阅读说明：本技术 一种以双环铂为有效成分的药物的检测方法 (Method for detecting medicine with dicycloplatin as effective component ) 是由 杨旭清 崔维川 郑建强 于 2014-08-13 设计创作，主要内容包括：本发明提供一种以双环铂为有效成分的药物的检测方法,所述药物为双环铂原料药或双环铂针剂,所述检测方法包括：对双环铂原料药或干燥后的双环铂针剂进行X射线粉末衍射分析,获得第一衍射图谱；确认所述第一衍射图谱中2θ角为10.3-10.7°处和/或2θ角为11.4-11.7°处是否具有衍射峰；若所述第一衍射图谱中2θ角为10.3-10.7°处具有衍射峰和2θ角为11.4-11.7°处不具有衍射峰中的一种或两种情况存在,则所述药物中含有双环铂。本发明提供的检测方法能够对含有双环铂的药物进行有效的定性和/或定量分析。(The invention provides a detection method of a medicine with dicycloplatin as an effective component, wherein the medicine is a dicycloplatin bulk drug or a dicycloplatin injection, and the detection method comprises the following steps: performing X-ray powder diffraction analysis on a dicycloplatin bulk drug or a dried dicycloplatin injection to obtain a first diffraction pattern; confirming whether the first diffraction pattern has diffraction peaks at the 2 theta angle of 10.3-10.7 degrees and/or at the 2 theta angle of 11.4-11.7 degrees; if one or both of a diffraction peak at a2 theta angle of 10.3-10.7 degrees and no diffraction peak at a2 theta angle of 11.4-11.7 degrees in the first diffraction pattern are present, dicycloplatin is contained in the drug. The detection method provided by the invention can be used for effectively carrying out qualitative and/or quantitative analysis on the medicament containing dicycloplatin.)

1. A dicycloplatin crystal, wherein in a diffraction pattern obtained by subjecting the crystal to X-ray powder diffraction analysis, a diffraction peak is present at a2 θ angle of 10.3 to 10.7 ° and/or a diffraction peak is absent at a2 θ angle of 11.4 to 11.7 °.

2. The crystal of claim 1, wherein the X-ray powder diffraction analysis is performed in transmission mode.

3. A method for detecting a medicament with dicycloplatin as an active ingredient comprises the step of directly carrying out mass spectrometry after the medicament is subjected to flow injection and sample injection.

4. The method of claim 3, wherein the mass spectrometry analysis is performed in negative ion mode.

5. The method of claim 3, wherein the drug comprises dicycloplatin if a molecular ion peak of dicycloplatin is detected.

6. A method for detecting the presence and/or strength of hydrogen bonds between carboplatin and cyclosuccinic acid in a solution, wherein said method is performed using nuclear magnetic resonance titration.

7. The method of claim 6, wherein the method comprises fixing the amount of cyclosuccinic acid and varying the amount of carboplatin added and determining the 1HNMR chemical shift of the carboxylicylic acid.

8. The method of claim 6 or 7, wherein hydrogen bonding occurs between the cyclosuccinic acid and carboplatin if the chemical shift of the carboxyhydride of the cyclosuccinic acid shifts from high field to low field with increasing amount of the cyclosuccinic acid.

9. The method of claim 6 or 7, further comprising calculating a binding constant for cyclosuccinic acid to carboplatin.

10. A dicycloplatin crystal form has X-ray powder diffraction pattern as shown in figure 1, a or b.

11. A detection method of a medicament taking dicycloplatin as an active ingredient is characterized in that the medicament is a dicycloplatin bulk drug, and the detection method comprises the following steps:

performing X-ray powder diffraction analysis on a dicycloplatin bulk drug to obtain a first diffraction pattern;

confirming whether the first diffraction pattern has diffraction peaks at the 2 theta angle of 10.3-10.7 degrees and/or at the 2 theta angle of 11.4-11.7 degrees;

if one or both of a diffraction peak at a2 theta angle of 10.3-10.7 degrees and no diffraction peak at a2 theta angle of 11.4-11.7 degrees in the first diffraction pattern are present, dicycloplatin is contained in the drug.

