CN106706832A - Method for determining content of trospium chloride - Google Patents
Method for determining content of trospium chloride Download PDFInfo
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- CN106706832A CN106706832A CN201510477440.0A CN201510477440A CN106706832A CN 106706832 A CN106706832 A CN 106706832A CN 201510477440 A CN201510477440 A CN 201510477440A CN 106706832 A CN106706832 A CN 106706832A
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Abstract
The invention discloses a method for determining the content of trospium chloride. The method adopts ultra-high performance liquid chromatography for determining the content of trospium chloride and comprises the following steps: carrying out ultra-high performance liquid chromatography detection of a prepared control sample solution and a prepared to-be-detected sample solution, to obtain ultra-high performance liquid chromatograms; and calculating the obtained chromatograms by an external standard method with a peak area, taking the ultra-high performance liquid chromatogram of the control sample solution obtained in the step 2) as a control, and calculating the content of trospium chloride in the to-be-detected sample. The method can prolong the service life of a chromatographic column, also can avoid the damage of high pH to trospium chloride, and makes the detection result accurate; and moreover, the method can separate impurity peaks from trospium chloride well. In addition, the method has high sensitivity and low determination cost.
Description
Technical field
The invention belongs to medicine detection field, it is related to a kind of method for determining trospium chloride content.
Background technology
Trospium chloride is a kind of muscarinic receptor antagonist, and the effect by blockage of acetylcholine to M-ChR is entered
And influence to mediate the organ of nerve impulse, including bladder by acetylcholine.The parasympathetic nerve tissue effect of trospium chloride
Reduce the muscular tone in bladder.Trospium chloride be treatment with urge incontinence, urgent urination, frequent micturition bladder excessive
The fiest-tire medication of movable disease (overactive bladder, OAB), has god compared with other anti-muscarinic receptor medicines
Through the advantage of system Small side effects, its structural formula is as follows：
The report of the current analysis method on trospium chloride raw material and related preparations is few, wherein former on trospium chloride
It is analyzing purity to expect that the assay method of content is used.Titration measuring content is time-consuming more long, it is necessary to 10-20 minutes,
In addition when preparation of traditional Chinese medicine assay, it is impossible to exclude the interference of auxiliary material, and when solution concentration is relatively low,
Electrode potential accuracy of measurement is limited when sensitivity is by low concentration.
At present, liquid chromatogram is one of most active analysis method in modern chromatographic techniques.In recent years, with science skill
The progress and industrial development of art, requirement of each field to liquid chromatography technology are increasingly improved.First it is with productivity
Continue to develop, the analysis of batch samples needs to complete in a short period of time, next to that in biological sample and natural product
In the analysis of thing sample, the complexity of sample proposes requirement higher etc. to separating power.Therefore, one kind is developed
Liquid chromatography technology-Ultra Performance Liquid Chromatography (Ultra Performance Liquid based on little particle filler
Chromatography, UPLC) and it is applied successfully to each analysis industry.Yet there are no using UPLC to analyze now
The relevant report of the content of trospium chloride raw material and preparation.
The content of the invention
It is an object of the invention to provide a kind of method for determining trospium chloride content.
The present invention provides a kind of method for determining trospium chloride content, and methods described is to be surveyed using ultra-performance liquid chromatography
Determine the content of trospium chloride, comprise the following steps：
1) trospium chloride control sample solution and testing sample solution are prepared respectively；
2) by step 1) the control sample solution for preparing and the testing sample solution carry out ultra high efficiency liquid phase
Chromatogram detection, obtains Ultra Performance Liquid Chromatography figure；
3) by gained chromatogram using external standard method with calculated by peak area, with step 2) obtained by control sample solution
Ultra Performance Liquid Chromatography figure calculates the content of trospium chloride in the testing sample as control, wherein,
The testing sample is the material containing trospium chloride.
Step 2) described in detect condition be：Chromatographic column used is reverse-phase chromatographic column, more preferably anti-phase C18
Or C8 chromatographic columns；
Mobile phase is the mixed liquor being made up of phosphate aqueous solution and acetonitrile of pH value 2.0~7.0；Wherein, the phosphoric acid water
The concentration of solution is 0.01mol/L~0.06mol/L, and the phosphate aqueous solution is (65~85) with the volume ratio of acetonitrile：
(15~35)；
The flow velocity of mobile phase used is 0.4ml/min~0.8ml/min；
Detection wavelength is 210nm~220nm.
In an embodiment of the present invention, the pH value is 2.0,3.0,5.0,7.0, and the pH value is preferably 3.0~5.0.
