CN113237988B

CN113237988B - Method for detecting content of degraded impurity aldol dimer in oxycodone liquid preparation

Info

Publication number: CN113237988B
Application number: CN202110482052.7A
Authority: CN
Inventors: 程梦威; 贾惠艳
Original assignee: BEIJING JESKONT PHARMACEUTICAL INTERNATIONAL CO LTD
Current assignee: BEIJING JESKONT PHARMACEUTICAL INTERNATIONAL CO LTD
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-12-31
Anticipated expiration: 2041-04-30
Also published as: CN113237988A

Abstract

The invention provides a method for detecting the content of degraded impurity aldol dimer in an oxycodone liquid preparation, belonging to the technical field of drug analysis. The invention adopts the high performance liquid chromatography for detection, selects a chromatographic column with octadecylsilane chemically bonded silica as a filler by accurately controlling the detection conditions, controls the composition and proportion of a mobile phase, simultaneously adopts a gradient elution method and controls a gradient elution program, can detect the content of an impurity dimer generated by degradation in an oxycodone hydrochloride liquid preparation, has high sensitivity and strong specificity in the detection method, can ensure effective detection of genotoxic impurity dimer in the oxycodone hydrochloride liquid preparation, and provides a powerful control strategy for the product quality control of the medicaments, thereby providing guarantee for the product quality safety and effectiveness. Moreover, the detection method is accurate, stable, durable, scientific and reliable, and can well determine the content of the oxycodone aldol dimer in the oxycodone liquid preparation.

Description

Method for detecting content of degraded impurity aldol dimer in oxycodone liquid preparation

Technical Field

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting the content of degraded impurity aldol dimer in an oxycodone liquid preparation.

Background

The main active ingredient of the oxycodone hydrochloride liquid preparation is oxycodone hydrochloride, the chemical name of oxycodone is 7, 8-dihydro-14-hydroxycodeinone, and the molecular formula is as follows: c₁₈H₂₁NO₄Molecular weight: 315.37, the structural formula is:

oxycodone was first produced by thebaine in germany in 1916 and is a non-proprietary drug. In 2017, it is the 52 th most commonly used prescription in the U.S., with over 1500 ten thousand prescriptions. In combination with some existing abuse deterrent agents, such as naloxone, it is an opioid drug used for the treatment of moderate to severe pain, and is also a common narcotic analgesic drug on the market for intraoperative or cancerous pain. It is usually taken orally, onset of pain relief usually begins within 15 minutes, and lasts up to 6 hours using an immediate release formulation. In the uk, they are available by injection, and some oral solution products are on the market, and combinations are also used with acetaminophen (acetaminophen), ibuprofen, naloxone and aspirin.

In the process of developing the quality of products, the Oxycodone hydrochloride liquid preparation is used as an analgesic, and due to the existence of high-concentration Oxycodone, the Oxycodone hydrochloride liquid preparation can be further condensed to form an impurity Aldol Dimer (Oxycodone Aldol Dimer) under the conditions of illumination and long-term normal temperature, and the pharmacological and toxicological test literature of the impurity at present learns that the impurity can be a toxic impurity and can generate genetic toxicity, the impurity exceeds a certain limit and can generate great harm to human bodies, and the production conversion and condensation process of the Aldol Dimer is shown as the following formula:

according to the international human registration coordination (ICH) Q3A requirement, the general requirement of the State drug administration for drug safety and effectiveness, and controllable quality, and the related guiding principle of drug quality research, the related impurities of the drug are effectively controlled to ensure the controllable product quality, but the polar compound for degrading the high molecular weight of the impurity aldol dimer is not suitable for detection by adopting a common isocratic elution method, and the polar compound is not easily separated from the main component oxycodone, or the peak is easily seriously trailing after separation, so that the sensitivity of a detection sample is influenced. Therefore, it is necessary to develop a durable method for detecting the content of degraded impurity aldol dimer in oxycodone liquid preparation with high impurity quantification accuracy, good peak symmetry and high indication.

Disclosure of Invention

The invention aims to provide a method for detecting the content of degraded impurity aldol dimer in an oxycodone liquid preparation, which is accurate, simple, convenient and durable, and has high impurity quantification accuracy and good peak symmetry.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for detecting the content of degraded impurity aldol dimer in an oxycodone liquid preparation, which comprises the following steps:

providing a test solution, a reference solution and a sensitivity solution; the test solution is an oxycodone liquid preparation; the reference solution is a diluted solution of the test solution, and the concentration of oxycodone in the reference solution is 50-100 mug/mL; the sensitivity solution is a diluted solution of a test sample solution, and the concentration of oxycodone in the sensitivity solution is less than or equal to 30 mug/mL;

respectively carrying out high performance liquid chromatography detection on the test solution, the reference solution and the sensitivity solution to obtain a test chromatogram, a reference chromatogram and a sensitivity chromatogram, so that the signal-to-noise ratio of the peak height of an oxycodone chromatogram in the sensitivity chromatogram is more than or equal to 10, and the tailing factor of the peak height of the oxycodone chromatogram in the reference chromatogram is 0.8-1.5; quantifying the content of degraded impurity aldol dimer in the oxycodone liquid preparation by adopting an external standard method or a self-contrast method, thereby detecting and obtaining the real content of the degraded impurity aldol dimer in the oxycodone liquid preparation;

the conditions of the high performance liquid chromatography detection comprise:

a chromatographic column: a high performance liquid chromatography column with octyl silane bonded silica gel as a filler;

column temperature: 55-60 ℃;

mobile phase: taking a sodium octane sulfonate buffer solution-acetonitrile mixture as a mobile phase A, and taking an acetonitrile-water mixture as a mobile phase B;

performing gradient elution by using the mobile phase, wherein the procedure of the gradient elution is as follows:

flow rate: the peak retention time of oxycodone is 6.8-7.2 min;

a detector: an ultraviolet detector with the detection wavelength of 280 nm;

sample introduction amount: 10 μ L.

Preferably, the oxycodone liquid preparation comprises an oxycodone oral solution or an oxycodone injection.

Preferably, the concentration of the test solution is 10-20 mg/mL.

