CN107976489A - A kind of method of residual pyridine in measure Pregabalin - Google Patents
A kind of method of residual pyridine in measure Pregabalin Download PDFInfo
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- CN107976489A CN107976489A CN201610940235.8A CN201610940235A CN107976489A CN 107976489 A CN107976489 A CN 107976489A CN 201610940235 A CN201610940235 A CN 201610940235A CN 107976489 A CN107976489 A CN 107976489A
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Abstract
The present invention proposes a kind of method for measuring residual pyridine in Pregabalin, and this method includes：Residual pyridine in the Pregabalin is analyzed by HPLC analytical method, to obtain chromatogram；And based on the chromatogram, determine the content of residual pyridine in the Pregabalin, wherein, the high performance liquid chromatography uses the following conditions：Chromatographic column is ZORBAX Eclipse XDB C18,4.6 × 250mm, 5 microns, detector DAD, Detection wavelength is 240nm, column temperature is 33 DEG C~37 DEG C, and mobile phase A is mutually the dipotassium hydrogen phosphate buffer solution of 10mmol/L, and the pH of the dipotassium hydrogen phosphate buffer solution is 6.8~7.2, Mobile phase B is mutually methanol, flow velocity is 0.8mL/min~1.2mL/min, elutes and uses isocratic elution, and the volume ratio of A phases and B phases is 68 in the isocratic elution:32~72:28, run time 20min.Using this method, can content that is easy, accurate, delicately, specificity measuring residual pyridine in Pregabalin bulk pharmaceutical chemicals, so as to effectively control drug effect, quality and the pharmaceutical safety of Pregabalin bulk pharmaceutical chemicals.
Description
Invention field
The present invention relates to biomedicine field, specifically, measuring residual pyridine in Pregabalin the present invention relates to a kind of
Method.
Technical background
Pregabalin is a kind of new γ-aminobutyric acid (GABA) receptor stimulating agent, local for treating neuropathic pain
Ictal insane pain and anxiety disorder.In December, 2004 U.S. Food and Drug Administration ((FDA) approval be used for treat diabetic keratopathy week
Enclose the neuropathic pain and banding spore rash post herpetic neuralgia of neuropathy.Its structural formula isBe white or
The crystalline solid of off-white color, has the characteristic of soluble easily in water, alkaline and acid aqueous solution.
When synthesizing Pregabalin, people can usually select pyridine, and as reaction dissolvent, the residual of pyridine can be to product
Security and validity bring hidden danger, it is therefore desirable to which pyridine residual quantity is controlled.
The boiling point of pyridine is 115.2 DEG C, and the method for generally use GC headspace injection carries out residual pyridine in medicine
Measure.But the dissolubility due to Pregabalin bulk pharmaceutical chemicals in organic solvent is poor, and residual solvent pyridine to be measured
For alkalescent, poor using peak shape during Headspace Analysis, the rate of recovery is also bad.It is general when in addition using ion chromatograph analysis
Auspicious Bahrain's degree of ionization is larger, solvent pyridine peak to be measured bag can be entered, disturb the measure of pyridine.
The content of the invention
It is contemplated that solve at least some of the technical problems in related technologies.For this reason, the present invention
One purpose is in a kind of method for proposing simplicity, accurately, delicately detecting residual pyridine in Pregabalin.
In the first aspect of the present invention, the present invention proposes a kind of method for measuring residual pyridine in Pregabalin.According to
The embodiment of the present invention, the described method includes：By HPLC analytical method to residual pyridine in the Pregabalin
Analyzed, to obtain chromatogram；And based on the chromatogram, determine the content of residual pyridine in the Pregabalin,
Wherein, the high performance liquid chromatography uses the following conditions：Chromatographic column is ZORBAX Eclipse XDB-C18,4.6 × 250mm, 5
Micron, detector DAD, Detection wavelength 240nm, column temperature are 33 DEG C~37 DEG C, and mobile phase A is mutually the phosphoric acid hydrogen of 10mmol/L
Dipotassium buffer solution, the pH of the dipotassium hydrogen phosphate buffer solution are 6.8~7.2, and Mobile phase B is mutually methanol, flow velocity 0.8mL/min
~1.2mL/min, elutes and uses isocratic elution, and the volume ratio of A phases and B phases is 68 in the isocratic elution:32~72:28, fortune
The row time is 20min.Using detection method according to embodiments of the present invention, this method is good with specificity, pyridine and adjacent color
Spectral peak separating degree is more than 1.5；Precision is good, and relative standard deviation is less than 2.0%；Accuracy rate is high, and the rate of recovery is 90%~110%
Between, meet Chinese Pharmacopoeia to residual solvent in bulk pharmaceutical chemicals or the requirement in relation to substance detecting method.The method can accurately determine
Measure the pyridine in Pregabalin bulk pharmaceutical chemicals in the range of 8ppm to 300ppm.
