Method for detecting impurities in norepinephrine bitartrate injection

Abstract

The invention provides a method for detecting impurities in norepinephrine bitartrate injection, and relates to the technical field of medicine detection. The specific chromatographic conditions are as follows: an Avantor ACE Excel 3C18-PFP chromatographic column is adopted, a 0.08% sodium heptanesulfonate solution is taken as a mobile phase A, a mixed solution of acetonitrile-0. 09% sodium heptanesulfonate solution is taken as a mobile phase B, gradient elution is performed, the column temperature is 35 DEG C, the detection wavelength is 280 nm, the flow velocity is 1.0 mL.min <-1 >, and the sample injection volume is 20 mu L. Under the detection method provided by the invention, the separation degree of norepinephrine bitartrate and each impurity peak is greater than 1.5, the linear relationship with the chromatographic peak area in a corresponding range is good, and the correlation coefficients are greater than 0.9999; the quantification limit of norepinephrine bitartrate, three process impurities (namely impurities A-C) and a degradation impurity (namely impurity D) is 0.006-0.04 [mu] g.mL <-1 >; the average recovery rates (n is equal to 3) of the low-concentration level, the medium-concentration level and the high-concentration level of the impurities A-D are 100.23%-101.47%, 100.00%-100.35%, 100.04%-100.21% and 99.90%-100.55% respectively, and the RSD (n is equal to 3) is 0.3%-1.5%, 0.1%-0.4%, 0.1%-0.2% and 0.2%-0.7% respectively. The method provided by the invention is strong in specificity, high in sensitivity and good in accuracy, and can be used as a control and quality evaluation method for process impurities and degraded impurities in the norepinephrine bitartrate injection.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting impurities in norepinephrine bitartrate injection. Background Art

[0002] Norepinephrine bitartrate, chemical name (R)-4-(2-amino-1-hydroxyethyl)-1,2-benzenediol bitartrate, is an adrenergic receptor agonist. It increases myocardial contractility, constricts blood vessels and raises blood pressure by stimulating α receptors and β receptors. It is clinically used to treat hemorrhagic shock, cardiogenic shock, septic shock, infectious shock, etc. caused by a sharp drop in blood pressure, and has a definite effect. The related substance items of norepinephrine bitartrate injection are only included in the "Chinese Pharmacopoeia" (ChP2020), and the related substance standards only control impurities B and impurity D, among which impurity B (process impurity) is controlled by external standard method, and impurity D (degradation impurity) is controlled by main component self-control method without correction factor; the related substance items of raw materials are included in the "Chinese Pharmacopoeia" (ChP2020) and the "British Pharmacopoeia" (BP 2024). BP 2024 only controls impurity A for the related substance items of raw materials.

[0003] The production process of norepinephrine bitartrate is to use catechol as the starting material and prepare it through chloroacetylation, amination, hydrogenation and splitting reactions. Common process impurities include impurities A to C, and degradation impurities include impurity D. There are literature reports on the current research on norepinephrine bitartrate APIs and preparations related substances, but there are problems such as the small number of known impurities controlled and the wide limits, and the quality of products between batches cannot be effectively distinguished. In order to strictly control the quality of APIs and preparations, the present invention, based on domestic and foreign pharmacopoeia standards and relevant literature, combined with the four guiding principles of the 2020 edition of the "Chinese Pharmacopoeia", established a method for simultaneously determining the contents of four impurities A to D in norepinephrine bitartrate injection by the main component self-control method of HPLC plus correction factors, providing a more comprehensive basis for improving the quality standards of norepinephrine bitartrate injection. The chemical structural formula of impurities A to D is shown below:

[0004]

[0005] Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the shortcomings of the prior art, the present invention provides a method for detecting the content of impurities A to D in norepinephrine bitartrate injection and controlling the impurity limit by using HPLC combined with a correction factor principal component self-control method.

[0008] (II) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] A method for detecting impurities in norepinephrine bitartrate injection, wherein the impurities are impurities A to D, and the specific structural formula is as follows:

[0011]

[0012] The method utilizes high performance liquid chromatography for detection, and specifically comprises the following steps:

[0013] (1) Prepare reference solution and test solution;

[0014] (2) Determine chromatographic conditions: Use Avantor ACE Excel 3C 18 -PFP chromatographic column; mobile phase A: sodium heptane sulfonate solution; mobile phase B: acetonitrile and mobile phase A mixed in a certain proportion; gradient elution was performed with a volume ratio of mobile phase A: mobile phase B = 95-40:5-60;

[0015] (3) Testing the reference solution and the test solution under the above-mentioned liquid chromatography conditions;

[0016] (4) Based on the peak areas of impurities A to D and the peak area of ​​norepinephrine bitartrate in the chromatogram, the contents of impurities A to D were calculated respectively using the principal component self-reference method with correction factors.