12. A detection method of a medicine with dicycloplatin as an active ingredient is characterized in that the medicine is a dicycloplatin injection, and the detection method comprises the following steps:

freeze-drying the dicycloplatin injection to prepare dicycloplatin powder;

performing X-ray powder diffraction analysis on the dicycloplatin powder to obtain a first diffraction pattern;

confirming whether the first diffraction pattern has diffraction peaks at the 2 theta angle of 10.3-10.7 degrees and/or at the 2 theta angle of 11.4-11.7 degrees;

if one or both of a diffraction peak at a2 theta angle of 10.3-10.7 degrees and no diffraction peak at a2 theta angle of 11.4-11.7 degrees in the first diffraction pattern are present, dicycloplatin is contained in the drug.

13. The detection method according to claim 11 or 12, wherein if the first diffraction pattern has diffraction peaks at both of 10.3 to 10.7 ° in terms of 2 θ and 11.4 to 11.7 ° in terms of 2 θ, the detection method further comprises:

preparing a dicycloplatin standard substance and impurities into mixed powder, and performing X-ray powder diffraction analysis to obtain a second diffraction pattern, wherein the mass percentage of the impurities in the mixed powder is X%;

respectively obtaining the integral intensity A of the diffraction peak at the 2 theta angle of 10.3-10.7 degrees in the first diffraction pattern₁₁And integrated intensity A of diffraction peak at 2 theta angle of 11.4-11.7 DEG₁₂And obtaining the integrated intensity A of the diffraction peak at the 2 theta angle of 10.3-10.7 DEG in the second diffraction pattern, respectively₂₁And integrated intensity A of diffraction peak at 2 theta angle of 11.4-11.7 DEG₂₂Calculating A₁₂/A₁₁And A₂₂/A₂₁；

If A₁₂/A₁₁≤A₂₂/A₂₁The content of impurities in the medicine is less than or equal to X percent, if A₁₂/A₁₁＞A₂₂/A₂₁If the content of impurities in the medicine is more than X%;

wherein the impurities are one or two of carboplatin and cyclosuccinic acid.

14. The detection method according to claim 13, wherein the impurity is carboplatin, the X% is 0.1 to 1%, and if a₁₂/A₁₁Less than or equal to 0.67, the content of carboplatin in the medicament is less than or equal to 1 percent; if A₁₂/A₁₁If the content is more than 0.67, the content of the carboplatin in the medicine is more than 1 percent.

15. The detection method according to claim 11 or 12, wherein the X-ray powder diffraction analysis is performed using an X-ray powder diffractometer equipped with a transmissive rotary sample stage, the method comprising: using monochromatic CuK_αRadiation, tube pressure 40kV, tube flow 40mA, 2 theta scan range 6-55 deg., scan speed 0.6s/step, step size 0.015 deg./step.

16. The detection method according to claim 12, further comprising:

diluting a double-ring platinum injection, performing reverse-phase high performance liquid chromatography detection, and respectively determining the mass contents of carboplatin and cyclosuccinic acid in the double-ring platinum injection by taking a carboplatin solution of 0.025-0.999mg/mL and a cyclosuccinic acid solution of 0.101-3.99mg/mL as standard solutions.

17. The detection method according to claim 16, wherein the mass concentration of dicycloplatin in the diluted dicycloplatin injection is 1mg/mL, and a carboplatin solution of 0.7mg/mL and a cyclosuccinic acid solution of 0.3mg/mL are used as standard solutions.

18. The detection method according to claim 12, further comprising:

diluting a double-ring platinum injection, performing reversed-phase high performance liquid chromatography detection, and respectively determining the molar contents of carboplatin and cyclosuccinic acid and the molar ratio of carboplatin to cyclosuccinic acid in the double-ring platinum injection by using a carboplatin solution of 0.067-2.7mmol/L and a cyclosuccinic acid solution of 0.7-27.8mmol/L as standard solutions.

19. The detection method according to claim 18, wherein the molarity of dicycloplatin in the diluted dicycloplatin injection is 2mmol/L, and a carboplatin solution of 2mmol/L and a cyclosuccinic acid solution of 2mmol/L are used as standard solutions.

