CN110161152A - The detection method of impurity in a kind of Sinomenine - Google Patents

Abstract

The present invention provides a kind of detection methods of impurity in Sinomenine, it is detected using HPLC, its testing conditions are as follows: use octadecylsilane chemically bonded silica for the chromatographic column of filler, mobile phase is made of mobile phase A and Mobile phase B, and mobile phase A is acetonitrile, and Mobile phase B is phosphate buffer, the volume ratio of mobile phase A and Mobile phase B is 5~15:95~85, flow velocity is 0.8~1.2ml/min, and Detection wavelength is 210~260nm, and column temperature is 25~40 DEG C.The present invention uses HPLC detection method, and pass through control testing conditions, fast and accurately the impurity in Sinomenine can be detected, and method is stablized, high sensitivity, as a result reliable, effective control to Sinomenine quality can be reinforced, help to improve the safety and validity of clinical application.

Description

The detection method of impurity in a kind of Sinomenine

[technical field]

The invention belongs to pharmaceutical technology fields, and in particular to the detection method of impurity in a kind of Sinomenine.

[background technique]

Sinomenine has the pharmacological actions such as anti-inflammatory, analgesia, decompression and anti-arrhythmia, and existing several formulations are answered at present For clinic, for treating rheumatic and rheumatoid arthritis etc., as Sinomenine enteric coatel tablets, Sinomenine are applied with Scattered, Zhengqing Fengtongning Retard Tablets, Zhengqingfengtongning injection etc..In recent years, Sinomenine effect and application increasingly by To attention.

Traditional Sinomenine production is used using alkalization water extraction technique and is added water and white lime, alkali in cocculi Change a period of time, a certain amount of technical benzene is then added, the refluxing extraction in water-bath is finally acidified, stands, crystallization.This method Obtained Sinomenine is generally light grey, canescence and yellow powder, contains many impurity.Patent CN201110361882.0 discloses a kind of method for preparing Sinomenine, and caulis sinomenii coarse powder is used to add HCL wetting, seep Filter, Leachate site obtains Sinomenine crude product by extraction, crystallization, then plus ethyl alcohol or purified water be heated to reflux to dissolution plus It is colourless to filtrate to enter active carbon, filtration, cooling, crystallization, filtering, ethanol washing filter cake, obtains Sinomenine fine work, wherein hydrochloric acid Although cucoline purity after purification reaches 94-99%, but still inevitably contain a certain amount of impurity.

Since Sinomenine is unstable in aqueous solution, in production process by aoxidize, high temperature, illumination are influenced, It is also possible to generate catabolite, can also generate impurity during the production of preparation and storage.

There is certain influence to the drug effect and finished product stability of Sinomenine bulk pharmaceutical chemicals and preparation in the presence of impurity. However the fresh defects inspecting and method of quality control for being related to Sinomenine less in disclosed prior art, therefore, it is necessary to Research and develop a kind of fast and accurately detection method of impurity in Sinomenine.

[summary of the invention]

The object of the invention is that solve the deficiencies in the prior art, and provide a kind of inspection of impurity in Sinomenine Survey method can fast and accurately detect impurity and its content in Sinomenine, control quality.

The purpose of the present invention is what is be achieved through the following technical solutions:

The detection method of impurity, is detected, testing conditions using HPLC in a kind of Sinomenine are as follows: uses 18 Alkyl silane bonded silica gel is the chromatographic column of filler, and mobile phase is made of mobile phase A and Mobile phase B, and mobile phase A is acetonitrile, Mobile phase B is phosphate buffer, and the volume ratio of mobile phase A and Mobile phase B is 5~15:95~85, flow velocity is 0.8~ 1.2ml/min, Detection wavelength are 210~260nm, and column temperature is 25~40 DEG C.

Preferably, the phosphate-buffered salt in the Mobile phase B is sodium dihydrogen phosphate or potassium dihydrogen phosphate, phosphorus in Mobile phase B The content of hydrochlorate is 0.5~0.8%, and the phosphate buffering liquid concentration unit is g/100mL.

