CN102565205B - Quality detection method of methyhaaltrexone bromide - Google Patents

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

A kind of quality detection method of methylnaltrexone bromide

technical field

The invention relates to a method for controlling the quality of methylnaltrexone bromide, which belongs to the technical field of chemical drug analysis.

Background technique

Methylnaltrexone bromide (methylnaltrexone, MNTX) is a selective mu opioid receptor antagonist that acts as a quaternary ammonium salt that limits the ability of methylnaltrexone bromide to cross the blood-brain barrier, making methylnaltrexone bromide Naltrexone acts as a peripherally acting opioid receptor antagonist, eg, in the tissues of the gastrointestinal tract, thereby reducing the constipating effects of opioids without affecting the analgesic effects of opioids on the central nervous system.

Methylnaltrexone bromide has the following characteristics: 1) It acts on peripheral μ receptors, but does not activate central opioid receptors, which may be a competitive antagonism; 2) It is safe, and a series of tests have not found that it has serious side effects, and common adverse effects The reactions are abdominal pain, diarrhea, flatulence and dizziness; 3) the effect is fast, and it can take effect in a few minutes; 4) the treatment of constipation is better than that of laxatives and softeners; It can also be combined with opioid receptors on the road, and can also be combined with opioid receptors in the whole body through blood distribution. Therefore, methylnaltrexone bromide can not only treat gastrointestinal side effects of opioids, but also treat parenteral adverse reactions; 6) no Through the blood-brain barrier, does not weaken the central analgesic effect of opiates.

The compound name of methylnaltrexone bromide is: bromide-17-(cyclopropylmethyl)-4,5α-epoxy-3,14-dihydroxy-17-methyl-6-oxomorphinan, which The structural formula is as follows:

Chinese patent 200680022957.X discloses a high-performance liquid chromatography assay method and assay conditions of methylnaltrexone bromide, the method and conditions are mainly used for the determination of S isomers, for related substances of methylnaltrexone bromide ( That is, the inspection and content determination of impurities) have not yet been published in the literature.

Contents of the invention

The object of the present invention is to provide a kind of quality detection method of bromidenaltrexone product. The method for detecting the quality of methylnaltrexone bromide of the present invention adopts high performance liquid chromatography (HPLC), and is suitable for inspection and content determination of related substances in the methylnaltrexone bromide synthesis process. And the quality control standard of methylnaltrexone bromide was formulated according to the quality detection method of the present invention.

The detection method of the present invention can effectively monitor the related substances of methylnaltrexone (including impurities introduced in the synthesis process, degradation products of methylnaltrexone and unreacted raw materials), and the method has high precision and strong specificity. The limit of impurities is strictly controlled in accordance with the drug registration management method and other relevant guidelines to improve the safety of drug use.

The high performance liquid chromatography condition of the present invention is: use octadecylsilane bonded silica gel as filler; With 0.1-0.2% trifluoroacetic acid solution-methanol (volume ratio 70-80: 20-30) as mobile phase; The wavelength is 230nm.

In the above HPLC detection method, the concentration of the trifluoroacetic acid solution used is preferably 0.15%; the volume ratio of the trifluoroacetic acid solution to methanol in the mobile phase is preferably 78:22.

For the above HPLC detection method, the flow rate is preferably 1 mL/min, and the column temperature is generally 30°C.

High performance liquid chromatography of the present invention measures the further specific assay method of bromidenaltrexone related substance as follows:

Take the sample to be tested, add mobile phase to prepare 1-2mg/ml test solution;

Get the test solution diluted 100 times as the control solution 1;

Weigh an appropriate amount of naltrexone hydrochloride reference substance, add mobile phase to make a solution of 0.004-0.008mg/ml as contrast solution 2;

High performance liquid chromatography (Chinese Pharmacopoeia version two appendix V D in 2005) is measured: with octadecylsilane bonded silica gel as filler, with 0.15% trifluoroacetic acid solution-methanol (volume ratio 78: 22) as mobile phase , the detection wavelength is 230nm, and the number of theoretical plates is not less than 3000 based on the peak of methylnaltrexone bromide;

Measure need testing solution and contrast solution 1,2, inject liquid chromatograph respectively and detect;

The content standard of related substances of methylnaltrexone bromide is: if there is an impurity peak in the chromatogram of the test product solution, the amount of known impurity naltrexone hydrochloride must not exceed 0.4% of the sample; a single unknown impurity must not exceed 0.1% of the sample; The sum of each impurity shall not exceed 1% of the sample.

