CN111551645B

CN111551645B - Method for detecting hydroxychloroquine sulfate related substances and application thereof

Info

Publication number: CN111551645B
Application number: CN202010360985.4A
Authority: CN
Inventors: 颜国明; 汪青青; 陈春燕; 李�杰; 袁少卿; 尹超
Original assignee: Shanghai Zhongxi Sunve Pharmaceutical Co Ltd
Current assignee: Shanghai Zhongxi Sunve Pharmaceutical Co Ltd
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2022-11-08
Anticipated expiration: 2040-04-30
Also published as: CN111551645A

Abstract

The invention discloses a method for detecting hydroxychloroquine sulfate related substances. The method comprises separating and detecting by reverse liquid chromatography; wherein, the chromatographic conditions are as follows: the mobile phase is a mixed solvent consisting of a water-soluble organic solvent and a buffer solution; wherein the buffer solution is an ammonium acetate solution or an ammonium formate solution; the pH value of the buffer solution is 9.0-10.5. The detection method can simultaneously detect 7 impurities included in the hydroxychloroquine sulfate raw material and the hydroxychloroquine sulfate raw material quality standard in the 9.8 edition European pharmacopoeia in the preparation, and has the advantages of good separation effect, high sensitivity, no blank interference, low detection cost, high analysis speed and the like.

Description

Method for detecting hydroxychloroquine sulfate related substances and application thereof

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting hydroxychloroquine sulfate related substances and application thereof.

Background

Hydroxychloroquine sulfate is used for treating discoid lupus erythematosus, systemic lupus erythematosus, rheumatoid arthritis and the like. The structural formula is as follows:

the chemical name is 2- [ [4- [ (7-chloro-4-quinolinyl) amino ] pentyl ] ethylamino ] -ethanolic sulfate. The related substances introduced in the hydroxychloroquine sulfate synthesis and degraded in storage need to be controlled in both bulk drugs and preparations.

At present, in the first volume E1-97 of the existing national standard of the hydroxychloroquine sulfate raw material, the drug standard of the Ministry of health (Ministry of second), the detection method of related substances is thin-layer chromatography, and the problems of poor specificity and low sensitivity exist. With the progress of analytical techniques and equipment, liquid chromatography is increasingly used for quality control of pharmaceutical products, including identification, analysis of related substances, content measurement, and the like. The existing analytical technology and method for detecting hydroxychloroquine sulfate in foreign pharmacopoeia by using liquid chromatography, for example, ph.eur.monograph 2849 of european pharmacopoeia 9.8 version, ultra-high performance liquid chromatography, octadecylsilane chemically bonded silica gel as a filler, 254nm of detection wavelength, phosphate buffer (1.26G of potassium dihydrogen phosphate is taken and put in 900mL of water, 0.15G of sodium heptanesulfonate is added, pH is adjusted to 7.0 by triethylamine, and diluted to 1L by water) and a mobile phase system of methanol, gradient elution is carried out, impurity B and impurity C are taken as specific impurities, and potential impurities a, D, E, F and G (chemical structural formulas and names of impurities a to G are shown in table 1) are simultaneously included, the method can effectively separate impurity B, impurity C and hydroxychloroquine (shown in fig. 1, fig. 1 is introduced from specification of EP official control hydroxychloroquine system applicability control in european pharmacopoeia website), the method can effectively separate impurity B, impurity C and can realize a strong interference peak detection from an unknown impurity baseline analytical reference, such as an unknown peak after the baseline of the unknown impurity C is separated from EP 000qchloroquine system, and the unknown peak of the reference peak after the baseline analysis is carried out; and the quality requirements of slave equipment, consumables and reagents of the ultra-high performance liquid chromatography are all higher than those of a common liquid chromatograph, and the detection cost is higher.

TABLE 1 chemical structural formulas and names of impurities A-G

Disclosure of Invention

The invention provides a method for detecting hydroxychloroquine sulfate related substances and application thereof, aiming at solving the problems of low specificity, low sensitivity and few detectable impurities of the detection method in the prior art. The detection method can simultaneously detect 7 impurities (impurities A-G, shown in table 1) included in the quality standard of the hydroxychloroquine sulfate raw material and the hydroxychloroquine sulfate bulk drug in the 9.8 edition of the Ozhou pharmacopoeia in the preparation, and has the advantages of good separation effect, high sensitivity, no blank interference, low detection cost, high analysis speed and the like.

The invention solves the technical problems through the following technical scheme.

