Quantitative detection method for D-norepinephrine bitartrate in norepinephrine bitartrate injection

Abstract

The invention discloses a quantitative detection method for D-norepinephrine bitartrate in a norepinephrine bitartrate injection, and relates to the technical field of medicine detection. The specific chromatographic conditions are as follows: an Avantor ACE Excel3AQ chromatographic column (150mm * 4.6 mm, 3mu m) is adopted, a mobile phase is a 2% sulfobutyl beta-cyclodextrin sodium solution, the flow velocity is 0.5 mL.min <-1 >, the column temperature is 20 DEG C, the detection wavelength is 280 nm, and the sample size is 10 mu L. The chromatographic conditions provided by the invention can effectively separate L-norepinephrine bitartrate from D-norepinephrine bitartrate, and the separation degree is greater than 2.0; according to the method disclosed by the invention, the linear relationship between the D-type concentration and the peak area is good (r is equal to 1.0000) within the range of 0.1953-488.2471 [mu] g.mL <-1 >; the average sample adding recovery rates of the low mass concentration, the medium mass concentration and the high mass concentration are 99.95%, 100.31% and 100.00% (n is equal to 3) respectively, and the RSD is 0.3%, 0.4% and 0.6% (n is equal to 3) respectively; the detection limit of D-norepinephrine bitartrate by using the method is lt; and 10.0%.

Description

Technical Field

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

[0002] Norepinephrine bitartrate, chemical name (R)-4-(2-amino-1-hydroxyethyl)-1,2-benzenediol bitartrate monohydrate, is an adrenergic receptor agonist that can strongly stimulate α and β receptors. Stimulating α receptors causes extreme vasoconstriction, increases blood pressure, and increases coronary blood flow; stimulating β receptors can strengthen myocardial contraction and increase cardiac output. It is a first-line clinical drug, mainly used to resist shock, such as shock caused by anesthesia, toxic shock, cardiogenic shock, etc.

[0003] There is a chiral carbon atom in the structure of norepinephrine bitartrate. Clinically, the left-handed L-type norepinephrine bitartrate (hereinafter referred to as L-type) is its active ingredient, and the right-handed D-type norepinephrine bitartrate (hereinafter referred to as D-type) is ineffective and is its optical enantiomer. Because the D-type has differences in receptor ligand interaction and biological function, the D-type usually has a certain effect on the efficacy of the L-type in pharmacology, pharmacokinetics and even toxicology, so the D-type content needs to be strictly controlled. The raw materials and preparations of this drug have been listed in China, but the current quality standards do not include D-type inspection items. As an injection, the concentration of the main component of norepinephrine bitartrate injection is certain and the concentration is low. It is relatively complicated to process samples using the normal phase detection method. It is very necessary to develop a simple, specific and accurate detection method. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the shortcomings of the prior art, the present invention takes norepinephrine bitartrate injection as the research object, refers to relevant guiding principles, and provides a quantitative detection method of D-type norepinephrine bitartrate in norepinephrine bitartrate injection based on HPLC, which provides effective guarantee for the quality control of norepinephrine bitartrate injection.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] A method for quantitatively detecting D-norepinephrine bitartrate in norepinephrine bitartrate injection, the method comprising the following steps:

[0009] (I) Determine the chromatographic conditions

[0010] Chromatographic column: Avantor ACE Excel 3AQ column; mobile phase: sulfobutyl beta-cyclodextrin sodium aqueous solution; flow rate 0.45-0.55 mL min -1 ; Column temperature 15-25℃; Detection wavelength 280nm; Injection volume 10μL;

[0011] (II) Solution preparation

[0012] (1) Test solution: commercially available norepinephrine bitartrate injection, appropriate amount;

[0013] (2) Control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well;

[0014] (3) D-type reference solution: Take about 2 mg of D-type norepinephrine bitartrate, weigh accurately, place in a 10 mL volumetric flask, add water to dissolve and dilute to the mark, shake well;

[0015] (iii) testing the control solution, D-type reference solution, and test solution separately under the above-mentioned liquid chromatography conditions;

[0016] (iv) Based on the peak area of ​​D-norepinephrine bitartrate and the peak area of ​​L-norepinephrine bitartrate in the chromatogram, the content of D-norepinephrine bitartrate is calculated using the principal component self-reference method.

[0017] Furthermore, the specification of the chromatographic column is 4.6 mm×150 mm, 3 μm.