According to an embodiment of the invention, the method for residual pyridine can also be further in said determination Pregabalin bulk pharmaceutical chemicals
Including at least one following additional technical feature：
According to an embodiment of the invention, the pH of the dipotassium hydrogen phosphate buffer solution is 7.0.Inventor in experiments it is found that,
The pH of dipotassium hydrogen phosphate buffer solution is 7.0, and the buffer capacity of buffer salt is stronger, and pyridine peak symmetrical factor is 1.31, and peak shape is preferable.
According to an embodiment of the invention, the flow velocity is 1.0mL/min.Inventor in experiments it is found that, flow velocity is
1.0mL/min, the retention time 6.11min of pyridine in determinand, it is suitable to retain, and pyridine peak is efficiently separated with other peak energy.
According to an embodiment of the invention, the volume ratio 70 of A phases and B phases in the isocratic elution:30.Inventor is in test
It was found that the volume ratio 70 of A phases and B phases in isocratic elution:30, the separating degree of pyridine is 4.57, and separating degree is high, and symmetrical factor is
1.28, peak shape is good, some impurity in Pregabalin are detected, which provides pyridine detection sensitivity good enough.
According to an embodiment of the invention, the column temperature is 35 DEG C.Inventor in experiments it is found that, column temperature be 35 DEG C, pyridine
Peak symmetrical factor 1.33, peak shape is preferable.
According to an embodiment of the invention, the Pregabalin bulk pharmaceutical chemicals are provided in the form of test solution, wherein,
The test solution is dipotassium hydrogen phosphate-methanol solution of Pregabalin bulk pharmaceutical chemicals, and is based on every milliliter of test sample
Solution, the content of Pregabalin bulk pharmaceutical chemicals is 10mg, wherein, the dipotassium hydrogen phosphate-methanol solution is the A phase and B phases
Mixed solution, the volume ratio of the A phases and B phases is 7:3.Inventor is found by experiment that Pregabalin bulk pharmaceutical chemicals are in above-mentioned phosphorus
Solvability is preferable in sour hydrogen dipotassium-methanol solution, meanwhile, the sample introduction concentration of Pregabalin bulk pharmaceutical chemicals can be protected in 10mg/mL
Card pyridine detection sensitivity good enough.
According to an embodiment of the invention, the dosage of the test solution is 20 microlitres, to the measure of pyridine content more
Truly, reliably, accurately.
According to an embodiment of the invention, it is described to be based on the chromatogram, determine pyridine in the Pregabalin bulk pharmaceutical chemicals
Content is determined by the following formula：
Wherein, AiFor the peak area of pyridine in test solution；ASFor pyridine peak area in contrast solution；WTFor test sample
Sample weighting amount, mg；WSTo prepare the quality that contrast solution weighs pyridine, mg；DTFor the extension rate of test sample；DSFor control
The extension rate of solution；The contrast solution is dipotassium hydrogen phosphate-methanol solution of pyridine, and is based on every milliliter of control
Solution, the content of pyridine is 2 micrograms.According to an embodiment of the invention, determined using aforesaid way residual in Pregabalin bulk pharmaceutical chemicals
The content of pyridine is stayed, accuracy rate is high, as a result more true and reliable.
In the second aspect of the present invention, the present invention proposes a kind of method for measuring residual pyridine in Pregabalin.According to
The embodiment of the present invention, the method specifically include：
(1) chromatographic condition
Chromatographic column is ZORBAX Eclipse XDB-C18,4.6 × 250mm, 5 micron,
Detector is DAD,
Detection wavelength is 240nm,
Column temperature is 35 DEG C,
Mobile phase A is mutually the dipotassium hydrogen phosphate cushioning liquid of 10mmol/L, and the pH of the dipotassium hydrogen phosphate cushioning liquid is
7.0
Mobile phase B is mutually methanol,
Flow velocity is 1.0mL/min,
Gradient is isocratic elution, and the volume ratio of A phases and B phases is 70 in isocratic elution:30,
Run time is 20min,
(2) blank solution is prepared
The volume ratio of methanol and A phases is 3:7 mixed solution as the blank solution,
(3) test solution is prepared
Accurately weighed test sample 100mg, into 10mL volumetric flasks, is dissolved with blank solution, and is ultrasonically treated, and is diluted to
Scale, shakes up, to obtain the final product the test solution,
(4) contrast solution is prepared
Accurately weighed pyridine 20mg, into 100mL volumetric flasks；Dissolved, and be ultrasonically treated with blank solution, be diluted to quarter
Degree, shakes up, up to storing solution is compareed；
Precision pipettes control storing solution 1.0mL into 100mL volumetric flasks, is diluted to scale with blank solution, shakes up, obtains pair
According to solution,
(5) by 20 microlitres of test solution injecting chromatographs, chromatogram is obtained, is calculated according to the chromatogram for examination
The content of pyridine in product solution,
Wherein, the content of pyridine in test solution is calculated as follows, takes the average value of 2 measurement results as survey
Determine result：
In formula：Ai is the peak area of pyridine in test solution；
ASFor continuously into the average value of pyridine peak area in 6 pin contrast solutions；
WTFor the sample weighting amount of test sample, mg；
WSTo prepare the quality that contrast solution weighs pyridine, mg；
DTFor the extension rate of test sample；
DSFor the extension rate of contrast solution.