Preferably, the preparation process of the reference solution and the sensitivity solution is independent as follows: and mixing the diluent with the test solution to obtain a reference solution or a sensitivity solution.

Preferably, the diluent comprises 0.85 mass percent of phosphoric acid aqueous solution, 0.85 mass percent of sulfuric acid aqueous solution, 0.01-0.03 mol/L hydrochloric acid aqueous solution or 0.01-0.03 mol/L acetic acid aqueous solution.

Preferably, the preparation process of the sodium octane sulfonate buffer solution in the mobile phase a comprises: and mixing ammonium dihydrogen phosphate, anhydrous sodium octane sulfonate, triethylamine and water, and adjusting the pH value to 1.95-2.05 by using phosphoric acid.

Preferably, the concentration of the sodium octane sulfonate buffer solution is 0.02-0.04 mol/L.

Preferably, the volume ratio of the sodium octane sulfonate buffer solution to the acetonitrile in the mobile phase A is (1050-1155): (95-200);

preferably, the volume ratio of acetonitrile to water in the mobile phase B is 400: 100.

Preferably, the specification of the chromatographic column is Waters C8

4 μm, 3.9 × 150mm chromatography column.

The invention provides a method for detecting the content of degraded impurity aldol dimer in an oxycodone liquid preparation, which adopts a high performance liquid chromatography for detection, through precisely controlling the detection conditions, selecting a chromatographic column with octadecylsilane chemically bonded silica as a filler, controlling the composition and proportion of a mobile phase, meanwhile, the content of impurity dimers generated by degradation in the oxycodone hydrochloride liquid preparation (including oral solution or injection) can be detected by adopting a gradient elution method and controlling a gradient elution program, the detection method has high sensitivity and strong specificity, can ensure that the genotoxic impurity dimers in the oxycodone hydrochloride liquid preparation are effectively detected, and the accuracy of impurity quantification and peak symmetry are greatly improved, and a powerful control strategy is provided for the product quality control of the medicines, so that the safety and the effectiveness of the product quality are guaranteed.

Compared with the existing detection technology (oxycodone hydrochloride injection import registration standard), the detection method provided by the invention improves the detection sensitivity of the target detection substance aldol dimer; the peak parameter of the chromatographic peak of the detected target substance is greatly improved, the peak symmetry of the impurity and the oxycodone serving as an active ingredient is better, and the quantification of the impurity is more accurate and reliable; the method adopts a gradient elution method for analysis in a shorter time, greatly improves the detection efficiency, and has better performance on the excellent performance.

The detection method has better durability, slightly changes parameters such as the temperature of a chromatographic mobile phase and a chromatographic column, the flow rate of the chromatographic column and the like, cannot cause the conditions that a detected target peak cannot be separated from other impurities or the peak parameters do not meet target requirements and the like, but the existing detection method (oxycodone hydrochloride injection import registration standard) has a fragile chromatographic system, slightly changes chromatographic conditions, can cause the conditions of detection sensitivity reduction and the like, and has harsher conditions.

The detection method has good reproducibility and repeatability, and is beneficial to improving the production efficiency.

Drawings

FIG. 1 is a chromatogram of a stock solution of a test sample in comparative example 1;

FIG. 2 is a chromatogram of a control solution of comparative example 1;

FIG. 3 is a linear regression of oxycodone hydrochloride for group A;

FIG. 4 is a linear regression plot of oxycodone hydrochloride for group B;

FIG. 5 is a linear regression plot of group A oxycodone aldol dimer;

FIG. 6 is a linear regression plot of oxycodone aldol dimer in group B;

FIG. 7 is a 0 hour sample chromatogram of the dimer methodology-solution stability-test solution;

FIG. 8 is a 6 hour sample chromatogram of a dimer methodology-solution stability-test solution;

FIG. 9 is a sample chromatogram of a dimer methodology-solution stability-test solution for 9 hours;

FIG. 10 is a 12 hour sample chromatogram of a dimer methodology-solution stability-test solution;

FIG. 11 is a 17 hour sample chromatogram of a dimer methodology-solution stability-test solution;

FIG. 12 is a 24 hour sample chromatogram of a dimer methodology-solution stability-test solution;

FIG. 13 is a dimer methodology-durability test-normal condition self-control chromatogram;

FIG. 14 is a dimer methodology-durability test dimer control solution chromatogram;

FIG. 15 is a chromatogram of a dimer methodology-durability test-test sample solution;

FIG. 16 is a dimer methodology-durability test-test chromatogram at a wavelength of 278 nm;

FIG. 17 is a dimer methodology-durability test-test chromatogram at a wavelength of 282 nm;

FIG. 18 is a dimer methodology-durability test-column temperature 58 ℃ test chromatogram;

FIG. 19 is a dimer methodology-durability test-column temperature 62 ℃ test chromatogram;

FIG. 20 is a dimer methodology-durability test-low flow rate (1.2mL/min) test chromatogram;

FIG. 21 is a dimer methodology-durability test-high flow rate (1.4mL/min) test chromatogram;

FIG. 22 is a dimer methodology-durability test-pH1.95 test chromatogram;

figure 23 is a dimer methodology-durability test-different chromatography column test chromatogram.

Detailed Description

column temperature: 55-60 ℃;

flow rate: the peak retention time of oxycodone is 6.8-7.2 min;

a detector: an ultraviolet detector with the detection wavelength of 280 nm;

sample introduction amount: 10 μ L.

In the present invention, the required raw materials or equipment are commercially available products well known to those skilled in the art unless otherwise specified.

The invention provides a test solution, a reference solution and a sensitivity solution. In the present invention, the test solution is an oxycodone liquid preparation; the oxycodone liquid preparation preferably comprises an oxycodone oral solution or an oxycodone injection. In the invention, the concentration of the test solution is preferably 10-20 mg/mL, and more preferably 15 mg/mL. The source of the oxycodone liquid preparation is not particularly limited in the present invention, and any commercially available product well known in the art may be used; in the embodiment of the invention, the oxycodone liquid preparation is

A commercially available liquid formulation of oxycodone hydrochloride.

