CN102565205B - Quality detection method of methyhaaltrexone bromide - Google Patents
Quality detection method of methyhaaltrexone bromide Download PDFInfo
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Abstract
The invention provides a quality detection method of methyhaaltrexone bromide, belonging to the technical field of chemical drug analysis. The quality detection method of methyhaaltrexone bromide provided by the invention adopts high performance liquid chromatography, and is suitable for inspections and content determinations of relevant substances in a synthesis process of methyhaaltrexone bromide. The quality detection method of the relevant substances of methyhaaltrexone bromide provided by the invention realizes effective monitoring of impurities introduced in the synthesis process and degradation products of methyhaaltrexone bromide, and has high accuracy and strong specificity.
Description
Technical field
The present invention relates to a kind of methylnaltrexone bromide method of quality control, belong to chemicals analysis technical field.
Background technology
Methylnaltrexone bromide (methyl naltrexone, MNTX) be a kind of selectivity mu opioid receptor antagonists, as a kind of quaternary ammonium salt, limited methylnaltrexone bromide by the ability of blood-brain barrier, make methylnaltrexone bromide as a kind of peripheral action opiate receptor antagonist, as act in stomach intestinal tissue, therefore reduced the constipation effect of opioid drug, do not affect simultaneously opioid drug to central nervous system analgesic activity.
Methylnaltrexone bromide has following characteristics: 1) acting on peripheral mu acceptor, do not activate maincenter opiate receptor, may be competitive antagonism; 2) safety, series of experiments does not all find that it has serious toxic and side effect, common adverse reactions is stomachache, diarrhoea, flatulence and dizzy; 3) effect is fast, several minutes can onset; 4) treatment constipation is than the logical purgatives of stool and softening agent good effect; 5) action pathway is not unique, can be directly on intestines and stomach opiate receptor be combined onset, also can be combined with opiate receptor through the blood whole body that distributes, therefore, methylnaltrexone bromide not only can be treated the gastrointestinal side-effect of opioid drug, also can treat the outer bad reaction of stomach and intestine; 6) not by blood-brain barrier, do not weaken the Central Analgesic Effect of opiate.
The compound name of methylnaltrexone bromide is called: bromination-17-(cyclopropyl methyl)-4,5 α-epoxy-3, and 14-dihydroxy-17-methyl-6-oxo morphinan, its structural formula is as follows:
Chinese patent 200680022957.X discloses a kind of high-performance liquid chromatogram determination method and condition determination of methylnaltrexone bromide, the method and condition are mainly used for the mensuration of S isomeride, for inspection and the assay of the related substance (being impurity) of methylnaltrexone bromide, also there is no at present disclosed bibliographical information.
Summary of the invention
The quality determining method that the object of this invention is to provide a kind of methylnaltrexone bromide product.Methylnaltrexone bromide quality determining method of the present invention adopts high performance liquid chromatography (HPLC), is applicable to inspection and the assay of the related substance in methylnaltrexone bromide synthesis process.And quality determining method according to the present invention has been formulated the quality control standard of methylnaltrexone bromide.
Detection method of the present invention can realize effective monitoring to methylnaltrexone bromide related substance (comprising impurity, methylnaltrexone bromide catabolite and the unreacted raw material introduced in building-up process), and precision of method is high, specificity is strong.L.o.i is strictly controlled to the security of using in order to improve medicine by medicine registration management way and other relevant governing principle.
High-efficient liquid phase chromatogram condition of the present invention is: with octadecylsilane chemically bonded silica, be filling agent; Take 0.1-0.2% trifluoroacetic acid solution-methyl alcohol (volume ratio 70-80: 20-30) be mobile phase; Detection wavelength is 230nm.
Above-mentioned HPLC detection method, the concentration of trifluoroacetic acid solution used is preferably 0.15%; In mobile phase, the volume ratio of trifluoroacetic acid solution and methyl alcohol is preferably 78: 22.
Above-mentioned HPLC detection method, flow velocity is preferably 1mL/min, and column temperature is generally 30 ℃.