The dosage of the 1 pin contrast solution is 20 microlitres.
Using detection method according to embodiments of the present invention, can it is easy, accurate, delicately measure in Pregabalin bulk pharmaceutical chemicals
The content of residual pyridine, so as to effectively control the quality of Pregabalin bulk pharmaceutical chemicals.
Brief description of the drawings
Fig. 1 is according to embodiments of the present invention 1 using test sample mark-on solution under the conditions of Intersil ODS-3 chromatographic columns
Chromatogram；
Fig. 2 is using test sample under the conditions of ZORBAX Eclipse XDB-C18 chromatographic columns according to the embodiment of the present invention 1
The chromatogram of mark-on solution；
Fig. 3 is the ultra-violet absorption spectrum of according to embodiments of the present invention 1 pyridine；
Fig. 4 is the ultra-violet absorption spectrum of according to embodiments of the present invention 1 Pregabalin；
Fig. 5 be according to embodiments of the present invention 1 Detection wavelength be 240nm, 245nm, 250nm under the conditions of, test sample mark-on
The chromatogram of solution；
Fig. 6 is the pyridine contrast solution under according to embodiments of the present invention 1 different pH (6.8~7.2) buffer conditions
Chromatogram；
Fig. 7 is that according to embodiments of the present invention 1 mobile phase ratio is K2HPO4:MeOH=70:Test sample under the conditions of 30 adds
Mark the chromatogram of solution.
Specific embodiment mode
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to text in the art
Offer described technology or condition or carried out according to product description.Reagents or instruments used without specified manufacturer,
For can be with conventional products that are commercially available.
Embodiment 1
In the present embodiment, inventor describe in detail measure Pregabalin in residual pyridine method development process with
And system suitability standard selection.
The selection of 1.1 chromatographic columns
Method initial stage of development, inventor is in Detection wavelength 254nm, 35 DEG C of column temperature, mobile phase H2O:MeOH=70:30, etc.
Degree elution 20min, sample size be under 20 microlitres of chromatographic condition investigated Intersil ODS-3 (250*4.6mm, 5 microns),
The chromatographic column of ZORBAX Eclipse XDB-C18 (4.6*250mm, 5 microns) different brands, to general in test sample mark-on solution
The retention time of auspicious Bahrain, the retention time of peak shape and pyridine, peak shape, peak height etc. influence, specific to investigate the result such as institute of table 1
Show, for Fig. 1 to investigate chromatogram obtained by Intersil ODS-3 chromatographic columns, Fig. 2 is investigation ZORBAX Eclipse XDB-C18 colors
Compose chromatogram obtained by column.Test sample mark-on solution is formulated as taking Pregabalin bulk pharmaceutical chemicals about 100mg, accurately weighed to hold to 10mL
In measuring bottle, with contrast solution dissolving and constant volume, shake up to obtain the final product, prepare 1 part, the preparation of contrast solution is the same as 2.1.
Table 1：Test sample mark-on solution investigates result
The results show that using Intersil ODS-3 chromatographies, Pregabalin retention time is 6.15min, general by table 1 and Fig. 1
Occurs negative peak before auspicious Bahrain's main peak, the retention time of pyridine is 9.72min, and pyridine peak height is 9110, and the peak shape of pyridine is trailed.
Table 1 and Fig. 2 are the results show that using ZORBAX Eclipse XDB-C18 chromatographic columns, Pregabalin retention time is
4.67min, occurs negative peak before Pregabalin main peak, the retention time 6.19min of pyridine, the peak height of pyridine is 12425, pyridine
Peak shape is trailed.
Above-mentioned investigation result is contrasted, it can be deduced that, using ZORBAX Eclipse XDB-C18 chromatography column conditions
Peak height compared to the pyridine using Intersil ODS-3 chromatography column conditions is improved, and the sensitivity of its method is more preferable, therefore is selected
ZORBAX Eclipse XDB-C18 chromatographic columns are selected to carry out the exploitation of the analysis method of residual pyridine in follow-up Pregabalin.
1.2 Detection wavelengths determine
According to Fig. 3, the maximal ultraviolet absorption of pyridine in 256nm, absorbed between 235~250nm it is all stronger, it is general
The maximal ultraviolet absorption of auspicious Bahrain is in 196nm, as shown in Figure 4.Under the conditions of 1.1 selected chromatographic columns, inventor is further right
Detection wavelength is investigated, and Detection wavelength is respectively that the results are shown in Figure 5 for the investigation of 240nm, 245nm, 250nm, investigates knot
Fruit shows that Detection wavelength is 240nm, 245nm, there is negative peak, Detection wavelength 240nm, Pregabalin master before Pregabalin main peak
The peak shape at peak is normal, occurs without negative peak, it is contemplated that should ensure that the response of pyridine will also take into account Pregabalin and occur without down enough
Peak, therefore wavelength of the final choice 240nm as detection.