Preferably, it is detected using UV-vis spectroscopy detector.

Preferably, sampling volume is 5~20ul.

Preferably, mobile phase is acetonitrile~0.78%NaH₂PO₄(8:92) (V/V) solution, flow velocity 1.0ml/min, column temperature 30 DEG C, Detection wavelength 210nm.

Preferably, the chromatographic column is selected from Waters XTerra RP18, Agilent ZORBAX Eclipse Plus C18, Agilent TC-C18 or SHIMADZU InertSustain C18 chromatographic column.

Preferably, the measurement of impurity is carried out using principal component Self-control method.

Preferably, specifically includes the following steps:

(1) preparation of test solution: taking Sinomenine sample, and addition Mobile phase B solution, which is dissolved and diluted, to be made often The solution of the Sinomenine Han 0.2~2mg in 1ml；

(2) preparation of contrast solution: the test solution for taking step (1) to obtain, it is molten that addition Mobile phase B is diluted to test sample The solution of liquid concentration 0.05~0.2%, as contrast solution；

(3) isometric test solution and contrast solution are measured, HPLC detection is carried out.

Preferably, the step (1), step (2) Mobile phase B are 0.78%NaH₂PO₄Solution；The step (3) is specifically examined Survey condition are as follows: with acetonitrile~0.78%NaH₂PO₄(8:92) (V/V) solution be mobile phase, flow velocity 1ml/min, 30 DEG C of column temperature, Sampling volume 10ul, detector be UV-vis spectroscopy detector, Detection wavelength 210nm, precision measure test solution with it is right According to each 10ul of solution, it is injected separately into liquid chromatograph, 3 times of record chromatogram to principal component Sinomenine peak retention time, Complete the detection of impurity.

The present invention uses HPLC detection method, and by control testing conditions, can be fast and accurately in Sinomenine Impurity detected, and method is stablized, as a result reliably high sensitivity can reinforce effective control to Sinomenine quality System, helps to improve the safety and validity of clinical application.

[Detailed description of the invention]

Fig. 1 is the amount of impurities of 3 lot number Y-1611013 sample of experimental example and the detection chromatogram of content；

Fig. 2 is the amount of impurities of 3 lot number Y-1611014 sample of experimental example and the detection chromatogram of content；

Fig. 3 is the amount of impurities of 3 lot number Y-1611015 sample of experimental example and the detection chromatogram of content；

Fig. 4 is the amount of impurities of 3 lot number Y-1611016 sample of experimental example and the detection chromatogram of content；

Fig. 5 is the amount of impurities of 3 lot number Y-1611017 sample of experimental example and the detection chromatogram of content.

[specific embodiment]

The invention will be further described with embodiment with reference to the accompanying drawing.

The present invention provides a kind of detection methods of impurity in Sinomenine, use HPLC to be detected, detector bar Part are as follows: use octadecylsilane chemically bonded silica (ODS) for the chromatographic column of filler, mobile phase is by mobile phase A and Mobile phase B group Be acetonitrile at, mobile phase A, Mobile phase B is phosphate buffer, the volume ratio of mobile phase A and Mobile phase B be 5~15:95~ 85, flow velocity is 0.8~1.2ml/min, and Detection wavelength 210-260nm, column temperature is 25~40 DEG C.

Phosphate-buffered salt in preferable mobile phase B is sodium dihydrogen phosphate or potassium dihydrogen phosphate, and phosphatic content is in water 0.5~0.8%, phosphate solution concentration unit is g/100mL, i.e., contains 0.5~0.8g phosphate in every 100mL water.

Chromatographic column can be selected Waters XTerra RP18, Agilent ZORBAX Eclipse Plus C18, Agilent TC-C18 or SHIMADZU InertSustain C18 chromatographic column.

