CN113466392B

CN113466392B - Method for detecting dexrazoxane and related substances thereof

Info

Publication number: CN113466392B
Application number: CN202110933445.5A
Authority: CN
Inventors: 张瑜; 丁兆; 胡和平; 李彬
Original assignee: Sichuan Huiyu Haiyue Pharmaceutical Technology Co ltd; SICHUAN HUIYU PHARMACEUTICAL CO Ltd
Current assignee: Sichuan Huiyu Haiyue Pharmaceutical Technology Co ltd; SICHUAN HUIYU PHARMACEUTICAL CO Ltd
Priority date: 2021-08-14
Filing date: 2021-08-14
Publication date: 2023-04-07
Anticipated expiration: 2041-08-14
Also published as: CN113466392A

Abstract

The invention discloses a method for detecting dexrazoxane and related substances thereof, which adopts a high performance liquid chromatography method to qualitatively or/and quantitatively detect the dexrazoxane and/or the related substances in dexrazoxane or a preparation thereof, wherein the detection conditions of the liquid chromatography comprise: a chromatographic column: a C18 or equivalent chromatographic column; mobile phase: mobile phase A and mobile phase B, wherein the mobile phase A is phosphate buffer solution, and the mobile phase B is phosphate buffer solution: and (3) gradient elution is carried out on a mixed solution of acetonitrile with the volume ratio of 80-90. The method can effectively detect the dexrazoxane and 9 related substances in the dexrazoxane simultaneously, and the verification experiment of the system applicability, specificity, accuracy, precision, linear range, quantitative limit detection limit and durability shows that the method can achieve the purpose of accurate quantitative analysis and is beneficial to carrying out stricter quality control on the dexrazoxane.

Description

Method for detecting dexrazoxane and related substances thereof

Technical Field

The invention relates to the field of detection methods, in particular to a method for detecting dexrazoxane and related substances thereof.

Background

Dexrazoxane (dexrazoxane), also known as dexrazoxane, is the d-isomer of propyleneimine (razoxane) and is the only FDA approved adjuvant in the united states for the treatment of DIC (disseminated intravascular coagulation or differential intravascular coagulation, DIC).

The dexrazoxane can rapidly penetrate through cell membranes, is hydrolyzed into TCRE-198 in cells, and is chelated with iron in the cells, so that the compound of ferric ions and anthracyclines such as doxorubicin is reduced, the formation of free radicals is prevented, and the cardiotoxicity of the anthracyclines such as doxorubicin is reduced. The dexrazoxane can also inhibit the cytotoxic effect of topoisomerase II and has an enhancing or antagonistic effect on the cytotoxicity of certain antitumor drugs in animal models. Research shows that the dexrazoxane can reduce the incidence rate of cardiotoxicity by about two thirds, does not influence the effect of chemotherapy or the overall survival rate, and is clinically used for reducing or alleviating cardiotoxicity caused by anthracycline chemotherapy.

As an important anti-tumor adjuvant, the quality of the dexrazoxane directly affects the health of patients, so the quality control of the dexrazoxane is particularly important. However, at present, the quality detection standards of the dexrazoxane and the preparation thereof are not accepted by all national standards, which is not beneficial to ensuring the safety and stability of the product.

The detection of the dexrazoxane and related substances thereof has the following problems:

first, common chromatographic conditions are difficult to preserve. The degraded impurities of the dexrazoxane contain carboxyl and amino bonds with very strong polarity, the retention and separation are difficult under the condition of common reversed phase chromatography, and the problems of interference detection and background absorption interference response, such as the addition of an ion pair reagent for improving the retention, are usually considered.

Second, there is no violet absorption curve. The dexrazoxane does not contain a conjugate bond, and partially degraded impurities even have no obvious absorption curve, so that the wavelength selection difficulty of ultraviolet detection is higher, and the detection result of a product is easy to distort and the quality is not conserved.

Thirdly, the dexrazoxane is sensitive to alkaline conditions and oxidation conditions, but the existing literature data only reports the degradation process under alkaline hydrolysis conditions, and the oxidation process of the dexrazoxane is not reported, so that the currently known dexrazoxane related substances are mostly limited to degradation impurities and are few in types, and the limitation in the dexrazoxane quality control process is caused.

Therefore, how to realize accurate detection of the dexrazoxane and related substances thereof and how to carry out more comprehensive quality control on the dexrazoxane are still to be explored.

