CN1621834A

CN1621834A - Use of high efficiency liquid chromatography in measuring content of related substances in Milrinone

Info

Publication number: CN1621834A
Application number: CN 200410098724
Authority: CN
Inventors: 赵志全
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2004-12-15
Filing date: 2004-12-15
Publication date: 2005-06-01

Abstract

The present invention provides the application of high efficiency liquid phase chromatographic process in measuring the relevant matters and their content in milrinone. The flowing phase may consist of borax buffering liquid, methanol and water in the proportion of 1-5 to 20-50 to 50-80, preferably in the proportion of 1-3 to 20-40 to 60-70, and the borax buffering liquid has the preferable concentration of 0.05 M and pH value of 7.0.

Description

High performance liquid chromatography is the application in its related substances in measuring milrinone

Technical field

The present invention relates to the application of high performance liquid chromatography, in particular, relate to high performance liquid chromatography application in its related substances in measuring milrinone.

Background technology

Milrinone chemistry 2-methyl by name-6-oxygen-1,6-dihydro-(3, the two pyridines of 4-)-5-formonitrile HCN.Press dry product and calculate, wherein contain C ₁₂H ₉N ₃O is no less than 98.5%.Be off-white color or little yellow crystalline powder, odorless, almost insoluble in water or ethanol, molten in the watery hydrochloric acid part omitted.

Milrinone is applicable to congestive heart failure short-term intravenous administration.The Mi Weinong parenteral solution can be with after an amount of physiological saline or the must dilution, (injected too fast in 10 minutes by the slow intravenous injection of 37.5 μ g/kg-50 μ g/kg earlier, premature ventricualr contraction may appear), continue after this to adjust consumption according to clinical effect with 0.375 μ g-0.75 μ g/kg/min drip-feed.

Owing to the bad reaction of milrinone mainly is to be produced by the impurity in its product, strict control its related substances wherein has very important significance.Though the method for its related substances in the present useful tlc determination milrinone, but the peak area that can only control single related substance peak (except solvent peak, the lactic acid peak) is not more than 1/5 of contrast solution main peak area, and summation is not more than 1.5% of reference substance main peak area.If can use a kind of new assay method, make related substance peak summation be not more than 0.5% of reference substance main peak area, improve the quality standard of milrinone, will have important practical significance.

Summary of the invention

The invention provides high performance liquid chromatography application in its related substances in measuring milrinone.

In above-mentioned high performance liquid chromatography, the moving phase of use can be made up of borate buffer solution, first alcohol and water.The concentration of described borate buffer solution is preferably 0.05M, and pH is preferably 7.0.

In above-mentioned high performance liquid chromatography, the ratio of described borate buffer solution, first alcohol and water is preferably (1-5): (20-50): (50-80), and more preferably (1-3): (20-40): (60-70).

High performance liquid chromatography is compared with thin-layered chromatography, has following advantage:

(1) it is not proper to adopt " filtration " to handle need testing solution in the thin-layered chromatography, and solution was clarified after sample added the methyl alcohol sonicated, and it is muddy to add the i.e. appearance of moving phase dilution, must ultrasonicly again can dissolve fully; Moving phase in this method employing milrinone standard preparation under the related substance item is directly handled sample, dissolves fully through the ultrasonic sample that makes, and uses the moving phase constant volume again, obtains the solution of clear;

(2) high effective liquid chromatography for measuring sample size, the row method of going forward side by side are learned checking, have obtained good linear relationship, and reappearance and the recovery are good, in a few days the having good stability of solution.

Description of drawings

Fig. 1 is sour destructive test high-efficient liquid phase chromatogram;

Fig. 2 is an alkali destructive test high-efficient liquid phase chromatogram;

Fig. 3 is a heat damage experiment high-efficient liquid phase chromatogram;

Fig. 4 is an oxidation destructive test high-efficient liquid phase chromatogram.

Embodiment

Further explain the present invention with embodiment below, but protection scope of the present invention is not limited to these embodiment.

The foundation of embodiment 1 chromatographic condition

Instrument: high performance liquid chromatograph (U.S. Waters), join 515 pumps, 2487 detecting devices, chem workstation.

Reagent: milrinone and reference substance (content is 99.7%); Methyl alcohol; Chromatographically pure; Water; Pure water; Borax; Boric acid.

