WO2023123146A1 - Ultra-high performance liquid chromatography–mass spectrometry method for detecting genotoxic impurity 14-bromo-4'-epi-daunorubicin - Google Patents

Abstract

An ultra-high performance liquid chromatography–mass spectrometry method for detecting a genotoxic impurity 14-bromo-4'-epi-daunorubicin, comprising the following steps: respectively testing a test solution and a reference solution by using ultra-high performance liquid chromatography–mass spectrometry, recording a generated chromatogram, calculating a peak area according to an external standard method, and measuring the content of 14-bromo-4'-epi-daunorubicin in a drug. According to the detection method, sample derivatization pretreatment is not required, the time required for detection is shortened, and the method has the advantages of high detection speed, high sensitivity and accurate analysis result.

Description

A method for the determination of genotoxic impurity 14-bromo-4'-epidaunorubicin by ultra-high performance liquid chromatography-mass spectrometry technical field

The invention belongs to the field of drug analysis and detection, and in particular relates to an ultra-high performance liquid chromatography-mass spectrometry method for measuring 14-bromo-4'-epidaunorubicin.

Background technique

Epirubicin hydrochloride (Linezolid, formula I) is a synthetic oxazolidinone antibiotic. Its mechanism of action is to directly intercalate between DNA nucleobase pairs, interfere with the transcription process, and prevent the formation of mRNA, thereby inhibiting DNA and RNA and For protein synthesis, compared with daunorubicin and doxorubicin, epirubicin hydrochloride has equal or slightly higher efficacy, but has relatively less toxicity to bone marrow and heart.

14-Bromo-4'-epidaunorubicin (formula II) is a process impurity of epirubicin hydrochloride and a decarboxylated ketone product. Its structure is similar to that of daunorubicin hydrochloride, and it contains a genotoxicity warning structure. It has been identified and evaluated as a class 3 impurity in ICH M7. According to ICH M7, its TTC is 1.5μg/day, and the maximum daily dose of epirubicin hydrochloride is 270mg, so the limit of 14-bromo-4'-epidaunorubicin is 5.6ppm.

Due to the low limit of 14-bromo-4'-epidaunorubicin (5.6ppm), the sensitivity of ordinary high performance liquid chromatography cannot meet the requirements. Whether 14-bromo-4'-epidaunicin can be completely removed as a genotoxic impurity in the process needs to be tested and evaluated. At present, there is no information about 14-bromo-4'-epidaunin in epirubicin hydrochloride Report on the detection method of mycomycin.

Therefore, it is an urgent problem for those skilled in the art to develop a method with high sensitivity, small amount of sample, and rapid and accurate detection of the genotoxic impurity 14-bromo-4'-epidaunorubicin.

Contents of the invention

The invention provides a UPLC-MS detection method for 14-bromo-4'-epidaunorubicin in medicine, and the detection method adopts ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). For the analysis of 14-bromo-4'-epidaunorubicin, there is no need to derivatize the sample, which shortens the time required for detection. It has good linearity, strong specificity, good precision, high accuracy, and high sensitivity. The method has the advantages of rapidly and accurately measuring the content of 14-bromo-4'-epidaunorubicin.

In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:

An ultra-high performance liquid chromatography-mass spectrometry analysis method for 14-bromo-4'-epidaunorubicin, comprising the following steps: using ultra-high performance liquid chromatography-mass spectrometry to separately test the test solution and the reference substance The solution is detected, the chromatogram produced is recorded, and the peak area is calculated by the external standard method to detect the content of 14-bromo-4'-epidaunorubicin in the drug.

In the preferred technical scheme, the conditions of the ultra-high performance liquid chromatography are: adopt octadecylsilane bonded silica gel column, preferably Waters ACQUITY

BEH C18 chromatographic column, the column temperature is 25°C-45°C, preferably 30°C-45°C, more preferably 35°C-40°C, most preferably 40°C; gradient elution is carried out with mobile phase, and the mobile phase includes mobile phase A And mobile phase B, described mobile phase A is the aqueous solution of formic acid or acetic acid, preferably formic acid aqueous solution, its volume fraction is 0.01%～0.5%, preferably 0.05%～0.2%, more preferably 0.1%; Described mobile phase B is methyl alcohol Or acetonitrile, preferably acetonitrile.