Disclosure of Invention

The invention aims to provide a method for detecting dexrazoxane and/or related substances thereof, which can better control the quality of dexrazoxane bulk drugs or preparations.

The dexrazoxane has important practical significance for quality control as a widely applied therapeutic adjuvant, does not contain pi-pi conjugated bonds, and has maximum absorption only at the low wavelength of 208nm, so background interference can be caused by terminal absorption of a plurality of solvents and mobile phases, and common reagents such as methanol, ammonium formate and ion pair are not recommended.

CN108226309B discloses a method for detecting dexrazoxane and three degradation impurities, but the organic solvent used in the method is methanol, and the inventors have found through experiments that in actual detection, methanol has terminal absorption at the detection wavelength (208 nm) thereof, and a gradient peak appears in a chromatogram, which is easy to interfere with the detection of impurities nearby, reduces the response value, and is not favorable for accurate detection.

Through continuous trial and exploration, the inventor finally finds that the effective separation of the dexrazoxane and related substances can be realized by adopting the phosphoric acid buffer solution with a low pH value and the acetonitrile and combining a specific gradient program for elution, the separation effect is good, and the background interference problems such as terminal absorption and the like can not exist.

In addition, currently known substances related to the dexrazoxane include impurity A (IMP-A), impurity B (IMP-B), impurity C (IMP-C) and impurity D (IMP-D), which are all impurities easily generated by hydrolysis of the dexrazoxane under alkaline conditions (see a path in figure 1), and a process impurity NMP may exist. In order to perform more comprehensive quality control on the dorzolam, the inventor analyzes the oxidation process of the dorzolam, and identifies four oxidation impurities of the dorzolam by combining HPLC (high performance liquid chromatography) and MS (mass spectrometry), wherein the four oxidation impurities are oxidation impurity 1, oxidation impurity 2, oxidation impurity 3 (chromogenic impurity 1) and oxidation impurity 4 (chromogenic impurity 2), and the oxidation impurity 1 and the oxidation impurity 2 are shown in figure 2, wherein the oxidation impurity 3 and the oxidation impurity 4 are a pair of isomers and are chromogenic impurities, and the effective detection of the oxidation impurities is realized by the novel method disclosed by the invention.

The impurities and their corresponding structures referred to in the present invention are shown in table 1.

TABLE 1 Derizozokerite and impurity Structure List thereof

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In order to solve the problems, the invention provides a method for detecting the dexrazoxane and/or related substances in the dexrazoxane or the preparation thereof, which adopts high performance liquid chromatography to carry out qualitative or/and quantitative detection, wherein the detection conditions of the liquid chromatography comprise:

a chromatographic column: a C18 or equivalent chromatographic column;

mobile phase: mobile phase A and mobile phase B, wherein the mobile phase A is phosphate buffer solution, and the mobile phase B is phosphate buffer solution: the mixed solution with the acetonitrile volume ratio of 80-90 is subjected to gradient elution by adopting the following elution procedure:

TABLE 2

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The "preparation thereof" refers to a preparation of dexrazoxane, including but not limited to injection, tablet, capsule, granule, emulsion, liposome, spray, inhalant, microsphere, etc.

Further, mobile phase B was phosphate buffer: the mixed solution of acetonitrile in volume ratio of 85 to 15 is subjected to gradient elution by adopting the following procedure:

TABLE 3

Time/min	Mobile phase A/vol%	Mobile phase B/vol%
			0	100	0
7	100	0
			30	50	50

。

The time of the gradient elution procedure of the mobile phase is limited to 25-35 min, and does not represent that the gradient elution procedure is only 25-35 min, the limitation of the gradient elution procedure is considered as an open limitation, and the gradient elution procedure can also comprise other elution procedures after 25-35 min.

Further, the mobile phase employed a gradient elution procedure after 30min as follows:

TABLE 4

Time/min	Mobile phase A/vol%	Mobile phase B/vol%
			40	50	50
45	100	0
			55	100	0

。

In a specific embodiment of the present invention, the phosphate buffer is a dihydrogen phosphate-phosphate buffer.

Further, in the phosphate buffer: the concentration of the dihydric phosphate is 0.005 mol/L-0.015 mol/L, and the pH is adjusted to 1.5-2.5 by phosphoric acid.