The present invention measures the specific chromatographic condition of the high performance liquid chromatography of bromidenaltrexone content in the sample to be tested as above, and the preparation of need testing sample and contrast solution is as follows:

Take the sample to be tested, add mobile phase to prepare 1-2mg/ml test solution;

Take the pure product of methylnaltrexone bromide (reference substance), and add mobile phase to prepare a solution of 1-2 mg/ml as the reference substance solution.

The test solution and the reference solution were measured, respectively injected into the liquid chromatograph for detection, and the content was calculated by the external standard method.

The invention determines the detection method of the related substances in the methylnaltrexone bromide product, and carries out detailed methodological verification. The method validation results show that the method can effectively detect the synthetic starting materials, synthetic by-products, and forced degradation products of methylnaltrexone bromide, so that the impurities introduced in the synthesis process and their degradation products can be effectively monitored. The method High precision and strong specificity. The limit of impurities is strictly controlled in accordance with the drug registration management method and other relevant guidelines to improve the safety of drug use.

In addition, the present invention has also carried out methodological verification to the method for determining the content of methylnaltrexone bromide by HPLC, and the verification result shows that the specificity of the established method is strong, the precision is good, the measured result is accurate and reliable, and can be used for methyl bromide Determination of naltrexone content.

Description of drawings

Fig. 1 is when carrying out bromidenaltrexone related substance detection to X-1 batch number 0 month sample, the HPLC collection of illustrative plates of need testing solution;

Fig. 2 is when X-1 batch number 0 month sample is carried out bromidenaltrexone related substance detection, the HPLC collection of illustrative plates of control solution 1 (need testing solution dilution 100 times).

Detailed ways

One, the preparation of bromidenaltrexone test sample

Methylnaltrexone bromide samples to be tested can be prepared by various synthetic methods, including but not limited to the following methods:

Under nitrogen protection, add 11.6g (34.0mmol) naltrexone base, 175mL anhydrous acetone, 23.2mL dimethylformamide (DMF), 11.4mL (208mmol) methyl bromide into the three-necked flask, turn off the nitrogen, and seal the reaction at 25°C for 21 After the reaction was completed, the reaction solution was concentrated under reduced pressure to dryness, and 150 mL of acetone was added to wash it to obtain methylnaltrexone bromide, which was vacuum-dried at 80° C. for 6 hours to obtain 13.4 g of the product, with a yield of 90.17%.

2. Detection of related substances

(1) Selection and optimization of chromatographic conditions

(1) Selection of detection wavelength

It can be seen from the HPLC-DAD ultraviolet scanning spectrum of bromethylnaltrexone test product that the main peak of bromylnaltrexone and its main process impurities and degradation products have similar ultraviolet absorption characteristics, all of which have a maximum absorption at 282nm and a maximum absorption at 230nm. The absorption at the shoulder peak. In the methodological research, combined with HPLC-DAD detector, the detection wavelengths of 230nm, 280nm and 310nm were selected for research.

Method: Take the spectrum of each specific destruction sample, methylnaltrexone bromide to be tested, and starting material (naltrexone hydrochloride) spectrum in the methodology research spectrum, and observe the difference in the number and size of impurities at different wavelengths.

The results are shown in Table 1.

Table 1 Selection results of detection wavelength

Results: From the analysis of impurity types, the detection wavelength of 230nm is more than that of 280nm and 310nm; from the analysis of the rationality of impurity size, the detection of impurity size at 230nm is more reasonable, and the relative response value of impurities is too high due to the low response value of the main peak of some spectra at 310nm , which is not conducive to the correct evaluation of the relevant substances of this product. Therefore, it is more reasonable to choose 230nm detection wavelength.

(2) Selection of mobile phase

Referring to the physical and chemical properties of methylnaltrexone bromide, methylnaltrexone bromide is easily soluble in dimethyl sulfoxide, soluble in water, 0.1mol/L hydrochloric acid, and N-methylpyrrolidone, and slightly soluble in methanol and ethanol.

With reference to the solubility of methylnaltrexone bromide in the acidic aqueous solution, the acidic aqueous solution is selected as the water phase; while referring to the solubility of methylnaltrexone bromide in various solvents, methanol is selected as the organic phase.

A. Screening of acidic aqueous solution system: Use methylnaltrexone bromide to be tested as a sample, and use different acidic aqueous solution-methanol systems under the same chromatographic column to screen a mobile phase suitable for the detection of impurities in this product. For specific methods and results, see Table 2.