In a preferred technical solution, the column diameter of the octadecylsilane bonded silica gel column is 2.1mm-4.6mm, the column length is 50mm-150mm, and the particle diameter is 1.6μm-3.0μm; preferably the column diameter is 2.1mm-3.5 mm, column length 50mm～100mm, particle size 1.6μm～2.5μm; more preferably column diameter 2.1mm～3.0mm, column length 50mm～100mm, particle size 1.6μm～1.8um; most preferably, column diameter 2.1mm, column length 100mm, particle size 1.7μm.

In a preferred technical scheme, the gradient elution procedure is:

At 0-4.0min, mobile phase A is 80-20vt% and mobile phase B is 20-80vt% for elution;

4.0～5.0min, mobile phase A 20%vt hold, mobile phase B 80vt% hold for elution;

At 5.0-5.1min, mobile phase A 20-80vt%, mobile phase B 80-20vt% for elution;

From 5.1 to 7.0 minutes, keep mobile phase A at 80%vt, and mobile phase B at 20vt% in parallel.

In a preferred technical solution, the conditions of the mass spectrometer are: the instrument used is an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer, scanning mode: mass spectrometry multiple reaction monitoring; ion source: electrospray ion source; Ion source mode: positive mode; capillary voltage: 1-4KV, preferably 1-3KV, more preferably 2-3KV, most preferably 3KV; drying gas temperature: 300-650°C, preferably 450-600°C, more preferably 500-550°C , most preferably 500°C; drying gas flow rate: 1000L/Hr; cone voltage: 15-40V, preferably 20-40V, more preferably 30-35V, most preferably 35V; ion source temperature: 150°C; 4-bromo-4' -Epidaunorubicin parent ion: 606.10, quantitative ion: 321.10, qualitative ion: 459.10.

In a preferred technical solution, the drug is an epirubicin bulk drug, preferably epirubicin hydrochloride.

In the preferred technical scheme, the concentration of the test solution is 0.5 to 2 mg/mL (concentration of epirubicin/erubicin hydrochloride), preferably 1 to 2 mg/mL, more preferably 1 mg/mL; preferably Yes, the solvent of the test solution is 40% to 60% aqueous acetonitrile, more preferably 50% aqueous acetonitrile.

In a preferred technical solution, the flow rate of the mobile phase is 0.2-0.4 mL/min, preferably 0.3 mL/min.

In the preferred technical scheme, the concentration range of the reference substance solution is 0.5ng/mL～20ng/mL (concentration of epirubicin/eprubicin hydrochloride), more preferably 1.0ng/mL～14.1ng/mL mL, most preferably 1.0ng/mL～14.0ng/mL.

In a preferred technical solution, the test injection volume of the test solution and the reference solution is 1-10 μL, preferably 1-3 μL, more preferably 3 μL.

It should be noted that, in some specific embodiments of the present invention, the standard curve of 14-bromo-4'-epidaunorubicin is determined by the following method: preparing about 1.0ng/mL, 2.0ng/mL, 6.0ng/mL mL, 10.0ng/mL, 14.0ng/mL 14-bromo-4'-epidaunorubicin standard solution;

Inject 14-bromo-4'-epidaunomycin standard solution into the ultra-high performance liquid phase-mass spectrometer respectively for detection, record the spectrum, and The peak area of mycin and the corresponding 14-bromo-4'-edaunorubicin concentration were used to determine the 14-bromo-4'-edaunorubicin standard curve. For example, the concentration of 14-bromo-4'-epidaunorubicin can be used as the abscissa, and the peak area of 14-bromo-4'-edaunorubicin can be used as the ordinate to determine the concentration of 14-bromo-4'-epidaunorubicin. Standard curve F of epidaunorubicin.