In a particular embodiment of the invention, in the phosphate buffer: the concentration of the dihydric phosphate is 0.01mol/L, and the pH is adjusted to 1.8 to 2.25, preferably 2.15 to 2.25, and most preferably 2.2 with phosphoric acid.

In a specific embodiment of the present invention, the related substance is selected from one or more of impurity a, impurity B, impurity C, impurity D, oxidized impurity 1, oxidized impurity 2, colored impurity 1, colored impurity 2, and N-methylpyrrolidone (NMP).

The method can complete the separation and detection of 9 related substances, does not need to add an ion pair reagent, and can be completed by adopting a high performance liquid chromatograph commonly used in a laboratory, the used chromatographic column is commonly used C18, the buffer solution is a phosphate system, the method is simple, convenient, accurate and strong in specificity, and the method is an effective method for analyzing the related substances of the dexrazoxane.

Further, the liquid chromatography detection conditions further include one or more of the following i to iv:

i specification of chromatographic column: 4.6 multiplied by 250mm,3 to 5 μm;

ii column temperature: 28-32 ℃;

iii flow rate: 0.5 ml/min-1.0 ml/min;

iv detection wavelength: 200nm to 220nm.

Further, the liquid chromatography detection conditions further comprise one or more of the following i to iv:

i specification of chromatographic column: 4.6X 250mm,5 μm;

ii column temperature: 28 ℃ to 32 ℃, preferably 30 ℃;

iii flow rate: 0.7-0.9 ml/min, preferably 0.8ml/min;

iv detection wavelength: 208 + -2 nm.

Further, the amount of the sample is 5 to 50. Mu.L, preferably 5 to 15. Mu.L.

In a specific embodiment of the present invention, the sample size is 10. Mu.L.

For the columns of the present invention, the inventors have tried various commercial C18 columns that are tolerant of 100% aqueous phase, including but not limited to columns selected from the group consisting of YMC-Triart C18,

LP-C18, super C18, poroshell 120EC-C18, waters Xbridge C18, poroshell 120SB-Aq, zorbax bones-RP or other equivalent chromatographic columns can realize effective detection of the dexrazoxane and the 9 related substances in the invention, wherein YMC-Triart C18, and/or the like are preferred>

LP-C18, super C18 chromatography columns. In the specific embodiment of the present invention, an embodiment using YMC-Triart-C18 column is described.

Furthermore, the detection method also comprises the step of preparing a test solution, which comprises the following steps: mixing the test sample with a diluent.

Further, the diluent is: 0.005 mol/L-0.015 mol/L dihydric phosphate aqueous solution, and adjusting the pH value to 1.0-2.0 by using phosphoric acid.

Further, the diluent is: 0.01mol/L of dihydric phosphate aqueous solution, and adjusting the pH to 1.3-1.7, preferably 1.5, with phosphoric acid;

further, the temperature of the diluent or the test solution is 2 ℃ to 8 ℃, preferably 5 ℃.

Further, the dihydrogen phosphate is one or more selected from potassium dihydrogen phosphate, sodium dihydrogen phosphate and ammonium dihydrogen phosphate, and is preferably potassium dihydrogen phosphate and/or sodium dihydrogen phosphate.

In a specific embodiment of the present invention, the dihydrogen phosphate is sodium dihydrogen phosphate.

Further, the concentration of the sample solution is 1mg/mL to 20mg/mL, preferably 1mg/mL to 10mg/mL, more preferably 2mg/mL to 8mg/mL, and still more preferably 5mg/mL.

The detection method can calculate the detection result by methods commonly used in the field, such as an area normalization method, a self-comparison method, an internal standard method, an external standard method and the like.

In the specific implementation mode of the invention, the contents of the impurities A, B and C are calculated by adopting an external standard method, and other impurities are calculated by using a main component external standard contrast.

The invention has the beneficial effects that:

(1) The detection method of the invention realizes effective separation of the dexrazoxane and 9 related substances, can simultaneously control the quality of the dexrazoxane and various related substances, overcomes the problems of gradient peaks and the like in the existing method, and has more accurate detection result; and 4 newly-discovered oxidized impurities in the invention can be synchronously detected by adopting the detection method, the method is the method for analyzing the most amount of the dexrazoxane impurities and the best separation effect at present, can help the research of product quality, further identify the characteristics of the product, provide an effective means for the development and detection of the product, and more comprehensively ensure the quality safety of the dexrazoxane.