The further concrete assay method of high effective liquid chromatography for measuring methylnaltrexone bromide related substance of the present invention is as follows::
Get testing sample, add the need testing solution that mobile phase is formulated as 1-2mg/ml;
Get need testing solution and dilute 100 times of solution 1 in contrast;
Take Naltrexone Hydrochloride reference substance appropriate, add solution that mobile phase makes 0.004-0.008mg/ml solution 2 in contrast;
High performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2005) is measured: with octadecylsilane chemically bonded silica, be filling agent, 0.15% trifluoroacetic acid solution-the methyl alcohol (volume ratio 78: 22) of take is mobile phase, detection wavelength is 230nm, and number of theoretical plate calculates and is not less than 3000 by methylnaltrexone bromide peak;
Measure need testing solution and contrast solution 1,2, injection liquid chromatography detects respectively;
The content standard of methylnaltrexone bromide related substance is: in need testing solution chromatogram, if any impurity peaks, the amount of known impurities Naltrexone Hydrochloride must not surpass 0.4% of sample; Single unknown impuritie must not surpass 0.1% of sample; Each impurity summation must not surpass 1% of sample.
The present invention measure methylnaltrexone bromide content in testing sample high performance liquid chromatography concrete chromatographic condition as mentioned above, being formulated as follows of test sample and contrast solution:
Get testing sample, add the need testing solution that mobile phase is formulated as 1-2mg/ml;
Take methylnaltrexone bromide sterling (reference substance), add solution that mobile phase is formulated as 1-2mg/ml product solution in contrast.
Measure need testing solution and reference substance solution, injection liquid chromatography detects respectively, by external standard method, calculates content.
The present invention has determined the detection method of related substance in methylnaltrexone bromide product, and has carried out detailed methodology checking.Methodology the result shows, the method can effectively detect the mandatory down hydrolysis products of synthetic initiation material, synthesising by-product and methylnaltrexone bromide, thereby impurity and catabolite thereof that building-up process is introduced all can be realized effective monitoring, and precision of method is high, specificity is strong.L.o.i is strictly controlled to the security of using in order to improve medicine by medicine registration management way and other relevant governing principle.
In addition, the method that the present invention measures methylnaltrexone bromide content to HPLC method has also been carried out methodology checking, and the result shows that the specificity of set up method is strong, and precision is good, and measured result accurately and reliably, can be used for the assay of methylnaltrexone bromide.
Accompanying drawing explanation
Fig. 1 is when to X-1 lot number, within 0 month, sample carries out the detection of methylnaltrexone bromide related substance, the HPLC collection of illustrative plates of need testing solution;
Fig. 2 is when to X-1 lot number, within 0 month, sample carries out the detection of methylnaltrexone bromide related substance, the HPLC collection of illustrative plates of contrast solution 1 (100 times of need testing solution dilutions).
Embodiment
One, the preparation of methylnaltrexone bromide testing sample
Methylnaltrexone bromide testing sample can be prepared by various synthetic methods, includes but not limited to following method:
Under nitrogen protection; in there-necked flask, add 11.6g (34.0mmol) naltrexone alkali, 175mL anhydrous propanone, 23.2mL dimethyl formamide (DMF); 11.4mL (208mmol) Celfume; close nitrogen, 25 ℃ of confined reactions 21 days, have reacted rear reduced pressure concentration reactant liquor to dry; after adding the washing of 150mL acetone, suction filtration obtains methylnaltrexone bromide; 80 ℃ of vacuum drying 6h, obtain 13.4g product, yield 90.17%.
Two, related substance detects
(1) selection of chromatographic condition and optimization
(1) detect the selection of wavelength
From the HPLC-DAD UV scanning collection of illustrative plates of methylnaltrexone bromide product to be tested, methylnaltrexone bromide main peak and main technique impurity thereof, catabolite all have similar characteristic ultraviolet absorption, all at 282nm place, have absorption maximum, and the absorption at 230nm place is acromion.In methodological study, in conjunction with HPLC-DAD detecting device, select 230nm, 280nm and 310nm to detect wavelength and study.