The method for detecting 14-bromo-4'-epidaunorubicin by ultra-high performance liquid phase-mass spectrometry (UPLC-MS) provided by the present invention has the following advantages: sample pretreatment is simple; The elution time of 4'-epidaunorubicin is about 2.8 minutes; the resolution is good, and there is no interference with the main peak of the sample; the linear correlation coefficient is good, and 14-bromo-4'-epidaunorubicin is within the respective linear range All internal linear correlation coefficients r>0.990; high sensitivity, the lowest quantification limit of instrument detection is 1.00ng/mL, and the lowest detection limit is 0.30ng/mL; high recovery rate, epirubicin hydrochloride samples added 14-bromo-4'- The recovery rate of epidaunorubicin impurity is 101%～106%. The method has the characteristics of rapidity, high efficiency, high sensitivity, no matrix interference in sample detection, good durability, etc., and can be applied to the detection of 14-bromo-4'-epidaunorubicin impurities.

The reagents and solvents used in the present invention are all chromatographic grades.

It should be emphasized that the numerical values or numerical endpoints involved in the technical solutions of the present invention are not limited to the numbers themselves in their meaning or intended scope of protection. Those skilled in the art can understand that they include those that have been widely recognized in the art. Acceptable permissible error ranges, such as experimental errors, measurement errors, statistical errors, random errors, etc., are included within the scope of the present invention.

Compared with the prior art, the present invention has the following beneficial effects, but this should not be interpreted as that the detection method of the present invention only has the following effects:

(1) The detection method of the present invention has high sensitivity, small amount of sample, no need to derivatize the sample, easy to operate, and its detection limit (0.30ppm) and quantification limit (1.00ppm) are far lower than 14-bromo-4'- The limit of epidaunorubicin (5.6ppm).

(2) The detection method of the present invention has a good linear relationship within the range of 1.00ng/mL～14.03ng/mL, and the linear correlation coefficient r is 0.9999.

(3) The detection method of the present invention is accurate and feasible, the sample recovery rate is between 101.99%-106.05%, the RSD is between 0.69%-1.96%, and the instrument precision RSD is 0.75%.

(4) The present invention has developed a new detection method for the genotoxic impurity 14-bromo-4'-epidaunicin, and realized the detection of 14-bromo-4'-epidaunin in epirubicin hydrochloride for the first time The effective control of mycotoxin ensures the safety of medication for patients.

Description of drawings:

Fig. 1 is the chromatogram of the specificity of 14-bromo-4'-epidaunorubicin of embodiment 1

Fig. 2 is the chromatogram of the 14-bromo-4'-epidaunorubicin reference substance of embodiment 2

Fig. 3 is the linear equation standard curve of 14-bromo-4'-epidaunorubicin of embodiment 2

Fig. 4 is the chromatogram of the quantitative limit solution of embodiment 3

Fig. 5 is the chromatogram of the detection limit solution of embodiment 3

Fig. 6 is the chromatogram of the sample (lot number 1060-D190701) solution of embodiment 6

Fig. 7 is the chromatogram of the sample (lot number 1060-D190702) solution of embodiment 6

Fig. 8 is the chromatogram of the sample (batch number 1060-D190703) solution of embodiment 6

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are to further describe the content of the present invention in detail, but it should not be understood that the scope of the above subject of the present invention is limited to the following examples. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The instrument and model used in the present invention are Waters ACQUITY I UPLC Class-TQ-S micro ultra-high performance liquid chromatography-triple quadrupole mass spectrometer. Epirubicin hydrochloride used in the examples of the present invention is a raw material drug produced by Zhejiang Hisun Pharmaceutical Co., Ltd. The 14-bromo-4'-epidaunorubicin reference substance used in the examples of the present invention is a refined standard substance of Hisun Pharmaceutical Co., Ltd., and the content is 76.2% after calibration.

The verification of items such as specificity, detection limit and quantification limit, precision, linearity and range, and accuracy in the specific implementation mode shall be carried out in accordance with the "Technical Guidelines for the Validation of Analytical Methods for Quality Control of Chemical Drugs" and the "Quality Standards Established for Chemical Drugs". Standardized Process Technical Guidelines", "Technical Guidelines for Chemical Drug Impurity Research", and relevant guidelines in the appendix of the current version of "Pharmacopoeia of the People's Republic of China" for methodological validation.