Preferably, the volume ratio of the phosphate aqueous solution and acetonitrile is (65~70)：(30~35) or (70~85)：(15~30),
It is further preferred that the phosphate aqueous solution is 65 with the volume parts ratio of acetonitrile：35 or 70：30 or 85：15.
In an embodiment of the present invention, the flow velocity of the mobile phase is 0.4ml/min, 0.5ml/min, 0.6ml/min
Or 0.8ml/min.
In an embodiment of the present invention, the Detection wavelength concretely 210nm, 215nm, 220nm；Preferably,
The column temperature of the chromatographic column be 30~50 DEG C, it is further preferred that the column temperature of the chromatographic column be 30 DEG C, 40 DEG C, 35 DEG C,
50℃。
In an embodiment of the present invention, the reverse-phase chromatographic column is Waters UPLC BEH Shield RP18, Waters
UPLC BEH C18、Waters XBridge BEH Shield RP18、Agilent ZORBAX RRHD SB-C18
Or Waters UPLC BEH C8；Preferably, the particle diameter of filler is 1.7 μm~2.5 μm in the reverse-phase chromatographic column,
The column length of chromatographic column is 50mm~100mm, and aperture is 2.1mm~4.6mm.
In an embodiment of the present invention, methods described sample size is 1 μ l.
In an embodiment of the present invention, the concentration range of the control sample solution is 10.10 μ g/ml~100.98 μ g/ml.
The trospium chloride control sample solution go out peak position for 0.627min, 0.628min or
0.627min-0.628min。
The method that the present invention obtains trospium chloride content in testing sample with calculated by peak area using external standard method, its principle is
Concentration based on trospium chloride control sample solution detects that trospium chloride is corresponding in gained spectrogram with Ultra Performance Liquid Chromatography figure
The peak area at peak is linear, therefore the concentration, trospium chloride in known trospium chloride control sample solution correspond to peak
In peak area and testing sample on the premise of the peak area at same position correspondence peak, you can calculated by the linear relationship
Obtain the content of trospium chloride in testing sample.
In an embodiment of the present invention, the material containing trospium chloride is trospium chloride bulk drug and various trospium chlorides
Preparation；Preferably, the trospium chloride preparation is trospium chloride double release capsule, trospium chloride spansule, Qu Si
Oronain piece or trospium chloride sustained release tablets.
In an embodiment of the present invention, detector used is Waters ACQUITY UPLC H-Class Bio ultra high efficiencies
Liquid chromatograph.
A kind of method of measure trospium chloride content each condition determination that the present invention is provided is optimized, with
Lower technique effect：
1st, extension chromatographic column service life, accuracy are high：In liquid technology, service life and the use of chromatographic column are flowed
The pH of dynamic phase is directly related, and pH is lower, and the damage to chromatographic column is bigger, simultaneously because trospium chloride is sensitive to alkali,
Degraded is susceptible to when pH is higher, its assay is influenceed.Therefore in the methods of the invention, applicant is to mobile phase
PH is groped, and mobile phase pH finally is defined as into 2~7, preferably 3.0~5.0, can not only extend making for chromatographic column
With the life-span, destructions of the pH high to trospium chloride can be also avoided, and the method rate of recovery is between 98%~102%,
Method accuracy is higher.
2nd, cost is substantially reduced：Solvent usage amount of the invention is only 0.4~0.8ml mobile phases, and solvent usage amount is little；
Meanwhile, by can be seen that the inventive method can determine a sample in 1min in chromatogram, save detection time.
3rd, separating degree is high and sensitivity is greatly enhanced：The method that the present invention is provided, can be by impurity peaks and trospium chloride
Separate well, especially principal component reaches more than 4.1 with the separating degree of adjacent nearest impurity peaks, it is to avoid due to
Main peak overlaps the systematic error for causing area repeatability poor and bringing with impurity peaks, and system suitability is good；While this hair
The quantitative limit of bright method reaches 0.15ng, and sensitivity is greatly enhanced.
4th, linearly more than 0.99 is reached：The inventive method determines the scope of trospium chloride concentration in 10.10 μ g/ml-100.98
During μ g/ml, concentration is presented good linear relation with peak area, and correlation coefficient r can reach more than 0.99.
5th, repeatability and Intermediate precision are preferable：The repeatability and Intermediate precision of the inventive method are preferable, RSD values
Respectively less than 2%, the result of the test deviation for measuring is small.
