CN110596282B

CN110596282B - Nedaplatin compound, separation method and application

Info

Publication number: CN110596282B
Application number: CN201810605672.3A
Authority: CN
Inventors: 李悦; 还传静; 陆静; 蔡鹏俊
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2018-06-13
Filing date: 2018-06-13
Publication date: 2021-08-13
Anticipated expiration: 2038-06-13
Also published as: CN110596282A

Abstract

The invention discloses a nedaplatin compound, a separation method and application. The invention discloses a nedaplatin compound shown as a formula I or a salt thereof. The invention also disclosesA method for separating nedaplatin bulk drugs comprises the following steps: eluting the nedaplatin crude drug in a chromatographic column. The separation method can effectively separate related substances of the nedaplatin crude drug, has strong specificity, high sensitivity and strong practicability, can simultaneously further obtain mass spectrum fragment information of the drug through electrospray multistage mass spectrum detection, analyze a cracking way, verify an analysis process through high-resolution mass spectrum, and complete the structural identification and confirmation of unknown impurities in the nedaplatin crude drug, thereby controlling the drug quality of the nedaplatin and laying a good foundation for the research of the unknown impurities of the nedaplatin; the nedaplatin compound of the invention is a necessity for controlling the quality of nedaplatin raw material medicine.

Description

Nedaplatin compound, separation method and application

Technical Field

The invention relates to a nedaplatin compound, a separation method and application.

Background

The platinum drugs are anti-tumor drugs, and the platinum drugs on the market are divalent platinum compounds, and have cis-structures, a pair of amine ligands and a pair of anion leaving groups, so that the platinum drugs are hydrated in a cell environment with low chloride ion concentration, and the anion groups are partially or completely dissociated to form active hydrates, so that the platinum drugs are combined with DNA (deoxyribonucleic acid), mediate the transcription of the DNA and play a role in resisting tumors. Nedaplatin, a second-generation platinum drug, was marketed in japan in 1995, and is useful for the treatment of head and neck tumors, non-small cell lung cancer, and the like. Nedaplatin is not recorded in pharmacopoeia of various countries, and the research reports on impurities are few.

The method for evaluating the quality of Niuyaman (Niuyaman. Nedaplatin, university of major design [ D ],. 2014, Master thesis) reported that the method for detecting impurities in Nedaplatin raw material drugs adopts a liquid phase method, and adopts octadecylsilane bonded silica gel as a filling agent (CAPCELL PAK C184.6.6X 250mm, 5 μm), a column temperature of 40 ℃, a detection wavelength of 220nm and a mobile phase of octane sodium sulfonate solution-acetonitrile (91: 9). Due to the strong polarity of nedaplatin, the method adopts a conventional C18 column, an ion pair reagent is used as a mobile phase, the components are weakly retained on a chromatographic column, the peak emergence speed of the main component is fast, the separation of related substances is not facilitated, and the method is not suitable for an LC-MS method.

The LC-MS method integrates efficient separation of HPLC and specificity, sensitivity and rapidness of mass spectrum to carry out structural identification of unknown impurities, and is better than the HPLC method.

The technical requirement of drug registration for human beings, which is the guidance rule promulgated by the International harmonization (ICH), requires structural identification of impurities with the content of more than 0.10 percent in the bulk drug. The "guiding principle of analysis of impurities in medicine" in pharmacopoeia of the people's republic of China (2015 edition) "clearly indicates that" the structure of impurities with apparent content of 0.1% or more and impurities with strong biological action or toxic impurities with apparent content of less than 0.1% are determined or confirmed ". The high performance liquid chromatography is used as a separation system and is combined with mass spectrometry, so that a complex sample can be effectively separated, trace and trace components in the complex sample can be detected, and related molecular mass and structure information can be provided.

At present, no report for separating and detecting related substances of nedaplatin by adopting an LC-MS method is seen at home and abroad. Therefore, it is necessary to establish a method for LC-MS separation and detection of nedaplatin-related substances and identify nedaplatin-related substances.