Table 2 mobile phase system selection results

Results: Conditions 4 and 6 had better peak shapes and symmetry. Considering that bromidenaltrexone is a zwitterionic compound, adding ion-pairing reagents is helpful to the separation of the main peak and impurities. However, since condition 4 can detect fewer impurities, trifluoroacetic acid was selected as the ion-pairing reagent, and further verification tests were carried out on the mobile phase system to optimize the mobile phase conditions.

B. mobile phase ratio screening: take methylnaltrexone bromide to be tested, under the same chromatographic column, investigate the effect on the detection result when the amount of trifluoroacetic acid in the aqueous phase is 0.1%, 0.15%, 0.2%, and 0.3% respectively Influence, the results are shown in Table 3:

table 3:

Results: With the increase of the amount of trifluoroacetic acid, the retention time of brommethylnaltrexone chromatographic peaks changed slightly; the number of impurities detected by adding 0.15%, 0.2%, and 0.3% trifluoroacetic acid was the same, and all of them could meet the requirements of the determination of related substances of this product requirements, while the detection sensitivity of 0.1% trifluoroacetic acid is slightly lower. Choose 0.15% trifluoroacetic acid-methanol (78:22) as the mobile phase of this product.

(2) Methodological verification

Research on naltrexone hydrochloride (starting material) and its by-products

According to the chromatographic conditions formulated above, analyze the purchased starting material naltrexone hydrochloride and multiple batches of methylnaltrexone bromide to be tested, and observe whether the chromatographic conditions can effectively detect the starting material and synthetic by-products.

Exclusive inspection

Method: Take three batches of synthetic crude methylnaltrexone bromide and naltrexone hydrochloride, inject samples according to the above chromatographic conditions, and record the chromatograms. The results are shown in Table 4.

Table 4 crude product and starting material specificity result

Conclusion: Judging from multiple batches of synthetic crude products, the main impurities in the synthesis process can be effectively separated from methylnaltrexone; the retention time of the main peak of the starting material and its main impurity peaks is different from that of methylnaltrexone in the sample. The introduction of starting materials will not affect the detection of related substances. It can be seen from the DAD spectrum that the main peak is relatively pure, and this method has good specificity for the detection of starting materials and synthetic by-products.

(3) Quantitative determination of naltrexone hydrochloride

Naltrexone hydrochloride is the starting raw material of this product, and it itself has certain pharmacological activity and toxicity, and the inventive method limits naltrexone hydrochloride into the quality standard, and the limit is defined as must not cross the naltrexone hydrochloride in the relevant substance detection item. Control solution peak area (0.4%). Include it in the inspection items of related substances of methylnaltrexone bromide raw materials, and carry out quantitative methodological verification on naltrexone hydrochloride according to the proposed testing method for related substances.

(1) Linear

Take an appropriate amount of naltrexone hydrochloride reference substance, accurately weighed, add mobile phase to dissolve and constant volume to make a stock solution with a concentration of 0.01007 mg/mL. Accurately measure 2, 4, 6, 8, and 10 mL of the above-mentioned stock solution in a 10 mL volumetric bottle, add mobile phase to dilute to make solutions with concentrations of 0.002014, 0.004028, 0.006042, 0.008056, and 0.01007 mg/mL, according to the relevant substance chromatogram Conditions, 20 μl were injected into the liquid chromatograph to measure the peak area. Take the concentration (C) as the abscissa and the peak area (A) as the ordinate to plot, and calculate the regression equation and correlation coefficient. The results are shown in Table 5

table 5

The results showed that naltrexone hydrochloride showed a good linear relationship in the range of 0.002014mg/mL～0.01007mg/mL.

(2) Injection precision

Take the same test solution, inject 6 times continuously, and calculate the RSD value of the peak area obtained for 6 times. As a result, the RSD value of the peak area is 1.68% after 6 continuous injections, and the precision of injection is good.

(3) Repeatability of the method

Get 6 parts of test product, by the same personnel, adopt the same instrument, measure in parallel, the results are shown in Table 6:

Table 6: Repeatability test results of the method

(4) Intermediate precision test

Take 6 copies of the same batch of test samples, and two analysts use the same method to take parallel samples for determination and calculate the content. The results are shown in Table 7:

Table 7

(5) Recovery rate

Precisely measure about 10 mg of 3 portions of naltrexone hydrochloride, place them in 10 mL measuring bottles respectively, add mobile phase to dissolve and dilute to the mark, then precisely measure 0.5 mL and place in a 50 mL measuring bottle, add mobile phase to make up to the mark, shake Mix well, then accurately measure 6mL and place it in a 10mL measuring bottle, add mobile phase to the volume, shake well, and use it as a 100% test solution; take three parts each of 80% and 120% naltrexone hydrochloride, and prepare in the same way , 80% and 120% of the test solution. Take the above solution and measure it according to the proposed chromatographic conditions, measure the reference substance in the same way, and calculate the recovery rate.