6th, it is wide using scope：Because the release time of trospium chloride sustained release preparation is long, is determined using conventional method and discharged
That spends is time-consuming more long, and because drug release determination method is identical with content assaying method, therefore the present invention not only can be surveyed accurately
Determine the trospium chloride content in trospium chloride raw material and the various preparations of trospium chloride, it is also possible to fast and accurately determine bent
The release of oronain is taken charge of, is that the exploitation and drug release rate monitoring of slow releasing pharmaceutical formulation and technology greatly save the time.
Brief description of the drawings
Fig. 1 is the reverse-phase chromatography detection spectrogram of blank solvent in embodiment 1.
Fig. 2 is the reverse-phase chromatography detection spectrogram of blank auxiliary in embodiment 1.
Fig. 3 is the reverse-phase chromatography detection spectrogram of trospium chloride reference substance in embodiment 1.
Fig. 4 is the μ g/ml of A 0.5, the μ g/ml of impurity B 2.5, the system adaptation of the μ g/ml of trospium chloride 1.5 in embodiment 1
Property solution reverse-phase chromatography detection spectrogram.
Fig. 5 is the reverse-phase chromatography detection spectrogram of trospium chloride quantitative limit.
Fig. 6 is three reverse-phase chromatography detection spectrograms of the double release capsule test samples of the trospium chloride of lot number in embodiment 1.
Fig. 7 is the reverse-phase chromatography detection spectrogram of trospium chloride spansule test sample in embodiment 2.
Fig. 8 is the reverse-phase chromatography detection spectrogram of trospium chloride piece test sample in embodiment 3.
Fig. 9 is the reverse-phase chromatography detection spectrogram of trospium chloride raw material test sample in embodiment 4.
Figure 10 is the reverse-phase chromatography detection spectrogram of blank solvent in embodiment 5.
Figure 11 is the reverse-phase chromatography detection spectrogram of blank auxiliary in embodiment 5.
Figure 12 is the reverse-phase chromatography detection spectrogram of trospium chloride reference substance in embodiment 5.
Figure 13 is the reverse-phase chromatography detection spectrogram of trospium chloride sustained release tablets test sample in embodiment 5.
Specific embodiment
With reference to specific embodiment, the present invention is further elaborated, but the present invention is not limited to following examples.Institute
State method and be conventional method unless otherwise instructed.The raw material can be obtained from open commercial sources unless otherwise instructed
.
Reagent in following examples, unless stated otherwise, is chromatographically pure, and can from regular distributor available from：
Wherein trospium chloride reference substance, purchased from lark prestige；Impurity A (diphenylglycollic acid) is purchased from lark prestige；Impurity B (two
Mandelic acid tropane) it is purchased from lark prestige.
Embodiment 1：Trospium chloride content method checking in the double release capsules of external standard method trospium chloride
First, chromatographic condition：
Instrument：Waters ACQUITY UPLC H-Class Bio Ultra Performance Liquid Chromatography instruments；
Chromatographic column：Waters UPLC BEH Shield RP18,1.7 μm, 2.1 × 50mm；
Mobile phase：PH value be 3.0 by 0.03mol/L phosphate aqueous solutions (with triethylamine adjust pH value be 3.0) and
Acetonitrile with volume ratio be 65：35 mixed liquors being obtained by mixing；
The flow velocity of mobile phase：0.6ml/min；Column temperature：35℃；Detection wavelength：215nm；Sample size is 1 μ l.
2nd, experimental procedure：
1st, specificity
Take respectively the blank solvent (water) of the double release capsules of trospium chloride, blank auxiliary solution (see CN104739808A,
Embodiment 1), trospium chloride reference substance solution (50 μ g/ml) each 1 μ l successively with above-mentioned chromatographic condition detect, with determine
Blank solvent goes out peak position with blank auxiliary.
Acquired results are empty shown in Fig. 2 as shown in figure 1, blank solvent has solvent peak to produce between 0.2~0.4min
The non-appearance of white auxiliary material, shown in Fig. 3, trospium chloride has absworption peak at 0.627min, and blank solvent and blank auxiliary exist
The assay of this product is not disturbed without absorption at this.
2nd, system suitability
Precision weighs impurity A, impurity B and trospium chloride reference substance, with water as solvent, impurity A is configured to respectively
0.5 μ g/ml, the μ g/ml of impurity B 2.5, the system suitability solution of the μ g/ml of trospium chloride 1.5.
Acquired results as shown in figure 4, impurity B reaches 4.10 in 0.562min appearances with trospium chloride separating degree,
Impurity A reaches 5.49 in 0.871min appearances with trospium chloride separating degree, and impurity does not influence the content of trospium chloride
Determine, system suitability is good.