[0017] Furthermore, the specification of the chromatographic column is 4.6 mm×100 mm, the filler particle size is 3 μm, the column temperature is 33-37° C., and the preferred column temperature is 35° C.

[0018] Furthermore, in step (2), mobile phase A is a sodium heptane sulfonate solution having a mass fraction of 0.05-0.1% and a pH value of 2.5-2.7 adjusted with phosphoric acid; preferably, the pH value is 2.6.

[0019] Furthermore, in step (2), the mobile phase B is a mixed solution of acetonitrile and mobile phase A in a volume ratio of 40 to 50:50 to 60.

[0020] Furthermore, in step (2), gradient elution is performed with mobile phase A: mobile phase B = 95-40:5-60, and the specific gradient elution program is: 0 min, A:B = 95:5→15 min, A:B = 80:20→25 min, A:B = 40:60→30 min, A:B = 40:60→31 min, A:B = 95:5→45 min, A:B = 95:5.

[0021] Furthermore, in step (2), the gradient elution flow rate is 0.9-1.1 mL min -1 The preferred flow rate is 1.0 mL min -1 .

[0022] Furthermore, in step (3), the injection volume of the reference solution and the test solution is 20 μL.

[0023] Furthermore, the detection wavelength of the liquid chromatograph in step (3) is 280 nm.

[0024] Furthermore, in step (4), the correction factors of impurities A to D are 0.6, 0.1, 0.6 and 0.7 respectively.

[0025] (III) Beneficial effects

[0026] The present invention establishes a method for simultaneously determining the contents of three process impurities (i.e., impurities A to C) and degradation impurities (i.e., impurity D) in norepinephrine bitartrate injection based on the principal component self-control method of HPLC combined with a correction factor, and discusses its limits. The specific chromatographic conditions are: using an Avantor ACE Excel3C18-PFP chromatographic column, using 0.08% sodium heptane sulfonate solution as mobile phase A, acetonitrile-0.08% sodium heptane sulfonate solution mixed solution as mobile phase B, gradient elution, column temperature 35°C, detection wavelength 280nm, flow rate 1.0mL·min-1, and injection volume 20μL. Under the detection method provided by the present invention, the separation degree of norepinephrine bitartrate and each impurity peak is greater than 1.5, and has a good linear relationship with the chromatographic peak area within the corresponding range, and the correlation coefficient is greater than 0.9999; the quantitative limit of norepinephrine bitartrate and the four impurities is 0.006-0.04μg·mL -1 The average recoveries (n=3) of impurities A-D at low, medium and high concentration levels were 100.23%-101.47%, 100.00%-100.35%, 100.04%-100.21% and 99.90%-100.55%, respectively, and the RSDs (n=3) were 0.3%-1.5%, 0.1%-0.4%, 0.1%-0.2% and 0.2%-0.7%, respectively. The method provided by the present invention has strong specificity, high sensitivity and good accuracy, and can be used as a control and quality evaluation method for process impurities and degradation impurities in norepinephrine bitartrate injection.

[0027] The present invention determines the correction factor f of impurities A to D relative to norepinephrine for the first time by calculating the slope of the regression equation of norepinephrine bitartrate and impurities A to D, and uses the principal component self-control method with correction factor to detect the related substances of norepinephrine bitartrate injection, and compares it with the reference substance external standard method. The results show that the method is more practical and feasible, and avoids the use of impurity reference substances. Compared with the impurity external standard method, the detection method provided by the present invention is accurate, reliable, efficient, simple to operate, and has strong specificity. The present invention is based on the principal component self-control method with correction factor established by high performance liquid chromatography to determine the content of 4 kinds of impurities in norepinephrine bitartrate injection. The method controls more specific impurities, tightens the limit, and helps to distinguish the quality of products on the market. It can be used for the quality control of norepinephrine bitartrate injection, and provides further reference for the revision and improvement of the standards of the Chinese Pharmacopoeia. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a chromatogram of system suitability solution; (A) chromatogram of system suitability solution 1; (B) chromatogram of system suitability solution 2; (C) chromatogram of system suitability solution 3; wherein: 1 is sodium metabisulfite; 2 is impurity D; 3 is norepinephrine bitartrate; 4 is degradation impurity; 5 is impurity A; 6 is impurity B; 7 is impurity C.

[0029] Figure 2 It is a chromatogram of a specificity test; (A) blank solvent; (B) blank excipient solution; (C) acid destruction; (D) alkali destruction; (E) oxidation destruction; (F) high temperature destruction; (G) light destruction; (H) undestroyed; among them: 2 is impurity D; 3 is norepinephrine bitartrate; 6 is impurity B; 7 is impurity C. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example

[0032] 1. Instruments and reagents

[0033] 1.1 Instrument

[0034] Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA, main instrument used in the experiment); Waterse 2695 high performance liquid chromatograph (Waters Corporation, USA, instrument used for calibration factor determination); electronic balance (accuracy of one hundred thousandth, XPE205, Mettler Toledo); CNC ultrasonic cleaner (KQ-250DV, Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water meter (Milli-Q Reference, Merck, Germany).