In the invention, the reference solution is a diluted solution of a test solution, and the concentration of oxycodone in the reference solution is 50-100 mug/mL; the preparation process of the control solution is preferably as follows: mixing the diluent with the test solution to obtain a reference solution. In the invention, the diluent preferably comprises 0.85 mass percent of phosphoric acid aqueous solution, 0.85 mass percent of sulfuric acid aqueous solution, 0.01-0.03 mol/L hydrochloric acid aqueous solution or 0.01-0.03 mol/L acetic acid aqueous solution. The process of mixing the diluent and the sample solution is not particularly limited in the present invention, and the diluent and the sample solution may be uniformly mixed according to a process known in the art to obtain a control solution having the above concentration.

In the invention, the sensitivity solution is a diluted solution of a test solution, and the concentration of oxycodone in the sensitivity solution is less than or equal to 30 mug/mL, and more preferably 1.5 mug/mL; the preparation process of the sensitivity solution is preferably as follows: and mixing the diluent with the test solution to obtain a sensitivity solution. In the invention, the diluent preferably comprises 0.85 mass percent of phosphoric acid aqueous solution, 0.85 mass percent of sulfuric acid aqueous solution, 0.01-0.03 mol/L hydrochloric acid aqueous solution or 0.01-0.03 mol/L acetic acid aqueous solution. The process of mixing the diluent and the sample solution is not particularly limited in the present invention, and the diluent and the sample solution may be uniformly mixed according to a process known in the art to obtain a sensitive solution having the above concentration.

After a test solution, a reference solution and a sensitivity solution are obtained, respectively carrying out high performance liquid chromatography detection on the test solution, the reference solution and the sensitivity solution to obtain a test chromatogram, a reference chromatogram and a sensitivity chromatogram, so that the signal-to-noise ratio of the peak height of an oxycodone chromatogram peak in the sensitivity chromatogram is more than or equal to 10, and the tailing factor of the peak height of the oxycodone chromatogram peak in the reference chromatogram is 0.8-1.5; and quantifying the content of the degraded impurity aldol dimer in the oxycodone liquid preparation by adopting an external standard method or a self-contrast method, thereby detecting and obtaining the real content of the degraded impurity aldol dimer in the oxycodone liquid preparation.

In the present invention, the conditions of the high performance liquid chromatography detection include:

column temperature: 55-60 ℃;

flow rate: the peak retention time of oxycodone is 6.8-7.2 min;

a detector: an ultraviolet detector with the detection wavelength of 280 nm;

sample introduction amount: 10 μ L.

In the present invention, the specification of the column is preferably Waters C8

4 μm, 3.9 × 150mm chromatography column.

In the invention, the column temperature is preferably 56-58 ℃.

In the invention, the preparation process of the sodium octane sulfonate buffer solution in the mobile phase a is preferably as follows: and mixing ammonium dihydrogen phosphate, anhydrous sodium octane sulfonate, triethylamine and water, and adjusting the pH value to 1.95-2.05 by using phosphoric acid. In the present invention, the ratio of the ammonium dihydrogen phosphate to the anhydrous sodium octane sulfonate to the triethylamine is preferably 18.4 g: 3.99 g: 4 mL; the amount of water used is preferably such that the mixed solution is diluted to 3520 mL. The process of adjusting the pH value using phosphoric acid in the present invention is not particularly limited, and may be carried out according to a process known in the art. In the invention, the concentration of the sodium octane sulfonate buffer solution is preferably 0.02-0.04 mol/L, and more preferably 0.03 mol/L.

In the invention, the volume ratio of the sodium octane sulfonate buffer solution to the acetonitrile in the mobile phase A is preferably (1050-1155): (95-200), more preferably (1100-1150): (100-180), and further preferably 1100: 150;

in the present invention, in the mobile phase B, the volume ratio of acetonitrile to water is preferably 400: 100.

in the present invention, during the detection by high performance liquid chromatography, gradient elution is performed by using the mobile phase, and the procedure of gradient elution is preferably as follows:

in the invention, the flow rate of the mobile phase is such that the peak retention time of oxycodone is 6.8-7.2 min, and more preferably 7.0 min; the specific process for controlling the flow rate is not particularly limited in the present invention, and the flow rate may be controlled according to a process well known in the art.

In the invention, the detector used for the high performance liquid chromatography detection is an ultraviolet detector, and the detection wavelength is 280 nm; the present invention is not limited to a specific type of the detector, and a detector known in the art may be used.

In the invention, the sample amount of the high performance liquid chromatography detection is 10 mu L.

In the invention, the chromatograph used for the high performance liquid chromatography detection is preferably a Woltz E2695 chromatograph or an Agilent 1290 chromatograph.

In the invention, the process of quantifying the aldol dimer by adopting an external standard method preferably adopts an impurity reference substance to quantify impurities, and the impurity quantitative calculation method is as shown in formula (1):

W％＝(C_R*A_s)/(A_R*C_sf) 100% formula (1);

in the formula (1), W% represents the content of aldol dimer as an impurity;

C_R-represents the concentration of the weighed out reference sample of aldol dimer as an impurity (mg/mL);

A_S-represents the peak area of the chromatographic peak of the impurity aldol dimer in the chromatogram of the test sample (detection sample);

A_R-represents the area of aldol dimer peak in the control chromatogram;

C_S-represents the concentration (mg/mL) of the test sample oxycodone hydrochloride

In the present invention, the aldol dimer is quantified by a self-control method, preferably by adding a correction factor, and the impurity is quantitatively calculated by the following formula (2):

calculating the impurity: w% (C)_R*A_s)/(A_R*C_sF) 100% formula (2);

in the formula (2), W% represents the content of aldol dimer as an impurity;

A_R-represents the area of aldol dimer peak in the control chromatogram;

C_S-represents the concentration (mg/mL) of the test article oxycodone hydrochloride;

f-represents the relative correction factor for the impurity relative to oxycodone hydrochloride, RRF (i.e., the reciprocal of the response factor).

In the invention, the signal-to-noise ratio of the peak height of the oxycodone chromatographic peak in the sensitivity chromatogram is not less than 10, and the tailing factor of the oxycodone chromatographic peak in the contrast chromatogram is 0.8-1.5. The invention has no special limitation on the process of satisfying the peak height signal-to-noise ratio of the oxycodone chromatogram peak in the sensitivity chromatogram by adjusting parameters and comparing the tail factor of the oxycodone chromatogram peak in the chromatogram to be 0.8-1.5, and can change the concentration of a test sample or change the detection condition of the high performance liquid chromatography according to the well-known process in the field.