Disclosure of Invention

The invention aims to overcome the defects of lack of simultaneous separation, detection and identification and structure confirmation of related substances in nedaplatin raw material medicines in the prior art, and provides a nedaplatin compound, a separation method and application thereof. The separation method can effectively separate related substances of the nedaplatin crude drug, has strong specificity, high sensitivity and strong practicability, can simultaneously further obtain mass spectrum fragment information of the drug through electrospray multi-stage mass spectrum detection, analyze a cracking way, verify an analysis process through high-resolution mass spectrum, and finish the structural identification and confirmation of an unknown impurity in the nedaplatin crude drug, thereby controlling the drug quality of the nedaplatin and laying a good foundation for the research of the unknown impurity of the nedaplatin; the nedaplatin compound of the invention is a necessity for controlling the quality of nedaplatin raw material medicine.

The invention provides a nedaplatin compound or salt thereof as shown in formula I:

the nedaplatin salt shown in the formula I is a salt formed by the nedaplatin compound shown in the formula I and acid. The acid may be an inorganic or organic acid capable of forming a salt with an amine compound, as is conventional in the art.

The invention also provides a separation method of the nedaplatin bulk drug, which comprises the following steps:

eluting the nedaplatin bulk drug in a chromatographic column;

the chromatographic column is a HILIC chromatographic column;

the eluted mobile phase is a mixed solution of a mobile phase A and a mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B is 25: 75-35: 65 (for example, 25:75, 30:70 or 35: 65);

the mobile phase A is a n-hexylamine aqueous solution which is adjusted to have a pH value of 5.3-5.7 (such as 5.3, 5.5 or 5.7) by formic acid and has a volume fraction of 0.05% -0.2% (such as 0.05%, 0.10% or 0.2%); the mobile phase B is acetonitrile;

the nedaplatin bulk drug comprises nedaplatin and a nedaplatin compound or salt thereof shown in formula I,

in the separation method, the nedaplatin bulk drug can also comprise nedaplatin compounds or salts thereof except the compound shown in the formula I; preferably, the nedaplatin active pharmaceutical ingredient can be prepared by the following method: reacting diiododiammineplatinum with glycolic acid.

In the present invention, the nedaplatin-based compound refers to a pure compound or a mixture of compounds similar to nedaplatin.

In the separation method, the nedaplatin crude drug can be injected by adopting a conventional method in the field, and is preferably injected in the form of an aqueous solution of the nedaplatin crude drug; preferably, the nedaplatin bulk drug is dissolved in water to form an aqueous solution of the nedaplatin bulk drug.

The concentration of the aqueous solution of the nedaplatin crude drug can be a concentration conventional in the art, for example, 0.5g/L to 1.5g/L, and further, for example, 0.8g/L to 1.2g/L (e.g., 1.0 g/L).

The sample amount of the aqueous solution of the nedaplatin crude drug can be a sample amount conventional in the art, for example, 10 μ L to 50 μ L, and for example, 15 μ L to 30 μ L (for example, 20 μ L).

In the separation method, the HILIC column may be a HILIC column conventional in the art, preferably TSK GEL AMIDE-HR80 (4.6X 250mm, 5 μm) column.

In the separation method, high performance liquid chromatography which is conventional in the art, preferably, a UPLC-3100 model ultra high performance liquid chromatography-mass spectrometry system of Waters or a 2695 model high performance liquid chromatography-mass spectrometry system of Waters can be used.

In the separation method, the flow rate of the separation method can be the flow rate conventional in the field; for example, 0.8mL/min to 1.2mL/min, and for example, 1.0 mL/min.

In the separation process, the column temperature of the separation process may be a column temperature conventional in the art, for example, 20 ℃ to 40 ℃, and further for example, 25 ℃ to 35 ℃ (e.g., 25 ℃, 30 ℃, or 35 ℃).

In the separation method, the ultraviolet absorption wavelength detected by the separation method can be an ultraviolet absorption wavelength conventional in the art, such as 215-225 nm, and further such as 215nm, 220nm or 225 nm.