1.3 mobile phases, pH, the selection and optimization of mobile phase ratio
Inventor further investigates mobile phase, and it is mutually water phase to investigate the results show mobile phase A, and symmetrical factor is
1.68, selective flow phase A phase are 10mmol/L dipotassium hydrogen phosphate solution buffer salts, and symmetrical factor 1.50, is mutually with mobile phase A
Water is preferable compared to the peak shape of pyridine, and the reservation enhancing of pyridine on a column, therefore selective flow phase A phases are phosphoric acid hydrogen
Two potassium solution buffer salts.
Inventor further investigates the pH of buffer salt, and the results are shown in Figure 6 for investigation, investigates the results show pH=
6.8, symmetrical factor 1.33；PH=7.0, symmetrical factor 1.31；PH=7.2, symmetrical factor 1.34, its buffer salt delay
It is relatively strong to rush ability, pyridine peak shape is preferable, wherein pH=7.0, and pyridine peak symmetrical factor is 1.31, and peak shape is more preferable, therefore excellent
Select pH=7.0.
Above-mentioned investigation has determined that chromatographic column is ZORBAX Eclipse XDB-C18, Detection wavelength 240nm, pH=7.0's
10mmol/L dipotassium hydrogen phosphates-methanol is mobile phase, and under isocratic elution 20min, inventor is further to the ratio of mobile phase
Investigated, investigated the results show that mobile phase ratio is respectively K2HPO4:MeOH=68:32、K2HPO4:MeOH=70:30、
K2HPO4:MeOH=72:28, Pregabalin and pyridine peak shape are normal, and the separating degree effect of pyridine is preferable.Wherein Fig. 7 is shown
Show, mobile phase K2HPO4:MeOH=70:30, some impurity in Pregabalin are also detected, and the separating degree of pyridine is 4.57,
Symmetrical factor is 1.28, more preferable compared to sensitivity, therefore preferably the condition carries out the investigation of follow-up sensitivity, concrete outcome such as table
Shown in 2.
Table 2：Test sample mark-on solution investigates result
The investigation of 1.4 flow velocitys and column temperature
Condition is had determined that based on above-mentioned, and under the conditions of mobile phase ratio is preferable, further flow velocity is investigated,
When investigation the results show flow velocity is respectively 0.8mL/min, 1.0mL/min, 1.2mL/min, Pregabalin and pyridine peak shape are just
Often, the separating degree effect of pyridine is preferable.When based on flow velocity being 1.0mL/min, the retention time 6.11min of determinand pyridine,
It is more reasonable compared to retaining, and pyridine peak is efficiently separated with other peak energy, and pyridine peak is not disturbed, preferably 1.0mL/min.
Inventor further investigates column temperature for 25 DEG C, 33 DEG C, 35 DEG C, 37 DEG C, and it is 25 DEG C to investigate the results show column temperature, pyridine
Peak symmetrical factor 1.79, peak shape is bad, and column temperature is respectively under the conditions of 33 DEG C, 35 DEG C, 37 DEG C, and peak shape is preferable, and wherein column temperature is
35 DEG C, pyridine peak symmetrical factor 1.33 is more preferable compared to peak shape at other temperature, and be easy to cause chromatographic column in view of column temperature is excessive
The loss of stationary phase, therefore preferably 35 DEG C.
The selection of 1.5 test sample concentrations
Preferable test sample dilution is mobile phase solution, and above-mentioned preferable isocratic elution mobile phase ratio is K2HPO4:
MeOH=70:30, it is preferable to the solvability of test sample, therefore inventor selects methanol/K2HPO4The ratio of buffer solution is 30:70
Mixed solution as test sample dilution.On determining for test sample compound concentration and sample size, inventor is eventually through examination
Test, definite test sample concentration is 10mg/mL, and sample size is 20 microlitres, can ensure pyridine detection sensitivity good enough.
1.6 system suitability standards select
Based on pyridine in ZORBAX Eclipse XDB-C18 retention time 6.15min, symmetrical factor is 1.28 and 3.2
The test result of system suitability, selects separating degree >=1.5 of pyridine and adjacent peak, and the RSD of 6 peak areas of sample introduction≤
10.0%, and the signal-to-noise ratio >=10 of 10% contrast solution is used as system suitability standard.
Embodiment 2
In the present embodiment, describe in detail inventor be how based on determined by embodiment 1 chromatographic condition obtain color
Spectrogram, and the content of residual pyridine in Pregabalin how is calculated using chromatogram.