[0018] Furthermore, the mass percentage of the mobile phase sodium sulfobutyl beta-cyclodextrin aqueous solution is 2-5%. The specific preparation method is to weigh 20-50 g of sodium sulfobutyl beta-cyclodextrin, add 1000 mL of water to dissolve it, and adjust the pH value to 2.3-2.7 with phosphoric acid.

[0019] Further, the chromatographic conditions were Avantor ACE Excel 3AQ column; mobile phase: 2% sulfobutyl beta-cyclodextrin sodium aqueous solution, pH value adjusted to 2.5 with phosphoric acid; flow rate 0.5 mL·min -1 ; Column temperature 20℃; Detection wavelength 280nm; Injection volume 10μL.

[0020] (III) Beneficial effects

[0021] The present invention establishes a high performance liquid chromatography combined with a principal component self-control method to quantitatively analyze D-type norepinephrine bitartrate in norepinephrine bitartrate injection. The specific chromatographic conditions are: Avantor ACE Excel 3AQ chromatographic column (150mm×4.6mm, 3μm), mobile phase is 2% sulfobutyl beta-cyclodextrin sodium solution (pH adjusted to 2.5 with phosphoric acid), flow rate 0.5mL·min -1 , column temperature 20°C, detection wavelength 280nm, injection volume 10μL. The chromatographic conditions provided by the present invention can effectively separate L-type and D-type norepinephrine bitartrate, with a separation degree greater than 2.0; under the method of the present invention, the concentration of D-type is 0.1953~488.2471μg·mL -1 The linear relationship with the peak area is good within the range (r=1.0000); the average sample recovery rates of low, medium and high mass concentrations are 99.95%, 100.31%, 100.00% (n=3), respectively, and the RSDs are 0.3%, 0.4%, 0.6% (n=3), respectively; the detection limit of D-type norepinephrine bitartrate using the present invention is <10.0%. The method provided by the present invention is accurate, reliable, efficient, simple to operate, and has strong specificity. The main component self-control method established by the present invention measures the content of D-type norepinephrine bitartrate in norepinephrine bitartrate, and the result is more accurate, which improves the quality control standard of this variety and provides further reference for the revision and improvement of the standard of the "Chinese Pharmacopoeia". BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The chromatograms of system suitability are shown in Figure 1, where 1 is D-type and 2 is L-type.

[0023] Figure 2 The chromatograms are specific experimental diagrams, A. blank solvent; B. blank excipient solution; C. acid-destroyed solution; D. alkali-destroyed solution; E. oxidative-destroyed solution; F. high-temperature-destroyed solution; G. light-destroyed solution; H. undestroyed solution; 1. D-type; 2. L-type. DETAILED DESCRIPTION

[0024] 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 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.

[0025] Example

[0026] 1. Instruments and reagents

[0027] Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA), chromatographic column (Avantor ACEExcel3AQ column, 4.6 mm × 150 mm, 3 μm), XPE 205 electronic balance with one hundred thousandth precision (Mettler Toledo), and multi-parameter pH meter (Mettler Toledo Instruments, Ltd.). L-type norepinephrine bitartrate (abbreviated as L-type) reference substance (batch number: 100169-202305, purity: 94.3%, source: China Food and Drug Inspection Institute), D-type norepinephrine bitartrate (abbreviated as D-type) reference substance (batch number: 22-01-2424, purity: 96.78%, source: SINCOPHARMACHEM), norepinephrine bitartrate injection (batch number: 2405271, 2405281, 2405291, specification 4ml: 8mg, provided by A company). Sodium sulfobutyl beta-cyclodextrin and phosphoric acid were analytical grade, and water was ultrapure water.

[0028] 2 Methods and Results

[0029] 2.1 Chromatographic conditions

[0030] The chromatographic column was an Avantor ACE Excel 3AQ column (4.6 mm × 150 mm, 3 μm); the mobile phase was a 2% sodium sulfobutyl beta-cyclodextrin solution (20 g of sodium sulfobutyl beta-cyclodextrin was weighed, 1000 mL of water was added to dissolve it, and the pH value was adjusted to 2.5 with phosphoric acid), and the elution was isocratic; the flow rate was 0.5 mL·min -1 ; Column temperature 20℃; Detection wavelength 280nm; Injection volume 10μL. Test according to the general rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0031] 2.2 Solution preparation

[0032] 2.2.1 Blank solvent: water.

