阅读说明：本技术 一种在制剂生产过程中检测磷酸肌酸钠残留的方法 (Method for detecting creatine phosphate sodium residue in preparation production process ) 是由 王庆 廖凡惠 于 2019-11-16 设计创作，主要内容包括：本发明公开一种生产过程中检测磷酸肌酸钠残留的方法：该方法是采用高效液相色谱法进行检测,包括下述步骤。步骤1：配置磷酸肌酸钠样品溶液,所述的磷酸肌酸钠样品溶液包括磷酸肌酸钠供试液和磷酸肌酸钠对照液；步骤2：将步骤1所得的样品溶液注入高效液相色谱仪,采用等度流动相进行洗脱,并记录色谱图。所述检测方法采用五氟苯基键合硅胶色谱柱或者氨基柱。本发明的检测方法操作简单快速,克服现有技术的缺陷,磷酸肌酸钠主峰保留时间较短,使用过程中色谱系统耐用稳定,磷酸肌酸钠残留可以有效检出,测定方法专属性强,检测结果准确可信,为连续批生产过程中磷酸肌酸钠的残留检测提供了一种简便合理的检测方法。(The invention discloses a method for detecting creatine phosphate sodium residue in a production process, which comprises the following steps: the method adopts high performance liquid chromatography for detection and comprises the following steps. Step 1: preparing a creatine phosphate sodium sample solution, wherein the creatine phosphate sodium sample solution comprises a creatine phosphate sodium test solution and a creatine phosphate sodium control solution; step 2: and (3) injecting the sample solution obtained in the step (1) into a high performance liquid chromatograph, eluting by adopting an isocratic mobile phase, and recording a chromatogram. The detection method adopts a pentafluorophenyl bonded silica gel chromatographic column or an amino column. The detection method provided by the invention is simple and rapid to operate, overcomes the defects of the prior art, has short retention time of the main peak of the creatine phosphate sodium, is durable and stable in a chromatographic system in a using process, can effectively detect creatine phosphate sodium residue, is strong in specificity, is accurate and reliable in detection result, and provides a simple, convenient and reasonable detection method for detecting creatine phosphate sodium residue in a continuous batch production process.)

1. A method for detecting creatine phosphate sodium residue in a production process is characterized in that the method is carried out by adopting high performance liquid chromatography and comprises the following steps:

step 1: preparing a creatine phosphate sodium sample solution, wherein the creatine phosphate sodium sample solution comprises a creatine phosphate sodium test sample solution and a creatine phosphate sodium reference solution;

step 2: injecting the sample solution obtained in the step 1 into a high performance liquid chromatograph, performing isocratic elution by adopting a mobile phase, and recording a chromatogram;

the preparation method of the reference substance solution comprises the following steps: taking a proper amount of creatine phosphate sodium reference substance, dissolving the creatine phosphate sodium reference substance by using a diluent, and diluting the creatine phosphate sodium reference substance into a solution containing 0.1-2 mu g of creatine phosphate sodium per 1mL serving as a reference substance solution;

the chromatographic column of the high performance liquid chromatography is a pentafluorophenyl hybrid column or an amino column;

the mobile phase is a mixed solvent of a chromatographic organic solvent and a phosphate buffer system or a carbonate buffer system, and the proportion of the organic solvent is 70-85%.

2. The method of claim 1, wherein the HPLC column is Kinetex F5100A 4.6.6X 150mm 2.6 μm or Shimaduramide 4.6X 250mm 5 μm.

3. The detection method of claim 1, wherein the phosphate buffer comprises a potassium phosphate, sodium phosphate, ammonium phosphate system; the pH value of the phosphate buffer system is 5-8.

4. The detection method according to claim 1, wherein the organic solvent is chromatographic acetonitrile or chromatographic methanol, and the chromatographic acetonitrile or chromatographic methanol contains 0.1-1% ethanol.

5. The detection method according to claim 4, wherein the ratio of ethanol in chromatographic methanol or chromatographic acetonitrile is 0.1-1: 100.

6. The detection method according to claim 1, wherein the detection wavelength of the high performance liquid chromatography is 205 to 220 nm.

7. The detection method according to claim 1, wherein the column temperature of the high performance liquid chromatography is 35 to 45 ℃.