Method: access method is learned specificity in research collection of illustrative plates and respectively destroyed sample collection of illustrative plates, methylnaltrexone bromide product to be tested, initiation material (Naltrexone Hydrochloride) collection of illustrative plates, observes the difference of its impurity number and impurity size under different wave length.
The results are shown in Table 1.
Table 1 detects the selection result of wavelength
Result: from dopant species analysis, 230nm detects wavelength ratio 280nm, 310nm checked for impurities is many; From the analysis on its rationality of impurity size, 230nm checked for impurities size is more reasonable, and part collection of illustrative plates 310nm, because main peak response is lower, causes impurity relative response too high, is unfavorable for correctly assessing this product related substance situation.Therefore, select 230nm to detect wavelength more reasonable.
(2) selection of mobile phase
With reference to methylnaltrexone bromide physicochemical property, methylnaltrexone bromide is easily molten in dimethyl sulfoxide (DMSO), dissolves slightly soluble in methyl alcohol, ethanol in water, 0.1mol/L hydrochloric acid, 1-METHYLPYRROLIDONE.
Solubleness with reference to methylnaltrexone bromide in acidic aqueous solution, selects acidic aqueous solution as water; The dissolubility in each kind solvent with reference to methylnaltrexone bromide, selects methyl alcohol as organic phase simultaneously.
A. acidic aqueous solution screening system: adopt methylnaltrexone bromide product to be tested as sample, under same chromatographic column, adopt different acidic aqueous solution-methyl alcohol systems, be applicable to screen a mobile phase that this product impurity detects, concrete grammar and the results are shown in Table 2.
Table 2 flow phase system selection result
Result: condition 4 and condition 6 peak shapes and symmetry are better, considers that methylnaltrexone bromide is zwitterionic compound, adds ion-pairing agent to have certain help to main peak and impurity separated.But because the impurity that condition 4 can detect is less, therefore select trifluoroacetic acid as ion-pairing agent, and this flow phase system is carried out to further demonstration test, to optimize mobile phase condition.
B. mobile phase ratio screening: get methylnaltrexone bromide product to be tested, under same chromatographic column, the impact on testing result when the consumption of investigating respectively trifluoroacetic acid in water is 0.1%, 0.15%, 0.2%, 0.3%, the results are shown in Table 3:
Table 3:
Result: methylnaltrexone bromide chromatographic peak is with the increase of trifluoroacetic acid consumption, and retention time slightly changes; The trifluoroacetic acid checked for impurities number of interpolation 0.15%, 0.2%, 0.3% is identical, and all can meet the requirement of this product determination of related substances, and 0.1% trifluoroacetic acid detection sensitivity is lower slightly.Select 0.15% trifluoroacetic acid-methyl alcohol (78: 22) as this product mobile phase.
(2) methodology checking
Naltrexone Hydrochloride (initiation material) and synthesising by-product research
By the chromatographic condition of above-mentioned formulation, initiation material Naltrexone Hydrochloride and the many batches of methylnaltrexone bromide products to be tested bought are analyzed, observe this chromatographic condition and whether can effectively detect initiation material and synthesising by-product.
Specificity is investigated
Method: get three batches of synthetic methylnaltrexone bromide crude product and Naltrexone Hydrochlorides, by above-mentioned chromatographic condition sample introduction, record chromatogram, the results are shown in Table 4.
Table 4 crude product and initiation material specificity result
Conclusion: from many batches of synthetic crude products, in building-up process, major impurity all can be effectively separated with methylnaltrexone bromide; Initiation material main peak and major impurity peak retention time thereof are different from methylnaltrexone bromide retention time, and as introduced initiation material in sample, it can not affect related substance and detect.From DAD collection of illustrative plates, can find out that main peak is purer, this method is better to initiation material and synthesising by-product detection specificity.