UV-vis spectroscopy detector detector can be used in the present invention；Sampling volume is preferably 5~20ul；It is preferred that using The measurement of principal component Self-control method progress impurity content.The preparation method of test solution and contrast solution is preferred are as follows: 1, confession The preparation of test sample solution: taking Sinomenine sample, be added Mobile phase B solution dissolve and dilute be made in every 1ml contain 0.2~ The solution of 2mg；2, the preparation of contrast solution: the test solution for taking step (1) to obtain is added Mobile phase B and is diluted to test sample The solution of solution concentration 0.05%~0.2%, as contrast solution.Using principal component Self-control method, reference substance can not used Matter facilitates the measurement for carrying out impurity content.

The detection method of impurity in Sinomenine provided by the invention is specific to walk as one of preferred embodiment It is rapid as follows:

(1) preparation of test solution: Sinomenine sample is taken, 0.78%NaH is added₂PO₄Solution, which is dissolved and diluted, to be made Containing the solution of 1.0mg in every 1ml；

(2) preparation of contrast solution: the test solution for taking step (1) to obtain adds 0.78%NaH₂PO₄Solution dilution 1000 times；

(3) use octadecylsilane chemically bonded silica (ODS) for the chromatographic column of filler, with acetonitrile~0.78%NaH₂PO₄ (8:92) (V/V) solution is mobile phase, and flow velocity 1ml/min, 30 DEG C of column temperature, sampling volume 10ul, detector is ultraviolet-visible Spectrophotometric detector, Detection wavelength 210nm, precision measure test solution and each 10ul of contrast solution, are injected separately into liquid chromatogram Instrument records chromatogram to 3 times of principal component Sinomenine peak retention time, completes the detection of impurity.

Phosphate solution concentration unit is g/100mL in the present invention.

The method for detecting impurities of one Sinomenine raw material of experimental example is established

The methods of Sinomenine raw material strong illumination, high temperature, high humidity, acid/base hydrolysis and oxidation are destroyed, than To the impurity number of the detection of three kinds of chromatographic conditions.

1.1 instruments and material

Instrument: Shimadzu 2010AT HPLC chromatogram instrument, C18 chromatographic column (Waters XTerra RP18), electric jacket, illumination Chamber；

Reagent: acetonitrile, sodium dihydrogen phosphate, sodium heptanesulfonate, hydrochloric acid, sodium hydroxide, hydrogen peroxide, deionized water；

Material to be tested: Sinomenine (lot number: Y-1509004).

1.2 destructive sample preparations

1. Sinomenine raw material: taking Sinomenine raw material 10mg, be placed in 50ml measuring bottle, add water to be configured to every ml and contain The solution of 0.2mg.

2. acid destroys sample: taking Sinomenine raw material 10mg, be placed in 50ml measuring bottle, add 0.1mol/L hydrochloric acid solution 10ml is placed at room temperature for 1h, adds 0.1mol/L sodium hydroxide solution to neutralize, water is added to be settled to scale.

3. alkali destroys sample: taking Sinomenine raw material 10mg, be placed in 50ml measuring bottle, add 0.1mol/L sodium hydroxide molten Liquid 10ml, is placed at room temperature for 1h, adds 0.1mol/L hydrochloric acid solution to neutralize, water is added to be settled to scale.

4. Oxidative demage sample: taking Sinomenine raw material 10mg, be placed in 50ml measuring bottle, add 30% (v/v) peroxidating Hydrogen solution 5ml, is placed at room temperature for 1h, after boiling 1min, water is added to be settled to scale.

5. illumination sample: Sinomenine raw material 10mg is taken, is placed in 50ml measuring bottle, irradiates 12h under the strong light of 4500xL, Water is added to be settled to scale.

6. heat damage sample: Sinomenine raw material 10mg is taken, is placed in 50ml measuring bottle, 1h in 105 DEG C of baking ovens is placed in, it is cold But after, water is added to be settled to scale.