[0035] 1.2 Drug testing

[0036] Reference substances: norepinephrine bitartrate (batch number: 100169-202305, content 94.3%) and impurity A (batch number: 100154-202307, content 99.5%) were purchased from China Food and Drug Inspection Institute; impurity B (batch number: 20230517-01, content 95.67%) was purchased from Nanjing Huanuo Biotechnology Co., Ltd.; impurity C (batch number: DM20110128, content 94.27%) was purchased from Guangzhou Junmu Biotechnology Co., Ltd.; impurity D (batch number: 20220816, content 98.38%) was purchased from Jiangsu Wansipu Technology Co., Ltd.; sodium metabisulfite (batch number: R16210, content 66.1%) was purchased from USP.

[0037] Acetonitrile was of chromatographic grade and provided by Merck; sodium heptane sulfonate and phosphoric acid were of analytical grade and provided by Chengdu Kelong Chemical Co., Ltd.; the water used in the experiment was ultrapure water (18.20 MΩ·cm).

[0038] Norepinephrine bitartrate injection (batch numbers: 2405271, 2405281, 2405291; specifications: 4ml:8mg; provided by Company A), blank excipients were provided by Company A.

[0039] 2 Methods and Results

[0040] 2.1 Liquid chromatography conditions

[0041] Using Avantor ACE Excel 3C 18-PFP column (4.6mm×100mm, 3μm), 0.08% sodium heptane sulfonate solution (weigh 0.8g sodium heptane sulfonate, add 1000ml water to dissolve, adjust pH to 2.6 with phosphoric acid) as mobile phase A, acetonitrile-0.08% sodium heptane sulfonate solution (volume ratio = 50:50) as mobile phase B, gradient elution (A:B): 0min (95:5) → 15min (80:20) → 25min (40:60) → 30min (40:60) → 31min (95:5) → 45min (95:5). Column temperature 35℃, detection wavelength 280nm, flow rate 1.0mL·min -1 , the injection volume was 20 μL.

[0042] 2.2 Solution preparation

[0043] The solvents used in the preparation of the following solutions are all mobile phase A.

[0044] (1) Blank excipient solution: Take the blank excipient under the prescription.

[0045] (2) Test solution: Take an appropriate amount of the product.

[0046] (3) Control solution: Accurately measure 1 mL of the test solution and place it in a 100 mL volumetric flask. Dilute to the mark with solvent and shake well.

[0047] (4) Sensitivity solution: Accurately measure 5 mL of the control solution and place it in a 100 mL volumetric flask. Dilute to the mark with solvent and shake well.

[0048] (5) Norepinephrine bitartrate stock solution: Take about 2 mg of norepinephrine bitartrate, place it in a 10 mL volumetric flask, dissolve it with a solvent and dilute it to make a solution containing about 0.2 mg of norepinephrine bitartrate per 1 mL.

[0049] (6) Impurity A stock solution: Take about 2 mg of impurity A and place it in a 10 mL volumetric flask. Dissolve and dilute with solvent to make a solution containing about 0.2 mg of impurity A per 1 mL.

[0050] (7) Impurity B stock solution: Take about 2 mg of impurity B and place it in a 10 mL volumetric flask. Dissolve and dilute it with solvent to make a solution containing about 0.2 mg of impurity B per 1 mL.

[0051] (8) Impurity C stock solution: Take about 2 mg of impurity C and place it in a 10 mL volumetric flask. Dissolve and dilute it with solvent to make a solution containing about 0.2 mg of impurity C per 1 mL.

[0052] (9) Impurity D stock solution: Take an appropriate amount of Impurity D reference substance, weigh accurately, dissolve in 0.9% sodium chloride solution and dilute to make a solution containing about 4 mg per 1 mL, and shake well.

[0053] (10) Mixed stock solution of impurities A, B and C: Take appropriate amounts of impurities A, B and C, weigh them accurately, dissolve them in solvent and quantitatively dilute them to make a solution containing approximately 80 μg of impurity A, 40 μg of impurity B and 80 μg of impurity C per 1 mL, and shake well.

[0054] (11) Spiked test solution (impurities A, B, C): Accurately measure 1.8 mL of norepinephrine bitartrate injection solution and place it in a 2 mL volumetric flask; accurately measure 100 μL of the mixed stock solution of impurities A, B, and D under "2.2.11" and place it in the above 2 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0055] (12) Spiked test solution (impurities A, B, D) control solution: Accurately measure 1 mL of the spiked test solution in "2.2.12" into a 100 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0056] (13) Spiked test solution (impurity D): Accurately measure 1.8 mL of norepinephrine bitartrate injection solution and place it in a 2 mL volumetric flask; accurately measure 100 μL of the impurity D stock solution under "2.2.10" and place it in the above 2 mL volumetric flask, dilute to the mark with 0.9% sodium chloride solution, and shake well.