The invention provides a method for detecting hydroxychloroquine sulfate related substances, which comprises the following steps of performing separation detection by adopting a reverse liquid chromatography; wherein, the chromatographic conditions are as follows:

the mobile phase is a mixed solvent consisting of a water-soluble organic solvent and a buffer solution;

wherein the buffer solution is an ammonium acetate solution or an ammonium formate solution; the pH value of the buffer solution is 9.0-10.5.

In the reverse liquid chromatography, the stationary phase is preferably octylsilane bonded silica gel, octadecylsilane bonded silica gel, or silica microsphere bonded pentafluorophenyl, more preferably octadecylsilane bonded silica gel.

In the present invention, in the reverse liquid chromatography, the length of the column is preferably one of 100mm, 150mm or 250mm, more preferably 150mm.

In the present invention, the pH of the buffer solution is preferably 10.0 to 10.5.

In the present invention, the concentration of the buffer solution is preferably 0.01 to 0.05mol/L, more preferably 0.05mol/L.

In the invention, preferably, the pH value of the buffer solution is adjusted by dropwise adding triethylamine or an ammonia solution. The concentration of the triethylamine or aqueous ammonia solution may be conventional in the art, and may be, for example, 100% triethylamine or aqueous ammonia reagent or diluted triethylamine or aqueous ammonia reagent.

In the present invention, in the reverse liquid chromatography, the wavelength selected for detection is preferably 245 to 270nm, more preferably 254nm.

In the present invention, the flow rate of the mobile phase is preferably 0.5 to 1.0mL/min, more preferably 0.8mL/min.

In the present invention, in the reverse phase liquid chromatography, the temperature of the column is preferably 30 to 40 ℃, for example, 35 ℃.

In the present invention, the water-soluble organic solvent is preferably one or more of methanol, acetonitrile, tetrahydrofuran, acetone and isopropanol, more preferably a mixed solvent of methanol, acetonitrile, tetrahydrofuran, acetone or isopropanol, such as methanol, acetonitrile and isopropanol in a volume ratio of 70.

In the present invention, the elution mode is preferably gradient elution.

Wherein, preferably, the total volume of the mobile phase is 100%; in 0-3 min, the volume of the buffer solution is 53% -57%; in 3-15 min, the volume of the buffer solution is decreased from 53-57% to 50%; the volume of the buffer solution is reduced from 50 percent to 27 to 32 percent within 15 to 36 min; the volume of the buffer solution is gradually increased from 27-32% to 53-57% in 36-37 min; and in 37-45 min, the volume of the buffer solution is 53% -57%.

In a preferred embodiment, the total volume of the mobile phase is 100%; in 0-3 min, the volume of the buffer solution is 57%; in 3-15 min, the volume of the buffer solution is reduced from 57% to 50%; in 15-36 min, the volume of the buffer solution is decreased from 50% to 32%; the volume of the buffer solution is increased from 32% to 57% in 36-37 min; the volume of the buffer solution is 57% in 37-45 min.

In a preferred embodiment, the total volume of the mobile phase is 100%; in 0-3 min, the volume of the buffer solution is 55%; the volume of the buffer solution is reduced from 55% to 50% in 3-15 min; the volume of the buffer solution is reduced from 50% to 30% in 15-36 min; the volume of the buffer solution is increased from 30% to 55% within 36-37 min; the volume of the buffer solution is 55 percent in 37-45 min.

In a preferred embodiment, the total volume of the mobile phase is 100%; in 0-3 min, the volume of the buffer solution is 53%; in 3-15 min, the volume of the buffer solution is reduced from 53% to 50%; the volume of the buffer solution is reduced from 50% to 27% in 15-36 min; the volume of the buffer solution is increased from 27% to 53% in 36-37 min; the volume of the buffer solution is 53% in 37-45 min.

In a preferred embodiment, the total volume of the mobile phase is 100%; the volume of the buffer solution is reduced from 55% to 30% in 0-36 min; the volume of the buffer solution is increased from 30% to 55% within 36-37 min; the volume of the buffer solution is 55 percent in 37-45 min.

The invention also provides a method for detecting related substances in the hydroxychloroquine sulfate preparation, which adopts the method for detecting related substances in hydroxychloroquine sulfate.

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.

Compared with the existing analysis method, the method has the beneficial effects that:

in the invention, a method for simultaneously detecting hydroxychloroquine sulfate raw material and impurities included in the hydroxychloroquine sulfate raw material quality standard in the European pharmacopoeia 9.8 version in the preparation is established, and the method has the advantages of good separation effect, high sensitivity, no blank interference, low detection cost, high analysis speed and the like.

