CN115078553A

CN115078553A - Method for detecting various substances in cisplatin raw material by using liquid chromatography

Info

Publication number: CN115078553A
Application number: CN201910395272.9A
Authority: CN
Inventors: 徐阳; 姚永年; 窦思红; 刘海倩
Original assignee: Nanjing Pharmaceutical Factory Co ltd
Current assignee: Nanjing Pharmaceutical Factory Co ltd
Priority date: 2019-05-13
Filing date: 2019-05-13
Publication date: 2022-09-20

Abstract

The invention provides a method for detecting various substances in cis-platinum raw material by using liquid chromatography, wherein in the chromatography condition of cis-platinum specified in EP8.0, a methanol component is added into a mobile phase, the pH value of the mobile phase is adjusted to be about 3.6-3.8, and the flow rate is adjusted to be about 0.8 ml/min. The invention can separate and detect the trichloro-aminoplatin, the anti-platinum and the cisplatin at one time in the detection process of the same device, and can improve the separation degree between the peaks of the impurities and the cisplatin.

Description

Method for detecting various substances in cisplatin raw material by using liquid chromatography

Technical Field

The present invention relates to a technique for inspecting a substance by using a liquid chromatograph. Background

Chromatography, also known as chromatography, is a high-performance physical separation technique that is used in analytical chemistry in conjunction with appropriate detection means to produce chromatographic analysis.

Basic principle of chromatographic separation: in chromatography there are two phases, one stationary, which we call the stationary phase; the other phase is continuously passed through the stationary phase, which we call the mobile phase.

Chromatography is a method of separating various substances to be separated by utilizing differences in affinity such as partition coefficients, adsorption capacities, etc. of the substances in two phases.

The mobile phase (gas, liquid) containing the sample is forced through a stationary phase surface fixed in a column or on a plate, immiscible with the flow. As the mixture carried in the mobile phase flows through the stationary phase, the components of the mixture interact with the stationary phase.

Because the difference of the property and structure of each component in the mixture is different from the strength and intensity of the acting force generated between the stationary phases, the mixture is repeatedly distributed and balanced between the two phases along with the movement of the mobile phase, so that the time for retaining each component by the stationary phase is different, and the components sequentially flow out of the stationary phase according to a certain sequence. And the separation and detection of each component in the mixture are realized by combining with a proper post-column detection method.

In chromatography, the mobile phase may be gas or liquid, and thus, Gas Chromatography (GC) and Liquid Chromatography (LC) are classified. The stationary phase may be either a solid or a liquid coated on a solid.

Cisplatin (CDDP) is a heavy metal complex with divalent platinum bound centrally to two chlorine atoms and two ammonia molecules. It is a platinum-containing anti-cancer drug that was first approved for clinical use in the united states in 1978 and is rapidly outstanding for the treatment of cancer. Cisplatin is a cell cycle non-specific drug, has cytotoxicity, can inhibit the DNA replication process of cancer cells, damages the cell membrane structure of the cancer cells, and has strong broad-spectrum anticancer effect. The traditional Chinese medicine composition can clinically show curative effects on various solid tumors such as ovarian cancer, prostatic cancer, testicular cancer, lung cancer, nasopharyngeal carcinoma, esophageal cancer, malignant lymphoma, head and neck squamous cell carcinoma, thyroid cancer, osteogenic sarcoma and the like.

The chromatographic conditions for cisplatin in the current EP8.0 (european pharmacopoeia version 8.0) are as follows:

a chromatographic column: a chromatography column (4 μm, 4.0 × 250 mm) using octyl bonded silica gel as a packing material;

mobile phase: 0.108% sodium octane sulfonate solution (1.08 g sodium octane sulfonate, 1.70g tetrabutylammonium hydrogen sulfate and 2.72g potassium dihydrogen phosphate are dissolved in water, and diluted to 950ml with water, and the solution is adjusted to pH 5.9 with 1mol/L sodium hydroxide, and then diluted to 1000 ml).

Flow rate: 1.0 ml/min;

detection wavelength: 210 nm;

column temperature: 30 ℃;

sample injection amount: 20 mu L of the solution;

test solution: cisplatin (1 mg/ml);

control solution: cisplatin (0.002 mg/ml), antiplatin (0.02 mg/ml), and cisplatin (0.01 mg/ml), calculated by external standard method.