Chromatographic column: Hypersil BDS C ₁₈Post (250mm * 4.6mm, 5 μ m);

Moving phase: 0.05molL ^-1Borate buffer solution (pH=7.0): methyl alcohol: water=2: 30: 70;

Flow velocity: 1.0mlmin ^-1

Detect wavelength: 254nm;

Sample size: 20 μ l (quantity tube).

Number of theoretical plate calculates by the milrinone peak and is not less than 3000.

The range of linearity

It is an amount of to get 105 ℃ of milrinone reference substances that are dried to constant weight, and accurate the title decides, and adds the moving phase dissolving and be diluted to 1ml to contain the solution of 0.5mg as standard reserving solution.Precision is measured standard reserving solution 0.5,1.0,1.5,2.0,2.5,3.0,3.5m1 puts respectively in 7 10ml measuring bottles, is diluted to scale with moving phase, shakes up, and sample introduction 20 μ l write down chromatogram, with milrinone concentration (c, mgml respectively ^-1) be horizontal ordinate, corresponding peak area is an ordinate, carries out linear regression, gets regression equation to be: Y=51821577.14286X+80825.57143, r=0.9999 (n=7).The result shows that milrinone is at 0.025～0.175mgml ^-1In the scope, peak area and concentration linear relationship are good.

Recovery test

Precision is measured the milrinone sample 30mg (3 parts) that oneself knows content, puts respectively in 3 100ml measuring bottles, adds reference substance 10,20,30mg more respectively, adds an amount of moving phase dissolving and is diluted to scale, shakes up; Get 5ml respectively and put in the 25ml measuring bottle, be diluted to scale, shake up, measure respectively with moving phase, calculate recovery rate, getting average recovery rate is 100.5%, RSD=0.71% illustrates that accuracy is good, sees Table 1.

Table 1

Known sample amount (mg)	Add sample size (mg)	Detected level (mg)	The recovery (%)	Average recovery rate (%)	???RSD(％)
Known sample amount (mg)	Add sample size (mg)	Detected level (mg)	The recovery (%)	Average recovery rate (%)	???RSD(％)	??29.9	??10.2	????40.2	????100.2	????100.5	???0.71
????40.1	????100.0							????40.2	????100.2
????40.1	????100.0	????40.0	????99.8

????30.2	????20.7	????51.6	????101.4
		????51.6	????101.4	????51.6	????101.4
		????51.6	????101.4	????51.6	????101.4
		????51.6	????101.4	????30.2	????30.6	????60.9	????100.2
????60.6	????99.7					????60.9	????100.2
????60.6	????99.7	????61.0	????100.3

The precision test

According to aforementioned chromatographic condition, getting concentration is 0.1mgml ^-1Standard solution repeat sample introduction 6 times, calculating RSD is 0.11%, the result shows that its precision is good.

Sample size is measured

Precision is measured sample 50mg, puts in the 100ml measuring bottle, and it is an amount of to add moving phase, ultrasonicly makes dissolving and is diluted to scale with moving phase, shakes up, and precision is measured 5ml and put in the 25ml measuring bottle, is diluted to scale with moving phase, shakes up, as need testing solution; Precision is measured 20 μ l, injects high performance liquid chromatograph, the record chromatogram; Other the milrinone reference substance that is dried to constant weight of learning from else's experience 105 ℃ is an amount of, adds moving phase and makes the solution that contains 0.1mg among every 1ml, measure with method, with external standard method by calculated by peak area, promptly.3 batch sample measurement results see Table 2.

Table 2

Lot number	The bottle number	Content (%)	Mean value (%)	???RSD(％)
Lot number	The bottle number	Content (%)	Mean value (%)	???RSD(％)	????031201	????1	????100.1	??100.2	???0.13
????100.3							????100.1
????100.3	????2	????100.2
????100.4		????100.2
????100.4		????031202	????1	????100.2		??99.7	???0.52
????100.1				????100.2
????100.1	????2			????99.2

		????99.3
		????99.3	????031203	????1	????100.0	????99.8	????0.29
????100.0					????100.0
????100.0	????2	????99.4
????99.7		????99.4

The sample liquid study on the stability

Get 1 part of need testing solution, respectively 0,2,4,6,8,10h measures, and the record peak area calculates RSD=0.75%, shows that the moving phase stability of solution of milrinone is better, and in a few days error is less.