[0057] (14) Spiked test solution (impurity D) control solution: Accurately measure 1 mL of the spiked test solution in "2.2.14" into a 100 mL volumetric flask, dilute to the mark with 0.9% sodium chloride solution, and shake well.

[0058] (15) System suitability solution 1: Take 20 mg of norepinephrine bitartrate API and place it in a 10 mL volumetric flask. Add 0.1 mL of each impurity A-C stock solution, add solvent to dissolve and dilute to scale. Shake well to prepare a mixed solution containing approximately 2 mg of norepinephrine bitartrate and approximately 2 μg of each impurity A-C per 1 mL.

[0059] (16) System suitability solution 2: Take 10 mg of norepinephrine bitartrate reference substance and add 0.1 mol·L -1 Dissolve in 5 mL of hydrochloric acid solution, take 1 mL, add 0.1 mL of concentrated hydrogen peroxide solution, shake well, irradiate under ultraviolet light (254 nm) for 90 minutes, add 9 mL of solvent, and shake well.

[0060] (17) System suitability solution 3: Take appropriate amounts of impurity D reference substance and sodium metabisulfite reference substance, dissolve them in 0.9% sodium chloride solution and quantitatively dilute them to make a solution containing approximately 0.2 mg of impurity D and sodium metabisulfite per 1 mL.

[0061] 2.3 System suitability test

[0062] Take each solution prepared under "2.2", measure it according to the chromatographic conditions under "2.1", and record the chromatogram. As a result, in the chromatogram of system suitability solution 1, norepinephrine bitartrate, impurity A, impurity B and impurity C appear in sequence, and the separation degree between the norepinephrine bitartrate peak and the adjacent impurity peak is 10.5; in the chromatogram of system suitability solution 2, the separation degree between the norepinephrine bitartrate peak and the adjacent degradation impurity peak should be 3.2; in the chromatogram of system suitability solution 3, sodium metabisulfite and impurity D appear in sequence, and the separation degree between the sodium metabisulfite peak and the impurity D peak is 1.8. In the chromatogram of the sensitivity solution, the signal-to-noise ratio of the norepinephrine bitartrate peak height is 387. This shows that this method has good system applicability. See the chromatogram for details. Figure 1 .

[0063] 2.4 Specificity test

[0064] Take 20μL of blank solvent and blank excipient solution, respectively, and inject and analyze them. The results show that the blank solvent and blank excipient do not interfere with the detection of known impurities. Take 1.8mL of this product (batch number: 2405271) and place it in a 2mL volumetric bottle. Perform acid destruction (0.1mol·L -1 Hydrochloric acid solution 0.1mL, stand at room temperature for 2h), alkali destruction (0.1mol·L -1 Sodium hydroxide solution 0.1mL, stand at room temperature for 1h), oxidative destruction (0.3% hydrogen peroxide solution 0.2mL, stand at room temperature for 4h), light destruction (placed at 4500lx illumination for 8h), high temperature destruction (heated in a 121℃ oven for 1.5h), let cool (acid and alkali degradation solutions are neutralized first), dilute to scale with solvent, shake well, and the forced degradation test solution is obtained. The results of the forced degradation test show that the chromatographic conditions have good specificity, and the separation between the degradation impurities and the main peak produced by each destruction condition is greater than 1.5, see Figure 2 .

[0065] 2.5 Linear relationships and correction factors for known impurities

[0066] 2.5.1 Series of linear solutions of impurities A, B, and C

[0067] Accurately measure 5 ml of each of the norepinephrine bitartrate stock solution and impurity A, B, and C stock solutions under "2.2", place them in the same 25 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain R6; accurately measure 0.5 ml, 1.0 ml, 2.0 ml, 5.0 ml, and 10.0 ml of R6 solution, place them in 20 ml volumetric flasks, dilute to the mark with solvent, and shake well to obtain R1, R2, R3, R4, and R5 solutions.

[0068] 2.5.2 Series of linear solutions of impurity D

[0069] Take 2 mg of each of the reference substance of norepinephrine bitartrate and the reference substance of impurity D, place them in the same 5 mL volumetric flask, add 0.9% sodium chloride solution to dissolve and dilute to the mark, shake well, and use as L6. Accurately measure 0.025 mL, 0.1 mL, 1.0 mL, 2.0 mL, and 5.0 mL of L6 solution, place them in 10 mL volumetric flasks, dilute to the mark with 0.9% sodium chloride solution, shake well, and obtain L1, L2, L3, L4, and L5 solutions.