8. The detection method according to claim 1, wherein the flow rate of the high performance liquid chromatography is 0.5 to 1.5 mL/min.

9. The detection method according to claim 1, wherein the step 2 further comprises the steps of: precisely measuring the blank solution, injecting into a liquid chromatograph, recording the chromatogram, and calculating the impurity content according to the external standard method of the main component.

Technical Field

The invention relates to the technical field of medicines, in particular to a method for detecting creatine phosphate sodium serving as a myocardial protectant, and particularly relates to a method for detecting creatine phosphate sodium residue in a production process.

Background

Creatine phosphate (creatine phosphate) is an important energy supplying substance in myocardial metabolism and was isolated from mammalian muscle in 1928. Disodium creatine phosphate (disodium creatine phosphate) with chemical name of N- [ imino (phosphino) methyl ] -N-methylglycine disodium salt has a structural formula shown as the following formula, is a medicinal form, has myocardial protection function, has no side effect, and can be used as adjuvant therapy medicine for cardiology and surgery. Can be clinically used for treating striated muscle activity deficiency and used as the auxiliary treatment of myocardial diseases. In 1992, Italy Oghui pharmaceutical factory first developed creatine phosphate disodium salt to be put on the market, and because of its definite therapeutic effect and small side effect, creatine phosphate disodium salt is widely used in heart surgery and myocardial infarction treatment in developed countries such as Europe and America, and has good effect. In 1995, China began to import creatine phosphate disodium salt for injection.

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Creatine phosphate sodium can be subjected to collinear production with other products in the production process, and the residual quantity of the medicine can influence the medicine quality and medicine safety of the product per se and other products subjected to collinear production, so that in the link of medicine production, the residual quantity limit calculation is carried out according to medicine GMP (good manufacturing practice) guidelines, and a proper method is searched for detection.

At present, a relevant substance detection method is reported in the literature, but a residual detection method is rarely reported in the literature, and the relevant substance method reported in the literature cannot meet the requirement of rapid and large-scale detection of residual detection, so that the technical personnel in the field are dedicated to developing an effective method for detecting the residual creatine phosphate sodium in the production process.

Disclosure of Invention

In the batch production process of creatine phosphate sodium, the residual sample of cleaning usually has more than 15 sampling points in one batch, so the liquid phase monitoring and sample injection times are more, usually 24 hours of continuous detection are needed in one day, and in the process of continuous production of a plurality of batches, continuous detection is needed for a plurality of days, which requires stable detection conditions, durable chromatographic columns and rapid detection results.

Creatine phosphate sodium is difficult to keep on a common C18 column or a main peak is overlapped with a blank peak, so that an ion pair reagent is added into a common mobile phase to strengthen the retention of the creatine phosphate sodium, for example, in a detection method of creatine phosphate sodium in 'Chinese pharmacopoeia' 2015 edition, the mobile phase is pure water fluidity added with the ion pair reagent tetrabutyl ammonium hydroxide, the peak emergence time of the creatine phosphate sodium in the mobile phase is about 30min, and rapid detection cannot be realized. In other documents (Chinese medicine and clinic 2005, 5 (11), 865-. Therefore, the sample is usually washed immediately after being used in the daytime, and the chromatographic column is repaired to a certain extent, so that continuous sample injection for more than 2 days is difficult to realize.

In addition, a literature (CN 101034086B) reports that a mobile phase containing n-pentylamine has a low theoretical plate number and a high baseline noise, so that the detection limit and the quantification limit are too high in concentration to meet the requirement of method verification.

In view of the above-mentioned drawbacks of the prior art, the technical problems to be solved by the present invention are: the detection method is effective, rapid, stable and durable in chromatographic conditions.