The separation method can be used as a method for detecting and identifying related substances of nedaplatin raw material medicines or used for purifying to obtain a single platinum substance, and preferably, the separation method is used as an enrichment preparation method of nedaplatin compounds and is used for collecting the nedaplatin compounds. More preferably, the separation method is used as an enrichment preparation method of the nedaplatin compound shown in the formula I, and the nedaplatin compound shown in the formula I is collected.

In the invention, the related substances of the nedaplatin bulk drug refer to initial raw materials, intermediates, polymers, side reaction products and degradation impurities which may exist in the process of preparing and storing the drug.

The invention also provides an application of the nedaplatin compound or the salt thereof shown as the formula I as a standard substance in the quality control of nedaplatin.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the invention establishes an LC-MS detection method of nedaplatin bulk drug for the first time, which is used for detecting related substances and verifies the method. The method can be used for separating and detecting related substances in the nedaplatin bulk drug and carrying out structure identification on the related substances in the nedaplatin bulk drug by a mass spectrometry method. The method has strong specificity, high sensitivity and strong practicability, and can be applied to the quality control and process optimization of medicaments.

Drawings

FIG. 1 is a diagram of LC-MS obtained in example 1;

FIG. 2 is a first order mass spectrum of nedaplatin compounds of formula I;

FIG. 3 is a second order mass spectrum of nedaplatin compounds of formula I;

FIG. 4 is a first order mass spectrum of nedaplatin;

FIG. 5 is a secondary mass spectrum of nedaplatin;

FIG. 6 is a diagram of LC-MS obtained in example 2;

FIG. 7 is a diagram of LC-MS obtained in example 3;

FIG. 8 is a diagram of LC-MS obtained in example 4;

FIG. 9 is a diagram of LC-MS obtained in example 5;

FIG. 10 is a diagram of LC-MS obtained in example 6;

FIG. 11 is a diagram of LC-MS obtained in example 7;

FIG. 12 is a diagram of LC-MS obtained in comparative example 1;

FIG. 13 is a LC-MS graph obtained in comparative example 2;

FIG. 14 is a diagram of LC-MS obtained in comparative example 3;

FIG. 15 is a diagram of LC-MS obtained in comparative example 4;

FIG. 16 is a graph of LC-MS obtained in comparative example 5.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The reagents used in the present invention are as follows:

acetonitrile (HPLC grade, J.T.Baker Co.), formic acid (analytical grade, Shanghai Hu laboratory instruments Co., Ltd.), n-hexylamine (analytical grade, Shanghai Allantin Biotech Co., Ltd.); concentrated hydrochloric acid (analytically pure, Shanghai Hui laboratory instruments Co., Ltd.); solid sodium hydroxide (guaranteed reagent, Shanghai Aladdin Biotechnology Co., Ltd.); 30 wt% hydrogen peroxide (Shanghai laboratory instruments, Inc.); the water is Wahaha purified water (passing through a 0.22 mu m water film); the preparation method of the nedaplatin crude drug specifically refers to Chinese patent document with publication number CN 102417522A, and the specific synthetic route is as follows:

the instrument used in the invention is as follows:

an UPLC-3100 type ultra-high performance liquid chromatography-mass spectrometry system [ equipped with an ultraviolet detector, an ESI source and a MassLynx 4.1 data processing system ] and a Waters 2695 type high performance liquid chromatography-mass spectrometry system [ equipped with an ultraviolet detector, an ESI source and a MassLynx 4.1 data processing system ], all of which are products of Waters corporation of America;

mettler AE-163 electronic balance.

Example 1

1mg of nedaplatin raw material medicine is dissolved in 1mL of water, and the sample volume is 20 mu L.

Chromatographic conditions are as follows:

a chromatographic column: TSK GEL AMIDE-HR80 hydrophilic chromatographic column 4.6X 250mm, 5 μm

Detection wavelength: 220nm

Flow rate: 1mL/min

Column temperature: 30 deg.C

Mobile phase A: 0.1% n-hexylamine in water (formic acid adjusted pH to 5.50)

Mobile phase B: acetonitrile

The volume ratio of mobile phase a to mobile phase B was 30: 70.