3rd, sample introduction precision
The trospium chloride reference substance solution of 50 μ g/ml is taken, precision measures 1 μ l injection liquid chromatographs, records chromatogram,
Repeat sample introduction 6 times, calculate the RSD values of peak area.The results are shown in Table 1.
Table 1, sample introduction precision result
Conclusion：RSD is 0.26%, and sample introduction precision is good.
4th, linear relationship and scope
Compound concentration is the trospium chloride reference substance solution of 10.10,30.29,50.49,70.69 and 100.98 μ g/ml,
Chromatographic determination is carried out respectively.With reference substance concentration as abscissa, with peak area as ordinate, linear regression processing is carried out,
Calculate regression equation and coefficient correlation.The results are shown in Table 2.
Table 2, linear test result
Conclusion：When to show sample size be 1 μ l, trospium chloride in the concentration range of 10.10 μ g/ml~100.98 μ g/ml,
Its concentration is in good linear relation with peak area.
5th, recovery test
Trospium chloride reference substance storing solution is prepared, the double release capsule prescription ratios of trospium chloride are added
The blank auxiliary of (CN2015100790283, embodiment 1), prepares the solution that concentration is 40,50,60 μ g/ml,
Each concentration is parallel to prepare three parts, shakes up, and filters, and precision measures the μ l of subsequent filtrate 1 respectively, injects liquid chromatograph.
The another trospium chloride reference substance that takes is appropriate, accurately weighed, is made in every 1ml containing about the μ g of trospium chloride 50 with water dissolves and dilution
Solution, as contrast solution.By external standard method with the calculated by peak area rate of recovery.The results are shown in Table 3.
Table 3, recovery test result
Conclusion：Between 98.0%~102.0%, average recovery rate is 100.8% to average recovery rate under each concentration,
RSD is 0.74%, and the rate of recovery is good.
6th, replica test
The preparation of reference substance solution：Trospium chloride reference substance 12.5mg is taken, accurately weighed, it is 50 μ g/ml to be prepared into concentration
Reference substance solution.
The preparation of need testing solution：The double release capsules 20 of trospium chloride are taken, content is finely ground, and precision weighs suitable
Amount (being approximately equivalent to trospium chloride 12.5mg), is prepared into the need testing solution that concentration is 50 μ g/ml, parallel preparation 6
Part.
Precision measures reference substance solution and each 1 μ l of need testing solution, injects liquid chromatograph, chromatogram is recorded, by outer
Mark method is with the content of each part sample of calculated by peak area.The results are shown in Table 4.
Table 4, replica test result
Conclusion：RSD is 0.81%, and repeatability is good.
7th, Intermediate precision experiment
In different check data, using different analytical instrument, different testing crew, according to step 6 " repeatability
Experiment " method, by external standard method with the content of each part sample of calculated by peak area, and calculates the relative standard deviation of content
(RSD),.The results are shown in Table 5.
Table 5, Intermediate precision result of the test
Conclusion：RSD is 0.81%, as a result shows that this product assay method Intermediate precision is good.
8th, quantitative limit
Take trospium chloride reference substance appropriate, it is accurately weighed, it is dissolved in water and stepwise dilution, precision measures the μ l of solution 1,
Injection liquid chromatograph, records chromatogram.As quantitative limit when main peak peak height is about 10 times of baseline noise in chromatogram
Concentration.It is computed, quantifying for this product is limited to 0.15ng.See Fig. 5.
6 parts of solution of quantitative limit concentration are separately prepared, the relative standard deviation for surveying 6 parts of peak areas of solution main peak is less than
2.0%.The results are shown in Table 6.
Table 6, quantitative limit result of the test (n=6)
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | Average | RSD (%) |
| Peak area | 459 | 450 | 465 | 472 | 475 | 462 | 464 | 1.95 |
| Retention time (min) | 0.677 | 0.678 | 0.678 | 0.677 | 0.678 | 0.677 | 0.678 | 0.08 |
Above content assaying method the result shows：This method specificity is strong, reproducible, the degree of accuracy is high, quantitative
Limit is low.
Test agent carries out assay in 3 batches using the method to production, and specification is 60mg, and lot number is：
201407002、201407003、201408004。
The double release capsules 20 of trospium chloride are taken, it is accurately weighed, calculate average loading amount.Content is taken, is well mixed,
Finely ground, precision is weighed and (is approximately equivalent to trospium chloride 12.5mg) in right amount, prepares the solution that concentration is 50 μ g/ml, essence
It is close to measure 1 μ l injection liquid chromatographs, record chromatogram；The another trospium chloride reference substance that takes is appropriate, accurately weighed, plus
Water is made the solution containing 50 μ g in every 1ml, is measured in the same method.By external standard method with calculated by peak area, obtain final product.Determine knot
Fruit is shown in Table 7 and Fig. 6.
