8, each active component (vitamin B1, vitamin B6, hydrogen chlorine
Thiazine, dihydralazine sulfate, promethazine hydrochloride) peak shape it is good (Fig. 6), can accurate quantitative analysis, each activearm after 45min
The dissolution rate divided is in 85% or more (Fig. 5).
Embodiment 4
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, unlike, the dissolution medium used in the present embodiment is water.
As a result see Fig. 7 and Fig. 8.
In the dissolution medium of water, each active component (vitamin B1, vitamin B6, Hydrochioro, dihydralazine sulfate,
Promethazine hydrochloride) peak shape it is good (Fig. 8), can accurate quantitative analysis, after 45min the dissolution rate of each active component 85% with
Upper (Fig. 7).
Embodiment 5
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, unlike：
Detection wavelength is 215nm.
Obtained chromatogram is as shown in Figure 9, it is seen then that 5 peaks is obtained in Fig. 9,5 components are totally separated in solution, are passed through
Spectrogram shown in Fig. 9 is further analyzed, result shown in table 2 is obtained, it is seen then that with this condition, each peak can be effective
Separation, theoretical cam curve and separating degree are shown in Table 1 and meet the requirements.
The correlated characteristic at each peak in 1 Fig. 9 of table
Embodiment 6
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is same as Example 1, the difference is that sample repeats once.
Obtained chromatogram is as shown in Figure 10, it is seen then that 5 peaks are obtained in Figure 10,5 substances are totally separated in solution, point
Good from spending, with this condition, the analysis that a sample can be completed of 6.8 minutes used times, analysis time is short.
By spectrogram shown in Fig. 10 is further analyzed, result shown in table 2 is obtained, it is seen then that with this condition,
Each peak can efficiently separate, and theoretical cam curve and separating degree meet the requirements.Under other conditions unanimous circumstances, wavelength
After 215nm and 220nm comparisons, theoretical cam curve and separating degree are suitable, but 215nm belongs to lower end and absorbs, so excellent
It is 220nm to select wavelength.
The correlated characteristic at each peak in 2 Figure 10 of table
Embodiment 7
The present embodiment 1 with ultra-performance liquid chromatography measure FUFANG LIXUEPING PIAN in 5 kinds of component dissolution rates method with
Embodiment 5 is essentially identical, unlike：
Detection wavelength is 225nm.
Obtained chromatogram is as shown in figure 11, it is seen then that 5 peaks are obtained in Figure 11,5 substances are totally separated in solution, point
Good from spending, with this condition, the analysis that a sample can be completed of 6.8 minutes used times, analysis time is short.
By spectrogram shown in Figure 11 is further analyzed, result shown in table 3 is obtained, it is seen then that with this condition,
Each peak can efficiently separate, and theoretical cam curve and separating degree meet the requirements.Under other conditions unanimous circumstances, wavelength
After 225nm and 220nm comparisons, under the conditions of 220nm, more preferably, it is advantageous to wavelength to be for theoretical cam curve and separating degree
220nm。
The correlated characteristic at each peak in 3 Figure 11 of table
Experimental example 1
This experimental example 1 with ultra-performance liquid chromatography measure FUFANG LIXUEPING PIAN in 5 kinds of component dissolution rates method with
Embodiment 1 is essentially identical, unlike：
Chromatographic column：Waters Acquity BEH C18,1.8 μm of 3.0*100mm.
Obtained chromatogram is as shown in figure 12, it is seen then that 4 peaks are obtained in Figure 12,5 substances are not totally separated in solution,
Vitamin B1 and dihydralazine sulfate fail to detach.
By spectrogram shown in Figure 12 is further analyzed, result shown in table 4 is obtained, it is seen then that with this condition,
Each peak fails to be kept completely separate, and does not meet system requirements.It is Waters Acquity H-Class CYANO it is advantageous to chromatographic column
1.8um 3.0*100mm。
The correlated characteristic at each peak in 4 Figure 12 of table
Embodiment 8
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, unlike：
Flow velocity：0.60mL/min.
Obtained chromatogram is as shown in figure 13, it is seen then that 5 peaks are obtained in Figure 13,5 substances are totally separated in solution, point
It is good from spending.
By spectrogram shown in Figure 13 is further analyzed, result shown in table 5 is obtained, it is seen then that with this condition,
Each peak can efficiently separate, and theoretical cam curve and separating degree meet the requirements.Under other conditions unanimous circumstances, flow velocity is
0.53mL/min and 0.60mL/min comparison after, under the conditions of 0.53mL/min, theoretical cam curve and separating degree more preferably, so
Preferable flow rate is 0.53mL/min.
The correlated characteristic at each peak in 5 Figure 13 of table
Embodiment 9
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 7, unlike：
Flow velocity：0.46mL/min.
Obtained chromatogram is as shown in figure 14, it is seen then that 5 peaks are obtained in Figure 14,5 substances are totally separated in solution, point
It is good from spending.
