Analysis method for detecting content of dalbavancin and application thereof

Abstract

The invention provides an analysis method for detecting the content of dalbavancin, which is characterized in that dalbavancin is detected by using reverse high performance liquid chromatography, and a mobile phase is ammonium dihydrogen phosphate solution-acetonitrile. The Dalbavancin has many components and similar structures, the high performance liquid chromatography provided by the invention can effectively separate five components A0, A1, B0, B1 and B2, not only can accurately detect the content of Dalbavancin B0, but also can detect the total content of Dalbavancin; meanwhile, the method has the advantages of high specificity, high accuracy, relatively good precision, relatively good durability and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of chromatography analysis, and in particular to an analysis method for detecting the content of dalbavancin and an application thereof. Background Art

[0002] Dalbavancin, also known as daucomycin, is a new type of semisynthetic glycopeptide antibiotic with A40926 as the precursor. It belongs to the same class as vancomycin and ramoplanin and has the same mechanism of action. Currently, the U.S. Food and Drug Administration (FDA) has approved dalbavancin for the treatment of acute bacterial skin and skin structure infections (ABSSSI) in pediatric patients (from birth to under 18 years old). Dalbavancin is the first and only single-dose infusion regimen for the treatment of ABSSSI in pediatric patients from birth. It is used to treat ABSSSI caused by designated susceptible Gram-positive bacteria in pediatric patients, including infections caused by methicillin-resistant Staphylococcus aureus (MRSA). In the United States, dalbavancin has previously been approved for the treatment of ABSSSI in adults (18 years and older). In adult patients, dalbavancin is the first and only intravenous antibiotic with a single-dose regimen (1500 mg) and a double-dose regimen (1000 mg, 500 mg one week later). Dalbavancin has good bactericidal activity and safety against a range of Gram-positive bacteria in vitro in both pediatric and adult patients.

[0003] Dalbavancin is made from A 0 , A 1 , B 0 , B 1 , B 2 A mixture of five closely related active homologues, ingredient B 0 It is the main component of dalbavancin. Its homologues have the same core structure, except that the N-acylaminoglucuronic acid part (R 1 ) structure, there are additional methyl groups on the fatty acid side chains and / or terminal amino groups (R 2 ) is different from B 0 It has the same therapeutic effect. Its structural formula is as follows. The chemical formula and molecular weight of each component are shown in Table 1:

[0004]

[0005] Table 1

[0006] Components <![CDATA[R 1 ]]> <![CDATA[R 2 ]]> Molecular formula Molecular weight <![CDATA[A 0 ]]> <![CDATA[CH(CH 3 ) 2 ]]> H <![CDATA[C 87 H 98 N 10 O 28 Cl 2 ]]> 1802.7 <![CDATA[A 1 ]]> <![CDATA[CH 2 CH 2 CH 3 ]]> H <![CDATA[C 87 H 98 N 10 O 28 Cl 2 ]]> 1802.7 <![CDATA[B 0 ]]> <![CDATA[CH 2 CH(CH 3 ) 2 ]]> H <![CDATA[C 88 H 100 N 10 O 28 Cl 2 ]]> 1816.7 <![CDATA[B 1 ]]> <![CDATA[CH 2 CH 2 CH 2 CH 3 ]]> H <![CDATA[C 88 H 100 N 10 O 28 Cl 2 ]]> 1816.7 <![CDATA[B 2 ]]> <![CDATA[CH 2 CH(CH 3 ) 2 ]]> <![CDATA[CH 3 ]]> <![CDATA[C 89 H 102 N 10 O 28 Cl 2 ]]> 1830.7

[0007] Currently, there are few literatures on the detection methods of dalbavancin content, and most of them are about dalbavancin B. 0 The content detection method of A 0 , A 1, B 1 , B 2 The contents of other components were basically calculated by the area normalization method, and when calculating the total content of dalbavancin, the accuracy of the external standard method was significantly better than that of the area normalization method.