(2) The detection method verifies the established method through system applicability, specificity, accuracy, precision, linear range, quantitative limit detection limit and durability verification experiments, and the result shows that the method can achieve the aim of accurate quantitative analysis, and the system applicability, specificity and linearity are good; the precision is high, and related substances with lower content in the dexrazoxane can be accurately detected; the accuracy and the durability are good; the invention can achieve the aim of accurate quantitative analysis, can better control the quality of related products of the dexrazoxane and is worthy of popularization and application.

Drawings

FIG. 1 is a Derazoxane alkaline condition hydrolysis degradation pathway;

FIG. 2 is a Dexazoxan oxidation condition degradation pathway;

FIG. 3 is a detection spectrum of the chromatographic condition of Condition 1 (lower panel is schematic at magnified baseline);

FIG. 4 is a detection spectrum of the chromatographic conditions of Condition 2;

FIG. 5 is a chromatogram of a measurement of the chromatographic conditions of Condition 3;

fig. 6 is a detection spectrum of the chromatographic condition of condition 4 (pH = 1.8);

fig. 7 is a detection spectrum of the chromatographic condition of condition 5 (pH = 1.5);

FIG. 8 is a spectrum of a test without destroying the test solution;

FIG. 9 is a spectrum of a test solution of acid-destroyed test sample;

FIG. 10 is a spectrum of a test solution of an alkali-destroyed test sample;

FIG. 11 is a detection spectrum of a test solution for oxidative destruction;

FIG. 12 is a graph showing the detection of high temperature damage to a test solution;

FIG. 13 is a graph showing a detection spectrum of a test solution with high wet destruction;

FIG. 14 is a spectrum of a test solution destroyed by light;

FIG. 15 is an MS spectrum of oxidized impurity 1;

FIG. 16 is a MS spectrum of oxidized impurity 2;

FIG. 17 is a graph of oxidized impurity 1 and 2MS/MS fragments;

FIG. 18 is a graph of the oxidized impurity 1UV spectrum;

FIG. 19 is a graph of oxidized impurity 2UV spectra;

FIG. 20 is a dexrazoxane UV spectrum;

FIG. 21 is a MS spectrum of the colored impurity 1;

FIG. 22 is a MS spectrum of the chromogenic impurity 2;

FIG. 23 is a graph of the fragments of

chromogenic impurities

1 and 2 MS/MS;

FIG. 24 is a UV spectrum of the colored impurity 1;

FIG. 25 is a spectrum of the colored impurity 2 UV.

Detailed description of the invention

The detection method of the present invention is further described below by way of specific embodiments and experiments.

The dexrazoxane for injection used in the embodiment of the present invention is a self-made dexrazoxane for injection, and the formula is the same as that of a commercially available dexrazoxane for injection (trade name:

)。

the relevant experimental procedures in the examples are as follows:

resolution solution: weighing a proper amount of a dexrazoxane sample for injection, adding an impurity control solution with the impurity accounting for about 0.1-0.2%, and adding a diluent to prepare resolution solutions containing dexrazoxane with different concentrations.

Preparation of a reference solution: taking a proper amount of each substance reference substance, precisely weighing, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 100mg of the reference substance in every 1mL, and taking the solution as a reference substance solution stock solution; precisely measuring a proper amount of the reference solution stock solution, adding a diluent to dissolve the reference solution stock solution, and quantitatively diluting the reference solution stock solution to prepare a solution containing about 10 mu g of the reference solution per 1 mL.

Preparation of a test solution: weighing a proper amount of dexrazoxane sample for injection, adding a diluent to dissolve the dexrazoxane sample into a solution containing 5mg of dexrazoxane in 1ml, shaking up, filtering, and taking the filtrate as a test solution.

System applicability solution: weighing about 50mg of dexrazoxane reference substance, placing the reference substance into a 10mL volumetric flask, respectively adding 1mL of reference stock solutions of the impurity A, the impurity B and the impurity C, dissolving the reference stock solutions by using a solvent and fixing the volume to the scale.