Table 7, pilot scale sample size measurement result
| Sample lot number | 201407002 | 201407003 | 201408004 |
| Content (%) | 98.3 | 99.9 | 99.2 |
Conclusion：Three batches of sample sizes are qualified.
Embodiment 2：Trospium chloride content in external standard method trospium chloride spansule
First, chromatographic condition：
Instrument：Waters ACQUITY H-Class Bio Ultra Performance Liquid Chromatography instruments；
Chromatographic column：Waters UPLC BEH C18,1.7 μm, 2.1 × 100mm；
Mobile phase：PH value be 7.0 by 0.03mol/L phosphate aqueous solutions (with triethylamine adjust pH value be 7.0) and
Acetonitrile with volume ratio be 85：15 mixed liquors being obtained by mixing；
The flow velocity of mobile phase：0.8ml/min；Column temperature：30℃；Detection wavelength：220nm；Sample size is 1 μ l.
2nd, experimental procedure：
1st, the rate of recovery
Prepare trospium chloride reference substance storing solution, add trospium chloride spansule prescription ratio (CN103690506B,
Embodiment 3) blank auxiliary, prepare concentration be 40,50,60 μ g/ml solution, each concentration it is parallel prepare three
Part, shake up, filter, precision measures the μ l of subsequent filtrate 1 respectively, injects liquid chromatograph.Separately take trospium chloride reference substance
In right amount, it is accurately weighed, with water dissolves and the solution being made in every 1ml containing about the μ g of trospium chloride 50 is diluted, as right
According to solution.By external standard method with the calculated by peak area rate of recovery.The results are shown in Table 8.
The recovery test result of table 8
Conclusion：Between 98.0%~102.0%, average recovery rate is 99.7% to average recovery rate under each concentration,
RSD is 0.94%, and the rate of recovery is good.
2nd, replica test
The preparation of reference substance solution：Trospium chloride reference substance 12.5mg is taken, accurately weighed, it is 50 μ g/ml to be prepared into concentration
Reference substance solution.
The preparation of need testing solution：Trospium chloride spansule 20 is taken, content is finely ground, and precision is weighed in right amount
(being approximately equivalent to trospium chloride 12.5mg), is prepared into the need testing solution that concentration is 50 μ g/ml, 6 parts of parallel preparation.
Precision measures reference substance solution and each 1 μ l of need testing solution, injects liquid chromatograph, chromatogram is recorded, by outer
Mark method is with the content of each part sample of calculated by peak area.The results are shown in Table 9.
The replica test result of table 9
Conclusion：RSD is 0.48%, and repeatability is good.
3rd, assay
The preparation of reference substance solution：Trospium chloride reference substance 12.5mg is taken, the reference substance that concentration is 50 μ g/ml is prepared molten
Liquid.
The preparation of need testing solution：Trospium chloride spansule 20 is taken, content is finely ground, and precision is weighed in right amount
(being approximately equivalent to trospium chloride 12.5mg), prepares the need testing solution that concentration is 50 μ g/ml.
Precision measures reference substance solution and each 1 μ l of need testing solution, injects liquid chromatograph, chromatogram is recorded, by outer
Mark method is obtained final product with the content of each part sample of calculated by peak area.Control is double sample crosspointers with sample in table 10, with
Product prepare two parts, it is ensured that collimation, duplicate detection 2 times, the results are shown in Table 10 and Fig. 7.
Trospium chloride content results in table 10, the trospium chloride spansule of embodiment 2
As seen from table, the content of trospium chloride is qualified in the trospium chloride spansule for being measured using the chromatographic condition.
Embodiment 3：Trospium chloride content in external standard method trospium chloride piece
First, chromatographic condition：
Instrument：Waters ACQUITY H-Class Bio Ultra Performance Liquid Chromatography instruments；
Chromatographic column：Waters XBridge BEH Shield RP18,2.5 μm, 4.6 × 75mm；
Mobile phase：PH value be 2.0 by 0.01mol/L phosphate aqueous solutions (with triethylamine adjust pH value be 2.0) and
Acetonitrile with volume ratio be 65：35 mixed liquors being obtained by mixing；
The flow velocity of mobile phase：0.4ml/min；Column temperature：35℃；Detection wavelength：210nm；Sample size is 1 μ l.