Proved by methodology, the specificity, linearity, range, repeatability, detection limit, quantitative limit and accuracy of the method all meet the requirements of a medicine quality standard analysis method verification guiding principle in the four-part appendix of the 2015 edition of Chinese pharmacopoeia, so the method has important practical significance in the synthesis of hydroxychloroquine sulfate and the quality control of a preparation process.

Drawings

FIG. 1 is a reference map of a reference substance for the applicability of the hydroxychloroquine sulfate system in the European pharmacopoeia.

Fig. 2 is a diagram showing the hydroxychloroquine sulfate analysis method-impurity specificity in example 2.

Detailed Description

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

In the following examples and comparative examples, the HPLC apparatus used was an Agilent 1260 series HPLC apparatus.

Example 1

(1) Chromatographic conditions are as follows:

stationary phase: octane silane bonded silica; (manufacturer Agilent, model ZORBAX Extend C8,5um, 4.6x150mm)

The length of the chromatographic column is 150mm;

mobile phase: mobile phase a (buffer solution): 0.01mol/L ammonium acetate water solution, and 100 percent triethylamine solution is used for adjusting the pH value to 9.0; mobile phase B (water-soluble organic solvent): methanol: acetonitrile =80 (V/V).

Gradient elution was performed according to the following procedure:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	55	45
3	55	45
			15	50	50
36	30	70
			37	55	45
45	55	45

Flow rate: 0.8mL/min; detection wavelength: 254nm

Column temperature: 35 ℃; sample introduction amount: 10 μ l

(2) Preparation of mixed impurity reference solution:

weighing a hydroxychloroquine sulfate reference substance and appropriate amount of impurities of each component, and adding water: methanol: 10% sulfuric acid =500:500:4 (V/V/V) is used as a diluent to be dissolved and prepared into a mixed impurity reference substance solution containing hydroxychloroquine sulfate of 0.5mg/mL and impurities of each component of 0.5 mu g/mL.

(3) Preparing a test solution:

weighing a proper amount of hydroxychloroquine sulfate (bulk drug, batch number: HS20181103V, produced by Chinese and western three-dimensional pharmaceutical industry Co., ltd.), adding water: methanol: 10% sulfuric acid =500:500:4 (V/V/V) is used as a diluent to be dissolved and prepared into a test solution containing 0.5mg/mL of hydroxychloroquine sulfate.

(4) Measurement of

Respectively and precisely measuring 10 mu l of the mixed impurity reference substance solution and the test sample solution, and respectively injecting the mixed impurity reference substance solution and the test sample solution into a liquid chromatograph for measurement and calculation to obtain the compound impurity reference substance, wherein the separation degrees of hydroxychloroquine, the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G in the mixed impurity reference substance solution are all larger than 1.5.

Example 2

(1) Chromatographic conditions are as follows:

stationary phase: octadecylsilane chemically bonded silica silane-bonded silica; (manufacturer Agilent, model ZORBAX Extend C18,5um, 4.6x150mm)

The length of the chromatographic column is 150mm;

mobile phase: mobile phase a (buffer solution): 0.05mol/L ammonium acetate water solution, and 100 percent triethylamine solution is used for adjusting the pH value to 10.5; mobile phase B (water-soluble organic solvent): methanol: acetonitrile: isopropanol =70

Gradient elution was performed according to the following procedure:

Flow rate: 0.8mL/min; detection wavelength: 254nm

Column temperature: 35 ℃; sample introduction amount: 10 μ l

(3) Preparation of mixed impurity reference solution:

(3) Preparing a test solution:

weighing appropriate amount of hydroxychloroquine sulfate (bulk drug, batch number: HS20181103V, produced by three-dimensional pharmaceutical Co., ltd.) and adding water: methanol: 10% sulfuric acid =500:500:4 (V/V/V) is used as a diluent to be dissolved and prepared into a test solution containing 0.5mg/mL of hydroxychloroquine sulfate.

(4) Measurement of

And respectively and precisely measuring 10 mu l of the mixed impurity reference substance solution and the sample solution, and respectively injecting the mixed impurity reference substance solution and the sample solution into a liquid chromatograph for measurement and calculation to obtain the compound impurity reference substance, wherein the separation degrees of hydroxychloroquine, the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G in the mixed impurity reference substance solution are all larger than 1.5, and the separation effects are shown in figure 2 and table 2.