The instrument and detection condition used in the embodiment of the present invention are as follows:

The instrument and model are Waters ACQUITY I UPLC Class-TQ-S micro ultra-high performance liquid chromatography-triple quadrupole mass spectrometer.

Chromatographic conditions:

Mobile Phase A: Pipette 1.0mL of formic acid into a 1000mL beaker, stir evenly, filter and degas.

Mobile phase B: acetonitrile, chromatographic acetonitrile produced by Merck Company, degassed.

Column: Waters ACQUITY

BEH C18 2.1*100mm, 1.7μm;

Flow rate: 0.3mL/min;

Column temperature: 40°C;

Injection volume: 3μL;

Gradient elution, the gradient elution program is shown in the following table 1

Table 1 Gradient elution program

time (min)	Mobile phase A%	Mobile phase B%
0.0	80	20
4.0	20	80
5.0	20	80
5.1	80	20
7.0	80	20

Mass spectrometry conditions: scan mode: mass spectrometry multiple reaction monitoring; ion source: electrospray ion source; ion source mode: positive mode; capillary voltage: 3KV; drying gas temperature: 500°C; drying gas flow rate: 1000L/Hr; cone voltage: 35V; ion source temperature: 150°C. Desolvation temperature: 350°C; acquisition mode: MRM; 4-bromo-4'-epidaunorubicin parent ion: 606.10, quantifier ion: 321.10, and qualitative ion: 459.10.

The preparation method of the solution used in the embodiment of the present invention is all prepared as follows:

Diluent: 50% acetonitrile, measure 500mL of acetonitrile and 500mL of water and mix well.

Linear stock solution a: Accurately weigh 10.96mg of 14-bromo-4'-epidaunorubicin reference substance into a 100mL volumetric flask, add diluent to dissolve, dilute to the mark, and mix well; precisely pipette 6.0mL of the above solution To a 100mL volumetric flask, dilute to the mark with a diluent, mix well, then precisely pipette 1.0mL of the above solution into a 100mL volumetric flask, dilute to the mark with a diluent, and mix well. A linear stock solution a with a concentration of 50.1091 ng/mL was obtained. (Remarks: The content of the 14-bromo-4'-epidaunorubicin reference substance is 76.2%.)

Standard curve measurement solutions 1 to 5: Precisely pipette linear stock solution a 0.5mL, 1.0mL, 3.0mL, 5.0mL, 7.0mL into five 25mL volumetric flasks, dilute to the mark with diluent, mix well, and obtain standard Curve determination liquids 1 to 5, the concentrations are 1.0022ng/mL, 2.0044ng/mL, 6.0131ng/mL, 10.0218ng/mL, 14.0306ng/mL standard curve determination liquids respectively.

Sample solution: Accurately weigh an appropriate amount of epirubicin hydrochloride sample, dissolve and dilute it with a diluent to form a sample solution containing 1 mg/mL of epirubicin hydrochloride, and mix well. Configure 2 copies in parallel.

Example 1 Specificity

Blank solution: same diluent,

Standard solution: Determination solution 3 for the standard curve.

Sample solution: Weigh 25mg of epirubicin hydrochloride sample into a 25mL volumetric flask, dissolve it with diluent and adjust the volume to the mark.

Sample plus impurity solution: Weigh 25 mg of epirubicin hydrochloride sample into a 25 mL volumetric flask, dissolve with diluent, then add 3.0 mL of standard curve measurement solution 3, dilute to the mark with diluent, and mix well.

Each of the above solutions was injected with one needle, and the spectrum was recorded (Figure 1). It can be seen from the spectrum that there is no interference peak around the peak of 14-bromo-4'-epidaunorubicin, indicating that the method has good specificity, and the method can be used for this impurity detection.