The process for detecting the actual content of degraded impurity aldol dimer in the oxycodone liquid preparation is not particularly limited and can be carried out according to the process well known in the art.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Test solution: a commercially available oxycodone hydrochloride injection (labeled amount: 100.5%, specification: 10mg:1 mL);

control solution: diluting the test solution into a solution containing oxycodone hydrochloride 50 mu g per 1mL by using a phosphoric acid aqueous solution with the mass fraction of 0.85% as a reference solution;

sensitivity solution: diluting the test solution with 0.85% phosphoric acid aqueous solution by mass fraction to obtain a solution containing 1.5 μ g of oxycodone hydrochloride per 1mL as a sensitivity solution;

high performance liquid chromatograph: agilent 1290 chromatography;

a chromatographic column: high performance liquid chromatography column with octyl silane bonded silica gel as filler, wherein the specification of the chromatography column is Waters C8

4 μm, 3.9 x 150mm chromatography column;

mobile phase: taking sodium octane sulfonate buffer solution-acetonitrile (volume ratio is 1100: 150) as a mobile phase A, and taking acetonitrile-water (volume ratio is 400: 100) as a mobile phase B;

the preparation process of the sodium octane sulfonate buffer solution comprises the following steps: adding water to dilute and dissolve 18.4g of ammonium dihydrogen phosphate, 3.99g of anhydrous sodium octane sulfonate and 4mL of triethylamine to 3520mL, and then adjusting the pH value to 2.0 by using phosphoric acid; the concentration of the octane sodium sulfonate solution buffer solution is 0.03 mol/L;

the high performance liquid chromatography detection adopts gradient elution, and the gradient elution program is shown in table 1:

table 1 gradient elution procedure for hplc assay in example 1

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
20	80	20
			21	100	0
30	100	0

A detector: ultraviolet detector, detection wavelength: 280 nm;

column temperature: 60 deg.C

Sample introduction amount: 10 mu L of the solution;

the flow rate is adjusted by adopting the method to ensure that the peak retention time of the main component oxycodone is about 7 minutes, 10 mu L of each of the test solution, the reference solution and the sensitivity solution is precisely measured and respectively injected into a high performance liquid chromatograph, the chromatogram is recorded, and the limit of impurities in the test solution is as follows: the proportion of the degraded component of the aldol dimer is not more than 0.5 percent (mass percentage) of the content of oxycodone;

method for quantification of impurity limits (method using self-control + correction factor): and (3) quantifying impurities by adopting an impurity reference substance, wherein the impurity quantitative calculation method is as shown in a formula (1):

W％＝(C_R*A_s)/(A_R*C_sf) 100% formula (1);

in the formula (1), W% represents the content of aldol dimer as an impurity;

A_R-represents the area of aldol dimer peak in the control chromatogram;

calculating the impurity: w% (C)_R*A_s)/(A_R*C_sF) 100% formula (2);

in the formula (2), W% represents the content of aldol dimer as an impurity;

A_R-represents the area of aldol dimer peak in the control chromatogram;

f-represents the relative correction factor for the impurity relative to oxycodone hydrochloride, RRF (i.e. the reciprocal of the response factor)

The signal-to-noise ratio of the peak height of the chromatographic peak of the main component in the chromatogram of the sensitivity solution is more than or equal to 10, and the tailing factor of the chromatographic peak of the main component (oxycodone) in the chromatogram of the contrast solution is between 0.8 and 1.5; if a chromatographic peak with the retention time of 2.17-2.21 times of the main component exists in the chromatogram of the test solution, the peak area is larger than the main peak area (0.5%) of the chromatogram of the control solution.

Comparative example 1

Detecting a sample by adopting a method for detecting an oxycodone aldol dimer in oxycodone hydrochloride injection import registration standard (JX20160245), and comparing the content of the aldol dimer in a self-made preparation (self-made oxycodone hydrochloride injection) and a reference preparation (commercially available oxycodone hydrochloride injection), wherein the content of the aldol dimer in the test solution: i.e., oxycodone hydrochloride injection (lot: BM201, certified company: NAPPPHARMACEUTICALS LIMITED, specification 1mL:10 mg); specific detection conditions are shown in table 1:

TABLE 1 determination of aldol dimer by imported drug registration standard (JX20160245)

The detection method described in Table 1 is adopted to detect oxycodone hydrochloride bulk drugs (oxycodone hydrochloride, batch number: 15-0072, supplier: Macfarlan Smith Limited, UK), self-made preparations and reference preparations, the sample detection spectrum is 1-2, and the detection result is as follows:

TABLE 2 summary of dimer test results in crude drug, reference formulation and self-made formulation

Sample name	Batch number	Peak area of aldol dimer	Tailing factor	Content (%)
					Raw material medicine	15-00724	Not detected out	1.71	-
Reference formulation 1mL 10mg	BM201	18549	1.75	0.15％
					Self-made preparation (1mL:10mg)	8161003	19523	2.97	0.16％
Self-made preparation (2mL:20mg)	8161003	12543	1.65	0.12％

As can be seen from table 2 and fig. 1-2, when the aldol dimer detection is performed on the oxycodone hydrochloride bulk drug, the self-made product and the reference preparation according to the import registration standard, the symmetry of the chromatographic peaks of the aldol dimer and the oxycodone is poor, and the two chromatographic peaks have serious tailing phenomena, which causes the deviation of the measurement result; the method has long detection time, which is approximately 60 minutes.

Method verification

1. Linearity and range

1.1 Linearity of oxycodone hydrochloride

10mg of oxycodone hydrochloride control (oxycodone hydrochloride, batch number: 15-0072, supplier: Macfarlan Smith Limited, UK) was precisely weighed, placed in a 20mL measuring flask, dissolved and diluted to the scale with a diluent (0.85% by mass aqueous phosphoric acid), shaken well and used as a linear stock solution.

Precisely measuring linear stock solutions 0.1mL, 0.2mL, 0.5mL, 1.0mL and 1.5mL, respectively placing in 5 10mL measuring bottles, adding the diluent to dilute to scale, shaking up, and respectively using as No. 1-5 linear solutions.