In order to achieve the above object, the present invention provides a method for detecting creatine phosphate sodium residue in a production process, wherein the method is performed by high performance liquid chromatography, and comprises the following steps:

step 1: preparing a creatine phosphate sodium sample solution, wherein the creatine phosphate sodium sample solution comprises a creatine phosphate sodium test sample solution and a creatine phosphate sodium reference solution;

step 2: injecting the sample solution obtained in the step 1 into a high performance liquid chromatograph, performing isocratic elution by adopting a mobile phase, and recording a chromatogram;

the preparation method of the reference substance solution comprises the following steps: taking a proper amount of creatine phosphate sodium reference substance, dissolving the creatine phosphate sodium reference substance by using a diluent, and diluting the creatine phosphate sodium reference substance into a solution containing 0.1-2 mu g of creatine phosphate sodium per 1mL serving as a reference substance solution;

the chromatographic column of the high performance liquid chromatography is a pentafluorophenyl hybrid column (including a core-shell column) or an amino column;

the mobile phase is a mixed solvent of a chromatographic organic solvent and a phosphate buffer or carbonate buffer system, and the proportion of the organic solvent is 70-85%.

Further, the column of the high performance liquid chromatography is Kinetex F5100A 4.6.6X 150mm 2.6 μm or Shimaduramide 4.6X 250mm 5 μm.

The phosphate buffer comprises potassium phosphate, sodium phosphate and an ammonium phosphate system; the pH value of the phosphate buffer system is 5-8.

The organic solvent is chromatographic acetonitrile or chromatographic methanol, and the chromatographic acetonitrile or chromatographic methanol contains 0.1-1% of ethanol; in the absence of ethanol, the pattern peak shape is broad and the theoretical plate number is low.

The ratio of ethanol in chromatographic methanol or chromatographic acetonitrile is 0.1-1: 100.

The detection wavelength of the high performance liquid chromatography is 205-220 nm.

The temperature of a chromatographic column of the high performance liquid chromatography is 35-45 ℃.

The flow rate of the high performance liquid chromatography is 0.5-1.5 mL/min.

The step 2 specifically comprises the following substeps: precisely measuring the blank solution, injecting into a liquid chromatograph, recording the chromatogram, and calculating the impurity content according to the external standard method of the main component.

The detection method provided by the invention is simple and rapid to operate, overcomes the defects of the prior art, has short retention time of the main peak of the creatine phosphate sodium, is durable and stable in a chromatographic system in the using process, has long service life of a chromatographic column, can effectively detect creatine phosphate sodium residue, is strong in specificity, is accurate and reliable in detection result, and provides a simple, convenient and reasonable detection method for detecting creatine phosphate sodium residue in the continuous batch production process. The method is suitable for the determination of the sampling detection of the cleaning residual quantity in the production process of multiple batches of raw material medicines or preparations of creatine phosphate sodium.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a specification test blank map of a preferred embodiment of the present invention;

FIG. 2 is a spectrum of a specific assay control according to a preferred embodiment of the present invention;

FIG. 3 is a quantitative limit map of a preferred embodiment of the present invention;

FIG. 4 is a detection limit map according to a preferred embodiment of the present invention;

FIG. 5 is a test solution profile of precision testing according to a preferred embodiment of the present invention;

FIG. 6 is a diagram of a 50% limit test solution for an accuracy test according to a preferred embodiment of the present invention;

FIG. 7 is a 100% limit test solution profile of an accuracy test according to a preferred embodiment of the present invention;

FIG. 8 is a 150% limit test solution profile of an accuracy test according to a preferred embodiment of the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Example 1

Detection conditions and Instrument conditions

Instrument condition LC-2030C high performance liquid chromatograph (quaternary pump, degasser, UV detector, column oven, autosampler, system monitor, lcoolution) (shimadzu corporation, japan); the sample (Daisy pharmaceutical Co., Ltd., Shenghuaxi, Chongqing), acetonitrile, ethanol (merck chromatographic purity), other reagents analytical purity, and water fresh ultrapure water.

According to the determination of high performance liquid chromatography (China pharmacopoeia 2015 edition general rule 0512), InertSustain Amide is used as a chromatographic column, and acetonitrile-0.2% ethanol solution is used as a mobile phase A; 0.02mol/L ammonium bicarbonate (PH 7.0) is used as a mobile phase B, the proportion of A is 75%, gradient elution is carried out, the flow rate is 1.0mL per minute, the column temperature is 35 ℃, the detection wavelength is 210nm, and the sensitivity is as follows: 1.0 AUFS.

Specificity

Sample solution preparation

Blank solvent: two blank cotton swabs are taken, are wetted by purified water and are wrung out (the purified water is used for cleaning equipment), 10ml of mobile phase is added, ultrasonic treatment is carried out for 3min, and filter membrane filtration is carried out.