By spectrogram shown in Figure 14 is further analyzed, result shown in table 6 is obtained, it is seen then that with this condition,
Each peak can efficiently separate, and theoretical cam curve and separating degree meet the requirements.Under other conditions unanimous circumstances, flow velocity is
0.53mL/min and 0.46mL/min comparison after, under the conditions of 0.53mL/min, theoretical cam curve and separating degree more preferably, so
Preferable flow rate is 0.53mL/min.
The correlated characteristic at each peak in 6 Figure 14 of table
Embodiment 10
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, unlike：
Sample size：1μL.
The correlated characteristic at each peak in 7 Figure 15 of table
Obtained chromatogram is as shown in figure 15, obtains result shown in table 7, it is seen then that with this condition, each peak can be effective
Separation, theoretical cam curve and separating degree meet the requirements.Under other conditions unanimous circumstances, sample size is 1 μ L and sample size
After 2 μ L comparisons, the peak area that sample size is 1 μ L is the half for the peak area that sample size is 2 μ L, is not suitable for the smaller feelings of dissolution rate
Quantifying under condition is 2 μ L it is advantageous to sample size.
Embodiment 11
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, unlike：
Sample size：3μL.
Obtained chromatogram is as shown in figure 16, obtains result shown in table 8, it is seen then that with this condition, each peak can be effective
Separation, the theoretical cam curve of vitamin B6 is relatively low, and separating degree meets the requirements.Under other conditions unanimous circumstances, sample size 3
μ L and sample size are after 2 μ L are compared, and under the conditions of sample size is 2 μ L, theoretical cam curve higher is 2 μ L it is advantageous to sample size.
Each peak correlated characteristic in 8 Figure 16 of table
Embodiment 12
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, unlike：
The initial proportion of what Mobile phase B of mobile phase A is 98:2.
The program of gradient elution is as follows：
The correlated characteristic at each peak in 9 Figure 17 of table
Obtained chromatogram is as shown in figure 17, obtains the correlated characteristic at each peak result as shown in table 9, it is seen then that at this
Each peak can efficiently separate under part, and theoretical cam curve and separating degree meet the requirements.Under other conditions unanimous circumstances, flowing
After phase B original ratios are 2% and Mobile phase B original ratio is 6% comparison, under conditions of Mobile phase B original ratio is 6%,
Separating degree higher is 6% it is advantageous to Mobile phase B original ratio.
Embodiment 13
The method provided in this embodiment for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
It is substantially the same manner as Example 1, unlike：
The initial proportion of what Mobile phase B of mobile phase A is 90:10.
The program of gradient elution is as follows：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0-0.3 | 90 | 10 |
| 0.3-3.0 | 90-40 | 10-60 |
| 3.0-4.7 | 40 | 60 |
| 4.7-5.0 | 40-94 | 60-6 |
| 5.0-6.8 | 94 | 6 |
The correlated characteristic at each peak in 10 Figure 18 of table
Obtained chromatogram is as shown in figure 18, obtains result shown in table 10, it is seen then that with this condition, each peak can have
The theoretical cam curve of effect separation, vitamin B6 and vitamin B1 is relatively low, and separating degree meets the requirements.In other conditions unanimous circumstances
Under, mobile phase original ratio is 10% and mobile phase original ratio is the item for being 10% in mobile phase original ratio after 6% comparison
Under part, separating degree higher is 6% it is advantageous to mobile phase original ratio.
Experimental example 2
Chromatography detection, vitamin B1, dimension are carried out by 1 identical method of embodiment, but using extremely big day hair RC8MD digestion instruments
Raw element B6, Hydrochioro and promethazine hydrochloride：Dissolved corrosion is good in 4 dissolution mediums.
Dihydralazine sulfate：In four media, wild effect is shown, dissolution rate is small, unstable in the medium, goes out
Existing degradation impurity.Through inquiry, Chinese Pharmacopoeia, FDA and Japan's dissolution database do not find dihydralazine sulfate folk prescription solid system
The dissolution assay method of agent, in order to evaluate the dissolved corrosion of the component, we continue to have done following research：
The reason of degradation with generation small for evaluation dihydralazine sulfate dissolution rate, the instrument condition of dissolution is had studied, is analyzed
The material of dissolution instrument is the agitating paddle of glass and stainless steel, and the material of glass is relatively stable, and stainless steel
Agitating paddle may have an impact the dissolved corrosion of dihydralazine sulfate, then replace agitating paddle, and trial is stirred with glass bar and carried out
The acetate buffer solution 900mL of pH4.5 is placed in stripping rotor by contrast test, is put into for reagent, is simulated with glass bar
Stirring took solution 10mL to filter respectively at the 5th, 10,15,20,30,45 minute, and supplemented in process container immediately identical
Solution 10mL, takes subsequent filtrate, as test solution.Through UPLC dissolution determinations, the dissolution rate of test sample increases, and more
Stablize.Explanation：The material of extremely big day hair RC8MD has an impact this component measurement.