(3) Naltrexone Hydrochloride quantitative measurement
Naltrexone Hydrochloride is the initiation material of this product, himself there is certain pharmacologically active and toxicity, the inventive method is limited the quantity of Naltrexone Hydrochloride to be incorporated into quality standard, and limit must not be decided to be Naltrexone Hydrochloride contrast solution peak area (0.4%) in related substance detection.Listed in methylnaltrexone bromide bulk drug related substance check item, by drafting related substance detection method, Naltrexone Hydrochloride is carried out to the checking of the quantitative square science of law.
(1) linearity
Get Naltrexone Hydrochloride reference substance appropriate, accurately weighed, add mobile phase dissolving constant volume and make the storing solution that concentration is 0.01007mg/mL.Precision measures above-mentioned storing solution 2,4,6,8,10mL in 10mL measuring bottle respectively, add mobile phase dilution and make that concentration is respectively 0.002014,0.004028,0.006042,0.008056, the solution of 0.01007mg/mL, press related substance chromatographic condition, get respectively 20 μ l injection liquid chromatographies, measure peak area.The concentration (C) of take is horizontal ordinate, take peak area (A) as ordinate mapping, calculates regression equation and related coefficient.The results are shown in Table 5
Table 5
Result shows that Naltrexone Hydrochloride is good linear relation within the scope of 0.002014mg/mL～0.01007mg/mL.
(2) sample introduction precision
Get same need testing solution, continuous sample introduction 6 times, calculates the RSD value of 6 gained peak areas, 6 continuous sample introductions as a result, and peak area RSD value is 1.68%, sample introduction precision is good.
(3) repeatability of method
Get 6 parts of test samples, by same personnel, adopt identical instrument, replicate determination, the results are shown in Table 6:
Table 6: the replica test result of method
(4) precision test in the middle of
Get with 6 parts of a collection of test samples, by two analysts, by same procedure, parallel sampling and measuring, calculates content respectively.The results are shown in Table 7:
Table 7
(5) recovery
Precision measures 3 parts of Naltrexone Hydrochloride 10mg left and right, be placed in respectively 10mL measuring bottle, adding mobile phase dissolves and is diluted to scale, precision measures 0.5mL and puts in 50mL measuring bottle again, adds mobile phase and is settled to scale, shakes up, precision measures 6mL and puts in 10mL measuring bottle again, add mobile phase and be settled to scale, shake up, as 100% need testing solution; Get each three parts of 80% and 120% Naltrexone Hydrochlorides, with method preparation, obtain 80% and 120% need testing solution.Get above-mentioned solution and measure by the chromatographic condition of drafting, be measured in the same method reference substance, calculate recovery rate.
Table 8: Naltrexone Hydrochloride determination of recovery rates result
Result shows: 80%, the recovery of 100%, 120% sample is respectively 99.55%, 99.39%, 99.18%; The recovery of 9 duplicate samples is all between 98%～102%, and RSD is 0.70%, and method accuracy is good.
(6) stability of solution
Prepare as stated above need testing solution, place successively 0,2,4, after 6h, according to above-mentioned chromatographic process, measure, the results are shown in Table 9.
Table 9 solution stability testing result
Conclusion: need testing solution is good at 6 hours internal stabilities.
(7) detectability and quantitative limit
Method: get Naltrexone Hydrochloride reference substance appropriate, be mixed with certain density need testing solution, stepwise dilution again, difference sample introduction 20 μ l, the peak height of working sample solution main peak and the ratio of baseline noise, get 3 times of values of baseline noise as the minimum detectability of this product, get 10 times of values of baseline noise as the quantitative limit of this product.
Result: the lowest detection that records Naltrexone Hydrochloride is limited to 0.218808ng, is quantitatively limited to 0.72936ng.
(3) mensuration of related substance in methylnaltrexone bromide product to be tested
Get testing sample, add the need testing solution that mobile phase is configured to 1-2mg/ml;
Get need testing solution and dilute 100 times of solution 1 in contrast;
Take Naltrexone Hydrochloride reference substance appropriate, add solution that mobile phase makes 0.004-0.008mg/ml solution 2 in contrast;
According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2005), measure, with octadecylsilane chemically bonded silica, it is filling agent, 0.15% trifluoroacetic acid solution-the methyl alcohol (volume ratio 78: 22) of take is mobile phase, detection wavelength is 230nm, and number of theoretical plate calculates and is not less than 3000 by methylnaltrexone bromide peak;
High performance liquid chromatograph: Agilent 1100 chromatographic work stations, DAD (or VWD) detecting device.