Precisely transferring 10 mu L of each of the No. 1 to No. 5 linear solutions, respectively injecting into a liquid chromatograph, and recording a chromatogram.

The above process was independently performed by two testers a and B using different hplc (wharton E2695 or agilent 1290) and respectively designated as group a and group B. The linear solution dilution process and test results are shown in tables 3-5:

TABLE 3 Linear oxycodone hydrochloride solution preparation procedure

Number of solution	S-1#	S-2#	S-3#	S-4#	S-5#
						Linear stock solution sample volume	0.1	0.2	0.5	1.0	1.5
Dilution factor	10	10	10	10	10
						Limit of equivalent impurities (%)	0.05％	0.1％	0.25％	0.5％	0.75％
Concentration (μ g/ml)	5	10	25	50	75

TABLE 4 Linear test results for oxycodone hydrochloride (experimenter A, Vorteth E2695)

TABLE 5 Linear test results for oxycodone hydrochloride (experimenter B, Agilent 1290 chromatograph)

1.2 Linearity of aldol dimers

10mg of oxycodone aldol dimer reference substance (batch number: YGY-P1, source: Shanghai pharmaceutical industry research institute, LLC) is precisely weighed, placed in a 20mL measuring flask, dissolved and diluted to scale by adding a diluent (phosphoric acid aqueous solution with the mass fraction of 0.85%), shaken up and used as an aldol dimer linear storage solution.

Precisely measuring 0.03mL, 0.1mL, 0.2mL, 0.5mL, 1.0mL and 1.5mL of the aldol dimer linear stock solution, respectively placing the aldol dimer linear stock solution in 6 10mL measuring bottles, adding the diluent to dilute the solution to a scale, shaking up, and respectively taking the solution as No. 1-6 linear solutions.

Precisely transferring 10 μ L of the No. 1-6 linear solutions, injecting into a liquid chromatograph, and recording chromatogram. The processes are respectively carried out at different times by two testers A and B, different high performance liquid chromatographs are used for independently carrying out tests and are respectively marked as A group and B group, and the linear solution dilution process is shown in a table 6:

TABLE 6 aldol dimer Linear solution preparation procedure

Number of solution	S-1#	S-2#	S-3#	S-4#	S-5#	S-6#
							Storage solution sample volume (mL)	0.03	0.1	0.2	0.5	1.0	1.5
Dilution factor	10	10	10	10	10	10
							Limit of equivalent impurities (%)	0.015％	0.05％	0.1％	0.25％	0.5％	0.75％
Concentration (μ g/mL)	1.50	5	10	25	50	75

Taking the concentration as an abscissa and the peak area as an ordinate, respectively drawing linear curves for the group A and the group B, and respectively showing the test results in tables 7 and 8:

TABLE 7 aldol dimer linearity test results (experimenter A, Vorteth E2695)

TABLE 8 aldol dimer linearity test results (experimenter B, Agilent 1290 chromatograph)

Wherein, the linear regression graphs of the oxycodone hydrochloride and the aldol dimer are respectively shown in the figures 3-6, and the specific numerical values are shown in the table 9:

TABLE 9 calculation of aldol dimer correction factor

As can be seen from FIGS. 3 to 6, tables 4 to 5 and tables 7 to 9: the concentration of the aldol dimer is within the range of 1.3-65 mu g/mL, and the correlation coefficient of a linear regression equation is 1.0; the concentration of oxycodone hydrochloride is within the range of 4.7-71 mu g/mL, the correlation coefficient of a linear regression equation is 1.0, and the linear relation between the sample concentration and the peak area of the two components is good within the concentration range. The average correction factor of the oxycodone dimer and the oxycodone hydrochloride is 1.07, and the content of impurities in the oxycodone aldol dimer can be calculated by adopting a self-contrast method within the range of 0.8-1.2.

2. Limit of quantitation and limit of detection tests

2.1 limit of quantitation

Precisely transferring 0.03mL of the aldol dimer control stock solution in the 1.2, placing the sample in a 10mL measuring flask, adding a diluent solution (phosphoric acid aqueous solution with the mass fraction of 0.85%) to dissolve and dilute the sample to a scale, and shaking the sample uniformly to obtain an aldol dimer limit solution. 6 sample solutions (1S to 6S, respectively) were prepared in parallel by the method of 1.2 above, 10. mu.l of each sample solution was precisely measured and injected into a liquid chromatograph, and chromatograms were recorded, and the obtained data are shown in Table 10.

TABLE 10 summary of the quantitative limit test results

2.2 detection Limit

Precisely transferring 3mL of the aldol dimer quantitative limiting solution, placing the solution into a 10mL measuring flask, adding a diluent solution (phosphoric acid aqueous solution with the mass fraction of 0.85%) to dilute the solution to a scale, and shaking up the solution to be used as the aldol dimer detection limiting solution. 3 samples (respectively marked as 1S-3S) are prepared in parallel according to the method 1.2, 10 mu L of each sample is precisely measured and injected into a liquid chromatograph, 1 sample introduction time of each sample is carried out, a chromatogram is recorded, and the obtained results are shown in a table 11.

TABLE 11 summary of test results on detection limits

As can be seen from tables 10 and 11, the concentration of the quantitative limiting solution of oxycodone aldol dimer is 1.5. mu.g/ml, the average signal-to-noise ratio is not lower than 10, which is equivalent to the limit of being able to quantitatively detect the impurities being 0.015%, the RSD of 6 quantitative limiting solution is 1.19%, and the repeatability is good; the concentration of the detection limiting solution is 0.456 mug/mL, the signal-to-noise ratio of 3 parts of the detection limiting solution is not lower than 3, which is equivalent to the lowest limit of 0.0045 percent of the impurity which can be detected. In comparative example 1, the limit of the aldol dimer impurity in the imported registration standard is not more than 0.5%, and the test result shows that the method has high sensitivity, the concentration of the test solution is proper, and the content of the aldol dimer in the product can be quantitatively detected.