Example 1 screening of chromatographic conditions

Screening of organic phase and elution procedure:

condition 1:

a chromatographic column: YMC-Triart-C18, 4.6X 250mm,5 μm;

mobile phase: phase A: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution, pH adjusted to 2.2. + -. 0.05 with phosphoric acid), phase B: methanol, gradient elution procedure was:

TABLE 5

Column temperature: 30 deg.C

Detection wavelength: 208nm;

sample introduction amount: 10 mu L of the solution;

sample temperature: 5 ℃;

diluent agent: a phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution is used for regulating the pH value to 1.5 by phosphoric acid and is cooled to 2-8 ℃ for use);

test solution preparation: a resolution solution with the concentration of 1mg/mL (prepared by dexrazoxane for injection and added with a control solution of impurities A-D);

flow rate: 0.8mL/min.

The results of the detection are shown in FIG. 3.

As shown in fig. 3, the chromatographic conditions can satisfy basic separation detection, but the lower graph of fig. 3 shows that the gradient peak is obvious due to absorption of methanol by using terminal ultraviolet detection, and the detection of impurities nearby is easily interfered, and the chromatographic conditions need to be further adjusted.

Condition 2:

a chromatographic column: YMC-Triart-C18, 4.6X 250mm,5 μm;

mobile phase: phase A: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution, pH adjusted to 2.2. + -. 0.05 with phosphoric acid), phase B: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution, pH adjusted to 2.2 ± 0.05 with phosphoric acid) -acetonitrile (V/V =90:

TABLE 6

Time/min	Mobile phase A/vol%	Mobile phase B/vol%
			0	100	0
7	100	0
			25	0	100
35	0	100
			37	100	0
45	100	0

Column temperature: 30 ℃;

detection wavelength: 208nm;

sample introduction amount: 10 mu L of the solution;

temperature of the sample: 5 ℃;

diluent (b): a phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution is used for regulating the pH value to 1.5 by phosphoric acid and is cooled to 2-8 ℃ for use);

test solution preparation: resolution solution (control solution prepared with dexrazoxane for injection and added NMP) at a concentration of 1mg/mL

Flow rate: 0.8mL/min.

When the condition 2 is adopted for detection, as can be seen from fig. 4, the problem of gradient peak observation under the condition of amplifying the base line is obviously improved, the condition of impurity interference is reduced, and the acetonitrile as an organic phase is more favorable for accurate detection of the dexrazoxane and related substances, but the separation degree of the NMP and the dexrazoxane under the condition is smaller and is 1.81, and the condition is easy to be interfered, and the condition needs to be continuously optimized.

Condition 3:

and (3) chromatographic column: YMC-Triart-C18, 4.6X 250mm,5 μm;

mobile phase: phase A: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution, pH adjusted to 2.2. + -. 0.05 with phosphoric acid), phase B: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate in water, pH adjusted to 2.2 ± 0.05 with phosphoric acid) -acetonitrile (V/V = 85) gradient elution procedure:

TABLE 7

Time/min	Mobile phase A/vol%	Mobile phase B/vol%
			0	100	0
7	100	0
			30	50	50
40	50	50
			45	100	0
55	100	0

Column temperature: 30 ℃;

detection wavelength: 208nm;

sample introduction amount: 10 mu L of the solution;

concentration of the test solution: a resolution solution with the concentration of 5mg/mL (prepared by dexrazoxane for injection and added with reference solutions of impurities A-D and NMP);

flow rate: 0.8mL/min.

The results are shown in Table 8 and FIG. 5.

TABLE 8

Components	IMP-A	IMP-B	IMP-C	IMP-D	NMP	Dexrazoxane
							RT(min)	5.850	8.733	6.376	9.882	25.051	27.113
RRT	0.22	0.32	0.24	0.36	0.92	1.00
							Separation degree of front and rear peaks	3.7/2.0	4.1/3.3	2.0/2.8	3.3/28.3	2.2/5.6	5.6/25.8
Tailing factor	1.2	1.1	ND	0.9	1.1	0.8
							Number of theoretical plates	9222	12276	8525	10673	83604	76405

As can be seen from table 8 and fig. 5, when the detection is performed under condition 3, the problem of gradient peaks is significantly improved, the detection of impurities is not affected, the separation conditions of each impurity peak and the main peak are good, and the separation degree between NMP and dexrazoxane is improved to 5.6.