[0070] Different test personnel measured the standard curves of norepinephrine bitartrate and each known impurity on different liquid chromatographs at different times and calculated the regression equation, and the ratio of the linear slope of the main component to the linear slope of each impurity was used as the correction factor. Accurately measure 20μl of each of the above R1~R6 and L1~L6 solutions, inject and analyze according to the chromatographic conditions under "2.1", record the chromatogram, and perform linear regression analysis with the injection concentration as the horizontal axis (x) and the corresponding peak area as the vertical axis (y). The test results are shown in Table 1-2. The results show that norepinephrine bitartrate and the four impurities have a good linear relationship with the peak area within their respective linear ranges. The correction factors of impurities A~D are 0.6, 0.1, 0.6 and 0.7, respectively, and all need to be calculated with correction factors.

[0071] Table 1 Impurity AC linear relationship and correction factor obtained by different personnel and different liquid chromatographs

[0072]

[0073] Note: “—” indicates the main component, and no correction factor is required; Experimental condition 1 refers to the data obtained by experimenter 1 at time 1 using instrument 1; Experimental condition 2 refers to the data obtained by experimenter 2 at time 2 using instrument 2, and the same applies below.

[0074] Table 2 Linear relationship and correction factor of impurity D obtained by different personnel and different liquid chromatographs

[0075]

[0076] 2.6 Limit of detection (LOD) and limit of quantification (LOQ)

[0077] Take an appropriate amount of the linear stock solution of norepinephrine bitartrate and impurities A to D under item "2.5", dilute them step by step with solvent, and prepare the detection limit solution at a signal-to-noise ratio of 3:1; prepare the quantitative limit solution at a signal-to-noise ratio of 10:1. Accurately measure 20μl, inject them into the liquid chromatograph in sequence, and record the chromatogram. The test results are shown in Table 3.

[0078] Table 3 LOD and LOQ results of norepinephrine bitartrate and impurity AD

[0079] limit Norepinephrine Impurity A Impurity B Impurity C Impurity D <![CDATA[LOD / (μg·mL -1 )]]> 0.0124 0.00936 0.00180 0.00909 0.00501 <![CDATA[LOQ / (μg·mL -1 )]]> 0.0414 0.0312 0.00601 0.0303 0.0167

[0080] 2.7 Precision test

[0081] 2.7.1 Injection precision

[0082] Use the R3 solution under "2.5.1" to inject 6 times in succession and record the chromatogram each time. The RSD of the peak area of ​​each impurity is between 0.11% and 0.34%, indicating that the precision of the instrument is good.

[0083] 2.7.2 Repeatability

[0084] Take an appropriate amount of this product (batch number: 2405271) and prepare 6 portions of test solution spiked with (impurities A to C) in parallel according to the method under "2.2"; prepare 6 portions of test solution spiked with (impurity D) in parallel according to the method under "2.2"; prepare 6 portions of test reference solution spiked with (impurity A to C) in parallel according to the method under "2.2.13"; prepare 6 portions of test reference solution spiked with (impurity D) in parallel according to the method under "2.2.15".

[0085] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of impurities A to D by the principal component self-control method with correction factor. As a result, the 6 spiked test solutions were calculated by the external standard method and the principal component self-control method with correction factor, and the detection amount of impurities A to D obtained by the two methods was compared by paired sample t test. The results showed that there was no statistically significant difference in the results of the two methods (P>0.05), indicating that the method has good accuracy. The results are shown in Table 4.

[0086] Table 4 Repeatability test results of spiked test solution

[0087]

[0088] 2.7.3 Intermediate precision

[0089] Take this product (batch number: 2405271), prepare 6 test solutions in parallel according to the method described in section "2.2", and analyze the samples at different times, by different experimenters, and with different instruments. In the 12 test solutions involved in the two experiments, impurity A was not detected, the detection amount of impurity B was 0.006%, the detection amount of impurity C was 0.02%, the detection amount of impurity D was 0.87%, the largest unknown single impurity was not detected, and the total impurity (except impurity B and impurity D, other total impurities after correction) was 0.03%. The above results show that the detection method provided by the present invention has good intermediate precision.

[0090] 2.8 Accuracy test

[0091] 2.8.1 Accuracy of impurities A to C

[0092] Solvent: Mobile phase A.

[0093] Impurity A, B, C mixed reference solution: Accurately measure 100 μL of the impurity A, B, C mixed stock solution under "2.2.11", place it in a 2 mL volumetric flask, dilute to the scale with solvent, and shake well.