The system applicability is as follows: the separation degree between the antiplatin and trichloro-ammine platinum peaks is not less than 2.5, and the antiplatin peak and the sodium chloride peak can be well separated. The cisplatin peak retention time was about 3.8 minutes, the relative retention time sodium chloride peak was about 0.5, the antiplatinum was about 0.6, and the trichloro-ammine platinum was about 0.7.

This method has certain limitations, which require: the inverse platinum is less than or equal to 2.0 percent; the content of platinum trichloro-ammonia is less than or equal to 1.0 percent; other single impurities are less than or equal to 0.1 percent; the sum of other impurities is less than or equal to 0.5 percent

The structural formulae of the three substances:

cisplatin:

(ii) a And (3) reverse platination:

；

and (3) chloroplatinum:

。

in the import registration standard JX20090030, two sets of liquid phase systems different from the liquid phase systems are adopted for testing the antiplatin and the trichloro-platinum, the test products and the reference products are prepared differently, and the operation is complicated.

Disclosure of Invention

The purpose of the invention is as follows:

the defects that the chromatographic technique is complicated and various substances with similar structures are difficult to detect simultaneously in EP8.0 are overcome, and a novel cisplatin analysis method is provided, so that the analysis method is simple and easy to operate, and cisplatin, trichloroaminoplatinum and antiplatin can be effectively detected in the same liquid phase system. The technical scheme is as follows:

the method for detecting substances in cisplatin raw material by using liquid chromatography of the invention is characterized in that in the chromatographic conditions specified in EP 8.0:

(1) improving the components and the mixture ratio of a mobile phase:

mobile phase: taking sodium octane sulfonate, tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate and methanol (9-11% of methanol is added into the mobile phase); methanol protects the column for the organic phase (3 reagents in the original mobile phase cause column pressure to rise, peak profile to deteriorate, and peak tailing to exacerbate, and methanol is added to delay the adverse effects on the column).

The reason why an organic solvent other than methanol is not used: methanol is used as a reagent for maintaining the chromatographic column, and can be directly used for washing the chromatographic column after the experiment is finished, so that other reagents are prevented from being switched; the organic reagent in the experiment needs to be chromatographically pure (otherwise, the interference of the mixed peaks can occur), and the purity of the rest organic solvent is not enough.

(2) The pH value of the mobile phase is modified to be about 3.6-3.8 by adding weak acid (preferably dilute sulfuric acid which has the same ionic group as tetrabutylammonium hydrogen sulfate in the mobile phase, has less complex components and stronger acidity and can quickly adjust the pH value), and the cisplatin is most stable at the pH value of 3.7.

(3) The flow rate is modified to be about 0.8ml/min, and the speed of 0.8ml/min can better separate the cisplatin, the trichloro-ammine platinum, the cisplatin and other impurities, and improve the separation degree between the impurities and between the peaks of the impurities and the cisplatin.

The other test conditions and test procedures were essentially identical to those described in EP 8.0.

Has the advantages that:

the invention can separate the platinum trichloro-ammonia, the anti-platinum and the cisplatin at one time in the detection process of the same device, and the operation is simple. And the separation degree between impurities and between other impurities and cisplatin peaks can be improved, and the cisplatin peaks can be displayed more clearly.

Drawings

FIG. 1 is a chromatogram of 20. mu.L of control solution 1 obtained by the method of the present invention;

FIG. 2 is a chromatogram of 20. mu.L of control solution 2 obtained by the method of the present invention;

FIG. 3 is a chromatogram of a 20. mu.L sample of cisplatin (1 mg/ml) obtained by the method of the present invention;

FIG. 4 is a chromatogram of 20. mu.L of control solution 1 obtained by the detection method in EP 8.0;

FIG. 5 is a chromatogram of 20. mu.L of control solution 2 obtained by the detection method in EP 8.0;

FIG. 6 is a chromatogram of a 20. mu.L sample of cisplatin (1 mg/ml) obtained by the detection method in EP 8.0.