Experimental example 3
The method for measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography that this experimental example provides
It is substantially the same manner as Example 1, unlike：
Gradient elution is not used, using isocratic elution, the ratio of mobile phase A and Mobile phase B is 40:60.
The program of isocratic elution is as follows：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0-6.8 | 40 | 60 |
Obtained chromatogram is as shown in figure 19, and table 11 is the correlated characteristic at each peak in Figure 19.
The correlated characteristic at each peak in 11 Figure 19 of table
Obtained chromatogram is as shown in figure 19, it is seen then that 3 peaks are obtained in Figure 19,5 substances are not totally separated in solution,
Vitamin B1, vitamin B6 and dihydralazine sulfate fail to detach.
By spectrogram shown in Figure 19 is further analyzed, result shown in table 11 is obtained, it is seen then that in this condition
Under, each peak fails to be kept completely separate, and does not meet system requirements.It is gradient elution it is advantageous to mobile phase.
The result shows that, method provided in an embodiment of the present invention can be by vitamin B1, vitamin based on the above embodiments
5 kinds of B6, Hydrochioro, dihydralazine sulfate, promethazine hydrochloride component good separations, and energy accurate quantitative analysis, to calculate compound
The dissolution rate of each active component in Reserpine In Tablets solves the unstable detection problem of dihydralazine sulfate, to compound reserpine
The quality evaluation of piece drug has a very important significance.
To sum up, provided by the invention to measure 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography
Method, detection FUFANG LIXUEPING PIAN 5 kinds of main component concentration in process in leaching while can be quick, accurate and highly sensitive
Variation tendency and its content, solve dihydralazine sulfate not at the problem of improving Traditional liquid phase chromatographic sample long operational time
Stable detection problem；
The run time of each sample was changed to 6.8 minutes by 25 minutes, was that observation FUFANG LIXUEPING PIAN exists because dissolution measures
The variation tendency of 5 kinds of main component concentration in process in leaching, sample size are big (such as if parallel determination 12 times, it is necessary to 12 *
6 time point *, 4 leaching condition=288 samples), quickly analysis also saves previous one to time-consuming dissolution determination
Time makes dissolution measurement become simple and quick.
In short, this method have it is simple, quickly, the advantages such as high sensitivity, selectivity be good.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method measuring 5 kinds of component dissolution rates in FUFANG LIXUEPING PIAN with ultra-performance liquid chromatography, which is characterized in that
5 kinds of components are respectively：Vitamin B1, vitamin B6, Hydrochioro, dihydralazine sulfate, promethazine hydrochloride；
Described method includes following steps：
It takes FUFANG LIXUEPING PIAN to dissolve out prepare liquid, gradient elution is carried out with the mobile phase being made of mobile phase A and Mobile phase B；
Mobile phase A is the aqueous solution containing hexane sulfonate, wherein hexane sulfonate concentration is that 0.05%-0.1%, pH are
2.5-3.0；
Mobile phase B is the mixed solution of methanol and acetonitrile, and the volume ratio of methanol and acetonitrile in Mobile phase B is 4.5-5.5:1.
2. according to the method described in claim 1, it is characterized in that, the program of gradient elution is as follows：
。
3. according to the method described in claim 2, it is characterized in that, the program of gradient elution is as follows：
。
4. according to the method described in claim 1, it is characterized in that, flow rate of mobile phase is 0.46-0.60mL/min.
5. according to the method described in claim 1, it is characterized in that, sampling volume is 1-3 μ L.
6. according to the method described in claim 1, it is characterized in that, Detection wavelength is 215-225nm.
7. according to the method described in claim 1, it is characterized in that, using cyano column as chromatographic column.
8. according to the method described in claim 1, it is characterized in that, column temperature is 34-36 DEG C.
9. according to claim 1-8 any one of them methods, which is characterized in that FUFANG LIXUEPING PIAN dissolution prepare liquid passes through such as
Lower method obtains：
FUFANG LIXUEPING PIAN sample is dissolved with dissolution medium, obtains FUFANG LIXUEPING PIAN dissolution prepare liquid；
Wherein, the dissolution medium is water, hydrochloric acid solution, acetate buffer solution or phosphate buffer solution.
10. according to the method described in claim 9, it is characterized in that, hydrochloric acid solution pH be 1.9-2.1, acetate buffer solution
PH is 4.4-4.6, and phosphate buffer solution pH is 6.7-6.9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109580842A (en) * | 2019-01-17 | 2019-04-05 | 武汉生物化学制药有限公司 | A kind of measuring method of compound gallbladder ammonia tablet dissolution |
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| CN111912925A (en) * | 2020-09-02 | 2020-11-10 | 亚宝药业集团股份有限公司 | Method for measuring contents of various components in compound reserpine tablet |
| CN114660213B (en) * | 2022-04-25 | 2024-03-08 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Component content determination method of compound reserpine hydrochlorothiazide tablet |
| CN116148373A (en) * | 2022-11-08 | 2023-05-23 | 地奥集团成都药业股份有限公司 | Pediatric benorilate vitamin B 1 Method for measuring dissolution rate of particles |
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