[0008] In order to make the determination results of dalbavancin content accurate and reliable, it is urgent to establish an analytical method with high accuracy, high precision and good durability. Summary of the invention

[0009] The object of the present invention is to provide an analytical method for detecting the content of dalbavancin, which uses a reversed-phase high performance liquid chromatograph to measure the chromatograms of a reference solution and a test solution, so that A 0 , A 1 , B 0 , B 1 , B 2 The separation degree between them is ≥1.5. According to the external standard method, the B 0 Peak area calculation of A in the test solution 0 , A 1 , B 0 , B 1 , B 2 The total content of

[0010] In one aspect, the present invention provides an analytical method for detecting the content of dalbavancin, comprising detecting dalbavancin using reversed-phase high performance liquid chromatography, wherein the mobile phase is diammonium phosphate solution-acetonitrile and the elution procedure is gradient elution.

[0011] According to an embodiment of the present invention, the mobile phase includes mobile phase A and mobile phase B; mobile phase A is ammonium dihydrogen phosphate solution-acetonitrile in a volume ratio of 80:20, and mobile phase B is ammonium dihydrogen phosphate solution-acetonitrile in a volume ratio of (60-70):(30-40). Further preferably, mobile phase B is ammonium dihydrogen phosphate solution-acetonitrile in a volume ratio of 66:34.

[0012] According to an embodiment of the present invention, the concentration of the ammonium dihydrogen phosphate solution in the mobile phase A and the mobile phase B is 0.025 mol / L, and the pH value is 5.8-6.2.

[0013] According to an embodiment of the present invention, the procedure of mobile phase gradient elution is as follows:

[0014] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 4 40 60 30 5 95 30.1 95 5 40 95 5

[0015] According to an embodiment of the present invention, the above elution procedure can achieve better separation effect and baseline stability relative to other elution procedures. Wherein, the other elution procedures include, for example, changing the elution time, and / or the ratio of mobile phase A and B. Specifically, for example, when the elution procedure is the following elution procedure, there will be a phenomenon of peak concentration and large baseline fluctuation, which leads to a decrease in signal-to-noise ratio, a decrease in peak resolution, insufficient quantitative accuracy, and a weakening of the repeatability and stability of the method, affecting the accuracy and reliability of chromatographic detection.

[0016] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 4 5 95 22 5 95 22.1 95 5 30 95 5

[0017] For another example, when the elution procedure is as follows, it will result in main peak B 0 Not completely separated from the previous impurity peak.

[0018] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 4 25 75 30 5 95 30.1 95 5 35 95 5

[0019] According to an embodiment of the present invention, the chromatographic column is a chromatographic column filled with octadecyl bonded silica gel. Preferably, the chromatographic column has a specification of 4.6 mm×100 mm, 2.7 μm or 4.6 mm×250 mm, 3 μm. Further preferably, the chromatographic column has a specification of 4.6 mm×100 mm, 2.7 μm.

[0020] According to an embodiment of the present invention, the column temperature of the HPLC method is 35-45°C, and more preferably, the column temperature is 40°C.

[0021] According to an embodiment of the present invention, the flow rate of the high performance liquid chromatography is 0.4-0.6 ml / min, and more preferably, the flow rate is 0.5 ml / min.

[0022] According to an embodiment of the present invention, the injection volume of the high performance liquid chromatography is 10-20 μl, and more preferably, the injection volume is 10 μl.

[0023] According to an embodiment of the present invention, the detection wavelength of the high performance liquid chromatography is 200-220 nm, and more preferably, the detection wavelength is 210 nm.

[0024] According to an embodiment of the present invention, the analysis method comprises measuring a test solution and a reference solution, respectively injecting them into a liquid chromatograph, recording a chromatogram, and calculating the peak area according to an external standard curve method.

[0025] According to the embodiment of the present invention, the peak area obtained by linear solution detection is used to draw a standard curve, and the component A in the test sample is calculated by the standard curve. 0 , A 1 , B 0 , B 1 , B2 The content.

[0026] According to an embodiment of the present invention, a dalbavancin sample is dissolved in a diluent to obtain a test solution.

[0027] According to an embodiment of the present invention, the test solution contains about 0.1 mg of dalbavancin per 1 ml.

[0028] According to an embodiment of the present invention, the diluent is a mixed solution of acetonitrile and water, and more preferably, the diluent is acetonitrile-water (20:80).

[0029] According to an embodiment of the present invention, the high performance liquid chromatography method can achieve the dalbavancin component A 0 , A 1 , B 0 , B 1 , B 2 The separations among the five components were all greater than 1.5.