Detailed Description

Example 1:

this example illustrates the retention time of each peak may drift back and forth for different batches of samples and for variations in column efficiency and pH adjustment of the mobile phase.

The method of claim 5.

All solutions were in 0.9% sodium chloride solution.

A cisplatin sample is precisely weighed and prepared into a solution of 1 mg/ml.

Preparing cisplatin reference substance into 1mg/ml solution; preparing a reverse platinum reference substance into a solution of 0.1 mg/ml; a platinum trichloro-amide control was prepared as a 0.056mg/ml solution.

The cisplatin reference substance, the anti-platinum reference substance and the trichloro-platinum reference substance solution of 0.05ml, 5.0ml and 5.0ml are respectively taken and put into a 25ml measuring flask together, and are diluted to a scale mark by 0.9 percent sodium chloride solution and shaken up to be used as a reference substance solution 1. Wherein the concentrations of cisplatin, anti-platinum and trichloro-ammine platinum are 0.02mg/ml, 0.01mg/ml and 0.002mg/ml respectively.

The control solution 1, 5ml was measured and placed in a 20ml measuring flask, diluted to the mark with 0.9% sodium chloride solution as control solution 2.

The test procedure was as follows:

1) pretreatment of a chromatographic column: and after the base line is stable, taking 1 and 20 mu L of the reference substance solution, injecting the reference substance solution into a liquid chromatograph, and repeatedly injecting samples for 5-10 needles.

2) 20 mu L of the solvent and the independently prepared contraplatinum and trichloro-ammine platinum reference substance solution are respectively taken and injected into a liquid chromatograph for positioning. If an impurity peak with the same retention time as that in the reference substance solution 1 exists after the solvent peak is deducted from the chromatogram, calculating according to a standard method except the peak area; if the impurity peak area is smaller than the cisplatin peak area in the reference substance solution 2, the impurity peak area is ignored.

3) Injecting 20 μ L of reference solution 1, reference solution 2, and cisplatin sample into liquid chromatograph to obtain their chromatograms, shown in figure 1, figure 2, and figure 3.

Limit requirements for test data:

the antiplatin area should not be 0.5 times (1.0%) of the corresponding peak area in control solution 1;

the corresponding peak area (1.0%) in the control solution 1 should not be exceeded by trichloro-ammine platinum;

the other single impurity can not exceed the peak area (0.2%) of cisplatin in the control solution 1; the sum of other impurities should not exceed 1.5 times (0.3%) of the peak area of cisplatin in the control solution 1.

Comparing the peak time, peak type, unknown single impurity position and the like through a chromatogram, and testing the result:

in FIG. 1, the order of appearance is shown for cisplatin, cisplatin and cisplatin.

In FIG. 2, it is determined whether the unknown monohybrid in the sample needs to be calculated (e.g., the peak area of the unknown monohybrid peak is larger than the peak area) based on the size of the cisplatin peak (the third peak).

The solvent peaks overlap with the peaks of other impurities in FIGS. 1-3 compared to FIGS. 4-6, and integration is facilitated.

Comparing figure 3 and figure 6 alone, it can be seen that the separation of the main cisplatin peak from the impurities is significantly improved (manual integration of impurities is required in figure 6).

Claims

1. A method for detecting various substances in a cisplatin raw material by using liquid chromatography, which basically adopts the cisplatin chromatographic conditions and detection steps specified in EP8.0, and is characterized in that: the components of the mobile phase are improved, and the mobile phase adopts a mixed solution of sodium octane sulfonate, tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate and methanol.

2. The method for assaying multiple substances in a cisplatin raw material as claimed in claim 1, wherein: the mobile phase contained 9-11% methanol.

3. The method for assaying multiple substances in a cisplatin raw material as claimed in claim 1 or 2, wherein: adjusting the pH value of the mobile phase to 3.6-3.8.

4. The method for assaying multiple substances in a cisplatin raw material as claimed in claim 3, wherein: the mobile phase was adjusted to pH 3.7 by addition of dilute sulfuric acid.

5. The method for assaying multiple substances in a cisplatin raw material as claimed in claim 3 or 4, wherein: the modified flow rate was around 0.8 ml/min.