[0033] 2.2.2 Blank excipient solution: Take an appropriate amount of blank excipient under the prescription.

[0034] 2.2.3 Test solution: Take an appropriate amount of this product.

[0035] 2.2.4 Control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well.

[0036] 2.2.5 D-type reference substance solution: Take about 2 mg of D-type reference substance, weigh accurately, place in a 10 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0037] 2.2.6 System suitability solution: Take about 4 mg of L-type reference substance, weigh accurately, put it in a 2 mL volumetric flask, add 1 mL of L-type reference substance solution, dissolve it in water and dilute to the scale, shake well.

[0038] 2.3 System suitability test

[0039] Take the system suitability solution under "2.2.6", measure according to the chromatographic conditions under "2.1", and record the chromatogram. In the chromatogram of the system suitability solution, D-type and L-type peaks appear in sequence; the retention time of D-type is 11.101min, the retention time of L-type is 11.822min, the separation degree between the two is 2.2, and the theoretical plate number is 17154 calculated based on the L-type peak. This shows that this method has good system suitability. See the chromatogram for details. Figure 1 .

[0040] 2.4 Specificity test

[0041] Take the blank solvent and blank auxiliary material solution under "2.2" and analyze them according to the method under "2.1". The results show that the blank solvent and blank auxiliary material have no interference on the determination of the main component and D-type. Accurately measure 1.8mL of this product (batch number: 2405271) and place it in a 2mL volumetric bottle. Perform acid degradation (0.1mol·L -1 0.1 mL of hydrochloric acid solution, let stand at room temperature for 1 h), alkali degradation (0.1 mol·L -1 0.1mL of sodium hydroxide solution, let stand at room temperature for 10min), oxidative degradation (0.2mL of 0.3% hydrogen peroxide solution, let stand at room temperature for 10min), light degradation (placed at an illumination of 4500lx for 7h), high temperature degradation (heated in a water bath at 80℃ for 2h), let it cool to room temperature (acid and alkali degradation solutions are neutralized first), then dilute with water to the scale, 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 L-type and D-type and the degradation impurity peaks generated by the destruction can be well separated, which is suitable for the determination of the D-type content in norepinephrine bitartrate injection. See the chromatogram for details. Figure 2 .

[0042] 2.5 Linear relationship and correction factor

[0043] Take about 10 mg of each L-type and D-type reference substance, weigh accurately, place in a 20 mL volumetric flask, dilute with water to make a solution containing about 500 μg of each L-type and D-type per 1 mL, as L6 solution. Accurately measure an appropriate amount of L6 solution, dilute with water to make solutions containing about 1, 4, 50, 100, 250 μg of each L-type and D-type per 1 mL as L1, L2, L3, L4, L5 reference substance solutions. Take the mass concentration X (μg·mL -1) as the horizontal coordinate and the peak area Y as the vertical coordinate, draw the standard regression equation curve of the linear solution of L1~L6, and calculate the correction factor by the ratio of the linear slope of L-type and the linear slope of D-type (see Table 1). The results show that L-type and D-type have a good linear relationship with the peak area within their respective linear ranges. The correction factor of D-type is 0.99. When the correction factor is within 0.9~1.1, it can be calculated without adding the correction factor.

[0044] 2.6 Limit of detection and limit of quantification

[0045] The L6 solution under item "2.5" was gradually diluted, and the sample was analyzed according to the chromatographic conditions under item "2.1". The mass concentration corresponding to the signal-to-noise ratio (S / N) of 3 was the detection limit (LOD), and the mass concentration corresponding to the S / N of 10 was the quantification limit (LOQ). The measurement results are shown in Table 1.

[0046] Table 1 Linear relationship, correction factor, LOD, LOQ results of L-type and D-type

[0047] L-Type D-Type Regression equation Y=9666.1519X+4866.5080 Y=9753.2383X+5622.1933 <![CDATA[Linear range / (μg·mL -1 )]]> 0.1917～479.2326 0.1953～488.2471 r 1.0000 1.0000 Correction Factor — 0.99 <![CDATA[LOD / (μg·mL -1 )]]> 0.002 0.001 <![CDATA[LOQ / (μg·mL -1 )]]> 0.006 0.003

[0048] Note: “—” indicates the main component, and no correction factor is required.

[0049] 2.7 Precision test

[0050] 2.7.1 Injection precision

[0051] Take the D-type reference solution under "2.2.5", inject it 6 times in succession, and record the chromatogram each time. The RSD of the D-type peak area is 0.06%, indicating that the injection precision of this method is good.