[0094] Solution with 50% recovery rate: accurately measure 1.8mL of norepinephrine bitartrate injection, place in a 2mL volumetric flask, measure 50μL of the mixed stock solution of impurities A, B, and C under "2.2.11", place in a 2mL volumetric flask, dilute to the mark with solvent, shake well, and prepare 3 copies in parallel;

[0095] Solution with 100% recovery rate: accurately measure 1.8 mL of norepinephrine bitartrate injection, place in a 2 mL volumetric bottle, measure 100 μL of impurity A, B, and C mixed stock solution, place in a 2 mL volumetric bottle, dilute to the mark with solvent, shake well, and prepare 3 copies in parallel;

[0096] Recovery rate 150% Solution: Accurately measure 1.8 mL of norepinephrine tartrate injection, place in a 2 mL volumetric bottle, and measure the impurities

[0097] Mix 150 μL of the stock solution, place in a 2 mL volumetric flask, dilute to the mark with solvent, shake well, and prepare 3 copies.

[0098] Inject them into liquid chromatograph respectively, and calculate the measured amount and recovery rate by external standard method.

[0099] 2.8.3 Impurity D Accuracy

[0100] Solvent: 0.9% sodium chloride solution.

[0101] Impurity D reference solution: Accurately measure 100 μL of the impurity D stock solution under "2.2.10", place it in a 2 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0102] Recovery rate 20% solution: accurately measure 1.8 mL of norepinephrine bitartrate injection, place in a 2 mL volumetric bottle, measure 20 μL of impurity D stock solution, place in a 2 mL volumetric bottle, dilute to the mark with solvent, shake well, and prepare 3 copies in parallel;

[0103] Solution with 100% recovery rate: accurately measure 1.8 mL of norepinephrine bitartrate injection, place in a 2 mL volumetric bottle, measure 100 μL of impurity D stock solution, place in a 2 mL volumetric bottle, dilute to the mark with solvent, shake well, and prepare 3 copies in parallel;

[0104] Recovery rate 150% solution: Accurately measure 1.8 mL of norepinephrine bitartrate injection, place in a 2 mL volumetric flask, measure 150 μL of impurity D stock solution, place in a 2 mL volumetric flask, dilute to the scale with solvent, shake well, and prepare 3 copies.

[0105] Take 20ul of each of the above solutions and inject them into the liquid chromatograph respectively, and use the external standard method to calculate the measured amount and recovery rate. The average recovery rate of impurities A to C equivalent to the concentration level of 50% to 150% of the limit concentration is within 100.00% to 101.47%, and the RSD is less than 2.0%. The accuracy of this method is good. The average recovery rate of impurity D equivalent to the concentration level of 20% to 150% of the limit concentration is within 99.90% to 100.55%, and the RSD is less than 2.0%. The results are shown in Table 5. The above results show that the detection method provided by the present invention has good accuracy.

[0106] Table 5 Impurity A ~ D accuracy test results

[0107]

[0108] 2.9 Stability test

[0109] Take the system suitability solution 1, system suitability solution 2, system suitability solution 3 and the test solution under "2.2" respectively, place them at room temperature for 0, 2, 4, 8, 12, 18, and 24 hours, and analyze them according to the conditions under "2.1". The results are shown in Table 6. The RSD values ​​of the peak areas of impurities A to C in system suitability solution 1 are all less than 2.0%; the RSD values ​​of the peak areas of impurities D in system suitability solution 3 are all less than 2.0%; in the test solution, impurity A was not detected, the RSD values ​​of the peak areas of impurities B to D were all less than 3.0%, and other single impurities were not detected; the separation between norepinephrine bitartrate and the adjacent degradation product peaks in system suitability solution 2 was greater than 1.5. This shows that the solution has good stability.

[0110] Table 6 System suitability Impurity AD in solution Peak area RSD (%) within 24 hours

[0111] System Suitability Solution 1 System Suitability Solution 3 Test solution Impurity A 1.9 / Not detected Impurity B 1.3 / 1.1 Impurity C 0.5 / 0.8 Impurity D / 0.7 0.9

[0112] 2.10 Durability test

[0113] Take the R1-R6 and L1-L6 reference solutions under item "2.5", and examine the mobile phase pH value, flow rate (±0.1 mL·min-1), column temperature (±2°C), and initial ratio of the mobile phase according to the chromatographic conditions under item "2.1", and detect and calculate the relative correction factors of norepinephrine and four impurities. The results are shown in Table 7. Compared with the data measured under the conditions of "2.5", the correction factors of the four impurities relative to norepinephrine did not differ by more than ±0.02. This shows that the method of the present invention has good durability.

[0114] Table 7 Durability test results

[0115]

[0116] 2.11 Sample determination

[0117] Take 3 batches of norepinephrine bitartrate injection, prepare the test solution and control solution according to the method under "2.2", inject and measure according to the chromatographic conditions under "2.1", record the chromatogram, calculate the content of impurities A to D according to the main component self-control method with correction factors, and calculate the content of other single unknown impurities according to the main component self-control method. The test results show that the content of known impurities A to C is less than 0.1%, the content of impurity D is less than 10.0%, and the total amount of other impurities is within 0.5%. The results are shown in Table 8.