Mass spectrum conditions: electrospray ionization source, positive ion detection.

The HPLC fractions were passed through a UV detector and then detected by MS in 2:1 split.

The LC-MS chart obtained in this example is shown in FIG. 1, and the components are shown in Table 1, the main peak retention time is appropriate, and the peak shape is good. The related substances can be well separated from the main peak. As can be seen from the figure, the method can be used for detecting nedaplatin and related substances thereof, and can be applied to monitoring and quality control of a nedaplatin bulk drug synthesis process.

TABLE 1

Note: % area is the percentage content measured by area normalization.

The related substances in table 1, namely the nedaplatin compound shown in formula I of the present invention, have a content of more than 0.1% by area normalization method. The mass-to-charge ratio is 317 through mass spectrum detection, and the mass-to-charge ratio is M + H]⁺Peak, the molecular weight of the nedaplatin-based compound was determined to be 316. Further performing MS/MS analysis on the 317 peak to obtain a secondary mass spectrum, and simultaneously comparing the secondary mass spectrum with a primary mass spectrum and a secondary mass spectrum of nedaplatin, wherein the primary mass spectrum of the nedaplatin compound shown as the formula I is shown as a figure 2, the secondary mass spectrum of the nedaplatin compound shown as the formula I is shown as a figure 3, the primary mass spectrum of the nedaplatin is shown as a figure 4, and the secondary mass spectrum of the nedaplatin is shown as a figure 5.

Compared with the primary mass spectrum of nedaplatin, the primary mass spectrum of nedaplatin compounds shown in formula I has m/z 227.00 and m/z 209.98 as common fragment ions. The m/z 227.00, m/z 209.98 ions result from the cleavage and rearrangement of the Pt-O bond, and therefore the amine ligand moiety is consistent with nedaplatin. Compared with the molecular weight of nedaplatin, the relative molecular mass of the nedaplatin compound has the difference of 14Da, and when the nedaplatin compound is analyzed from a high-resolution mass spectrogram, a CO is separated from m/z 318 ions₂Neutral molecule, producing fragment ion CH of m/z 274.02₇O₂N₂Pt⁺Consistent with the inference. The structure of the nedaplatin compound shown in formula I and the secondary mass spectrum cracking pathway are shown as follows:

the cleavage pathway was verified by high resolution mass spectrometry, and the high resolution data is shown in table 2.

TABLE 2

The source of the nedaplatin-based compound represented by formula I is assumed to be a by-product produced by oxidizing sodium glycolate in the raw material to sodium oxalate.

Example 2

Experiments were conducted using similar experimental materials and instrument conditions and experimental procedures as in example 1, except that the volume ratios of mobile phase a and mobile phase B were adjusted to 25:75 and 35:65, respectively, and the resulting liquid phase diagram is shown in fig. 6, and the contents, the number of theoretical plates, the tailing factor, the degree of separation, and the like are shown in table 3. The change of the proportion of the mobile phase affects the retention time of each component, the retention time is in direct proportion to the proportion of the water phase, the influence on the theoretical plate number is small, the tailing factors are all less than 2.0, the separation degree is more than 1.5, and the separation method shows that the proportion of the mobile phase is within the variation range of +/-5 percent, and the separation method has good durability.

TABLE 3

Note: the content is the percentage content measured by an area normalization method.

Example 3

Experiments were conducted using similar experimental materials and instrument conditions and experimental procedures as in example 1, except that the mobile phase a had pH values of 5.30 and 5.70, respectively, and the obtained liquid phase diagram was as shown in fig. 7, and the contents, the number of theoretical plates, the tailing factor, the degree of separation, and the like thereof are shown in table 4. The test result shows that the change of the pH value of the mobile phase A is within the variation range of +/-0.2, the change of the theoretical plate number is small, and the influence on the detection of nedaplatin is small.