[0030] According to an embodiment of the present invention, the high performance liquid chromatography method can achieve the dalbavancin component A 0 , A 1 , B 0 , B 1 , B 2 And the separation and detection of impurity 1, impurity 2 and impurity 3.

[0031] According to an embodiment of the present invention, the high performance liquid chromatography method can achieve the dalbavancin component A 0 , A 1 , B 0 , B 1 , B 2 The quantitative detection concentration range and linear relationship are shown in Table 6.

[0032] In a second aspect of the present invention, a reagent combination for detecting the content of dalbavancin is provided, wherein the reagent combination comprises a mobile phase A and a mobile phase B; the mobile phase A is a diammonium phosphate solution-acetonitrile in a volume ratio of 80:20, and the mobile phase B is a diammonium phosphate solution-acetonitrile in a volume ratio of (60-70):(30-40). Further preferably, the mobile phase B is a diammonium phosphate solution-acetonitrile in a volume ratio of 66:34.

[0033] According to an embodiment of the present invention, the reagent combination further includes a diluent; preferably, the diluent is a mixed solution of acetonitrile and water, and further preferably, the diluent is acetonitrile-water (20:80).

[0034] According to an embodiment of the present invention, the reagent combination further comprises a dalbavancin component A 0 , A 1 , B0 , B 1 and B 2 further preferably, it also comprises impurity 1, impurity 2 and impurity 3 stock solutions; preferably, the concentration of the stock solution is 0.1-0.5 mg / ml.

[0035] According to an embodiment of the present invention, the reagent combination further comprises a dalbavancin component A 0 , A 1 , B 0 , B 1 and B 2 positioning solution; preferably, the concentration of the positioning solution is 1-10 μg / ml.

[0036] According to an embodiment of the present invention, the reagent combination further comprises a localization solution of impurity 1, impurity 2 and impurity 3; preferably, the concentration of the localization solution is 1-10 μg / ml.

[0037] According to an embodiment of the present invention, the reagent combination further comprises a dalbavancin component A 0 , A 1 , B 0 , B 1 and B 2 Linear solution.

[0038] According to an embodiment of the present invention, the B 0 The linear solution includes a series of linear gradients of B in the concentration range of 0.01 mg / ml-2 mg / ml. 0 According to an embodiment of the present invention, the B 0 The linear solution includes 5-7 linear gradient solutions with a concentration of 0.01 g / ml-2 mg / ml. According to a specific embodiment of the present invention, the B 0 The linear solutions include dalbavancin B at concentrations of 0.05 mg / ml, 0.08 mg / ml, 0.1 mg / ml, 0.12 mg / ml, and 0.16 mg / ml. 0 Solution.

[0039] According to an embodiment of the present invention, the B 2 Linear solution or B 1 The linear solution includes a series of linear gradients of B in the range of 0.0005 mg / ml to 0.025 mg / ml. 2 Solution or B 1 According to an embodiment of the present invention, the B 2 Linear solution or B 1The linear solution includes 5-7 linear gradient solutions with a concentration of 0.0005mg / ml-0.025mg / ml. According to a specific embodiment of the present invention, the B 2 Linear solution or B 1 The linear solutions include dalbavancin B at concentrations of 0.001 mg / ml, 0.003 mg / ml, 0.006 mg / ml, 0.01 mg / ml, and 0.02 mg / ml. 2 Dalbavancin B 1 Solution.

[0040] According to an embodiment of the present invention, the A 0 The linear solution includes a series of linear gradients of A in the range of 0.0002 mg / ml to 0.02 mg / ml. 0 According to an embodiment of the present invention, the A 0 The linear solution includes 5-7 linear gradient solutions with a concentration of 0.0002mg / ml-0.02mg / ml. According to a specific embodiment of the present invention, the A 0 The linear solutions included dalbavancin A at concentrations of 0.0003 mg / ml, 0.001 mg / ml, 0.003 mg / ml, 0.006 mg / ml, and 0.01 mg / ml. 0 Solution.

[0041] According to an embodiment of the present invention, the A 1 The linear solution includes a series of linear gradients of A in the range of 0.0005 mg / ml to 0.02 mg / ml. 1 According to an embodiment of the present invention, the A 1 The linear solution includes 5-7 linear gradient solutions with a concentration of 0.0005 mg / ml-0.02 mg / ml. According to a specific embodiment of the present invention, the A 1 The linear solutions include dalbavancin A at concentrations of 0.0005 mg / ml, 0.001 mg / ml, 0.003 mg / ml, 0.006 mg / ml, and 0.01 mg / ml. 1 Solution.