2nd, experimental procedure：
1st, the rate of recovery
Prepare trospium chloride reference substance storing solution, add trospium chloride tablet recipe ratio (CN201510363089.2,
Embodiment 2) blank auxiliary, prepare concentration be 40,50,60 μ g/ml solution, each concentration it is parallel prepare three
Part, shake up, filter, precision measures the μ l of subsequent filtrate 1 respectively, injects liquid chromatograph.Separately take trospium chloride reference substance
In right amount, it is accurately weighed, with water dissolves and the solution being made in every 1ml containing about the μ g of trospium chloride 50 is diluted, as right
According to solution.By external standard method with the calculated by peak area rate of recovery.The results are shown in Table 11.
The recovery test result of table 11
Conclusion：Between 98.0%~102.0%, average recovery rate is 100.2% to average recovery rate under each concentration,
RSD is 0.83%, and the rate of recovery is good.
2nd, replica test
The preparation of reference substance solution：Trospium chloride reference substance 12.5mg is taken, accurately weighed, it is 50 μ g/ml to be prepared into concentration
Reference substance solution.
The preparation of need testing solution：Trospium chloride piece 20 is taken, finely ground, precision is weighed and (is approximately equivalent to bent department in right amount
Oronain 12.5mg), it is prepared into the need testing solution that concentration is 50 μ g/ml, 6 parts of parallel preparation.
Precision measures reference substance solution and each 1 μ l of need testing solution, injects liquid chromatograph, chromatogram is recorded, by outer
Mark method is with the content of each part sample of calculated by peak area.The results are shown in Table 12.
The replica test result of table 12
Conclusion：RSD is 0.53%, and repeatability is good.
3rd, linear relationship and scope
Prepare trospium chloride reference substance solution with reference to the method for embodiment 1 carries out chromatographic determination respectively, and according to the method described above
Regression equation and coefficient correlation are calculated, regression equation is y=3350x-1139, and correlation coefficient r is 0.9998.
Result shows sample size when being 1 μ l, trospium chloride in the concentration range of 10.00 μ g/ml~100.88 μ g/ml,
Its concentration is in good linear relation with peak area.
4th, assay
With embodiment 2,13 and Fig. 8 is the results are shown in Table.
Trospium chloride content results in table 13, the trospium chloride piece of embodiment 3
As seen from table, the content of trospium chloride is qualified in the trospium chloride piece for being measured using the chromatographic condition.
Embodiment 4：Trospium chloride content in external standard method trospium chloride raw material
First, chromatographic condition：
Instrument：Waters ACQUITY H-Class Bio Ultra Performance Liquid Chromatography instruments；
Chromatographic column：Agilent ZORBAX RRHD SB-C18,1.8 μm, 2.1 × 50mm
Mobile phase：PH value be 3.0 by 0.03mol/L phosphate aqueous solutions (with triethylamine adjust pH value be 3.0) and
Acetonitrile with volume ratio be 70：30 mixed liquors being obtained by mixing；
The flow velocity of mobile phase：0.5ml/min；Column temperature：50℃；Detection wavelength：215nm；Sample size is 1 μ l.
2nd, experimental procedure：
1st, replica test
The preparation of reference substance solution：Trospium chloride reference substance 12.5mg is taken, the reference substance that concentration is 50 μ g/ml is prepared molten
Liquid.
The preparation of need testing solution：Trospium chloride raw material 12.5mg (purchased from Yangzijiang Pharmaceutical Group Co., Ltd) is taken,
Prepare the need testing solution that concentration is 50 μ g/ml, 6 parts of parallel preparation.
Precision measures reference substance solution and each 1 μ l of need testing solution, injects liquid chromatograph, chromatogram is recorded, by outer
Mark method the results are shown in Table 14 with the content of each part sample of calculated by peak area.
Table 14, replica test result
Conclusion：RSD is 0.05%, and repeatability is good.
2nd, Intermediate precision experiment
In different check data, using different analytical instrument, different testing crew, according to " repeating for step 1
Property experiment " method, by external standard method with the content of each part sample of calculated by peak area, and calculate the relative standard deviation of content
(RSD).The results are shown in Table 15.
Table 15, Intermediate precision result of the test
Conclusion：RSD is 0.06%, as a result shows that this product assay method Intermediate precision is good.
3rd, quantitative limit
With reference to the detection method of the quantitative limit of embodiment 1, determining for trospium chloride reference substance under the present embodiment chromatographic condition is drawn
Amount is limited to 0.149ng.