20. The detection method according to any one of claims 16 to 19, wherein the conditions of the reverse phase high performance liquid chromatography are as follows: a chromatographic column: AQ-C18, 4.6X 250mm, 5 μm; detection wavelength: 220 nm; sample introduction amount: 10 mu L of the solution; flow rate: 1.0 mL/min; mobile phase: phase A is 20mmol/L sodium dihydrogen phosphate buffer solution with pH of 3.0, and phase B is methanol; gradient conditions: 0-3min, 5% of phase B, 3-8min, 5-60% of phase B, 8-12min, and 60% of phase B.

Technical Field

The invention relates to a drug detection method, in particular to a detection method of a drug taking dicycloplatin as an active ingredient.

Background

Dicycloplatin (Dicycloplatin) is a relatively stable supramolecular compound consisting of carboplatin and cyclobutane diacid through four hydrogen bonds, and the chemical structure of Dicycloplatin (Dicycloplatin) is 1, 1-cyclobutane dicarboxylic acid, diammine platinum (II) and 1, 1-cyclobutane dicarboxylic acid (Bis- [1, 1-cyclobutane dicarboxylic acid diammine nanoplatin (II) ] complex). Research shows that dicycloplatin has the features of wide spectrum, high efficiency, low toxicity, low medicine resistance, low cross medicine resistance, high penetrability, etc. and is one new generation of supermolecule anticancer medicine. For example, the invention patent with publication number CN1311183A discloses acute toxicity, drug effect experiments and clinical tests on dicycloplatin, and the results show that dicycloplatin is not only low in toxicity but also high in anticancer activity, and has significant efficacy on secretory carcinoma, head and neck cancer, nasopharyngeal carcinoma, breast cancer, lung cancer, liver cancer, pancreatic cancer, gastric cancer, intestinal cancer, lymph cancer and the like. At present, no related method for effectively qualitatively and/or quantitatively determining dicycloplatin is reported.

Disclosure of Invention

The invention provides a method for detecting a medicament taking dicycloplatin as an effective component, which is used for carrying out effective qualitative and/or quantitative analysis on dicycloplatin.

The clinical efficacy of dicycloplatin as a new generation of supermolecule anticancer drug proves that the dicycloplatin provides a basis for popularization and application of the supermolecule anticancer drug, and is indispensable for realizing industrial production of the drug and establishing a related quality detection and monitoring system. The supermolecular structure of dicycloplatin comes from hydrogen bond bonding between carboplatin and cyclobutane diacid in molecules, and the inventor finds that most of test methods such as liquid chromatography-mass spectrometry (LC-MS), flow injection-mass spectrometry, infrared spectroscopy, capillary electrophoresis (CZE) and the like cannot accurately determine dicycloplatin in the medicine taking dicycloplatin as an effective component due to the supermolecular structural characteristics of dicycloplatin, particularly, dicycloplatin compounds cannot be distinguished from a physical mixture of carboplatin and cyclobutane diacid, and the measured result is easy to generate false images.

Therefore, the invention provides a detection method of a medicament taking dicycloplatin as an active ingredient, wherein the medicament is a dicycloplatin bulk drug, and the detection method comprises the following steps:

performing X-ray powder diffraction analysis on a dicycloplatin bulk drug to obtain a first diffraction pattern;

confirming whether the first diffraction pattern has diffraction peaks at the 2 theta angle of 10.3-10.7 degrees and/or at the 2 theta angle of 11.4-11.7 degrees;

if one or both of a diffraction peak at a2 theta angle of 10.3-10.7 degrees and no diffraction peak at a2 theta angle of 11.4-11.7 degrees in the first diffraction pattern are present, dicycloplatin is contained in the drug.

The inventors have found that the qualitative analysis of dicycloplatin is effectively carried out using X-ray powder diffraction analysis (XRPD). Wherein, in a diffraction pattern obtained in a transmission mode at room temperature, a diffraction peak at a2 theta angle of 10.3-10.7 degrees (about 10.51 degrees) can indicate that dicycloplatin exists, and the physical mixture of the cyclosuccinic acid, the carboplatin, the cyclosuccinic acid and the carboplatin does not have a diffraction peak at the position; furthermore, the absence of a diffraction peak at a2 θ angle of 11.4 to 11.7 ° (around 11.55 °) may also indicate the presence of dicycloplatin, where cyclosuccinic acid, carboplatin, a physical mixture of cyclosuccinic acid and carboplatin all have diffraction peaks. Therefore, X-ray powder diffraction analysis can be used to qualitatively detect, in particular, powdery dicycloplatin-containing drugs, such as dicycloplatin bulk drug.