3. Stability of solution

3.1 stability of aldol dimer control solution

Precisely transferring 1.0mL of the aldol dimer control stock solution in the 1.2, placing the sample in a 10mL measuring flask, adding a diluent solution (phosphoric acid aqueous solution with the mass fraction of 0.85%) to dilute the sample to a scale, shaking the sample uniformly, and taking the sample as an aldol dimer control stability investigation solution. Taking the reference substance stability investigation solution, placing the solution in a transparent colorless liquid phase vial, placing the solution under the condition of not shielding from light at room temperature, injecting samples after 0 hour, 3 hours, 6 hours, 11 hours, 17 hours and 24 hours respectively, recording a sample solution chromatogram at each time, calculating the ratio of the peak area in the chromatogram at each time point to the peak area at 0 hour, and obtaining the test results shown in the table 12:

TABLE 12 summary of aldol dimer control solution stability test results

Test investigation time (h)	Peak area	Peak area ratio to 0h
			0	55891	1.00
3	55825	1.00
			6	56009	1.00
11	56202	1.01
			17	56066	1.00
24	55865	1.00

3.2 examination of stability of test solutions

Taking oxycodone hydrochloride injection stock solution as a test sample stability investigation solution, placing the test sample stability investigation solution at a lightproof room temperature, injecting samples after 0 hour, 6 hours, 9 hours, 12 hours, 17 hours and 24 hours respectively, and recording sample solution maps at all times. And calculating the area of the aldol dimer peak and the oxycodone hydrochloride peak in the chromatogram at each time point, and comparing the areas with the 0-hour peak area respectively. The spectra of the samples at the time points are shown in FIGS. 7-12, and the test results are shown in Table 13:

TABLE 13 summary of stability test results for oxycodone and aldol dimer in test solutions

As can be seen from tables 12 and 13, when the test solution and the aldol dimer control solution are left at room temperature for 24 hours without being shaded, neither the oxycodone peak area nor the aldol dimer peak area changes significantly, the ratio of the peak area at different observation time points to the 0 hour peak area is 0.98-1.02, and the test results show that the two sample solutions are stable within 24 hours.

4. Recovery rate

(1) Aldol dimer stock solution

Taking 10mg of an aldol dimer reference substance, precisely weighing, placing in a 20mL measuring flask, adding a diluent solution (phosphoric acid aqueous solution with the mass fraction of 0.85%), dissolving and diluting to scale, shaking up, and taking the solution as an aldol dimer storage solution.

(2) Aldol dimer control solution

Precisely measuring 1.0mL of the aldol dimer stock solution, placing the aldol dimer stock solution into a 10mL measuring flask, adding a diluent (phosphoric acid aqueous solution with the mass fraction of 0.85%) to dilute the solution to a scale, and shaking up to obtain an aldol dimer control solution.

(3) Test solution (background solution): namely oxycodone hydrochloride injection stock solution.

(4) Sample recovery rate test solution preparation

Preparation of quantitative concentration-limiting sample-adding test solution

Taking 0.03mL of the aldol dimer stock solution, placing the aldol dimer stock solution in a 10mL measuring flask, adding the injection stock solution to dilute the solution to a scale, shaking the solution uniformly, and taking the solution as a quantitative limit concentration sample feeding test solution. Three samples were prepared in parallel according to the above preparation procedure and respectively labeled LL-1# -3 #. The preparation and loading process is shown in table 14:

TABLE 14 preparation of limiting concentration (0.015%) of sample-applied test solutions

② preparation of low-concentration sample-adding test solution

Taking 0.5mL of the aldol dimer stock solution, placing the aldol dimer stock solution in a 10mL measuring flask, adding the injection stock solution to dilute the solution to a scale, shaking up the solution to be used as a low-concentration sample-adding test solution. Three samples were prepared in parallel according to the above preparation procedure, and are respectively marked as L-1# -3 #. The preparation loading process is shown in table 15:

TABLE 15 preparation of Low concentration (0.25%) sample application test solution

Preparation of medium-concentration sample-adding test solution

Taking 1.0mL of the aldol dimer stock solutions respectively, placing the aldol dimer stock solutions into a 10mL measuring flask, adding the injection stock solution to dilute to a scale, shaking up, and taking the mixture as a medium-concentration sample-adding test solution. Three samples were prepared in parallel according to the above preparation procedure, and are respectively labeled as M-1# -3 #. The preparation loading process is shown in table 16:

preparation of test solutions loaded at concentrations (0.5%) in Table 16

Preparation of high-concentration sample-adding test solution

Taking 1.2mL of the aldol dimer stock solution, placing the aldol dimer stock solution in a 10mL measuring flask, adding the injection stock solution to dilute the solution to a scale, shaking up the solution to be used as a high-concentration sample-adding test solution. Three samples were prepared in parallel according to the above preparation procedure and respectively noted as H-1# -3 #. The preparation and loading process is shown in table 17:

TABLE 17 preparation of high concentration (0.6%) sample test solutions

Precisely measuring the sample adding test solution, injecting sample according to a formulated chromatographic condition, recording a chromatogram, and calculating the recovery rate of impurities according to an external standard method, wherein the results are shown in a table 18:

TABLE 18 summary of aldol dimer sample recovery test results

As can be seen from table 18: through the sample adding and recovery rate test of 4 concentrations of the impurity aldol dimer and 3 samples with each concentration, the average recovery rate of the impurity aldol dimer in 12 samples is 94.5%, the recovery rate is between 90% and 110%, the coefficient of variation of the recovery rate of 12 samples is 3.0%, and the test result shows that the method can accurately measure the content of the aldol dimer in the samples.

5. Precision degree

5.1 precision of sample introduction

Precisely transferring 10 mu L of aldol dimer reference solution in the '4 and recovery rate test', continuously feeding samples for 6 times according to the formulated chromatographic conditions, recording a chromatogram, calculating the RSD of the peak area of the 6-pin sample solution, and obtaining the test result shown in the table 19:

TABLE 19 summary of the results of the sample introduction precision tests

As can be seen from Table 19, the same aldol dimer control solution was continuously injected for 6 times, the RSD of the peak area was 0.13% and less than 2.0%, and the test results indicated that the precision of the apparatus was good.