Stock control solution (0.5 mg/mL): taking about 50mg (about 65mg according to hydrate) of the creatine phosphate sodium reference substance into a 100mL measuring flask, dissolving by using a mobile phase, fixing the volume to the scale, and shaking up.

Control solution (0.0012 mg/mL): precisely measuring 1.0mL of the reference stock solution in a 50mL measuring flask, fixing the volume to a scale, and shaking up; and precisely measuring 6mL of the solution in a 50mL measuring flask, fixing the volume to the scale, and shaking up.

Measurement method

Respectively injecting blank and control solutions, investigating the specificity of the method, and confirming that the blank solution does not interfere with the determination of creatine phosphate sodium by the verification method; the blank solution was not interfering with the main peak. The results of the special tests prove that: the retention time of the creatine phosphate sodium is 6.362min, and a blank solution has no interference on main peak detection. The experimental results are shown in figures 1 and 2.

Limit of detection of quantitative limit

And (4) quantitative limit: and adjusting the concentration of the next sample injection according to the peak height by injecting a low-concentration creatine phosphate sodium control solution. And when the signal-to-noise ratio is 10, the concentration is the quantitative limit concentration, the quantitative limit solution is repeatedly injected into 6 needles, and the RSD of the peak area is calculated and reported. As shown in FIG. 3, 0.0439. mu.g/ml was finally determined as the limit of quantitation when the S/N was about 11, and the area RSD was 1.7%.

Detection limit: and adjusting the concentration of the next sample injection according to the peak height by injecting a low-concentration creatine phosphate sodium reference solution. When the signal-to-noise ratio (S/N) is 3, the concentration is the detection limit, the detection limit solution is repeatedly injected into 6 needles, and the RSD of the peak area is calculated and reported. As shown in FIG. 4, the concentration of 0.0143. mu.g/ml was finally determined to be about 3.5 in terms of S/N as the detection limit. The area RSD was 3.3%. Quantitative limit detection limit test results:

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linearity

The concentration of the quantitative limit, 30%, 50%, 80%, 100%, 120%, 150% of the concentration of the surface residue limit was selected for validation, and each concentration was repeated 3 times to validate that the method could meet the requirements.

Linear stock (0.12 mg/mL): taking about 24mg (about 31mg according to hydrate) of the creatine phosphate sodium reference substance into a 200mL measuring flask, dissolving by using a mobile phase, fixing the volume to the scale, and shaking up.

Quantitative limiting solution: and determining the concentration of the solution according to the quantitative limit determination item.

Linear solution:

30% linear solution: taking 0.3mL of linear stock solution, putting the linear stock solution into a 100mL volumetric flask, fixing the volume to a scale, and shaking up;

50% linear solution: taking 0.5mL of linear stock solution, putting the linear stock solution into a 100mL volumetric flask, fixing the volume to a scale, and shaking up;

80% linear solution: taking 0.8mL of linear stock solution, putting the linear stock solution into a 100mL volumetric flask, fixing the volume to a scale, and shaking up;

100% linear solution: taking 1.0mL of linear stock solution, putting the linear stock solution into a 100mL volumetric flask, fixing the volume to a scale, and shaking up;

120% linear solution: taking 1.2mL of linear stock solution, putting the linear stock solution into a 100mL volumetric flask, fixing the volume to a scale, and shaking up;

150% linear solution: taking 1.5mL of the linear stock solution, placing the linear stock solution into a 100mL volumetric flask, fixing the volume to the scale, and shaking up.

The results of the linearity test are as follows:

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the correlation coefficient r of the regression line of the linear test result is 0.9998, and the ratio of the Y-axis intercept to the 100% response value is 3.0%, which shows that the linear relation is good and the method is reliable.

Accuracy of

The concentration of creatine phosphate sodium in the leacheate was tested using a validated test method by simulating actual wash conditions. Three concentrations of 50%, 100% and 150% can be selected, each concentration is repeatedly carried out for 3 times, and the recovery rate and the repeatability of the sampling method are verified so as to verify that the sampling method of the leacheate can meet the requirements.