Embodiment 2 linearity and range

Accurately measure the standard curve measurement solution 1-5, inject 3.0uL each, and record the spectrum of the reference substance (Figure 2).

Take the concentration (ng/mL) as the abscissa and the peak area as the ordinate to perform linear regression, and the linear correlation coefficient r should be greater than 0.990. Draw standard curve (Fig. 3), the results are shown in Table 2, obtain standard curve equation y=1372.6x-67.609, r=0.9999, show that 14-bromo-4'-epidaunorubicin solution is in 1.0ng/mL～14.0ng Good linearity in the /mL range.

Table 2 Linearity and range results

Concentration (ng/mL)	Peak area
1.0022	1353.481
2.0044	2703.27
6.0131	8131.966
10.0218	13590.599
14.0306	19278.536
Standard Curve Equation	Y=1372.6x-67.609
r	0.9999

Embodiment 3 detection limit and quantitative limit

The limit of quantification solution was configured in 6 parts and injected separately. The signal-to-noise ratio of each limit of quantification solution should be greater than 10, and the peak area RSD of 6 consecutive injections of the limit of quantification solution should not be greater than 10.0%. The limit of detection solution was configured in 3 parts and injected separately, and the signal-to-noise ratio of the limit of detection in each part should be greater than 3.

Quantitation limit solution preparation: Precisely pipette 0.5mL each of linear stock solution a into six 25mL volumetric flasks, dilute to the mark with diluent, and mix well. Inject 3.0 μL directly, and record the spectrum (Figure 4).

Detection limit solution: Precisely pipette 3.0mL each of the quantification limit solution into three 10mL volumetric flasks, dilute to the mark with diluent, and mix well. Inject 3.0 μL directly, and record the spectrum (Figure 5).

The test results are shown in Table 3: the signal-to-noise ratio of the detection limit solution is greater than 3, the signal-to-noise ratio of the quantification limit solution is greater than 10, and the peak area RSD of the 6-pin continuous injection of the quantification limit solution is less than 10.0%, indicating that the detection limit and the quantification limit meet Test requirements.

Table 3 Quantitation limit and detection limit results

Embodiment 4 accuracy (sample addition recovery rate)

Accurately weigh 25 mg of epirubicin hydrochloride and place it in a 25 mL volumetric flask, and prepare 9 parts in parallel. Divide into three groups (3 parts in each group), add 0.5mL linear stock solution a to the first group, add 3.0mL linear stock solution a to the second group, add 7.0mL linear stock solution a to the third group, and prepare the concentration 3 copies of 1.00ng/mL limit of quantitation solution, 6.01ng/mL spiked solution, and 14.03ng/mL spiked solution were injected into 3.0uL respectively, and the spectra were recorded. The results all met the requirements, and the method was explained. The accuracy is good. The results are shown in Table 4 below.

Table 4 Accuracy (recovery rate) result

Example 5 Precision

Accurately weigh 25 mg of epirubicin hydrochloride sample and place it in a 25 mL volumetric flask, add 3.0 mL of reference substance linear stock solution a to prepare a spiked solution with a concentration of 6.01 ng/mL, and prepare 6 copies in parallel. Inject 3.0 μL respectively, and record the spectrum. The laboratory results all meet the limit requirements, indicating that the precision of the method is good. The results are shown in Table 5 below.

Table 5 Results of precision experiments

Example 6 Sample Detection

Weigh 3 batches of API samples 1060-D190701, 1060-D190702, and 1060-D190703 of epirubicin hydrochloride, each 25 mg into a 25 mL volumetric flask. Two copies of each batch of samples were prepared in parallel. After the system is balanced, enter the blank solution, 2 injections of the linear solution, and 1 injection of the sample solution for detection, and record the spectra (Fig. 6, Fig. 7, Fig. 8). The results show that none of the three batches of samples 14-Bromo-4'-epidaunorubicin was detected, ie less than the detection limit, and the limits were reported as <1.00 ppm in all.