The preparation of 2.1 related solutions
Mobile phase：
A phases：Weigh about 1.74g anhydrous potassium dihydrogen phosphate, in the weighed beaker to 1L, add the dissolving of 1L ultra-pure waters, mix, use
It is 7.0 that the phosphoric acid solution or 0.1mol/L potassium hydroxide solutions of 0.1mol/L, which adjusts pH value, then with 0.2 μm of water system filter membrane mistake
Filter ultrasound to obtain the final product；
B phases：100% methanol；
Dilution (blank solution)：A phases:B phase=7:3 mixed liquor；
Compare storing solution：Pyridine about 20mg is taken into 100mL volumetric flasks, it is accurately weighed, scale is diluted to diluent, is shaken
It is even to obtain the final product；
Contrast solution：Pipette 1.0mL and compare storing solution into 100mL volumetric flasks, be diluted to scale with diluent, shake up i.e.
；
Sensitivity solution：1.0mL contrast solutions are pipetted into 10mL volumetric flasks, scale is diluted to diluent, shakes up i.e.
；
Test solution：Test sample about 100mg is taken, it is accurately weighed, into 10mL volumetric flasks, quarter is diluted to diluent
Degree, shakes up to obtain the final product；It is parallel to prepare 2 parts.
2.2 chromatographic condition
Chromatographic column：ZORBAX Eclipse XDB-C18,4.6 × 250mm, 5 micron,
Column temperature：35 DEG C,
Detection wavelength：240nm,
Flow velocity：1.0mL/min
Sampling volume：20 microlitres,
Mobile phase is prepared：A phases (0.01mol/L pH=7.0 dipotassium hydrogen phosphate cushioning liquids)：Weigh the anhydrous phosphorus of about 1.74g
Sour hydrogen dipotassium, in the weighed beaker to 1L, adds the dissolving of 1L ultra-pure waters, mixes, with the phosphoric acid solution or 0.1mol/ of 0.1mol/L
It is 7.0 that L potassium hydroxide solutions, which adjust pH value, then with 0.2 micron of water system membrane filtration ultrasound to obtain the final product；
B phases：Methanol,
Isocratic elution：A phases:B phase=70:30.
2.3 coherent detections operate
After baseline balance, under 2.2 chromatographic conditions, blank solution, sensitivity solution, contrast solution, test sample are taken
Solution, by 3 sequence sample introduction of table, records each collection of illustrative plates.
Table 3：Sample introduction sequence
| Solution title | Sample introduction needle number |
| Blank solution | 1 pin |
| 1 part of sensitivity solution | 1 pin |
| 1 part of contrast solution | 6 pins |
| 2 parts of test solution | Each 1 pin |
System suitability requirement：Pyridine peak signal-to-noise ratio is not less than 10 in sensitivity solution；Pyridine in first pin contrast solution
It is not less than 1.5 with the separating degree of adjacent peak；The RSD of 6 pin pyridine peak area of continuous sample introduction must not exceed 10.0%.Wherein system is fitted
With carrying out test sample detection and methodology validation after the sexual satisfaction requirement.
The computational methods of the content of 2.4 residual pyridines based on chromatogram
The content of pyridine in test sample is calculated according to the following formula, testing result is used as using the average value of 2 measurement results：
In formula：AiFor the peak area of pyridine in test solution；
ASFor continuously into the average value of pyridine peak area in 6 pin contrast solutions；
WTFor the sample weighting amount of Pregabalin test sample, mg；
WSTo prepare the quality that contrast solution weighs pyridine, mg；
DTFor the extension rate of test sample；
DSFor the extension rate of contrast solution,
Acceptable standard is pyridine content≤200ppm.
Embodiment 3
In the present embodiment, inventor measures the present invention methodological study of residual pyridine in Pregabalin.
3.1 system suitability
Blank solution, the preparation method of contrast solution are each to prepare 1 part with 2.1.
Operation：Under 2.2 chromatographic conditions, after system balancing, 1 pin of blank solution sample introduction, sensitivity solution sample introduction 1 are taken
Pin, 6 pin of contrast solution sample introduction, records chromatogram.Result of the test show 6 pin pyridine peak area of continuous sample introduction be followed successively by 72298,
74131st, 74240,73774,74888,74759, the RSD of 6 pin pyridine peak area of continuous sample introduction is 1.27%, is met the requirements；Spirit
Pyridine peak signal-to-noise ratio is 32.80 in sensitivity solution, is met the requirements；The separating degree of pyridine and adjacent peak is in the first pin of contrast solution
N/A, based on the peak for there was only pyridine in contrast solution, N/A is infinitely great, is met the requirements.Therefore, system suitability meets detection and wants
Ask, so as to continue the investigation of subsequent processes.
3.2 specificity
Blank solution, control storing solution, contrast solution, the preparation method of test solution are the same as 2.1；
The preparation of 100% test sample mark-on solution：Take test sample about 100mg, in the accurately weighed volumetric flask to 10mL, with pair
According to solution dissolving and constant volume, shake up to obtain the final product, prepare 1 part.