[0118] Table 8 Determination results of impurities A to D in norepinephrine bitartrate injection

[0119] Sample batch number C1002A2230102 C1002A2230201 C1002A2230301 Impurity A / % — — — Impurity B / % 0.006 0.005 0.006 Impurity C / % 0.02 0.02 0.03 Impurity D / % 0.87 0.88 0.89 Other maximum unknown impurities / % — — — Total amount of other impurities / % 0.03 0.03 0.03

[0120] Note: “—” indicates below the detection limit.

[0121] 3 Discussions

[0122] 3.1 Impurity Limit Study

[0123] The 2020 edition of the Chinese Pharmacopoeia controls known impurities by only using the external standard method to control impurity B and the self-control method without adding a correction factor to control impurity D, but there are the following problems: 1. The reference substance is difficult to purchase and increases the production cost of the enterprise; 2. The correction factor of impurity D is 0.7, which exceeds the range of 0.9 to 1.1. Therefore, when controlling impurity D, it should be combined with the corresponding correction factor to calculate and improve the accuracy of the result. The present invention studies the sources and derivations of impurities in the impurity spectrum of the starting materials, intermediate products and finished products of the synthesis process, and formulates corresponding control strategies based on the research results to control each known impurity below the acceptable limit. The impurity attribution analysis is carried out. Impurity A is an incomplete product of the intermediate product reaction. Referring to the ICH Q3B (R2) impurity limit control requirements and combining the foreign pharmacopoeia standards for the limit of impurity A, its limit is determined to be: impurity A ≤ 0.2%. Impurity B is a byproduct of the reduction reaction caused by the reduction of the intermediate product. It is mainly generated by the incomplete reduction reaction of the intermediate in the synthesis route during the reduction process (the third step). We focus on controlling it. Referring to the ICH Q3B (R2) impurity limit control requirements and combining the results of exploratory experiments, we determine its limit as: Impurity B ≤ 0.10%. Impurity C is generated due to the presence of methanol in the final purification step. It is a process impurity. Referring to the ICH Q3B (R2) impurity limit control requirements and combining the results of multiple batches of tests, we determine its limit as: Impurity C ≤ 0.2%. Impurity D is a degradation impurity. Referring to the ICH Q3B (R2) impurity limit control requirements and combining the long-term test data, accelerated test data and multiple batches of test results, we determine its limit as: Impurity D ≤ 10.0%.

[0124] 3.2 Screening of chromatographic conditions

[0125] Determination of detection wavelength: The present invention uses a diode array detector of a liquid chromatograph to scan norepinephrine bitartrate and each impurity at a wavelength of 190 to 400 nm. The results show that impurities A, C and D have maximum absorption at 280 nm, and impurity B has maximum absorption at 280 nm and 310 nm, respectively. In combination with the norepinephrine bitartrate API and preparation pharmacopoeia standards, a wavelength of 280 nm is used for related substance inspection, and different domestic production enterprise standards also use 280 nm as the detection wavelength for related substance determination. Finally, it is determined that the related substances of norepinephrine bitartrate are detected using a wavelength of 280 nm.

[0126] Determination of chromatographic column: The exploratory experiment found that in the system suitability solution, sodium metabisulfite and impurity D have similar polarity, so the separation degree between impurity D and sodium metabisulfite is used as an indicator to measure the performance of the chromatographic column in this experiment. We selected the chromatographic column Agilent InfinityLab Poroshell 120EC-C 18(100mm×4.6mm, 3μm), Thermo scientificHypersil BDS C 18 (4.6mm×150mm, 5μm), Dikma Technologies Spursil C 18 -EP (3.0mm×100mm, 3μm), Avantor ACE Excel 3C 18 -PFP (4.6mm×100mm, 3μm) was tested. The results showed that when using Avantor ACE Excel 3C 18 -PFP (4.6mm×100mm, 3μm) chromatographic column, the separation degree of impurity D and sodium metabisulfite reached 1.8, while the others failed to achieve good separation. Therefore, the present invention uses Avantor ACE Excel 3C 18 -PFP (4.6mm×100mm, 3μm) chromatographic column.

[0127] Determination of gradient elution program: The experiment explored the effects of four gradient elution programs on the separation degree of impurity D and sodium metabisulfite, and the results are shown in Table 9. When the gradient elution program is the chromatographic condition under item "2.1" (i.e., program 4), the peak shape of each impurity is the best, the separation degree of impurity D and sodium metabisulfite is 1.8, and the signal-to-noise ratio is 384, which is significantly higher than the separation degree and signal-to-noise ratio of programs 1-3. The above results show that the gradient elution program under the method of the present invention can achieve the best separation effect between impurities, and the accuracy and reliability of the analysis are higher.