TABLE 4

Example 4

Experiments were conducted using similar experimental materials and instrument conditions and experimental procedures as in example 1, except that in mobile phase a, the concentration of n-hexylamine aqueous solution was divided into 0.05% and 0.2%, and the resulting liquid phase diagram is shown in fig. 8, and the contents, the number of theoretical plates, the tailing factor, the degree of separation, and the like are shown in table 5. The test result shows that the concentration of the mobile phase buffer salt is within the variation range of 0.05-0.2%, the change of the theoretical plate number is not large, the separation degree is good, and the durability of the separation method is good.

TABLE 5

Example 5

Experiments were conducted using experimental materials and instrument conditions and experimental procedures similar to those of example 1, except that the column temperatures were 25 ℃ and 35 ℃ respectively, and the resulting liquid phase diagram is shown in FIG. 9, and the contents, the number of theoretical plates, the tailing factor, the degree of separation, and the like are shown in Table 6. The test result shows that the change of the column temperature has little influence on the theoretical plate number, the tailing factors are less than 2.0, the separation degree is more than 1.5, and the column temperature has less influence on the detection of nedaplatin within the variation range of +/-5 ℃.

TABLE 6

Example 6

Nedaplatin LC-MS method detection limit investigation

Precisely weighing about 1mg of nedaplatin raw material medicine in a 50mL volumetric flask, adding water to dissolve the nedaplatin raw material medicine, diluting the nedaplatin raw material medicine with water to a constant volume, further diluting the nedaplatin raw material medicine step by step to prepare a sample solution of 0.0004mg/mL, injecting 20 mu L of the sample solution, and recording a chromatogram.

Chromatographic conditions are as follows:

Detection wavelength: 220nm

Flow rate: 1mL/min

Column temperature: 30 deg.C

Mobile phase A: 0.1% n-hexylamine in water (formic acid adjusted pH to 5.50)

Mobile phase B: acetonitrile

The volume ratio of mobile phase a to mobile phase B was 30: 70.

The test results are shown in fig. 10, and the results show that when the signal-to-noise ratio is about 3 (n ═ 3), the detection limit of nedaplatin bulk drug is 8 ng.

Example 7

Experiments were conducted using experimental materials similar to example 1, with the exception that the ultraviolet absorption wavelengths in the chromatographic conditions were 215nm and 225nm, respectively, and the resulting liquid phase diagram is shown in FIG. 11, and the contents, theoretical plate number, tailing factor, degree of separation, and the like are shown in Table 7. The test result shows that the change of the ultraviolet absorption wavelength has little influence on the theoretical plate number, the tailing factors are less than 2.0, the separation degree is more than 1.5, and the ultraviolet absorption wavelength has little influence on the detection of nedaplatin.

TABLE 7

Comparative example 1

Chromatographic conditions are as follows:

a chromatographic column: capcell PAK C184.6X 250mm, 5 μm

Detection wavelength: 220nm

Flow rate: 0.7mL/min

Column temperature: 30 deg.C

Mobile phase A: 0.1% ammonia (formic acid adjusted pH to 6.00)

Mobile phase B: methanol

The volume ratio of mobile phase a to mobile phase B was 70: 30.

The LC-MS chart obtained in this example is shown in FIG. 12, and it is clear that the main component is hardly retained.

Comparative example 2

Chromatographic conditions are as follows:

a chromatographic column: capcell PAK C184.6X 250mm, 5 μm

Detection wavelength: 220nm

Flow rate: 0.7mL/min

Column temperature: 30 deg.C

Mobile phase A: 0.1% ammonium acetate (formic acid adjusted pH to 6.00)

Mobile phase B: acetonitrile

The volume ratio of mobile phase a to mobile phase B was 70: 30.

The LC-MS chart obtained in this example is shown in FIG. 13, and it is clear that the main component is hardly retained.