Table 8: Results of determination of recovery rate of naltrexone hydrochloride

The results showed that the recoveries of 80%, 100%, and 120% samples were 99.55%, 99.39%, and 99.18% respectively; the recoveries of the nine samples were all between 98% and 102%, and the RSD was 0.70%. good.

(6) Solution stability

Prepare the test solution according to the above method, place it for 0, 2, 4, and 6 hours in turn, and measure it according to the above chromatographic method. The results are shown in Table 9.

Table 9 solution stability test result

Conclusion: The test solution has good stability within 6 hours.

(7) Limit of detection and limit of quantitation

Method: Take an appropriate amount of naltrexone hydrochloride reference substance, prepare a certain concentration of the test solution, then dilute it step by step, inject 20 μl of the sample respectively, measure the ratio of the peak height of the main peak of the sample solution to the baseline noise, and take 3 times of the baseline noise The value is taken as the minimum detection limit of this product, and the value of 10 times of the baseline noise is taken as the quantification limit of this product.

Results: The lowest detection limit of naltrexone hydrochloride was 0.218808ng, and the quantification limit was 0.72936ng.

(3) Determination of related substances in methylnaltrexone bromide test article

Take the sample to be tested, add mobile phase to configure the test solution of 1-2mg/ml;

Get the test solution diluted 100 times as the control solution 1;

Weigh an appropriate amount of naltrexone hydrochloride reference substance, add mobile phase to make a solution of 0.004-0.008mg/ml as contrast solution 2;

Measure according to high performance liquid chromatography (Chinese Pharmacopoeia 2005 edition two appendix V D), use octadecylsilane bonded silica gel as filler, take 0.15% trifluoroacetic acid solution-methanol (volume ratio 78: 22) as mobile phase, the detection wavelength is 230nm, and the number of theoretical plates is not less than 3000 based on the peak of methylnaltrexone bromide;

High-performance liquid chromatography: Agilent 1100 chromatography workstation, DAD (or VWD) detector.

Measure need testing solution and contrast solution 1,2, inject liquid chromatograph respectively, X-1, X-2, X-3 three batches of sample related substance determination results are shown in Table 10:

Table 10 Determination results of related substances

It can be seen from the above that the method for detecting related substances of methylnaltrexone bromide of the present invention can effectively monitor both the impurities introduced in the synthesis process and their degradation products, and the method has high precision and strong specificity.

3. Determination of the content of methylnaltrexone bromide

(1) Determination method

(1) Instruments and reagents

High performance liquid chromatography: Agilent 1100 chromatography workstation, DAD (or VWD) detector;

Reference substance: pure methylnaltrexone bromide XJNQT-DZP, self-made, content 100%;

Reagents: methanol chromatographically pure (J.T.Baker), trifluoroacetic acid (Lancaster, 99%, FA031424).

(2) Chromatographic conditions

Chromatographic column: Akasil C18, 250×4.6mm, 5μm

Detection wavelength: 230nm; column temperature: 30°C; flow rate: 1mL/min.

Mobile phase: 0.15% trifluoroacetic acid solution-methanol (volume ratio 78:22)

(3) Preparation of solution

Take the sample, add mobile phase to configure it as a 1mg/ml solution, shake well, and filter to obtain the test solution; prepare the reference solution in the same way.

(4) Determination method

Measure according to the above-mentioned high-performance liquid chromatography, accurately measure the reference substance solution and the test solution, and inject them into the liquid chromatograph for determination, and calculate the content by the external standard method.

(2) Methodological verification

The chromatographic conditions under related substances of methylnaltrexone bromide were used, and the specificity of the method, system durability, detection limit, solution stability and other aspects have been investigated under the related substances. The HPLC method was used for the content determination of methylnaltrexone bromide, focusing on verifying the rationality of wavelength selection, linear relationship and linear range, repeatability and precision of the method, etc.