TABLE 2

Serial number	Peak name	Retention time (min)	Symmetry factor	Degree of separation	Number of plates
						1	Impurity A	4.105	1.36	3.87	13238
2	Impurity B	6.259	1.25	4.38	17467
						3	Impurity C	7.216	1.36	7.02	13452
4	Impurity E	8.863	1.15	2.49	25564
						5	Impurity D	9.677	1.33	13.51	7941
6	Hydroxychloroquine sulfate	14.837	1.3	15.05	32066
						7	Impurity G	21.664	1.08	13.97	54582
8	Impurity F	27.437	0.9	10.22	134750

Note: the symmetry factor calculation formula is as follows:

methodology examined as follows:

(1) Specificity and system adaptability

Respectively taking a proper amount of hydroxychloroquine sulfate, an impurity A, an impurity B, an impurity C, an impurity D, an impurity E, an impurity F and an impurity G, and using water: methanol: 10% sulfuric acid =500:500:4 (V/V/V) is used as a diluent to be dissolved and prepared into a mixed impurity reference substance solution containing hydroxychloroquine sulfate of 0.5mg/mL and impurities of each component of 0.5 mu g/mL, and the result of the measurement shows that the separation degree of each component is more than 1.5.

(2) Linearity

Respectively taking a proper amount of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, and using water: methanol: 10% sulfuric acid =500:500:4 (V/V/V) is used as a diluent to be dissolved and prepared into series of standard solutions of 20 percent, 50 percent, 100 percent, 150 percent and 200 percent respectively by taking the hydroxychloroquine sulfate test sample solution (0.5 mg/mL) with the concentration of 0.1 percent as the reference. And (5) injecting and measuring according to the drawn-up chromatographic conditions, and performing linear regression on the mass concentration (X, mg/mL) of the corresponding solvent by using the peak area (Y). The obtained impurities have good linear relation in the concentration range of 0.1-1.0 mu g/mL, and the correlation coefficients are all larger than 0.999.

(3) Precision degree

Weighing appropriate amount of hydroxychloroquine sulfate (bulk drug, batch number: HS20181103V, produced by three-dimensional pharmaceutical Co., ltd.) impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, adding water: methanol: 10% sulfuric acid =500:500:4 (V/V/V) is used as a diluent to be dissolved and prepared into a mixed impurity reference solution containing hydroxychloroquine sulfate of 0.5mg/mL and impurities of each component of 0.5 mu g/mL, 6 parts are prepared in parallel for determination, and the result shows that the sample injection is performed in parallel 6, and the RSD of each impurity is less than 3 percent.

(4) Sensitivity of the probe

Weighing appropriate amount of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, and using water: methanol: 10% sulfuric acid =500:500:4 (V/V/V) is used as a diluent to be dissolved and prepared into a mixed impurity reference substance solution containing 0.25 mu g/mL of each component impurity, and the signal-to-noise ratio of each component is more than 10 when the determination is carried out.

(5) Accuracy of

Weighing a proper amount of hydroxychloroquine sulfate (bulk drug, batch number: HS20181103V, produced water from Chinese and western three-dimensional pharmaceutical limited company: methanol: 10% sulfuric acid =500: 4 (V/V/V).

As proved by methodology verification of specificity, linearity, precision, sensitivity and accuracy, verification results all meet the requirements of the verification guiding principle of the medicine quality standard analysis method in the appendix of the four parts of the 2015 edition of Chinese pharmacopoeia.

Example 3

Example 3 differs from example 1 only in that: gradient elution was performed according to the following procedure:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	57	43
3	57	43
			15	50	50
36	32	68
			37	57	43
45	57	43

Example 4

Example 4 differs from example 1 only in that: gradient elution was performed according to the following procedure:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	53	47
3	53	47
			15	50	50
36	27	73
			37	53	47
45	53	47

The procedure gradient elution pattern of examples 3 and 4 was comparable to that of examples 1 and 2.

Example 5

Example 5 differs from example 1 only in that: gradient elution was performed according to the following procedure:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	55	45
36	30	70
			37	55	45
45	55	45

In example 5, impurities a to G were simultaneously detected, and the separation effect thereof is shown in table 3.

TABLE 3

Serial number	Peak name	Retention time (min)	Symmetry factor	Degree of separation	Number of plates
						1	Impurity A	4.101	1.33	3.87	13176
2	Impurity B	5.232	1.30	10.52	16380
						3	Impurity C	7.487	1.40	5.38	12632
4	Impurity E	8.797	1.15	5.25	25188
						5	Impurity D	10.938	1.25	8.73	5381
6	Hydroxychloroquine sulfate	15.027	1.33	9.72	30694
						7	Impurity G	21.549	1.26	0.98	61588
8	Impurity F	27.437	0.9	10.22	134750

Comparative example 1

Comparative example 1 differs from example 1 only in mobile phase a (buffer solution): 0.05mol/L ammonium acetate solution in water, and the pH is adjusted to 8.5 with 100% triethylamine solution.