Screening of pH value:

condition 4:

a chromatographic column: YMC-Triart-C18, 4.6X 250mm,5 μm;

mobile phase: phase A: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution, pH adjusted to 1.8 with phosphoric acid), phase B: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution, pH adjusted to 1.8 with phosphoric acid) -acetonitrile (V/V =85 15), gradient elution procedure same as table 7;

column temperature: 30 ℃;

detection wavelength: 208nm;

sample injection amount: 10 mu L of the solution;

concentration of the test solution: a resolution solution with the concentration of 1mg/mL (prepared by dexrazoxane for injection and added with a control solution of impurities A-D);

flow rate: 0.8mL/min.

Condition 5:

a chromatographic column: YMC-Triart-C18, 4.6X 250mm,5 μm;

mobile phase: phase A: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution, pH adjusted to 1.5 with phosphoric acid), phase B: phosphate buffer (0.01 mol/L sodium dihydrogen phosphate aqueous solution, pH adjusted to 1.5 with phosphoric acid) -acetonitrile (V/V = 85) with the same gradient elution procedure as in table 7;

column temperature: 30 ℃;

detection wavelength: 208nm;

sample introduction amount: 10 mu L of the solution;

flow rate: 0.8mL/min.

The results of the tests under

conditions

4 and 5 are shown in Table 9, FIGS. 6 and 7.

TABLE 9

As is clear from the results of table 9, fig. 6 and fig. 7, each impurity has already completed its peak in the pure water system, and as the pH decreases, the retention time of each impurity and the main component moves forward, and when the pH =1.8, the separation degree of the impurity D from the front peak is not good, but the impurity D can be used for separation and detection of the dexrazoxane, the impurity a and the impurity B; when the pH is =1.5, the impurities B and D are overlapped, and the peaks are poor, but the method can be used for separating and detecting the dexrazoxane, the impurity A and the impurity C.

The preferred chromatographic conditions finally determined are condition 3.

Example 2 forced degradation test analysis

In order to verify that the detection method can be applied to the quality control of the dexrazoxane at each stage, a forced degradation test is carried out on a dexrazoxane sample for injection, and the experimental method comprises the following steps:

acid destruction: weighing about 55mg of dexrazoxane sample for injection, placing the sample into a 10ml measuring flask, adding 4ml of 1M hydrochloric acid aqueous solution, and placing the sample at room temperature for 24 hours;

alkali destruction: weighing about 55mg of a dexrazoxane sample for injection, putting the sample into a 10ml measuring flask, adding 3ml of 0.1M NaOH aqueous solution, and standing for 10 minutes at room temperature;

and (3) oxidative destruction: weighing about 55mg of a dexrazoxane sample for injection, putting the sample into a 10ml measuring flask, adding 1.5ml of 0.3% hydrogen peroxide solution, and standing at room temperature for 1.5h;

high-temperature destruction: taking a proper amount of dexrazoxane sample for injection, and destroying the sample at the high temperature of 80 ℃ for 5 days;

high-humidity destruction: weighing about 55mg of dexrazoxane sample for injection, placing the sample into a 10ml measuring flask, and destroying the sample for 18 days by high humidity (25 ℃, RH 92.5%);

light damage: taking a proper amount of dexrazoxane sample for injection at 5000lux and ultraviolet of 84uw/cm ² The destruction took 18 days.

Each sample of the forced degradation test was examined by the method of the present invention (Condition 3), and the results are shown in Table 10 and FIGS. 8 to 14.

TABLE 10

* Note: the oxidized

impurities

1 and 2 are a pair of isomers, the peak-to-valley ratio between the

oxidized impurities

1 and 2 is 7.3 which meets the requirement that the pharmacopoeia is more than 2.0, and the separation can be achieved.

The results in Table 10 show that the method of the present invention can be used for detecting injection dexrazoxane samples which are not destroyed, acid destroyed, alkali destroyed, oxidation destroyed, high temperature destroyed, high humidity destroyed and illumination destroyed, and can be used for analyzing the dexrazoxane and 9 related substances efficiently and accurately, and can be widely used for quality control in the production, storage and use processes of the dexrazoxane.