Claims (10)

1. An ultra-high performance liquid chromatography-mass spectrometry analysis method for 14-bromo-4'-epidaunorubicin, characterized in that it comprises the following steps: using ultra-high performance liquid chromatography-mass spectrometry to test the samples respectively solution and the reference substance solution were detected, the chromatogram produced was recorded, and the peak area was calculated by the external standard method to detect the content of 14-bromo-4'-epidaunorubicin in the drug.
2. The detection method according to claim 1, characterized in that, the condition of the ultra-high performance liquid chromatography is: adopt octadecylsilane bonded silica gel chromatographic column, preferably Waters ACQUITY

   

   BEH C18 chromatographic column, the column temperature is 25°C-45°C, preferably 30°C-45°C, more preferably 35°C-40°C, most preferably 40°C; gradient elution is carried out with mobile phase, and the mobile phase includes mobile phase A And mobile phase B, described mobile phase A is the aqueous solution of formic acid or acetic acid, preferably formic acid aqueous solution, its volume fraction is 0.01%～0.5%, preferably 0.05%～0.2%, more preferably 0.1%; Described mobile phase B is methyl alcohol Or acetonitrile, preferably acetonitrile.
3. The detection method according to claim 2, characterized in that, the column diameter of the octadecylsilane bonded silica gel chromatography column is 2.1 mm to 4.6 mm, the column length is 50 mm to 150 mm, and the particle diameter is 1.6 μm to 3.0 μm; Preferably, the column diameter is 2.1mm-3.5mm, the column length is 50mm-100mm, and the particle size is 1.6μm-2.5μm; more preferably, the column diameter is 2.1mm-3.0mm, the column length is 50mm-100mm, and the particle size is 1.6μm-1.8μm; most preferably, The column diameter is 2.1 mm, the column length is 100 mm, and the particle size is 1.7 μm.
4. detection method according to claim 2, is characterized in that, the program of described gradient elution is:

   At 0-4.0min, mobile phase A is 80-20vt% and mobile phase B is 20-80vt% for elution;

   4.0～5.0min, mobile phase A 20%vt hold, mobile phase B 80vt% hold for elution;

   At 5.0-5.1min, mobile phase A 20-80vt%, mobile phase B 80-20vt% for elution;

   From 5.1 to 7.0 minutes, keep mobile phase A at 80%vt, and mobile phase B at 20vt% in parallel.
5. The detection method according to any one of claims 1 to 4, wherein the conditions of the mass spectrometer are: the instrument used is an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer, and the scanning mode is mass spectrometry Multiple reaction monitoring; ion source: electrospray ion source; ion source mode: positive mode; capillary voltage: 1-4KV, preferably 1-3KV, more preferably 2-3KV, most preferably 3KV; drying gas temperature: 300-650°C, Preferably 450-600°C, more preferably 500-550°C, most preferably 500°C; drying gas flow rate: 1000L/Hr; cone voltage: 15-40V, preferably 20-40V, more preferably 30-35V, most preferably 35V; Source temperature: 150°C; 4-bromo-4'-epidaunorubicin precursor ion: 606.10, quantitative ion: 321.10, qualitative ion: 459.10.
6. The detection method according to any one of claims 1-5, wherein the drug is an epirubicin bulk drug, preferably epirubicin hydrochloride.
7. The detection method according to any one of claims 1 to 6, wherein the concentration of the test solution is 0.5 to 2 mg/mL, preferably 1 to 2 mg/mL, more preferably 1 mg/mL; preferably, The solvent of the test solution is 40%～60% acetonitrile aqueous solution, more preferably 50% acetonitrile aqueous solution.
8. The detection method according to any one of claims 2-7, characterized in that the flow rate of the mobile phase is 0.2-0.4 mL/min, preferably 0.3 mL/min.
9. The detection method according to any one of claims 1-8, characterized in that the concentration range of the reference solution is 0.5 ng/mL-20 ng/mL, more preferably 1.0 ng/mL-14.1 ng/mL.
10. The detection method according to any one of claims 1 to 9, characterized in that the test injection volume of the test solution and the reference solution is 1 to 10 μL, preferably 1 to 3 μL, more preferably 3 μL .

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