4th, assay
Precision measures reference substance solution and each 1 μ l of need testing solution, injects liquid chromatograph, chromatogram is recorded, by outer
Mark method is obtained final product with the content of each part sample of calculated by peak area.Control is double sample crosspointers with sample in table 16, with
Product prepare two parts, it is ensured that collimation, duplicate detection 2 times, the results are shown in Table 16 and Fig. 9.
Table 16, the trospium chloride material content measurement result of embodiment 4
As seen from table, the content of trospium chloride is qualified in the trospium chloride raw material for being measured using the chromatographic condition.
Embodiment 5：Trospium chloride content in external standard method trospium chloride sustained release tablets
First, chromatographic condition：
Instrument：Waters ACQUITY H-Class Bio Ultra Performance Liquid Chromatography instruments；
Chromatographic column：Waters UPLC BEH C8,1.7 μm, 2.1 × 50mm；
Mobile phase：PH value be 5.0 by 0.06mol/L phosphate aqueous solutions (with triethylamine adjust pH value be 5.0) and
Acetonitrile with volume ratio be 70：30 mixed liquors being obtained by mixing；
The flow velocity of mobile phase：0.5ml/min；Column temperature：40℃；Detection wavelength：215nm；Sample size is 1 μ l.
2nd, experimental procedure：
1st, specificity
Embodiment 1 according to patent CN101596170A prepares trospium chloride sustained release tablets, blank auxiliary () respectively,
Blank solvent, blank auxiliary solution, trospium chloride reference substance solution (50 μ g/ml) each 1 μ l are taken successively with above-mentioned chromatogram
Condition detected, peak position is gone out with determine blank solvent and blank auxiliary.Shown in Figure 10, blank solvent is in 0.2~0.4min
Between there is solvent peak to produce, shown in Figure 11, blank auxiliary have between 0.2~0.5min auxiliary material peak produce, Tu12Suo
Show, trospium chloride has absworption peak at 0.628min, blank solvent and blank auxiliary are not disturbed in this place without absorption
The assay of this product.
2nd, replica test
Take trospium chloride reference substance appropriate, it is accurately weighed, add water and be made the contrast solution containing 50 μ g in every 1ml；Take
Trospium chloride sustained release tablets 20, accurately weighed, finely ground, precision is weighed and (is approximately equivalent to trospium chloride 12.5mg) in right amount,
Prepare the solution that concentration is 50 μ g/ml, 6 parts of parallel preparation.
Precision measures reference substance solution and each 1 μ l of need testing solution, injects liquid chromatograph, chromatogram is recorded, by outer
Mark method is obtained final product with the content of each part sample of calculated by peak area.The results are shown in Table 17.
Table 17, replica test result
Conclusion：RSD is 0.71%, and repeatability is good.
3rd, Intermediate precision experiment
In different check data, using different analytical instrument, different testing crew the, according to " weight of above step 2
Renaturation is tested " method, by external standard method with the content of each part sample of calculated by peak area, and calculate the relative standard deviation of content
(RSD).The results are shown in Table 18.
Table 18, Intermediate precision result of the test
Conclusion：RSD is 0.98%, as a result shows that this product assay method Intermediate precision is good.
4th, recovery test
Prepare trospium chloride reference substance storing solution, add trospium chloride sustained-release tablet recipe ratio (CN101596170A,
Embodiment 1) blank auxiliary, prepare concentration be 40,50,60 μ g/ml solution, each concentration it is parallel prepare three
Part, shake up, filter, precision measures the μ l of subsequent filtrate 1 respectively, injects liquid chromatograph.Separately take trospium chloride reference substance
In right amount, it is accurately weighed, with water dissolves and the solution being made in every 1ml containing about the μ g of trospium chloride 50 is diluted, as right
According to solution.By external standard method with calculated by peak area, 19 are the results are shown in Table.
Table 19, recovery test result
Conclusion：Between 98.0%~102.0%, average recovery rate is 100.0% to average recovery rate under each concentration,
RSD is 0.46%, and the rate of recovery is good.
5th, assay
Trospium chloride sustained release tablets 20 are taken, accurately weighed, finely ground, precision is weighed and (is approximately equivalent to trospium chloride in right amount
12.5mg), the solution that concentration is 50 μ g/ml is prepared, precision measures 1 μ l injection liquid chromatographs, records chromatogram；
The another trospium chloride reference substance that takes is appropriate, accurately weighed, adds water and is made the solution containing 50 μ g in every 1ml, is measured in the same method.