7. blank sample: each of the above sample preparation placebo solution is a (to remove raw material, by each preparation of above-mentioned test Solution is a).

1.3 testing conditions

Condition one: using octadecylsilane chemically bonded silica for the chromatographic column (Waters XTerra RP18) of filler, With acetonitrile~0.78%NaH₂PO4 (8:92) (V/V) solution be mobile phase, 30 DEG C of column temperature, sampling volume 10ul, Detection wavelength 210nm, flow velocity 1ml/min, detector are UV-vis spectroscopy detector；

Condition two: use octadecylsilane chemically bonded silica for the chromatographic column (Waters XTerra RP18) of filler； Using acetonitrile as mobile phase A, 0.78% sodium dihydrogen phosphate is Mobile phase B, by progress gradient elution shown in table 1；Sampling volume 10ul, Detection wavelength 210nm, detector are UV-vis spectroscopy detector.Number of theoretical plate is calculated not by Sinomenine peak Lower than 5000.

Table 1

Time (min)	A (%)	B (%)
			0	2	98
5	2	98
			10	5	95
25	12	88
			66	50	50

Condition three: using octadecylsilane chemically bonded silica for the chromatographic column (Waters XTerra RP18) of filler, (contain 0.1% triethylamine, with phosphoric acid tune with the 0.01mol/L potassium dihydrogen phosphate aqueous solution of the sodium heptanesulfonate of 0.005mol/L PH2.5)-acetonitrile (85:15) is mobile phase；Flow velocity is 1ml/min, and detector is UV-vis spectroscopy detector, sampling volume 10ul, Detection wavelength 210nm, 30 DEG C of column temperature.

1.4 testing result

Testing result is as shown in table 2, from table 2 it can be seen that Sinomenine material sample detects one under condition two A time is the impurity peaks of 21.753min；Acid destroys sample and impurity peaks is not detected under the conditions of three；Alkali destroys sample and exists Condition once detects 2 impurity peaks, is located at 4.872min and 32.005min；Oxidative demage sample is in three conditions Under detect impurity peaks, the impurity peaks quantity that conditional one and condition two detect is 9, more than condition three detect To 8 impurity peaks quantity；Heat damage sample once detects 2 impurity peaks in condition, be located at 9.905min and At 10.180min, 1 impurity peaks is detected under condition three, is located at 8.253min；Illumination sample is once detected in condition 2 impurity peaks, are located at 9.164min and 9.777min.

2 defects inspecting number of table and retention time

1.5 conclusion

In conventional HPLC detection method, it can continuously change the polarity of mobile phase using gradient elution, relative to etc. Degree elution is conducive to separate and measure, and this experiment is if other conditions are the same, and condition one uses isocratic elution, Condition two uses gradient elution, and the impurity number detected in the case of condition one is more instead.Moreover, being flowed under regular situation Add triethylamine in phase, maskable silicone hydroxyl significantly improves peak shape, eliminates hangover, addition sodium heptanesulfonate can by with sample The effect of molecule improves peak shape to prevent the effect of sample molecule and silicone hydroxyl, and this experiment condition three is added in mobile phase Sodium heptanesulfonate and triethylamine detect obtained impurity number and are also not so good as condition one, therefore optimal detection condition are as follows: use Octadecylsilane chemically bonded silica is the chromatographic column of filler, with acetonitrile~0.78%NaH₂PO₄(8:92) solution is mobile phase, Flow velocity is 1.0ml/min, and 30 DEG C of column temperature, sampling volume 10ul, detector is UV-vis spectroscopy detector, Detection wavelength 210nm。

Two Sinomenine solution destructive testing of experimental example

The test of 2.1 high temperatures

It takes Sinomenine raw material about 5.0g to be placed in 250ml round-bottomed flask, 200ml deionized water is added, flow back 1h, cold But to after room temperature, precision measures 1ml into 100ml measuring bottle, adds deionized water to scale, shakes up, as high temperature for examination Product；

The test of 2.2 Oxidative demages

It takes Sinomenine raw material about 50mg to be placed in 10ml measuring bottle, adds 2ml deionized water dissolving, shake up, be added 30% Hydrogen peroxide 3ml, place 2h, after boiling 1min, add deionized water to scale, precision measures 1ml into 50ml measuring bottle, adds Ionized water shakes up, to scale as Oxidative demage test sample；

2.3 blank test

Raw material is removed, it is a by each preparation solution of above-mentioned test, respectively as respective blank control liquid.