Detectability is measured

Get 2.5 μ gml ^-1The milrinone reference substance solution, survey its detectability (S/N=2) with dilution method, by 2 times of snr computation, under this chromatographic system condition, detect and be limited to 2.5 * 10 ^-5Mgml ^-1

The sample determination of related substances

It is an amount of that precision is measured milrinone, makes 0.5mgml respectively with moving phase ^-1Need testing solution and 2.5 μ gml ^-1Contrast solution.Under aforementioned chromatographic condition, get contrast solution 20 μ l and inject high performance liquid chromatograph, regulate instrumental sensitivity, make the major component peak be about 20% of full scale.Precision is measured need testing solution 20 μ l sample introductions, and the record chromatogram is to 3 times of major component peak retention time.Need testing solution is measured each related substance peak area summation as showing the related substance peak, must not be greater than contrast solution main peak area (0.5%).3 batch samples all detect multiple related substance in (031201,031202,031203), and the total amount of related substance is all less than 0.5%.

The stability experiment of embodiment 2 high performance liquid chromatographies

High performance liquid chromatography is compared with thin-layered chromatography, has advantage highly sensitive, that accuracy is good, not only can be qualitative for related substance, and can be quantitative, can better control the quality of milrinone.The concrete related substance of relevant these product is not seen concrete report in the literature, thus designed tests such as acid, alkali, heat, oxidation destruction, to investigate reliability, the stability of this method system.

The interference of blank solvent

It is an amount of to get moving phase, filters, and gets filtered fluid and measures in accordance with the law, and solvent is noiseless to the milrinone determination of related substances.

The acid destructive test

Get the about 20mg of milrinone, add 0.1mol/L hydrochloric acid solution 10ml, water-bath reflux 20min, cooling adds moving phase to 50ml, shakes up, and filters, and gets filtered fluid and checks that in accordance with the law high-efficient liquid phase chromatogram is seen Fig. 1.The result show acid this method is not had influence.

The alkali destructive test

Get the about 20mg of milrinone, add 0.1mol/L sodium hydroxide solution 10ml, water-bath reflux 20min, cooling adds moving phase to 50ml, shakes up, and filters, and gets filtered fluid and checks that in accordance with the law high-efficient liquid phase chromatogram is seen Fig. 2.The result shows that alkali does not have influence to this method.

Heat-rupture test

Get the about 20mg of milrinone, add water 10ml, reflux 30min, cooling adds moving phase to 50ml, shakes up, and filters, and gets filtered fluid and checks that in accordance with the law high-efficient liquid phase chromatogram is seen Fig. 3.The result shows that high temperature does not have influence to this method.

Oxidation destroys the part test

Get the about 20mg of milrinone, add 10%H ₂O ₂Solution 10ml, water-bath reflux 20min, cooling adds moving phase to 50ml, shakes up, and filters, and gets filtered fluid and checks that in accordance with the law high-efficient liquid phase chromatogram is seen Fig. 4.The result shows that oxygenant does not have influence to this method.

Conclusion

By accompanying drawing 1～4 as seen, the minimum milrinone that can check 0.01 μ g/ml at least of this law; The milrinone peak can separate with the product peak after destroying, and degree of separation meets the requirements.

Comparative Examples

Get three batches of milrinones, use high performance liquid chromatography and tlc determination respectively, the results are shown in Table 3.

Table 3

As seen: high performance liquid chromatography is compared with thin-layered chromatography, have highly sensitive, accuracy is high, not only can quantitative measurement to impurity, and disturb little characteristics.

Claims

High performance liquid chromatography related substance in measuring milrinone detect or assay in application.
2. application as claimed in claim 1 is characterized in that, the moving phase of using in the described high performance liquid chromatography is made up of borate buffer solution, first alcohol and water.
3. application as claimed in claim 2 is characterized in that, the concentration of described borate buffer solution is 0.05M.
4. application as claimed in claim 3 is characterized in that, the ratio of described borate buffer solution, first alcohol and water is (1-5): (20-50): (50-80).
5. application as claimed in claim 3 is characterized in that, the ratio of described borate buffer solution, first alcohol and water is (1-3): (20-40): (60-70).
6. as each application among the claim 2-5, it is characterized in that the pH of described borate buffer solution is 7.0.