Measure need testing solution and contrast solution 1,2, difference injection liquid chromatography, X-1, X-2, X-3 tri-batch sample determination of related substances the results are shown in Table 10:
Table 10 determination of related substances result
Therefore building-up process is introduced to impurity to methylnaltrexone bromide related substance detection method of the present invention and catabolite all can be realized effective monitoring, precision of method is high, specificity is strong.
Three, the assay of methylnaltrexone bromide
(1) assay method
(1) instrument and reagent
High performance liquid chromatograph: Agilent 1100 chromatographic work stations, DAD (or VWD) detecting device;
Reference substance: methylnaltrexone bromide sterling XJNQT-DZP, self-control, content 100%;
Reagent: methyl alcohol chromatographically pure (J.T.Baker), trifluoroacetic acid (Lancaster, 99%, FA031424).
(2) chromatographic condition
Chromatographic column: Akasil C18,250 * 4.6mm, 5 μ m
Detect wavelength: 230nm; Column temperature: 30 ℃; Flow velocity: 1mL/min.
Mobile phase: 0.15% trifluoroacetic acid solution-methyl alcohol (volume ratio 78: 22)
(3) preparation of solution
Sample thief, adds mobile phase and is configured to 1mg/ml solution, shakes up, and filters and obtain need testing solution; With legal system for reference substance solution.
(4) determination method
According to above-mentioned high effective liquid chromatography for measuring, precision measures reference substance solution, need testing solution, injects respectively hplc determination and get final product, and by external standard method, calculates content.
(2) methodology checking
Adopt the chromatographic condition under methylnaltrexone bromide related substance item, under related substance item, the aspects such as the specificity of method, system durability, detectability, stability of solution are investigated.Assay by HPLC method for methylnaltrexone bromide, rationality, linear relationship and the range of linearity that emphasis checking wavelength is selected, the repeatability of method and precision etc.
(1) detect the rationality of wavelength
HPLC-DAD gained UV scintigram by methylnaltrexone bromide, have maximum absorption band, but absorption value is lower near 282nm, is acromion absorption value higher at 230nm, therefore select 230nm as the detection wavelength of methylnaltrexone bromide assay.
(2) linear relationship is investigated
Get reference substance 100mg, accurately weighed, add mobile phase dissolving constant volume and make the storing solution that concentration is 10mg/mL.Precision measures above-mentioned storing solution, is quantitatively diluted to 0.2,0.4,0.6,0.8,1.0, the solution of 2.0mg/mL, gets respectively 20 μ l injection liquid chromatographies, measures peak area.The concentration (C) of take is horizontal ordinate, take peak area (A) as ordinate mapping, calculates regression equation and related coefficient.The results are shown in Table 11:
Table 11 linear relationship result
Conclusion: methylnaltrexone bromide concentration is good linear relation within the scope of 200.56 μ g/mL～2005.6 μ g/mL.
(3) repeatability of method
Get test sample, by same personnel, adopt identical instrument, replicate determination 6 duplicate samples, the results are shown in 12.
The replica test result of table 12 method
(4) precision test in the middle of
Get with a collection of test sample, by two analysts, by same procedure, parallel sampling and measuring is 6 parts respectively, calculates content.The results are shown in Table 13.
Precision test result in the middle of table 13
Conclusion: the middle precision of method is good.
(5) quantitative limit
Get the solution under assay item, stepwise dilution, difference sample introduction 20 μ l, the peak height of working sample solution main peak and the ratio of baseline noise, get 10 times of values of baseline noise as the quantitative limit of this product.Result: what record this product is quantitatively limited to 80.88ng.
(3) sample determination result
Get test agent in three batches, by above-mentioned definite HPLC method, measure.Reference substance solution, need testing solution sample size are 20 μ l, by external standard method, calculate content, the results are shown in Table 14.