[0052] 2.7.2 Repeatability

[0053] Take an appropriate amount of this product (batch number: 2405271) and prepare the test solution according to the method in "2.2.3" and below. Prepare 6 samples in parallel, inject and measure, and calculate the content according to the D-type principal component self-control method. In the six test solutions tested, D-type was detected, and the average content of D-type was 4.53%, with an RSD of 0.27%, indicating that the method has good repeatability.

[0054] 2.7.3 Intermediate precision

[0055] Take an appropriate amount of this product (batch number: 2405271) and prepare the test solution according to the method under "2.2.3". Prepare 6 copies in parallel, and analyze the samples at different times, by different experimenters, and using different instruments. The results show that the D-type content detected in the 12 test solutions in the two experiments is basically the same. The average D-type content in the two experiments is 4.54%, and the RSD is 0.41%, indicating that the intermediate precision of this method is good.

[0056] 2.8 Accuracy test

[0057] Take about 8 mg of D-type reference substance, weigh accurately, place in a 2 mL volumetric flask, add water to dissolve and dilute to make each 1 mL contain about 4 mg·mL of D-type -1 The solution was used as the D-type accuracy reserve solution; 9 samples of 1.8mL of norepinephrine bitartrate injection (batch number: 2405271) with known D-type content were accurately measured and placed in 2mL volumetric bottles, and 3 samples of 50μL, 100μL, and 150μL of D-type accuracy reserve solution were accurately added to prepare recovery test sample solutions at low, medium, and high concentration levels. The average recovery of D-type was calculated by external standard method and principal component self-control method. The results are shown in Table 2. As can be seen from Table 2, according to the principal component self-control method, the average recovery of D-type was 99.95% to 100.31% (n=3), and the relative standard deviation (RSD) of the determination results was 0.3% to 0.6% (n=3). The statistical software SPSS25.0 was used to compare the D-type determination results obtained by the two methods through paired sample t-test. The results showed that there was no statistically significant difference between the two methods (P>0.05), indicating that this method has good accuracy.

[0058] Table 2 Comparison of the results of the main component self-control method and D-type external standard method for the test sample

[0059]

[0060]

[0061] 2.9 Stability test

[0062] Take the system suitability solution under "2.2.6" and the test solution under "2.2.3" and analyze them at different time points at room temperature. The system suitability solution is placed at room temperature for 75 hours, and the peak area change values ​​of L-type and D-type are both less than 2.0%. The system suitability solution is stable at room temperature for 75 hours. The test solution is placed at room temperature for 75 hours, and the RSD of the D-type detection amount is less than 3%. The test solution is stable within 75 hours at room temperature.

[0063] 2.10 Durability test

[0064] Take the system suitability solution under "2.2.6" and the test solution under "2.2.3", make appropriate changes to the conditions (flow rate, column temperature, mobile phase pH, different chromatographic columns), and the parameters are shown in Table 3. The separation degree of the main peak and the D-type peak, the theoretical plate number of the main peak, and the change of the D-type detection amount in the test solution under different conditions of the system suitability solution are investigated. The research results show that the separation degree of the main peak and the D-type peak in the chromatogram of the system suitability solution under different parameters is greater than 1.5, and the theoretical plate number is greater than 3000; in the chromatogram of the test solution, the maximum change value of the D-type detection amount is less than 0.05%. This shows that the method has good durability, as shown in Table 3.

[0065] Table 3 Durability test results

[0066]

[0067] Note: Column 1, Avantor ACE Excel 3AQ (4.6 mm × 150 mm, 3 μm), No.: ACE-LC-2023-0024; Column 2, Waters T3 (4.6 mm × 150 mm, 2.7 μm), number: Waters-LC-2023-0027.

[0068] 2.11 Sample content determination

[0069] Three batches of samples were tested according to the established method, and the D-form contents in the three batches of samples were 4.5%, 4.2%, and 4.1%, respectively.