For a solution containing dicycloplatin (e.g., a dicycloplatin injection), the inventors found that most detection conditions (e.g., liquid chromatography analysis, etc.) destroy hydrogen bonds in the dicycloplatin molecule, so that the dicycloplatin cannot exist in a supramolecular form, i.e., dicycloplatin is completely dissociated into carboplatin and cyclosuccinic acid in the solution, and thus the dicycloplatin in the dicycloplatin solution cannot be effectively detected qualitatively, for example, cannot be distinguished from a mixed solution of carboplatin and cyclosuccinic acid.

The inventor of the present invention has studied and found that, although dicycloplatin is completely dissociated into carboplatin and cyclosuccinic acid in a solution state (such as an aqueous solution), the solution has only a small amount of carboplatin and cyclosuccinic acid after being lyophilized, and in a diffraction pattern obtained by performing X-ray powder diffraction analysis on a water-soluble lyophilized powder of a dicycloplatin solution, a diffraction peak is present at a2 theta angle of 10.3 to 10.7 ° and/or a diffraction peak (hereinafter referred to as a characteristic peak) is absent at a2 theta angle of 11.4 to 11.7 °, that is, a water-soluble lyophilized powder of a dicycloplatin solution has the same characteristic peak as a dicycloplatin bulk drug; the water-soluble freeze-dried powder of the physical mixture of the cyclosuccinic acid and the carboplatin forms a small amount of dicycloplatin, but shows different characteristic peaks, namely no diffraction peak at a2 theta angle of 10.3-10.7 degrees and/or a diffraction peak at a2 theta angle of 11.4-11.7 degrees. Therefore, the use of XRPD can effectively perform qualitative detection of dicycloplatin in a dicycloplatin solution, thereby distinguishing it from a mixed solution of carboplatin and cyclosuccinic acid.

Therefore, the invention also provides a detection method of a medicament taking dicycloplatin as an active ingredient, wherein the medicament is a dicycloplatin injection, and the detection method comprises the following steps:

freeze-drying the dicycloplatin injection to prepare dicycloplatin powder;

performing X-ray powder diffraction analysis on the dicycloplatin powder to obtain a first diffraction pattern;

confirming whether the first diffraction pattern has diffraction peaks at the 2 theta angle of 10.3-10.7 degrees and/or at the 2 theta angle of 11.4-11.7 degrees;

if one or both of a diffraction peak at a2 theta angle of 10.3-10.7 degrees and no diffraction peak at a2 theta angle of 11.4-11.7 degrees in the first diffraction pattern are present, dicycloplatin is contained in the drug.

In the invention, the medicament contains dicycloplatin which is an effective component of the medicament; in addition, the medicament may also contain carboplatin and/or cyclosuccinic acid which is not combined into dicycloplatin or is formed by the dissociation of dicycloplatin, and the carboplatin and/or the cyclosuccinic acid are defined as impurities in the invention. Specifically, the invention can carry out qualitative detection on dicycloplatin in the medicine by three ways, namely: 1) if the first diffraction pattern has a diffraction peak at a2 theta angle of 10.3-10.7 degrees, the medicament contains dicycloplatin; 2) if the first diffraction pattern has no diffraction peak at the 2 theta angle of 11.4-11.7 degrees, the medicament contains dicycloplatin; 3) if the first diffraction pattern has a diffraction peak at a2 theta angle of 10.3-10.7 degrees and no diffraction peak at a2 theta angle of 11.4-11.7 degrees, dicycloplatin is contained in the drug. And, the above cases 2) and 3) indicate that the drug does not contain the impurities.