As is clear from the results of the detection of the oxidative destruction, four kinds of main oxidized impurities are newly generated, and the four kinds of oxidized impurities having RRTs of 0.26, 0.28, 0.73, and 0.79 under the conditions of the preferred detection method of the present invention are designated as oxidized impurity 1, oxidized impurity 2, oxidized impurity 3 (colored impurity 1), and oxidized impurity 4 (colored impurity 2) in this order, depending on the retention time. The inventors further analyzed and identified four new impurities generated in the oxidative destruction, see fig. 15 to 25, and identified the structures of the oxidized impurity 1, the oxidized impurity 2, the chromogenic impurity 1 and the chromogenic impurity 2, see fig. 2, and the analysis results are as follows:

the oxidation of dexrazoxane occurs mainly on tertiary amino groups, as a result of typical nitrogen oxidation reactions of tertiary amino groups and rearrangement reactions of N-oxides, because dexrazoxane has two tertiary amino groups, thereby generating two pairs of isomeric oxidation impurities:

oxidized impurities 1 and 2: o atoms attack N atoms to form N ^- Oxide, two impurities appeared at 7.1min and 7.4min on the chromatogram can be seen on the MS spectrum, and the M/z under the + ESI condition is 285.1195, 285.1199, and is [ M + H ]] ⁺ Ion peak of (2). Further secondary MS/MS fragment analysis is carried out, and a structural fragment with the M/z of 171.0764 appears, namely a fragment formed by the breakage of a tertiary amino group and an adjacent bridging C bond [ M ]]Thus, the structure of the compound can be obtained. In addition, since the UV absorption spectra of the two compounds are similar to that of dexrazoxane, the main structure of the compound is not significantly changed, and the final analysis results in two oxidized impurities (oxidized impurity 1 and oxidized impurity 2 are respectively selected from one of the two compounds and are not the same compound) as shown in table 11.

TABLE 11

Color impurities 1 and 2: n-oxygen compounds are unstable and O competes for electrons and then forms OH ^- Root detachment, C occurrence of empty orbit and adjacent N ⁺ Conjugated pi bonds are formed, resulting in rearrangement and ultimately formation of large conjugated bonds with other lactam groups, resulting in chromogenic groups. The MS spectrum can show two impurities appearing at 20.0min and 21.5min on the chromatogram, and the M/z under the + ESI condition is 267.1095, 267.1095 and is [ M] ⁺ Ion peak of (2). Further performing secondary MS/MS fragment analysis, wherein structural fragments with M/z of 155.0816 appear, and the structural fragments are formed by bridging a charged center N and adjacent bridgesFragments formed by breaking C bonds [ M ]]Therefore, the structure of the compound can be obtained. In addition, according to the UV absorption spectrogram analysis of the two compounds, the two impurities show significant absorption around 375nm, which proves that the impurities rearrange to generate large conjugated bonds and generate chromogenic groups (corresponding to complementary color red or yellow after 375nm absorption and consistent with actual appearance color), and finally two chromogenic impurities (chromogenic impurity 1 and chromogenic impurity 2 are respectively selected from one of the two compounds and are not the same compound) as shown in table 12 are obtained.

The injection dexrazoxane hydrochloride is added into the dexrazoxane injection formula, the pH value of the detection environment is about 2.0, and OH is detected ^- Is negligible, so that the anions corresponding to the

chromogenic impurities

1 and 2 in the solution are Cl during detection ^- . If the pH value of the crude drug and the preparation of the dexrazoxane for injection is adjusted by hydrochloric acid/sodium hydroxide in the preparation, storage or transportation process of the drug, the pH value of the final preparation product is between 1.0 and 2.5, and OH is at the moment ^- Negligible, the anion remaining predominantly Cl ^- 。

TABLE 12

Methodological validation (condition 3):

table 13 analytical methods verification summary

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The result shows that the system applicability and specificity of the method are good, the linearity is good, the linear range, the detection limit and the quantitative limit all meet the requirements, the accuracy and the durability are good, and the method is proved to be suitable for detecting the dexrazoxane and related substances thereof.

The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.