Comparative example 3

Chromatographic conditions are as follows:

Detection wavelength: 220nm

Flow rate: 1mL/min

Column temperature: 30 deg.C

Mobile phase A: 0.1% ammonia (formic acid adjusted pH to 5.50)

Mobile phase B: methanol

The volume ratio of mobile phase a to mobile phase B was 80: 20.

The LC-MS chart obtained in this example is shown in fig. 14, and it is understood from the chart that the main peak and the impurity peak cannot be separated.

Comparative example 4

Chromatographic conditions are as follows:

Detection wavelength: 220nm

Flow rate: 1mL/min

Column temperature: 30 deg.C

Mobile phase A: 20mM ammonium acetate (pH adjusted to 5.50 with ammonia)

Mobile phase B: methanol

The volume ratio of mobile phase a to mobile phase B was 80: 20.

As shown in fig. 15, the LC-MS chart obtained in this example shows that the main peak and the impurity peak cannot be separated from each other.

Comparative example 5

Chromatographic conditions are as follows:

Detection wavelength: 220nm

Flow rate: 1mL/min

Column temperature: 30 deg.C

Mobile phase A: 0.1% ammonia (formic acid adjusted pH to 5.50)

Mobile phase B: methanol

The volume ratio of mobile phase a to mobile phase B was 30: 70.

The LC-MS chart obtained in this example is shown in FIG. 16, and it is clear that the main component is hardly retained.

Claims

1. A method for separating nedaplatin bulk drug is characterized by comprising the following steps:

eluting the nedaplatin bulk drug in a chromatographic column;

wherein the chromatographic column is a HILIC chromatographic column which is TSK GEL AMIDE-HR 80;

the eluted mobile phase is a mixed solution of a mobile phase A and a mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B is 25: 75-35: 65;

the mobile phase A is a n-hexylamine aqueous solution with the pH value of 5.3-5.7 adjusted by formic acid and the volume fraction of 0.05% -0.2%; the mobile phase B is acetonitrile;

the flow rate of the separation method is 0.8mL/min to 1.2 mL/min;

the column temperature of the separation method is 20-40 ℃;

the ultraviolet absorption wavelength in the detection of the separation method is 215-225 nm;

2. the method for separating nedaplatin drug substance according to claim 1, wherein the nedaplatin drug substance is prepared according to the following method: reacting diiododiammineplatinum with glycolic acid;

and/or the nedaplatin bulk drug is injected in the form of aqueous solution of the nedaplatin bulk drug.

3. The method for separating nedaplatin drug substance according to claim 2, wherein the concentration of the aqueous solution of nedaplatin drug substance is 0.5g/L to 1.5 g/L;

and/or the sample volume of the aqueous solution of the nedaplatin crude drug is 10-50 muL.

4. The method for separating nedaplatin drug substance according to claim 3, wherein the concentration of the aqueous solution of nedaplatin drug substance is 0.8-1.2 g/L;

and/or the sample volume of the aqueous solution of the nedaplatin crude drug is 15-30 muL;

and/or the flow rate of the separation method is 1.0 mL/min;

and/or the column temperature of the separation method is 25-35 ℃;

and/or the ultraviolet absorption wavelength detected by the separation method is 220 nm.

5. The method for separating nedaplatin crude drug according to claim 1, wherein the HILIC chromatographic column has a particle size of 5 μm, and the column specification is as follows: 4.6X 250 mm;

and/or the volume ratio of the mobile phase a to the mobile phase B is 25:75, 30:70 or 35: 65;

and/or the mobile phase a has a pH of 5.3, 5.5 or 5.7;

and/or the volume fraction of mobile phase a is 0.05%, 0.10% or 0.2%.

6. The method for separating nedaplatin crude drug according to claim 1, wherein the method is used as a method for detecting and identifying related substances of nedaplatin crude drug or for purifying to obtain a single platinum substance, and the platinum substance is nedaplatin or a nedaplatin compound represented by formula I.

7. The method for separating nedaplatin crude drug according to claim 6, wherein the separation method is used as an enrichment preparation method of nedaplatin compounds represented by formula I, and the collection of nedaplatin compounds represented by formula I is completed.