Further, the present inventors have found that the impurity, particularly carboplatin, exhibits a diffraction peak at a2 θ angle of 11.4 to 11.7 ° for a drug containing dicycloplatin as an active ingredient. Accordingly, the present invention also provides a method for quantitatively detecting said impurities in a pharmaceutical, comprising: if the first diffraction pattern has diffraction peaks at both of angles 2 theta of 10.3-10.7 ° and angles 2 theta of 11.4-11.7 °, the detection method further comprises:

preparing a dicycloplatin standard substance and impurities into mixed powder, and performing X-ray powder diffraction analysis to obtain a second diffraction pattern, wherein the mass percentage of the impurities in the mixed powder is X%;

respectively obtaining the integral intensity A of the diffraction peak at the 2 theta angle of 10.3-10.7 degrees in the first diffraction pattern₁₁And integrated intensity A of diffraction peak at 2 theta angle of 11.4-11.7 DEG₁₂And obtaining the integrated intensity A of the diffraction peak at the 2 theta angle of 10.3-10.7 DEG in the second diffraction pattern, respectively₂₁And integrated intensity A of diffraction peak at 2 theta angle of 11.4-11.7 DEG₂₂Calculating A₁₂/A₁₁And A₂₂/A₂₁；

If A₁₂/A₁₁≤A₂₂/A₂₁The content of impurities in the medicine is less than or equal to X percent, if A₁₂/A₁₁＞A₂₂/A₂₁If the content of impurities in the medicine is more than X%;

wherein the impurities are one or two of carboplatin and cyclosuccinic acid.

In the present invention, the content of impurities in the drug can be evaluated by using the content of carboplatin in the drug (the impurity is carboplatin) as an evaluation index of impurities in the drug, and particularly, the X% may be 0.1 to 1%. At this time, if A₁₂/A₁₁Less than or equal to 0.67, the content of carboplatin in the medicament is less than or equal to 1 percent; if A₁₂/A₁₁If the content is more than 0.67, the content of carboplatin in the medicine is more than 1 percent; further, if A₁₂/A₁₁Less than or equal to 0.55 percent, the content of carboplatin in the medicament is less than or equal to 0.5 percent; if A₁₂/A₁₁If the content is more than 0.55 percent, the content of the carboplatin in the medicine is more than 0.5 percent; still further, if A₁₂/A₁₁Less than or equal to 0.5, the content of carboplatin in the medicament is less than or equal to 0.1 percent; if A₁₂/A₁₁If the content is more than 0.5 percent, the content of the carboplatin in the medicine is more than 0.1 percent. In the invention, the detection limit of the content of carboplatin in the medicament can reach 0.1%.

In a specific embodiment of the present invention, the X-ray powder diffraction analysis is performed using an X-ray powder diffractometer equipped with a transmissive rotary sample stage, the method comprising: using monochromatic CuK_αRadiation, tube pressure 40kV, tube flow 40mA, 2 theta scan range 6-55 deg., scan speed 0.6s/step, step size 0.015 deg./step. The first and second diffraction patterns are both obtained in transmission mode at room temperature.

Further, the detection method of the present invention further comprises quantitative detection of a drug containing dicycloplatin as an active ingredient, that is:

the method comprises the steps of preparing a dicycloplatin raw material medicine into a solution, diluting a dicycloplatin injection, and then carrying out reversed-phase high performance liquid chromatography detection, wherein a carboplatin solution of 0.025-0.999mg/mL and a cyclobutane acid solution of 0.101-3.99mg/mL are used as standard solutions, and the mass contents of carboplatin and cyclobutane acid in the dicycloplatin injection are respectively determined.

Further, the mass concentration of dicycloplatin in the solution or the diluted dicycloplatin injection is 1mg/mL, and a carboplatin solution of 0.7mg/mL and a cyclosuccinic acid solution of 0.3mg/mL are used as standard solutions. The determination of the mass content can be carried out by an external standard method.

Other quantitative detection methods may also be employed in the present invention, for example:

preparing a dicycloplatin bulk drug into a solution, diluting a dicycloplatin injection, and then carrying out reversed-phase high performance liquid chromatography detection, and simultaneously determining the molar contents of carboplatin and cyclosuccinic acid and the molar ratio of carboplatin to cyclosuccinic acid in the dicycloplatin injection by taking a 0.067-2.7mmol/L carboplatin solution and a 0.7-27.8mmol/L cyclosuccinic acid solution as standard solutions.