[0070] 3 Discussion

[0071] 3.1 Selection of mobile phase

[0072] Publication No. CN110133163A discloses a method for detecting the ratio of multiple norepinephrine bitartrate components in a drug, wherein the separation degree of D-type and L-type is 1.52 when the mobile phase is selected as an inorganic phase 2% sulfobutyl beta-cyclodextrin sodium solution and an organic phase acetonitrile at a ratio of 98.5:1.5. On this basis, we studied the effect of the inorganic phase 2% sulfobutyl beta-cyclodextrin sodium solution mixed with different organic phases as the mobile phase on the separation degree of D-type and L-type, and the results are shown in Table 4. When the pure inorganic phase of 2% sodium sulfobutyl beta-cyclodextrin solution is used as the mobile phase, the separation degree between D-type and L-type reaches 2.2, while when 2% sodium sulfobutyl beta-cyclodextrin solution is mixed with different organic phases as the mobile phase, the separation degree is only 1.28-1.36. The results show that the addition of the organic phase has a significant adverse effect on the separation degree of the detected substance. Therefore, the present invention selects a pure inorganic phase (2% sodium sulfobutyl beta-cyclodextrin solution) as the mobile phase.

[0073] Table 4 Effect of different mobile phases on the separation of D-type and L-type

[0074]

[0075] We further explored the concentration of sodium sulfobutyl beta-cyclodextrin solution, and the results are shown in Table 5. When the concentration of sodium sulfobutyl beta-cyclodextrin solution is between 1-3%, the D-type tailing factor ranges from 0.95 to 1.04; when the concentration of sodium sulfobutyl beta-cyclodextrin solution is above 5%, the D-type tailing factor increases to above 1.16; the above results show that the separation effect is better when the concentration of sodium sulfobutyl beta-cyclodextrin solution is between 1-3%, and when the concentration of sodium sulfobutyl beta-cyclodextrin solution is 2%, the D-type tailing factor is 1.0, and the peak shape is good. Therefore, the present invention selects 2% sodium sulfobutyl beta-cyclodextrin solution as the mobile phase.

[0076] Table 5 Effect of different mass fractions of sodium sulfobutyl beta-cyclodextrin solution on enantiomeric tailing factor

[0077] Quality score 1％ 2％ 3％ 5％ 7.5％ Tailing Factor 0.95 1.00 1.04 1.16 1.25

[0078] We further explored the pH value of the 2% sodium sulfobutyl beta-cyclodextrin solution, and the results are shown in Table 6. When the pH value is adjusted to 3.0, the D-type tailing factor is 1.2; when the pH value is adjusted to 2.0, the D-type tailing factor is 0.8; and when the pH value is adjusted to 2.3-2.7, the D-type tailing factor ranges from 0.95-1.05, which can achieve a good separation effect; when the pH value is adjusted to 2.5, the D-type tailing factor is 1.0, and the peak shape is good. Therefore, the mobile phase pH value selected in the present invention is 2.5.

[0079] Table 6 Effect of different pH values ​​of 2% sulfobutyl beta-cyclodextrin sodium solution on D-type tailing factor

[0080] pH=2.0 pH = 2.3 pH=2.5 pH=2.7 pH=3.0 Tailing Factor 0.8 0.95 1.0 1.05 1.2

[0081] 3.2 Selection of impurity quantification method

[0082] Impurity quantification methods usually include external standard method (impurity reference method), principal component self-reference method with correction factor, principal component self-reference method without correction factor, and peak area normalization method.

[0083] The external standard method is more accurate in quantitative determination and is the most direct method for effective control of impurities. The correction factor should be strictly determined in the main component self-control method with correction factor added. The premise of the main component self-control method without correction factor added is to assume that the response factors of impurities and main components are basically the same. When the relative response factor is within the range of 0.9 to 1.1, the main component self-control method can be used. When it exceeds the range of 0.9 to 1.1, the impurity reference method or the main component self-control method with correction factor added should be used. The peak area normalization method is simple and fast, but it is generally not recommended to use it because the response factors of impurities and main components are different, the amount of impurities and the amount of main components are not necessarily in the same linear range, and the integration precision and accuracy of the instrument for trace impurities and constant main components are different. The method disclosed in Publication No. CN110133163A uses the area normalization method when calculating the D-type content. In this experiment, on the premise of fully verifying the correction factor, the results of the external standard method and the principal component self-reference method were compared (the results are shown in Table 2). It is recommended to use the principal component self-reference method to calculate the content of D-type, and the results are more accurate than those of the area normalization method.

[0084] 3.3 Wavelength selection

[0085] The L-type and D-type were fully scanned in the range of 200nm to 400nm using a diode array detector. It was found that the main components L-type and D-type both had maximum absorption at 280nm, and the auxiliary materials had no interference at this wavelength. Therefore, the test used 280nm as the detection wavelength.