By external standard method with calculated by peak area, obtain final product.Control is double sample crosspointers with sample in table 20, and same sample prepares two parts,
Guarantee collimation, duplicate detection 2 times, measurement result is shown in Table 20 and Figure 13.
The assay result of trospium chloride in table 20, the trospium chloride sustained release tablets of embodiment 5
As seen from table, the content of trospium chloride is qualified in the trospium chloride sustained release tablets for being measured using the chromatographic condition.
Claims (10)
1. a kind of method for determining trospium chloride content, methods described is to use ultra-performance liquid chromatography to determine bent department's chlorine
The content of ammonium, comprises the following steps：
1) trospium chloride control sample solution and testing sample solution are prepared respectively；
2) by step 1) the control sample solution for preparing and the testing sample solution carry out ultra high efficiency respectively
Liquid chromatographic detection, obtains Ultra Performance Liquid Chromatography figure；
3) by gained chromatogram using external standard method with calculated by peak area, with step 2) obtained by control sample solution
Ultra Performance Liquid Chromatography figure calculates the content of trospium chloride in the testing sample as control, wherein,
The testing sample is the material containing trospium chloride.
2. method according to claim 1, it is characterised in that step 2) described in the condition that detects be：Institute
It is reverse-phase chromatographic column with chromatographic column, more preferably anti-phase C18 or C8 chromatographic columns；
Mobile phase is the mixed liquor being made up of phosphate aqueous solution and acetonitrile of pH value 2.0~7.0；Wherein,
The concentration of the phosphate aqueous solution is the parts by volume of 0.01mol/L~0.06mol/L, the phosphate aqueous solution and acetonitrile
Number is than being (65~85)：(15~35)；
The flow velocity of mobile phase used is 0.4ml/min~0.8ml/min；
Detection wavelength is 210nm~220nm.
3. method according to claim 2, it is characterised in that the pH value is 3.0~5.0.
4. the method according to any one of claim 2~3, it is characterised in that the phosphate aqueous solution and acetonitrile
Volume ratio be (65~70)：(30~35) or (70~85)：(15~30), it is further preferred that the phosphoric acid water
Solution is 65 with the volume parts ratio of acetonitrile：35 or 70：30 or 85：15.
5. the method according to any one of claim 2~4, it is characterised in that the flow velocity of the mobile phase is 0.4
Ml/min, 0.5ml/min, 0.6ml/min or 0.8ml/min.
6. the method according to any one of claim 2~5, it is characterised in that the Detection wavelength be 210nm,
215nm、220nm；Preferably, the column temperature of the chromatographic column is 30~50 DEG C, it is further preferred that the chromatographic column
Column temperature be 30 DEG C, 35 DEG C, 40 DEG C, 50 DEG C.
7. the method according to any one of claim 2~6, it is characterised in that the reverse-phase chromatographic column is Waters
UPLC BEH Shield RP18、Waters UPLC BEH C18、Waters XBridge BEH Shield RP18、
Agilent ZORBAX RRHD SB-C18 or Waters UPLC BEH C8；Preferably, the reverse-phase chromatographic column
The particle diameter of middle filler is 1.7 μm~2.5 μm, and the column length of chromatographic column is 50mm~100mm, and aperture is 2.1mm~4.6mm.
8. according to any described method in claim 2~7, it is characterised in that sample size is 1 μ l.
9. the method according to any one of claim 2~8, it is characterised in that the control sample solution it is dense
Degree scope is 10.10 μ g/ml~100.98 μ g/ml.
10. the method according to any one of claim 1~9, it is characterised in that the material containing trospium chloride
It is trospium chloride bulk drug and various trospium chloride preparations；Preferably, the trospium chloride preparation is that trospium chloride pair is released
Put capsule, trospium chloride spansule, trospium chloride piece or trospium chloride sustained release tablets.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109884205A (en) * | 2019-03-12 | 2019-06-14 | 康诚科瑞医药研发（武汉）有限公司 | The quantitative detecting method of trospium chloride in a kind of blood plasma |
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Address after: 100176, No. two, No. 36, Hai Lu, Beijing economic and Technological Development Zone, Beijing Applicant after: Staidson (Beijing) Bio-pharmaceuticals Co., Ltd. Applicant after: Beijing Choutet doctor medicine technology Ltd Address before: 100176, No. two, No. 36, Hai Lu, Daxing District economic and Technological Development Zone, Beijing Applicant before: Staidson (Beijing) Bio-pharmaceuticals Co., Ltd. Applicant before: BEIJING STAIDSON NEW DRUG RESEARCH CO., LTD. |
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Application publication date: 20170524 |