Table 14HPLC method is measured three batches of pilot scale sample size results
The present invention has set up the method for HPLC method mensuration methylnaltrexone bromide content, and the method has been carried out to methodology checking, and the result shows that set up method specificity is strong, precision is good, measured result accurately and reliably, can be used for the assay of methylnaltrexone bromide.
Claims (2)
1. the detection method of a methylnaltrexone bromide, adopt high performance liquid chromatography to detect the methylnaltrexone bromide related substance in methylnaltrexone bromide testing sample, described related substance comprises impurity, methylnaltrexone bromide catabolite and the unreacted raw material of introducing in building-up process, it is characterized in that, the method for measuring methylnaltrexone bromide related substance comprises the following steps:
1) get testing sample, add the need testing solution that mobile phase is formulated as 1-2mg/ml;
2) get need testing solution and dilute 100 times of solution 1 in contrast;
3) take Naltrexone Hydrochloride reference substance, add solution that mobile phase is mixed with 0.004-0.008mg/ml solution 2 in contrast;
4) measure need testing solution and contrast solution 1,2, by following chromatographic condition, carry out high-performance liquid chromatogram determination: with octadecylsilane chemically bonded silica, be filling agent, 0.15% trifluoroacetic acid solution-methyl alcohol that the volume ratio of take is 78: 22 is mobile phase, detection wavelength is 230nm, high performance liquid chromatography flow velocity is 1mL/min, the column temperature of performance liquid chromatographic column is 30 ℃, and number of theoretical plate calculates and is not less than 3000 by methylnaltrexone bromide peak;
5) chromatogram recording is analyzed.
2. a detection method for methylnaltrexone bromide, adopts the methylnaltrexone bromide content in high effective liquid chromatography for measuring methylnaltrexone bromide testing sample, it is characterized in that, the method for measuring methylnaltrexone bromide content comprises the following steps:
1) get testing sample, add mobile phase and be formulated as 1mg/ml solution, shake up, filter and obtain need testing solution; With method, get methylnaltrexone bromide sterling and prepare reference substance solution;
2) measure need testing solution and reference substance solution, according to following chromatographic condition, carry out high-performance liquid chromatogram determination: with octadecylsilane chemically bonded silica, be filling agent, 0.15% trifluoroacetic acid solution-methyl alcohol that the volume ratio of take is 78: 22 is mobile phase, detection wavelength is 230nm, high performance liquid chromatography flow velocity is 1mL/min, the column temperature of performance liquid chromatographic column is 30 ℃, and number of theoretical plate calculates and is not less than 3000 by methylnaltrexone bromide peak;
3), according to the chromatogram of measuring, by external standard method, calculate the methylnaltrexone bromide content of methylnaltrexone bromide testing sample.
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| CN101208344A (en) * | 2005-05-25 | 2008-06-25 | 普罗热尼奇制药公司 | (R)-N-methylnaltrexone, processes for its synthesis and its pharmaceutical use |
| CN101646676A (en) * | 2006-11-22 | 2010-02-10 | 普罗基因制药公司 | 7,8-is saturated-4, (S)-N-steric isomer of 5-epoxy-morphinanium analogs |
| CN101685084A (en) * | 2008-09-22 | 2010-03-31 | 重庆医药工业研究院有限责任公司 | Method for detecting methylnaltrexone bromide and impurity thereof by chromatography |
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| CN101208344A (en) * | 2005-05-25 | 2008-06-25 | 普罗热尼奇制药公司 | (R)-N-methylnaltrexone, processes for its synthesis and its pharmaceutical use |
| CN101646676A (en) * | 2006-11-22 | 2010-02-10 | 普罗基因制药公司 | 7,8-is saturated-4, (S)-N-steric isomer of 5-epoxy-morphinanium analogs |
| CN101685084A (en) * | 2008-09-22 | 2010-03-31 | 重庆医药工业研究院有限责任公司 | Method for detecting methylnaltrexone bromide and impurity thereof by chromatography |
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