Operation：Under 2.2 chromatographic conditions, blank solution, test solution, contrast solution, test sample mark-on solution are taken
Each 1 pin of sample introduction, and corresponding chromatogram is recorded, specific test result is as shown in table 4.
Table 4：Specificity result
| Solution title | Pyridine retention time (min) | Pyridine peak area | Pyridine separating degree |
| Blank solution | / | / | / |
| Test solution | / | / | / |
| Contrast solution | 6.11 | 74482 | / |
| Test sample mark-on solution | 6.11 | 73139 | 7.24 |
Table 4 is investigated the results show that blank solution is noiseless to pyridine in contrast solution and test solution, is met the requirements；
The separating degree minimum value at pyridine peak and Pregabalin peak is 7.24 in test solution, contrast solution, test sample mark-on solution, symbol
Close and require；The retention time at pyridine peak is consistent with contrast solution in test sample mark-on solution, is 6.11min；Test sample mark-on
Pyridine peak area is 73139 in solution, and compared with test solution, the peak area of pyridine has increase in test sample mark-on solution,
Meet the requirements.Because the method specificity is good.
3.3 precision
The preparation of the repeated solution of analysis：Take test sample about 100mg, it is molten with compareing in the accurately weighed volumetric flask to 10mL
Liquid dissolving and constant volume, shake up to obtain the final product, and parallel to prepare 6 parts, which is prepared by analyzing personnel's first；
The preparation of Intermediate precision solution：Take test sample about 100mg, it is molten with compareing in the accurately weighed volumetric flask to 10mL
Liquid dissolving and constant volume, shake up to obtain the final product, it is parallel prepare 6 parts, by different analysis personnel in the different time in same laboratory
Proceeded as follows using different instruments.
Analyze repetitive operation：Under 2.2 chromatographic conditions, 6 parts of repeated solution of analysis are taken, each 1 pin of sample introduction, according to being
The content of pyridine in the repeated solution of 6 parts of analyses of mean value calculation of peak area is compareed in applicability of uniting.
Intermediate precision operates：Under 2.2 chromatographic conditions, 6 parts of Intermediate precision solution are taken, each 1 pin of sample introduction, according to being
The content of pyridine in 6 parts of Intermediate precision solution of mean value calculation of peak area is compareed in applicability of uniting.
Analyze in the repeated solution of 6 parts of analyses of repetitive test the results show, the RSD values of pyridine content are 0.52%, symbol
Close and require.
Intermediate precision result of the test shows that the RSD values of pyridine content in 6 parts of Intermediate precision solution are 1.24%, at the same time
The RSD of pyridine content is 0.92% in 12 parts of solution of analysis repeatability and Intermediate precision, analysis repeatability and intermediate precision
The absolute difference for spending pyridine content average value is 0.60ppm, is met the requirements.Therefore method precision is good.
3.4 linear relationship
Blank solution, control storing solution, the preparation method of contrast solution are each to prepare 1 part with 2.1；
The preparation of linear storing solution：Precision pipettes control storing solution 25mL into 100mL volumetric flasks, is diluted to diluent
Scale, shakes up to obtain the final product；
The preparation of quantitative limit solution：Contrast solution 4ml is taken to be diluted to scale into 100mL volumetric flasks with diluent, shake up
To obtain the final product.
The preparation of linear solvent：Precision pipettes the linear storing solution or contrast solution of different volumes as shown in table 5 in not
In the volumetric flask of same volume, scale is diluted to diluent, is shaken up, it is each to prepare 1 part up to the linear solvent under each concentration.
Table 5
| Concentration level | 10% | 50% | 100% | 150% |
| Linear storing solution (mL) | / | 1 | 2 | 3 |
| Contrast solution (mL) | 1 | / | / | / |
| Volumetric flask (mL) | 10 | 50 | 50 | 50 |
Operation：Under 2.2 chromatographic conditions, 1 pin of blank solution sample introduction is taken, each concentration level lower linear solution is taken and quantifies
Solution is limited, successively from low concentration to high concentration, each concentration sample introduction 2 times, records chromatogram.Specific test result is shown in Table 6, with pyrrole
Pyridine peak area average value makees its concentration one-variable linear regression, equation of linear regression y=37784.335x-1294.439, phase
Relation number r is 1.000, is met the requirements；The absolute value of the Y intercept of pyridine peak area ratio corresponding with 100% limit concentration
For 1.74%, meet the requirements.This method pyridine is linear good in the range of 0.08156 μ g/mL to 3.0585 μ g/mL.