The related substances test of 2.4 raw materials

It takes Sinomenine raw material about 2.5mg to be placed in 10ml measuring bottle, adds deionized water to scale, obtain 0.25mg/ml's Test solution.

Above-mentioned sample is subjected to HPLC detection, HPLC testing conditions: uses octadecylsilane chemically bonded silica for filler Chromatographic column (Waters XTerra RP18), with acetonitrile~0.78%NaH₂PO₄(8:92) (V/V) solution is mobile phase, column temperature 30 DEG C, sampling volume 10ul, Detection wavelength 210nm, flow velocity 1ml/min, detector is UV-vis spectroscopy detector.

According to HPLC chromatogram, the stability of Sinomenine solution is investigated, analyses whether to generate new impurity, detection knot Fruit is as shown in table 3.From table 3 it can be seen that Sinomenine passes through high temperature and Oxidative demage, new impurity is produced, especially Through Oxidative demage, it is more to generate amount of impurities.

Table 3

The impurity analysis of three Sinomenine raw material of experimental example

Raw material progress impurity research is chosen to be pressed according to " chemicals impurity investigative technique guideline " (version in 2005) The Light absorbing impurty standard of Sinomenine bulk pharmaceutical chemicals and preparation is studied and established to maximum daily dose method, determine impurity spectrum and it is miscellaneous Matter limit (reporting limit, limit of identification and Quality Control limit).Structural Identification or confirmation are carried out to the impurity beyond limit of identification, looked for Preparation raw material is used as to impurity number and content are least.

3.1 instruments and material

Instrument: Shimadzu 2010AT HPLC chromatogram instrument, the C18 chromatographic column that octadecylsilane chemically bonded silica is filler (Waters XTerra RP18)；

Reagent: acetonitrile, sodium dihydrogen phosphate, deionized water；

Material to be tested: Sinomenine (lot number: Y-1611013, Y-1606014, Y-1606015, Y-1606016, Y- 1606017)。

3.2 method

The measurement of amount of impurities and content is carried out using principal component Self-control method；5 batch Sinomenine samples are taken respectively Appropriate product add 0.78%NaH₂PO₄Solution dissolves and dilutes the solution being made in every 1ml containing about 1.0mg, as test solution； Precision measures test solution 1ml, sets in 100ml measuring bottle, adds 0.78%NaH₂PO₄Solution is diluted to scale, shakes up, then accurate Test solution 1ml is measured, sets in 10ml measuring bottle, adds 0.78%NaH₂PO₄Solution is diluted to scale, shakes up, molten as compareing Liquid.Using octadecylsilane chemically bonded silica as filler, with acetonitrile~0.78%NaH₂PO₄(8:92) solution is mobile phase, flow velocity For 1ml/min, 30 DEG C of column temperature, sampling volume 10ul, detector is UV-vis spectroscopy detector, Detection wavelength 210nm, essence Close measurement test solution and each 10ul of contrast solution, are injected separately into liquid chromatograph, and record chromatogram to principal component peak retains 3 times of time record each impurity appearance situation.

3.3 test result

The testing result of Sinomenine raw material impurity quantity and content as shown in table 4 and Fig. 1-5 (survey respectively by each lot number Fixed two parallel samples).