[0042] According to an embodiment of the present invention, the stock solution, positioning solution and linear solution are all prepared based on a diluent.

[0043] The third aspect of the present invention provides application of the method of the first aspect and the reagent combination of the second aspect in the detection and quality control of dalbavancin hydrochloride injection preparations.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] Dalbavancin has many components with similar structures. The high performance liquid chromatography method provided by the present invention can 0 , A 1 , B 0 , B 1 , B 2 The effective separation of the five components not only enables accurate detection of dalbavancin B 0 The method can not only detect the content of dalbavancin, but also the total content of dalbavancin; at the same time, the method has the advantages of strong specificity, high accuracy, good precision and durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the HPLC chromatogram of Example 1;

[0047] Figure 2 This is the HPLC chromatogram of acetonitrile-water (20:80) in the method validation experiment;

[0048] Figure 3 This is the HPLC chromatogram of the reference solution in the method validation experiment;

[0049] Figure 4 This is the HPLC chromatogram of the test solution in the method validation experiment;

[0050] Figure 5 This is the HPLC chromatogram of the spiked test solution in the method validation experiment;

[0051] Figure 6 It is the HPLC chromatogram of Comparative Example 1;

[0052] Figure 7 It is the HPLC chromatogram of Comparative Example 2;

[0053] Figure 8 It is the HPLC chromatogram of Comparative Example 3;

[0054] Fig. 9 It is the HPLC chromatogram of Comparative Example 4;

[0055] Fig.10 It is the HPLC chromatogram of Comparative Example 5. DETAILED DESCRIPTION

[0056] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0057] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0058] The dalbavancin hydrochloride for injection used in the following examples and verification examples comes from Livzon Pharmaceutical Factory of Livzon Group.

[0059] The dalbavancin working reference substance used in the following verification examples was from Fuzhou Fuxing Pharmaceutical Co., Ltd. of Livzon Group.

[0060] The impurities and components used in the following verification examples (A 0 , A 1 , B 1 , B 2 ) The reference substances were all from Tianjin WuXi AppTec. Impurities 1, 2, and 3 are degradation impurities with molecular formulas of C 70 H 69 Cl 2 N 9 O 22 , C 88 H 100 Cl 2 N 10 O 28 , C 82 H 90 Cl 2 N 10 O 23 The chemical structures of impurity 1, impurity 2 and impurity 3 are shown below:

[0061]

[0062]

[0063] The high performance liquid chromatograph used in the following examples and verification examples is Shimadzu (LC-20AD).

[0064] Example 1

[0065] Take an appropriate amount of 20% acetonitrile as the blank solution.

[0066] Take one bottle of dalbavancin hydrochloride for injection (500 mg), add 20% acetonitrile to dissolve the contents and dilute to make a solution containing approximately 0.1 mg of dalbavancin per 1 ml, which is used as the test solution.

[0067] Chromatographic conditions

[0068] Column: Agilent InfinityLab Poroshell 120EC-C18, 4.6 mm × 100 mm, 2.7 μm;

[0069] Detection wavelength: 210nm;

[0070] Column temperature: 40°C;

[0071] Flow rate: 0.5ml / min;

[0072] Mobile Phase:

[0073] Mobile phase A: 0.025 mol / L ammonium dihydrogen phosphate buffer solution-acetonitrile (80:20), pH = 5.8-6.2;

[0074] Mobile phase B: 0.025 mol / L ammonium dihydrogen phosphate buffer solution-acetonitrile (66:34), pH = 5.8-6.2;

[0075] The elution procedure is as follows:

[0076] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 4 40 60 30 5 95 30.1 95 5 40 95 5

[0077] Accurately measure 10 μl of blank solution and test solution and inject them into HPLC, and record the chromatogram. Figure 1 shown.

[0078] The results showed that in the chromatogram of the test solution, A 0 , A 1 , B 0 , B 1 , B 2 The peaks appeared in sequence, with retention times of 15.693min, 16.709min, 22.645min, 23.844min, and 24.667min, respectively. The separation between the five components was greater than 1.5. At the same time, it was found that the main peak B 0 It can be completely separated from its preceding impurity peak, with a resolution of 1.7.