Claims

1. A method for detecting dexrazoxane and/or related substances in dexrazoxane or a preparation thereof is characterized in that high performance liquid chromatography is adopted for qualitative or/and quantitative detection, and the detection conditions of the liquid chromatography comprise:

a chromatographic column: a C18 chromatographic column; specification of chromatographic column: 4.6 multiplied by 250mm,3 to 5 μm;

mobile phase: mobile phase A and mobile phase B, wherein the mobile phase A is phosphate buffer solution, and the mobile phase B is phosphate buffer solution: the mixed solution of acetonitrile in volume ratio of 85 to 15 is subjected to gradient elution by adopting the following procedure:

time/min Mobile phase A/vol% Mobile phase B/vol% 0 100 0 7 100 0 30 50 50

The mobile phase was eluted after 30min using the following gradient elution procedure:

time/min Mobile phase A/vol% Mobile phase B/vol% 40 50 50 45 100 0 55 100 0

Wherein the phosphate buffer is a dihydrogen phosphate-phosphate buffer; in the phosphate buffer: the concentration of the dihydric phosphate is 0.005 mol/L-0.015 mol/L, and the pH is adjusted to 2.15-2.25 by phosphoric acid;

column temperature: 28-32 ℃;

detection wavelength: 200 nm-220 nm;

the related substances are impurity A, impurity B, impurity C, impurity D, oxidized impurity 1, oxidized impurity 2, chromogenic impurity 1, chromogenic impurity 2 and N-methylpyrrolidone:

2. the assay of claim 1, wherein in the phosphate buffer: the concentration of the dihydric phosphate was 0.01mol/L, and the pH was adjusted to 2.2 with phosphoric acid.

3. The detection method according to any one of claims 1 to 2, wherein the liquid chromatography detection conditions further include: flow rate: 0.5 ml/min-1.0 ml/min.

4. The detection method according to any one of claims 1 to 2, wherein the liquid chromatography detection conditions further include:

i chromatographic column specification: 4.6X 250mm,5 μm;

ii column temperature: 30 ℃;

iii flow rate: 0.7-0.9 ml/min;

iv detection wavelength: 208 + -2 nm.

5. The detection method according to any one of claims 1 to 2, wherein the liquid chromatography detection conditions further include: flow rate: 0.8ml/min.

6. The detection method according to any one of claims 1 to 2, wherein the amount of the sample is 5 to 50. Mu.L.

7. The detection method according to any one of claims 1 to 2, wherein the amount of the sample is 5 to 15. Mu.L.

8. The detection method according to any one of claims 1 to 2, wherein the sample volume is 10. Mu.L.

9. The detection method according to any one of claims 1 to 2, wherein the column is selected from the group consisting of YMC-Triart C18,

LP-C18, super C18, poroshell 120EC-C18, waters Xbridge C18, poroshell 120SB-Aq, zorbax boxes-RP chromatography columns.

10. The detection method according to any one of claims 1 to 2, wherein the column is selected from the group consisting of YMC-Triart C18,

LP-C18, super C18 chromatography columns.

11. The detection method according to any one of claims 1 to 2, wherein the column is YMC-Triart C18.

12. The detection method according to claim 1, further comprising preparing a test solution, comprising the steps of: mixing the test sample with a diluent; the diluent is as follows: 0.005 mol/L-0.015 mol/L dihydric phosphate aqueous solution, and adjusting the pH value to 1.0-2.0 by using phosphoric acid.

13. The detection method according to claim 12, wherein the diluent is 0.01mol/L of an aqueous solution of dihydrogen phosphate.

14. The detection method according to claim 12, wherein the pH is adjusted to 1.3 to 1.7 with phosphoric acid.

15. The detection method according to claim 12, wherein the pH is adjusted to 1.5 with phosphoric acid.

16. The assay of claim 12, wherein the diluent or test solution is at a temperature of 2 ℃ to 8 ℃.

17. The assay of claim 12, wherein the diluent or test solution is at a temperature of 5 ℃.

18. The detection method according to any one of claims 1, 2, and 12, wherein the dihydrogen phosphate is one or more selected from potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate.

19. The method according to any one of claims 1, 2 and 12, wherein the dihydrogen phosphate is selected from potassium dihydrogen phosphate and/or sodium dihydrogen phosphate.

20. The detection method according to any one of claims 1, 2 and 12, wherein the dihydrogen phosphate is sodium dihydrogen phosphate.

21. The detection method according to claim 12, wherein the concentration of the sample solution is 1mg/mL to 20mg/mL.

22. The detection method according to claim 12, wherein the concentration of the sample solution is 1mg/mL to 10mg/mL.

23. The detection method according to claim 12, wherein the concentration of the sample solution is 2mg/mL to 8mg/mL.

24. The assay of claim 12, wherein the concentration of the test solution is 5mg/mL.