Further, the molarity of dicycloplatin in the solution or the diluted dicycloplatin injection is made to be 2mmol/L, and a carboplatin solution of 2mmol/L and a cyclosuccinic acid solution of 2mmol/L are used as standard solutions.

In the specific method of the invention, the conditions of the reversed-phase high performance liquid chromatography are as follows: a chromatographic column: AQ-C18, 4.6X 250mm, 5 μm; detection wavelength: 220 nm; sample introduction amount: 10 mu L of the solution; flow rate: 1.0 mL/min; mobile phase: phase A is 20mmol/L sodium dihydrogen phosphate buffer solution with pH of 3.0, and phase B is methanol; gradient conditions: 0-3min, 5% of phase B, 3-8min, 5-60% of phase B, 8-12min, and 60% of phase B.

The method for detecting the medicine with the dicycloplatin as the active ingredient can perform qualitative and/or quantitative analysis on the dicycloplatin in the medicine, can eliminate false images caused by other determination methods, can truly and intuitively perform effective determination on the dicycloplatin, and particularly can distinguish the dicycloplatin from a physical mixture of carboplatin and cyclosuccinic acid, and can effectively determine the content of impurities (calculated by the carboplatin) in the medicine, thereby laying a foundation for quality control of the dicycloplatin medicine.

Drawings

FIG. 1 is an X-ray powder diffraction pattern of sample 1 in example 1 of the present invention in transmission mode and reflection mode, wherein a is transmission mode and b is reflection mode;

FIG. 2 is an X-ray powder diffraction pattern of each sample of example 1 of the present invention;

FIG. 3 is an X-ray powder diffraction pattern of each sample of example 2 of the present invention;

FIG. 4 is a DSC curve of each sample of example 4 of the present invention;

FIG. 5 is an infrared spectrum of sample 1 of comparative example 1 of the present invention;

FIG. 6 is an infrared spectrum of sample 4 of comparative example 1 of the present invention;

FIG. 7 is a mass spectrum of a bicyclic platinum injection of comparative example 2 of the present invention in positive ion mode;

FIG. 8 is a mass spectrum of a bicyclic platinum injection of comparative example 2 of the present invention in the anion mode.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings and the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The samples adopted in the embodiment of the invention comprise:

dicycloplatin bulk drug (hereinafter referred to as sample 1): four batches, all purchased from Xingda scientific systems, Beijing, were numbered: 20130115-1, 20130115-2, 2013015-3, and 20130216;

cyclobutanedioic acid (sample 2 below): the purity is more than 99.5 percent;

carboplatin (hereinafter sample 3): the purity is more than 99 percent;

physical mixture of cyclosuccinic acid and carboplatin (sample 4 below): mixing 19mg of cyclosuccinic acid and 50mg of carboplatin, and slightly grinding the mixture into powder in a mortar, wherein the molar ratio of the cyclosuccinic acid to the carboplatin is about 1: 1; in the present invention, a mixture obtained by directly mixing cyclosuccinic acid and carboplatin is defined as a physical mixture of cyclosuccinic acid and carboplatin, and hydrogen bonds are not formed between the cyclosuccinic acid and the carboplatin in the mixture;

water-soluble lyophilized powder of dicycloplatin bulk drug (hereinafter referred to as sample 5): dissolving 60mg of dicycloplatin bulk drug with 2.5ml of deionized water, standing at room temperature for 2h, freezing in a refrigerator at-70 ℃ for 4h, transferring to a freeze dryer (the temperature of a cold trap is-69 ℃, and the vacuum is 10pa), and freeze-drying for 12h to prepare white freeze-dried powder;

water-soluble lyophilized powder of a physical mixture of cyclosuccinic acid and carboplatin (sample 6 below): preparing the physical mixture of the cyclobutane succinic acid and the carboplatin into freeze-dried powder according to the method;

dicycloplatin standard and injection: purchased from Xingda scientific systems, Beijing, wherein: the dicycloplatin injection is a 5mL ampoule, and the content of dicycloplatin is 1% (namely 1g/100 mL);

freeze-dried powder of dicycloplatin injection: the dicycloplatin injection is prepared into freeze dried powder according to the method.