Table 6：Linear result
3.5 accuracy
Blank solution, the preparation method of test solution are with 2.1, and wherein blank solution prepares 1 part, and test solution is matched somebody with somebody
2 parts of system；
The preparation of linear storing solution：Precision pipettes control storing solution 25mL into 100mL volumetric flasks, is diluted to diluent
Scale, shakes up to obtain the final product, prepares 1 part；
The preparation of accuracy solution：About 500mg test samples are taken, it is accurately weighed, it is placed in 50mL volumetric flasks, it is parallel to weigh 9
Part；After weighing up, 30~40mL diluents are first added into each volumetric flask, sample ultrasonic are dissolved, then according to the form below 7 is distinguished again
The linear storing solution of different volumes is pipetted in above-mentioned 9 volumetric flasks, each concentration prepares 3 parts.Again quarter is diluted to diluent
Degree, shakes up to obtain the final product.
Table 7
| Concentration level | 50% mark-on solution | 100% mark-on solution | 150% mark-on solution |
| Linear storing solution (mL) | 1.0 | 2.0 | 3.0 |
| Volumetric flask (mL) | 50 | 50 | 50 |
Operation：Under 2.2 chromatographic conditions, 1 pin of blank solution sample introduction is taken, takes 2 parts of test solutions respectively into 1 pin, it is each dense
Horizontal lower each 1 pin of sample introduction of accuracy solution of degree, records chromatogram.
Wherein, calculated respectively according to the following formula before mark-on and pyridine content, solvent recovery in test solution after mark-on
Rate：
Theoretical amount=CS×V
In formula：CSConcentration for the linear storing solution of pyridine added in test sample mark-on solution, mg/mL；
V is the volume of the linear storing solution of pyridine added in each test sample mark-on solution, mL；
CS+TFor the pyridine residual quantity measured in each test sample mark-on solution；
CiFor the pyridine residual quantity measured in test solution；
Average value for the pyridine residual quantity measured in test solution；
AiFor pyridine peak area in test solution；
ASFor pyridine average peak area in 2 pin contrast solutions；
AS+TFor the peak area of pyridine in each test sample mark-on solution；
WTFor the sample weighting amount of test sample in test solution, mg；
WSFor the sample weighting amount of pyridine in contrast solution, mg；
WS+TFor the sample weighting amount of test sample in each test sample mark-on solution, mg；
DTFor the extension rate of test solution；
DSFor the extension rate of contrast solution；
DS+TFor the extension rate of test sample in each test sample mark-on solution.
Result of the test is as shown in table 8, and result of the test is shown in the 50% of limit concentration, under 100%, 150%, pyridine recycling
Rate monodrome scope is 91.20%~104.23%, between 90%~110%, is met the requirements；The RSD of pyridine rate of recovery monodrome
For 4.50%, meet the requirements.Therefore this method accuracy is good.
Table 8：Accuracy result
3.6 durabilities are investigated
3.6.1 solution stability testing
The preparation method of contrast solution prepares 1 part with 2.1；
The preparation of 100% test sample mark-on solution：Take test sample about 100mg, in the accurately weighed volumetric flask to 10mL, with pair
According to solution dissolving and constant volume, shake up to obtain the final product, prepare 1 part.
Operation：Under 2.2 chromatographic conditions, contrast solution, 100% test sample mark-on solution is taken to be placed in room temperature, respectively
1 pin of sample introduction when 0h, 7h, 12h, 19h, 24h, 48h and 72h, records chromatogram.At room temperature, in the 72h of investigation, Ge Geshi
Between to put pyridine peak area and 0h peak area ratios scope in contrast solution be 0.94~1.00 (between 0.90~1.10)；
In 100% test sample mark-on solution pyridine peak area and 0h peak area ratios scope for 0.94~0.99 (0.90~1.10 it
Between), meet the requirements.Contrast solution and test solution interior stabilization when 72 is small at room temperature.
3.6.2 chromatographic condition changes experiment
The preparation method of blank solution, sensitivity solution and contrast solution with 2.1, blank solution, sensitivity solution and
Contrast solution respectively prepares 1 part.
Operation：Using the chromatographic condition under 2.2 as basic condition, single factor test change is carried out, is specifically shown in Table 9.Each condition
Under, after baseline balance, each 1 pin of sample introduction of blank solution, sensitivity solution, contrast solution is taken, records chromatogram.
Table 9
Result of the test is as shown in table 10, and result of the test is shown under each chromatographic condition of change, sensitivity solution pyridine peak
Signal-to-noise ratio minimum value is 22.85；The RSD maximums of 3 pin pyridine peak area of continuous sample introduction are 1.15%；In the first pin of contrast solution
Separating degree minimum value >=1.5 of pyridine and adjacent peak, meet the requirements, the good tolerance of this method.It is wherein molten based on compareing
There was only the peak of pyridine in liquid, the separating degree of pyridine and adjacent peak is infinitely great in the first pin of contrast solution.
Table 10：Durability result
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment of the present invention or example.In the present specification, schematic expression of the above terms is not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
Combined in an appropriate manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this area
Art personnel can be tied the different embodiments or example described in this specification and different embodiments or exemplary feature
Close and combine.