5.2 repeatability test

6 parts of test sample solution are prepared in parallel, the content (%) of the aldol dimer in the 6 parts of test sample is calculated by respectively adopting a self-control method and an external standard method according to a proposed aldol dimer detection method, the repeatability of the aldol dimer detection method and the difference of the two calculation methods are inspected, and the test result is shown in a table 20.

TABLE 20 summary of repeatability test results

Sample number ID	Calculation by self control method (%)	External standard method calculation (%)
			S-1#	0.23	0.22
S-2#	0.23	0.22
			S-3#	0.23	0.22
S-4#	0.23	0.22
			S-5#	0.23	0.22
S-6#	0.23	0.22
			Average (%)	0.23	0.22
RSD(％)	0.82％	0.82％

The contents of the aldol dimer in the 6 parts of test solution are calculated by adopting two calculation methods, the two calculation results are consistent, and the RSD value of the 6 parts of sample solution is 0.82 percent and is less than 2.0 percent, which indicates that the method has good repeatability.

5.2 intermediate precision

Another instrument is used, different testers are used at different times, another 6 parts of test sample solutions are prepared in parallel according to the preparation method of each sample in the 5.2 repeatability test, the chromatogram is recorded according to the formulated chromatographic conditions, the dimer content (%) in the 12 parts of test samples in the intermediate precision test and the 5.2 repeatability test is calculated by respectively adopting an external standard method and a self-comparison method, and the results are shown in table 21.

TABLE 21 summary of intermediate precision test results

As can be seen from table 21: the aldol dimer is calculated by 12 samples respectively by adopting an external standard method and a self-contrast method, the average values are 0.23 percent and 0.22 percent respectively, and the calculation results of the two impurities are consistent. The impurity content RSD of the two calculation methods is respectively 0.84 percent and 1.37 percent, and both are less than 2.0 percent, the precision of the method is good according to the test result, and the impurity content can be accurately calculated by using an external standard method and a self-contrast method.

6. Durability test

Under the preliminarily prepared liquid chromatography conditions, the column temperature, the organic phase ratio and the pH value were varied to examine the durability of the chromatography conditions, and the variation of the chromatography conditions is shown in Table 22:

TABLE 22 durability test chromatographic Condition parameter variations

Item of parameter	Variation parameter
		Normal condition	Process conditions of example 1
Detection wavelength	Target conditions. + -. 2nm
		Flow rate of flow	Target flow rate. + -. 0.1ml/min
Temperature of chromatographic column	Target temperature + -2 deg.C
		pH of mobile phase	1.95～2.05
Different brands of similar chromatographic columns	Agilent ZORBAX SB C8(4.6×150mm，3.5μm)

Test solution: namely oxycodone hydrochloride injection stock solution.

② self-contrast solution: measuring 0.5mL of injection stock solution, placing in a 100mL measuring flask, adding diluent solution (phosphoric acid aqueous solution with mass fraction of 0.85%) to dilute to scale, and shaking up to obtain the final product.

③ aldol dimer control solution: precisely weighing 10mg of oxycodone aldol dimer reference substance, precisely weighing, placing into a 20mL measuring flask, adding a diluent solution (phosphoric acid aqueous solution with the mass fraction of 0.85%) to dilute to a scale, and shaking up. Precisely transferring 2mL of the solution, placing the solution into a 20mL measuring flask, adding a diluent solution (phosphoric acid aqueous solution with the mass fraction of 0.85%) to dilute the solution to a scale, and shaking up the solution to obtain the product.

And (3) recording chromatograms by using the prepared sample solution according to proposed chromatographic conditions, calculating the content (%) of the dimer in the sample to be tested by respectively adopting an external standard method and a self-comparison method, and counting the relative retention time of the aldol dimer, wherein the test result is shown in a table 23, and the chromatogram of each sample is respectively shown in fig. 13-23.

TABLE 23 durability test oxycodone aldol dimer content measurement results summary Table

As can be seen from Table 23, the chromatographic condition parameters slightly changed, the content of the aldol dimer in the test sample was not significantly different from the normal condition, and the results calculated by the external standard method and the self-control method were substantially consistent, respectively 0.20% and 0.22%, indicating that the method of the present invention is accurate and reliable for detecting the aldol dimer in the oxycodone liquid preparation. And the chromatographic conditions slightly change, the relative retention time of the aldol dimer changes from 2.07 to 2.21, the specific chromatographic column brand model is adopted, the peak-out time range of the oxycodone hydrochloride is specified, and the peak-out position of the aldol dimer can be positioned by adopting the relative retention time.

7. Comparison of oxycodone aldol dimer detection results

The measurement of aldol dimer was carried out on 6 samples and one reference preparation (commercially available oxycodone hydrochloride injection, lot # BM201, CA792 carrier: NAP PHARMACEUTICAL LIMITED) and a self-made preparation in total of two specifications by the method of example 1 and the method of comparative example 1, imported drug registration Standard (Standard No.: JX20160245), respectively, and the measurement results of each sample batch are shown in Table 24:

TABLE 24 summary of aldol dimer test results for two test methods

As can be seen from Table 24, the home-made formulation and the reference formulation were tested by the standard methods of import registration and example 1, respectively, and the contents of aldol dimer were consistent. The method of the embodiment 1 adopts a self-contrast method and an external standard method for calculation, and the detection results are consistent; the imported registration standard adopts an external standard method and a self-contrast method for calculation, and the result difference is large, because the peak type of the main component of oxycodone in the self-contrast solution in the imported registration standard method is trailing, the integral is not accurate, and the result difference is large. The method for preparing the oxycodone aldol dimer overcomes the defect of poor peak patterns of oxycodone hydrochloride and the aldol dimer, and ensures accurate integration result, thereby ensuring the accuracy and reliability of detection result.

8 specificity test

8.1 specificity test under conventional conditions

(1) Diluent solution: i.e., 0.85% phosphoric acid solution.

(2) The test solution is the stock solution of the injection.

(3) Preparing dimer peak positioning solution, namely precisely weighing 5mg of an aldol dimer reference substance (batch number: YGY-P1 purity: 87.04%, Shanghai pharmaceutical industry research institute Co., Ltd.), placing the aldol dimer reference substance in a 10mL measuring flask, adding a diluent solution to dilute the solution to a scale, shaking up the solution to be used as a dimer peak positioning storage solution, precisely weighing 1.0mL of the peak positioning storage solution, placing the solution in a 10mL measuring flask, adding the diluent to dilute the solution to the scale, and shaking up the solution to be used as the dimer peak positioning solution.