(1) Rationality of detection wavelength

From the UV scanning diagram obtained by HPLC-DAD of bromidenaltrexone, there is a maximum absorption peak near 282nm, but the absorption value is low, and the shoulder peak absorption value is higher at 230nm, so 230nm is selected as the determination of bromidenaltrexone content Detection wavelength.

(2) Linear relationship inspection

Take 100 mg of the reference substance, weigh it accurately, add mobile phase to dissolve and make a stock solution with a concentration of 10 mg/mL. Accurately measure the above stock solution, quantitatively dilute it into 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mg/mL solutions, inject 20 μl into the liquid chromatograph respectively, and measure the peak area. Take the concentration (C) as the abscissa and the peak area (A) as the ordinate to plot, and calculate the regression equation and correlation coefficient. The results are shown in Table 11:

Table 11 Linear relationship results

Conclusion: The concentration of methylnaltrexone bromide showed a good linear relationship in the range of 200.56 μg/mL to 2005.6 μg/mL.

(3) Repeatability of the method

Take the test product, and use the same instrument to measure 6 samples in parallel by the same person, see 12 for the results.

The repeatability test result of table 12 method

(4) Intermediate precision test

Take the same batch of test samples, and use the same method by two analysts to measure 6 samples in parallel and calculate the content. The results are shown in Table 13.

Table 13 Intermediate precision test results

Conclusions: The intermediate precision of the method is good.

(5) limit of quantification

Take the solution under the content determination item, dilute it step by step, inject 20 μl of samples respectively, measure the ratio of the peak height of the main peak of the sample solution to the baseline noise, and take 10 times of the baseline noise as the quantitative limit of this product. Results: The quantification limit of this product was determined to be 80.88ng.

(3) Sample measurement results

Three batches of pilot test samples were taken and determined by the HPLC method determined above. The sample volume of the reference substance solution and the test solution was 20 μl, and the content was calculated by the external standard method. The results are shown in Table 14.

Table 14HPLC method measures three batches of pilot test sample content results

The present invention establishes a method for measuring the content of bromethylnaltrexone by HPLC, and the method is verified by methodology. The verification result shows that the established method has strong specificity, good precision, and the measured results are accurate and reliable, and can be used for Determination of bromidenaltrexone content.

Claims (2)

1. a detection method of methylnaltrexone bromide, adopts high performance liquid chromatography to detect the related substances of methylnaltrexone bromide in the sample of methylnaltrexone bromide to be tested, and said related substances include the Impurities, methylnaltrexone bromide degradation products and unreacted raw materials are characterized in that the method for determining related substances of methylnaltrexone bromide comprises the following steps: 1) Take the sample to be tested, add mobile phase to prepare a 1-2mg/ml test solution; 2) Take the test solution diluted 100 times as the control solution 1; 3) Weigh naltrexone hydrochloride reference substance, add mobile phase to prepare a solution of 0.004-0.008 mg/ml as control solution 2; 4) Measure the test solution and control solutions 1 and 2, and carry out high-performance liquid chromatography determination according to the following chromatographic conditions: use octadecylsilane bonded silica gel as filler, and use 0.15% Tris at a volume ratio of 78:22 Fluoroacetic acid solution-methanol is the mobile phase, the detection wavelength is 230nm, the flow rate of high-performance liquid chromatography is 1mL/min, the column temperature of high-performance liquid chromatography is 30°C, and the number of theoretical plates calculated based on the peak of methylnaltrexone bromide is not less than 3000; 5) Analyze the measured chromatogram. 2. a detection method of methylnaltrexone, adopt high performance liquid chromatography to measure the content of methylnaltrexone in the sample of methylnaltrexone to be tested, it is characterized in that, the method for measuring the content of methylnaltrexone Include the following steps: 1) Take the sample to be tested, add mobile phase to prepare a 1mg/ml solution, shake well, and filter to obtain the test solution; take the pure product of methylnaltrexone bromide in the same way to prepare the reference solution; 2) Take the test solution and the reference solution, and perform HPLC determination according to the following chromatographic conditions: use octadecylsilane bonded silica gel as filler, and use 0.15% trifluoroacetic acid with a volume ratio of 78:22 Solution-methanol is the mobile phase, the detection wavelength is 230nm, the flow rate of high-performance liquid chromatography is 1mL/min, the column temperature of high-performance liquid chromatography is 30°C, and the number of theoretical plates is not less than 3000 based on the peak of methylnaltrexone bromide; 3) According to the determined chromatogram, calculate the content of methylnaltrexone bromidexone in the sample to be tested by the external standard method.

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