Although the embodiment of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (9)
- A kind of 1. method for measuring residual pyridine in Pregabalin, it is characterised in that pass through HPLC analytical method pair Residual pyridine is analyzed in the Pregabalin, to obtain chromatogram；And based on the chromatogram, determine the Puri The content of residual pyridine in Bahrain, wherein, the high performance liquid chromatography uses the following conditions：Chromatographic column is ZORBAX Eclipse XDB-C18,4.6 × 250mm, 5 micron,Detector is DAD,Detection wavelength is 240nm,Column temperature is 33 DEG C~37 DEG C,Mobile phase A is mutually the dipotassium hydrogen phosphate buffer solution of 10mmol/L, and the pH of the dipotassium hydrogen phosphate buffer solution is 6.8~7.2,Mobile phase B is mutually methanol,Flow velocity is 0.8mL/min~1.2mL/min,Gradient is isocratic elution, and the volume ratio of A phases and B phases is 68 in isocratic elution:32~72:28,Run time is 20min.
- 2. according to the method described in claim 1, it is characterized in that, the pH of the dipotassium hydrogen phosphate buffer solution is 7.0.
- 3. according to the method described in claim 1, it is characterized in that, the flow velocity is 1.0mL/min.
- 4. the according to the method described in claim 1, it is characterized in that, volume ratio 70 of A phases and B phases in the isocratic elution:30.
- 5. according to the method described in claim 1, it is characterized in that, the column temperature is 35 DEG C.
- 6. according to the method described in claim 1, it is characterized in that, in the liquid phase chromatography analytical method, the Puri bar Woods bulk pharmaceutical chemicals are provided in the form of test solution, wherein, the test solution is the phosphoric acid of Pregabalin bulk pharmaceutical chemicals Hydrogen dipotassium-methanol solution, and every milliliter of test solution is based on, the content of Pregabalin bulk pharmaceutical chemicals is 10mg, wherein, Dipotassium hydrogen phosphate-the methanol solution is the mixed solution of the A phases and B phases, and the volume ratio of the A phases and B phases is 7:3.
- 7. according to the method described in claim 6, it is characterized in that, the dosage of the test solution is 20 microlitres.
- 8. according to the method described in claim 1, it is characterized in that, described be based on the chromatogram, the Pregabalin is determined Middle residual pyridine is determined by the following formula：Wherein：AiFor the peak area of pyridine in test solution；ASFor pyridine peak area in contrast solution；WTFor the sample weighting amount of test sample, mg；WSTo prepare the quality that contrast solution weighs pyridine, mg；DTFor the extension rate of test sample；DSFor the extension rate of contrast solution；The contrast solution is dipotassium hydrogen phosphate-methanol solution of pyridine, and is based on every milliliter of contrast solution, pyridine Content is 2 micrograms.
- A kind of 9. method for measuring residual pyridine in Pregabalin, it is characterised in that including：(1) chromatographic conditionChromatographic column is ZORBAX Eclipse XDB-C18,4.6 × 250mm, 5 micron,Detector is DAD,Detection wavelength is 240nm,Column temperature is 35 DEG C,Mobile phase A is mutually the dipotassium hydrogen phosphate cushioning liquid of 10mmol/L, and the pH of the dipotassium hydrogen phosphate cushioning liquid is 7.0,Mobile phase B is mutually methanol,Flow velocity is 1.0mL/min,Gradient is isocratic elution, and the volume ratio of A phases and B phases is 70 in isocratic elution:30,Run time is 20min,(2) blank solution is preparedThe volume ratio of methanol and A phases is 3:7 mixed solution as the blank solution,(3) test solution is preparedAccurately weighed test sample 100mg, into 10mL volumetric flasks, is dissolved with blank solution, and is ultrasonically treated, and is diluted to quarter Degree, shakes up, to obtain the final product the test solution,(4) contrast solution is preparedAccurately weighed pyridine 20mg, into 100mL volumetric flasks, is dissolved with blank solution, and is ultrasonically treated, and is diluted to scale, Shake up, up to storing solution is compareed；Precision pipettes control storing solution 1.0mL into 100mL volumetric flasks, is diluted to scale with blank solution, shakes up, must compare molten Liquid,(5) by 20 microlitres of test solution injecting chromatographs, chromatogram is obtained, it is molten that test sample is calculated according to the chromatogram The content of pyridine in liquid,Wherein, the content of pyridine in test solution is calculated as follows, takes the average value of 2 measurement results to be tied as measure Fruit：Wherein：AiFor the peak area of pyridine in test solution；ASFor continuously into the average value of pyridine peak area in 6 pin contrast solutions；WTFor the sample weighting amount of test sample, mg；WSTo prepare the quality that contrast solution weighs pyridine, mg；DTFor the extension rate of test sample；DSFor the extension rate of contrast solution；The dosage of the 1 pin contrast solution is 20 microlitres.
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| CN113740471B (en) * | 2021-11-08 | 2022-02-11 | 南京威凯尔生物医药科技有限公司 | Method for detecting milobalin and enantiomer impurities thereof by high performance liquid chromatography |
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