Table 4

The limit of impurities of 5 bulk pharmaceutical chemicals of table

From table 4 and table 5 it is found that detecting 4 lot numbers (Y-1611013, Y-1606014, Y- in Sinomenine raw material 1606015, Y-1606016) amount of impurities is 2, and the amount of impurities of 1 lot number (Y-1606017) is 1, the above lot number Single impurity content was less than for 0.10% (Sinomenine maximum daily dose is less than 2g) in raw material, was below " chemicals Impurity investigative technique guideline " impurity limit of identification (as shown in table 5) specified in (version in 2005), it does not need to do impurity Structural Identification can choose the less Y-1606014 of total impurities content or the less Y-1606017 of impurity number as preparation original Material.

Above-described is only embodiments of the present invention, it should be noted here that for those of ordinary skill in the art For, without departing from the concept of the premise of the invention, improvement can also be made, but these belong to protection model of the invention It encloses.

Claims (9)

1. the detection method of impurity in a kind of Sinomenine, it is characterised in that detected using HPLC, testing conditions are as follows: Use octadecylsilane chemically bonded silica for the chromatographic column of filler, mobile phase is made of mobile phase A and Mobile phase B, mobile phase A For acetonitrile, Mobile phase B is phosphate buffer, and the volume ratio of mobile phase A and Mobile phase B is 5~15:95~85, flow velocity 0.8 ~1.2ml/min, Detection wavelength are 210~260nm, and column temperature is 25~40 DEG C.

2. the detection method of impurity in Sinomenine as described in claim 1, it is characterised in that the phosphorus in the Mobile phase B Acid buffering salt is sodium dihydrogen phosphate or potassium dihydrogen phosphate, and phosphatic content is 0.5~0.8% in the Mobile phase B, the phosphorus Phthalate buffer concentration unit is g/100mL.

3. the detection method of impurity in Sinomenine as described in claim 1, it is characterised in that use UV-vis spectroscopy Detector is detected.

4. the detection method of impurity in Sinomenine as described in claim 1, it is characterised in that sampling volume be 5~ 20ul。

5. the detection method of impurity in Sinomenine as described in claim 1, it is characterised in that the mobile phase is acetonitrile ~0.78%NaH₂PO₄(8:92) (V/V) solution, flow velocity 1.0ml/min, 30 DEG C of column temperature, Detection wavelength 210nm.

6. the detection method of impurity in Sinomenine as described in claim 1, it is characterised in that the chromatographic column is selected from Waters XTerra RP18, Agilent ZORBAX Eclipse Plus C18, Agilent TC-C18 or SHIMADZU InertSustain C18 chromatographic column.

7. the detection method of impurity in Sinomenine as claimed in any one of claims 1 to 6, it is characterised in that: using it is main at The measurement for dividing Self-control method to carry out impurity.

8. the detection method of impurity in Sinomenine as claimed in claim 7, it is characterised in that specifically includes the following steps:

(1) preparation of test solution: taking Sinomenine sample, and addition Mobile phase B solution, which is dissolved and diluted, to be made in every 1ml The solution of the Sinomenine containing 0.2~2mg；

(2) preparation of contrast solution: the test solution for taking step (1) to obtain, it is dense that addition Mobile phase B is diluted to test solution The solution of degree 0.05~0.2%, as contrast solution；

(3) isometric test solution and contrast solution are measured, HPLC detection is carried out.

9. the detection method of impurity in Sinomenine as claimed in claim 8, it is characterised in that the step (1), step (2) Mobile phase B is 0.78%NaH₂PO₄Solution；The specific testing conditions of the step (3) are as follows: with acetonitrile~0.78%NaH₂PO₄ (8:92) (V/V) solution is mobile phase, and flow velocity 1ml/min, 30 DEG C of column temperature, sampling volume 10ul, detector is ultraviolet-visible Spectrophotometric detector, Detection wavelength 210nm, precision measure test solution and each 10ul of contrast solution, are injected separately into liquid chromatogram Instrument records chromatogram to 3 times of principal component Sinomenine peak retention time, completes the detection of impurity.