(4) Raw material test solution: weighing 40mg of aldol dimer reference substance, precisely weighing, placing in a 5mL measuring flask, adding 4mL of diluent solution for dissolving, and shaking up to be used as a raw material test solution.

(5) Dimer loading test solution preparation: placing 2mL of the test solution of the raw material medicine into a 5mL measuring flask, adding 0.5mL of the dimer peak positioning storage solution into the measuring flask, adding a diluent solution to dilute to a scale, and shaking up to obtain the test solution.

Precisely measuring 10 μ l of each solution, injecting sample according to the preset chromatographic conditions, recording chromatogram, and showing the results of specificity test in Table 25:

TABLE 25 summary of aldol dimer specificity test results

Sample name	Aldol dimer retention time (min)	Number of theoretical plate	Tailing factor
				Diluent	-	-	-
Test solutions	15.711	1.202421e+005	1.18
				Dimer peak localization solution	15.715	1.184805e+005	0.94
Bulk drug test solution	-	-	-
				Dimer sample application test solution	15.697	1.181964e+005	0.93

As can be seen from Table 25, the diluent does not interfere with the measurement of dimer in the sample, the dimer is well separated from the adjacent impurity peaks, and the specificity of the aldol dimer detection method meets the requirements.

8.2 specificity test under Strong degradation conditions

Forced destruction and degradation are carried out on a sample to be tested, the purpose is to confirm whether the method is exclusive or not, further know the distribution condition of impurities of the sample under each harsh degradation condition and know the inherent stability of the product; the specificity of the method was verified by peak purity detection to ensure that no impurities co-eluted with the aldol dimer peak, and the sample destruction process for each strong degradation condition is shown in table 26:

TABLE 26 sample destruction procedure for each of the Strong degradation conditions

The prepared sample solutions are injected according to the formulated chromatographic conditions, chromatograms are recorded, the damaged sample solutions are subjected to aldol dimer chromatographic peak purity investigation and are simultaneously compared with undamaged samples, and the test results are summarized as shown in table 27:

TABLE 27 summary of test results for strong degradation of aldol dimer assay

As can be seen from Table 27, neither the diluent solvent nor the chromatographic peak produced under each destruction condition interfered with the detection of aldol dimer, indicating that the specificity of the method met the requirements. And degrading the samples by acid, alkali, illumination, oxidation and high temperature, wherein the peak purity of the aldol dimer chromatographic peak in each sample is greater than a specified peak purity factor, so that co-eluted substances are not found in the aldol dimer chromatographic peak. The aldol dimer contained in the sample was degraded to some extent under each of the destructive conditions, indicating that the aldol dimer was less stable under the destructive conditions.

In addition, by combining the impurity localization (adopting relative retention time) of the impurity aldol dimer, the retention time of the main peak of the oxycodone serving as the active ingredient of the liquid preparation is about 7.1 minutes, and the retention time of the aldol dimer is about 15.7 minutes, under the chromatographic conditions of the above examples, the separation degree between the degraded impurity aldol dimer and the oxycodone impurity is good, the chromatographic peak symmetry of the impurity aldol dimer is good, the tailing factor is 0.94 and is close to 1.0, and meanwhile, the analysis time is about 30 minutes, so that the analysis time is shortened, and the requirement of daily impurity quality control is met.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting the content of degraded impurity aldol dimer in an oxycodone liquid preparation is characterized by comprising the following steps:

respectively carrying out high performance liquid chromatography detection on the test solution, the reference solution and the sensitivity solution to obtain a test chromatogram, a reference chromatogram and a sensitivity chromatogram, so that the signal-to-noise ratio of the peak height of an oxycodone chromatogram in the sensitivity chromatogram is more than or equal to 10, and the tailing factor of the peak height of the oxycodone chromatogram in the reference chromatogram is 0.8-1.5; quantifying the content of degraded impurity aldol dimer in the oxycodone liquid preparation by adopting a self-contrast method, thereby detecting and obtaining the real content of the degraded impurity aldol dimer in the oxycodone liquid preparation;

column temperature: 55-60 ℃;

mobile phase: taking a sodium octane sulfonate buffer solution-acetonitrile mixture as a mobile phase A, and taking an acetonitrile-water mixture as a mobile phase B; the volume ratio of the sodium octane sulfonate buffer solution to the acetonitrile in the mobile phase A is 1100: 150; the concentration of the sodium octane sulfonate buffer solution is 0.02-0.04 mol/L, and the pH value is 1.95-2.05;

the volume ratio of acetonitrile to water in the mobile phase B is 400:100, respectively;

flow rate: the peak retention time of oxycodone is 6.8-7.2 min;

a detector: an ultraviolet detector with the detection wavelength of 280 nm;

sample introduction amount: 10 μ L.

2. The test method according to claim 1, wherein the oxycodone liquid preparation comprises an oxycodone oral solution or an oxycodone injection.

3. The detection method according to claim 2, wherein the concentration of the sample solution is 10 to 20 mg/mL.

4. The detection method according to claim 1 or 3, wherein the control solution and the sensitivity solution are prepared independently by: and mixing the diluent with the test solution to obtain a reference solution or a sensitivity solution.

5. The detection method according to claim 4, wherein the diluent comprises 0.85% by mass of an aqueous phosphoric acid solution, 0.85% by mass of an aqueous sulfuric acid solution, 0.01 to 0.03mol/L of an aqueous hydrochloric acid solution, or 0.01 to 0.03mol/L of an aqueous acetic acid solution.

6. The detection method according to claim 1, wherein the preparation process of the sodium octane sulfonate buffer solution in the mobile phase A comprises the following steps: and mixing ammonium dihydrogen phosphate, anhydrous sodium octane sulfonate, triethylamine and water, and adjusting the pH value to 1.95-2.05 by using phosphoric acid.

7. The detection method according to claim 1, wherein the chromatographic column has a specification of Waters C8

4 μm, 3.9 × 150mm chromatography column.