[0128] Program 1 (A:B): 0min(100:0)→10min(95:5)→30min(40:60)→31min(40:60)→40min(100:0);

[0129] Program 2 (A:B): 0 min (95:5) → 10 min (70:30) → 25 min (40:60) → 35 min (40:60) → 40 min (95:5);

[0130] Program 3 (A:B): 0 min (95:5) → 15 min (70:30) → 25 min (40:60) → 35 min (40:60) → 40 min (95:5) → 45 min (95:5);

[0131] Program 4 (A:B): 0 min (95:5) → 15 min (80:20) → 25 min (40:60) → 30 min (40:60) → 31 min (95:5) → 45 min (95:5).

[0132] Table 9 Effect of different gradient elution procedures on the separation of impurity D and sodium metabisulfite

[0133] Elution procedure Separation Signal-to-Noise Ratio Procedure 1 1.5 276 Procedure 2 1.6 284 Procedure 3 1.5 302 Procedure 4 1.8 384

[0134] 4 Conclusion

[0135] The present invention determines the correction factor f of impurities A to D relative to norepinephrine for the first time by calculating the slope of the regression equation of norepinephrine bitartrate and impurities A to D, and uses the principal component self-control method with correction factor to detect the related substances of norepinephrine bitartrate injection, and compares it with the reference substance external standard method. The results show that the method is more practical and feasible, and avoids the use of impurity reference substances. Compared with the impurity external standard method, the detection method provided by the present invention is accurate, reliable, efficient, simple to operate, and has strong specificity. The present invention is based on the principal component self-control method with correction factor established by high performance liquid chromatography to determine the content of 4 kinds of impurities in norepinephrine bitartrate injection. The method controls more specific impurities, tightens the limit, and helps to distinguish the quality of products on the market. It can be used for the quality control of norepinephrine bitartrate injection, and provides further reference for the revision and improvement of the standards of the Chinese Pharmacopoeia.

[0136] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting impurities in norepinephrine bitartrate injection, wherein the impurities are impurities A to D, and the specific structural formula is as follows: The method utilizes high performance liquid chromatography for detection, and specifically comprises the following steps: (1) Prepare reference solution and test solution; (2) Determine chromatographic conditions: Use Avantor ACE Excel 3C 18 -PFP chromatographic column; mobile phase A: sodium heptane sulfonate solution; mobile phase B: acetonitrile and mobile phase A mixed in a certain proportion; gradient elution was performed with a volume ratio of mobile phase A: mobile phase B = 95-40:5-60; (3) Testing the reference solution and the test solution under the above-mentioned liquid chromatography conditions; (4) Based on the peak areas of impurities A to D and the peak area of ​​norepinephrine bitartrate in the chromatogram, the contents of impurities A to D were calculated respectively using the principal component self-reference method with correction factors.

2. A method for detecting impurities in norepinephrine bitartrate injection according to claim 1, characterized in that: The size of the chromatographic column is 4.6 mm × 100 mm, the filler particle size is 3 μm, and the column temperature is 33-37 °C.

3. A method for detecting impurities in norepinephrine bitartrate injection according to claim 1, characterized in that: In step (2), the mobile phase A is a sodium heptane sulfonate solution having a mass fraction of 0.05-0.1% and adjusted to a pH value of 2.5-2.7 with phosphoric acid.

4. A method for detecting impurities in norepinephrine bitartrate injection according to claim 1, characterized in that: In step (2), the mobile phase B is a mixed solution of acetonitrile and mobile phase A in a volume ratio of 40 to 50:50 to 60.

5. A method for detecting impurities in norepinephrine bitartrate injection according to claim 1, characterized in that: In step (2), gradient elution is performed with mobile phase A: mobile phase B = 95-40:5-60, and the specific gradient elution program is: 0 min, A:B = 95:5→15 min, A:B = 80:20→25 min, A:B = 40:60→30 min, A:B = 40:60→31 min, A:B = 95:5→45 min, A:B = 95:

5.

6. A method for detecting impurities in norepinephrine bitartrate injection according to claim 1, characterized in that: The gradient elution flow rate in step (2) was 0.9-1.1 mL min -1 .

7. A method for detecting impurities in norepinephrine bitartrate injection according to claim 1, characterized in that: In step (3), the injection volume of the reference solution and the test solution is 20 μL.

8. A method for detecting impurities in norepinephrine bitartrate injection according to claim 1, characterized in that: The detection wavelength of the liquid chromatograph in step (3) is 280 nm.

9. A method for detecting impurities in norepinephrine bitartrate injection according to claim 1, characterized in that: The correction factors of impurities A to D in step (4) are 0.6, 0.1, 0.6 and 0.7 respectively.

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