50% concentration: taking about 25mg (about 32.5mg according to hydrate) of creatine phosphate sodium reference substance, precisely weighing, placing in a 100ml measuring flask, adding a mobile phase for dissolving and diluting to a scale, shaking up, taking 1ml to place in a 50ml measuring flask, adding the mobile phase to the scale, shaking up, taking 6ml to place in a 50ml measuring flask, adding the mobile phase to the scale, and shaking up to obtain about 0.6ug of solution containing creatine phosphate sodium in each 1 ml; 3 parts are prepared in parallel.

Concentration of 100%: taking about 50mg (about 65mg according to hydrate) of creatine phosphate sodium reference substance, precisely weighing, placing in a 100ml measuring flask, adding mobile phase for dissolution and diluting to scale, shaking up, taking 1ml to place in a 50ml measuring flask, adding mobile phase to scale, shaking up, taking 6ml to place in a 50ml measuring flask, adding mobile phase to scale, shaking up to obtain about 1.2ug of solution containing creatine phosphate sodium in each 1 ml; 3 parts are prepared in parallel.

Concentration of 150%: taking about 75mg (about 97.5mg according to hydrate) of creatine phosphate sodium reference substance, precisely weighing, placing in a 100ml measuring flask, adding a mobile phase for dissolving and diluting to a scale, shaking up, taking 1ml to place in a 50ml measuring flask, adding the mobile phase to the scale, shaking up, taking 6ml to place in a 50ml measuring flask, adding the mobile phase to the scale, and shaking up to obtain about 1.8ug of solution containing creatine phosphate sodium in each 1 ml; 3 parts are prepared in parallel.

And after the system is stable, injecting a blank solution and a sample solution, and respectively calculating the recovery rate. The chromatograms obtained at different concentrations are shown in detail in fig. 6, 7 and 8. The accuracy test results are as follows:

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the recovery was 104.8% at the 50% level, 99.85% at the 100% level, 102.13% at the 150% level, and 102.27% on average. According to the 2010 version GMP guidelines for medicine production, the average value of the accuracy recovery rate is not less than 75%, the RSD is not more than 10%, and the average recovery rate and the RSD of the current method meet the requirements.

Precision degree

Test solution: taking 50mg (about 65mg according to the hydrate) of creatine phosphate sodium for injection, precisely weighing, placing in a 100ml measuring flask, adding the mobile phase for dissolution and diluting to the scale, shaking up, taking 1ml to place in a 50ml measuring flask, adding the mobile phase to the scale, shaking up, taking 6ml to place in a 50ml measuring flask, adding the mobile phase to the scale, shaking up, and preparing 6 parts by the same method.

The precision test solution spectrum is shown in figure 5. The results of the precision test are as follows:

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the result of the method precision experiment shows that 6 parts of test solution are repeatedly prepared, each part of solution is injected for 1 time, and the result RSD of the creatine phosphate content is 0.95 percent after 6 times of measurement, so that the detection method precision is good.

Stability of solution

The stability of the creatine phosphate sodium solution was demonstrated by analyzing the test solutions at room temperature and 10 c for different periods of time, respectively. The following points in time need to be considered: 0h, 2h, 4h, 6h, 8h and 12h, and if the solution is found to be unstable, the time interval is shortened. The appearance of the solution should be clear; the maximum deviation of the area of the main peak of the control solution at 0h is less than or equal to 5 percent, namely the solution is considered to be stable. The solution stability test results are as follows:

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as can be seen from the data in the table, the solution of the test sample is stable within 12 hours at room temperature or 10 ℃, and the test sample can be used within 12 hours after the sample is prepared.

The verification of the items such as specificity, linearity, accuracy, detection limit, precision, solution stability and the like proves that the method can meet the requirements of a drug cleaning residue detection method required by GMP.

Example 2 continuous production of 3 batches of residual test values

The sampling points were numbered C1-C16. First batch residue detection test results:

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ND indicates no detection, the same is as below.

The results of the second batch residual detection test are as follows:

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results of the third batch residual test:

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and in the three-batch continuous production process, the creatine phosphate sodium residue sampling detection result shows that the chromatographic condition is continuously detected for 3 days, the system is stable, the chromatographic column state is stable, and actually, under the condition of continuous operation for 30 days, the theoretical plate number of the chromatographic column is stable, and the conditions of double peaks, peak broadening and theoretical plate number reduction which are easily generated under the ion pair condition do not occur.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.