The retention times of impurities B, C and D coincide and cannot be separated.

Claims

1. A method for detecting hydroxychloroquine sulfate related substances is characterized by comprising the following steps of performing separation detection by adopting a reverse liquid chromatography, wherein the elution mode is gradient elution; wherein, the chromatographic conditions are as follows:

the mobile phase is a mixed solvent consisting of a water-soluble organic solvent and a buffer solution; the water-soluble organic solvent is methanol, acetonitrile and isopropanol in a volume ratio of 70;

when the water-soluble organic solvent is methanol, acetonitrile and isopropanol in a volume ratio of 70;

when the water-soluble organic solvent is methanol and acetonitrile of 80;

wherein the buffer solution is an ammonium acetate solution or an ammonium formate solution; the pH value of the buffer solution is 9.0-10.5;

the gradient elution is: based on the total volume of the mobile phase as 100 percent; in 0-3 min, the volume of the buffer solution is 53% -57%; in 3-15 min, the volume of the buffer solution is decreased from 53-57% to 50%; in 15-36 min, the volume of the buffer solution is reduced from 50% to 27% -32%; the volume of the buffer solution is increased from 27-32% to 53-57% within 36-37 min; in 37-45 min, the volume of the buffer solution is 53-57%;

wherein, in the reverse phase liquid chromatography, the length of the chromatographic column is one of 150mm or 250 mm;

wherein, in the reverse liquid chromatography, the wavelength selected for detection is 245-270 nm;

wherein, the related substances are 7, and the related substances are as follows:

2. the detection method according to claim 1, wherein in the reverse phase liquid chromatography, the length of the column is 150mm.

3. The detection method according to claim 1, wherein the buffer solution has a pH of 10.0 to 10.5.

4. The detection method according to claim 1, wherein the concentration of the buffer solution is 0.01 to 0.05mol/L.

5. The detection method according to claim 4, wherein the concentration of the buffer solution is 0.05mol/L.

6. The detection method according to claim 1, wherein the pH of the buffer solution is adjusted by adding triethylamine or an aqueous ammonia solution dropwise.

7. The detection method according to claim 1, wherein in the reverse phase liquid chromatography, the detection wavelength is 254nm;

and/or the flow rate of the mobile phase is 0.5-1.0 mL/min;

and/or in the reverse phase liquid chromatography, the temperature of a chromatographic column is 30-40 ℃.

8. The detection method according to claim 7, wherein in the reverse phase liquid chromatography, the flow rate of the mobile phase is 0.8mL/min;

and/or, in the reverse phase liquid chromatography, the temperature of the chromatographic column is 35 ℃.

9. The detection method according to claim 1, wherein the total volume of the mobile phase is 100%; in 0-3 min, the volume of the buffer solution is 57%; in 3-15 min, the volume of the buffer solution is reduced from 57% to 50%; in 15-36 min, the volume of the buffer solution is decreased from 50% to 32%; the volume of the buffer solution is increased from 32% to 57% in 36-37 min; in 37-45 min, the volume of the buffer solution is 57%;

or, based on the total volume of the mobile phase being 100%; in 0-3 min, the volume of the buffer solution is 55%; the volume of the buffer solution is reduced from 55% to 50% in 3-15 min; the volume of the buffer solution is reduced from 50% to 30% in 15-36 min; the volume of the buffer solution is increased from 30% to 55% within 36-37 min; the volume of the buffer solution is 55% in 37-45 min;

or, based on the total volume of the mobile phase being 100%; in 0-3 min, the volume of the buffer solution is 53%; in 3-15 min, the volume of the buffer solution is reduced from 53% to 50%; the volume of the buffer solution is reduced from 50% to 27% in 15-36 min; the volume of the buffer solution is increased from 27% to 53% in 36-37 min; in 37-45 min, the volume of the buffer solution is 53%;

or, based on the total volume of the mobile phase being 100%; the volume of the buffer solution is reduced from 55% to 30% in 0-36 min; the volume of the buffer solution is increased from 30% to 55% within 36-37 min; the volume of the buffer solution is 55 percent in 37-45 min.

10. A method for detecting a substance related to hydroxychloroquine sulfate in a preparation, which comprises carrying out the detection by the method for detecting a substance related to hydroxychloroquine sulfate as described in any one of claims 1 to 9.