[0086] 3.4 Column selection

[0087] The present invention explores the normal and reverse phase chiral columns, but in the experimental analysis, it is found that the detection sensitivity of L-type and D-type in the normal phase column is poorer than that in the reverse phase column. The polarity of L-type and D-type in the structure of norepinephrine bitartrate is relatively large. Using a normal phase column will cause tartaric acid to peak at the back, which is difficult to separate. There is overlap in the experiment. Therefore, the present invention uses a reverse phase chiral column.

[0088] 3.5 Limit Control Limit Study

[0089] The experiment used the proposed D-type detection method to detect three batches of samples and three batches of reference preparations provided by the manufacturer. The detection amounts of D-type were 4.5%, 4.2%, 4.1% and 4.7%, 3.9%, 3.8%, respectively. The D-type content was detected by conducting 0-day, long-term 6-month and accelerated 6-month tests on the validation batch of test products (batch number: 2405271). The results showed that the detection amounts of D-type were 4.5%, 5.6%, and 8.4%, respectively. Combined with the reference literature and the results of the three batches of samples and the long-term accelerated test results, the D-type content limit was proposed to be controlled within 10.0%.

[0090] 4 Conclusion This study established a method for determining the content of D-type norepinephrine bitartrate in norepinephrine bitartrate injection using high performance liquid chromatography (HPLC) combined with the principal component self-control method, and the results of this method were proved to be reliable through methodological verification. Compared with the impurity external standard method, this method is accurate, reliable, efficient, simple to operate, and has strong specificity. The common isomer area normalization method was not used in the experiment because although this method is simple and fast, the integration precision and accuracy of the main components and trace impurities of different instruments are different, which will affect the accuracy of the results. Therefore, compared with the area normalization method, the principal component self-control method established in this study was more accurate in measuring the content of D-type norepinephrine bitartrate in norepinephrine bitartrate, which improved the quality control standard of this variety and provided further reference for the revision and improvement of the standards of the Chinese Pharmacopoeia.

[0091] 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 quantitatively detecting D-norepinephrine bitartrate in norepinephrine bitartrate injection, characterized in that: The method adopts high performance liquid chromatography and specifically comprises the following steps: (I) Determine the chromatographic conditions Chromatographic column: Avantor ACE Excel 3AQ column; mobile phase: sulfobutyl beta-cyclodextrin sodium aqueous solution; flow rate 0.45-0.55 mL min -1 ; Column temperature 15-25℃; Detection wavelength 280nm; Injection volume 10μL; (II) Solution preparation (1) Test solution: commercially available norepinephrine bitartrate injection, appropriate amount; (2) Control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well; (3) D-norepinephrine bitartrate reference solution: Take about 2 mg of D-norepinephrine bitartrate, accurately weigh it, place it in a 10 mL volumetric flask, add water to dissolve and dilute to the mark, and shake well; (III) Using the above-mentioned liquid chromatography conditions, the control solution, the D-norepinephrine bitartrate reference solution, and the test solution were tested respectively; (iv) Based on the peak area of ​​D-norepinephrine bitartrate and the peak area of ​​L-norepinephrine bitartrate in the chromatogram, the content of D-norepinephrine bitartrate is calculated using the principal component self-reference method.

2. The method for quantitatively detecting D-type norepinephrine bitartrate in norepinephrine bitartrate injection according to claim 1, characterized in that: The specifications of the chromatographic column are 4.6mm×150mm, 3μm.

3. The method for quantitatively detecting D-type norepinephrine bitartrate in norepinephrine bitartrate injection according to claim 1, characterized in that: The mass percentage of the mobile phase sodium sulfobutyl beta-cyclodextrin aqueous solution is 2-5%. The specific preparation method is to weigh 20-50g of sodium sulfobutyl beta-cyclodextrin, add 1000mL of water to dissolve it, and adjust the pH value to 2.3-2.7 with phosphoric acid.

4. The method for quantitatively detecting D-type norepinephrine bitartrate in norepinephrine bitartrate injection according to claim 1, characterized in that: Chromatographic conditions: Avantor ACE Excel 3AQ column; mobile phase: 2% sulfobutyl beta-cyclodextrin sodium aqueous solution, pH = 2.5 adjusted with phosphoric acid; flow rate 0.5 mL·min -1 ; Column temperature 20℃; Detection wavelength 280nm; Injection volume 10μL.

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