[0079] Example 2 Methodology Verification Experiment

[0080] 1. Specificity test

[0081] Blank solution: Take an appropriate amount of 20% acetonitrile as the blank solution.

[0082] Blank excipient solution: Take 1 bottle of blank preparation (500 mg), add diluent to dissolve and transfer the entire amount to a 100 ml volumetric flask, add diluent to dilute to the mark, shake well, and use as the stock solution. Accurately measure 1 ml of the stock solution, place it in a 50 ml volumetric flask, add diluent to dilute to the mark, shake well, and use as the blank excipient solution.

[0083] Dalbavancin B 0 Positioning solution: Weigh 30 mg of dalbavancin working reference substance (containing about 25 mg of dalbavancin) into a 250 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, and use as dalbavancin B 0 The positioning solution also serves as the reference solution. Prepare 2 in parallel.

[0084] Impurity 1, impurity 2, impurity 3 and dalbavancin component (A 0 , A 1 , B 1 , B 2 ) Preparation of stock solution and positioning solution: Weigh appropriate amount of each impurity and component reference substance, place in a volumetric bottle, add diluent to dissolve and dilute to scale, shake well, as stock solution, accurately measure appropriate amount of the stock solution, place in a volumetric bottle, add diluent to dilute to scale, shake well, as positioning solution. Impurity 1, impurity 2, impurity 3 and dalbavancin component (A 0 , A 1 , B 1 , B 2 ) The preparation of stock solution and positioning solution is shown in Table 2.

[0085] Table 2 Solution preparation

[0086]

[0087] Test solution: Take 5 bottles of dalbavancin hydrochloride for injection, add an appropriate amount of diluent to dissolve the contents and transfer the entire amount to the same 250ml volumetric flask, dilute to the mark with diluent, shake well, and use as the test stock solution. Accurately measure 1ml of the test stock solution, place it in a 100ml volumetric flask, add diluent to dilute to the mark, shake well, and make a solution containing approximately 0.1mg of dalbavancin per 1ml as the test solution.

[0088] Spiked test solution: Take 5 bottles of dalbavancin hydrochloride for injection, add an appropriate amount of diluent to dissolve the contents and transfer the entire amount to the same 250ml volumetric flask, dilute to the mark with diluent, shake well, and use as the test stock solution. Measure 1ml of the test stock solution, 2.5ml of the impurity 1 stock solution, 1.5ml of the impurity 2 stock solution, and 1ml of the impurity 3 stock solution, respectively, place them in the same 100ml volumetric flask, dilute to the mark with diluent, shake well, and use as the spiked test solution.

[0089] Take 10 μl of each of the above prepared solutions and inject them into a high performance liquid chromatograph, perform detection according to the conditions described in Example 1, and record the chromatogram. The HPLC chromatogram of the blank solution acetonitrile-water (20:80) is shown in Figure 2 As shown; the HPLC chromatogram of the reference solution is as shown Figure 3 As shown; the HPLC chromatogram of the test solution is as shown Figure 4 As shown; the HPLC chromatogram of the spiked test solution is as shown Figure 5 The system suitability results are shown in Table 3, and the separation of the spiked test solution and the impurity location results are shown in Table 4.

[0090] Table 3 System suitability results

[0091]

[0092] From the above table, we can see that in the 5-pronged reference solution 1, the main peak B 0 The peak area RSD value is 0.22%. After adding the reference solution 1, the main peak B 0 The peak area of ​​the first 5 samples was compared with that of the reference solution 1. 0 The peak area RSD value of B in reference solution 2 was 0.40%, which was less than 2.0%. 0 The response factor of B in 5-pronged reference solution 1 0 The average response factor ratio is 99.5%, which is in the range of 98.0% to 102.0%. The method system of the present invention has good applicability.

[0093] Table 4 Separation of spiked test solution and location of impurities

[0094] name Retention time in the positioning solution (min) Retention time in spiked test solution (min) Separation Impurity 1 3.014 2.991 / <![CDATA[Dalbavancin A 0 > 15.102 15.108 41.6 <![CDATA[Dalbavancin A 1 > 16.071 16.078 3.6 Impurity 2 17.805 17.829 6.1 <![CDATA[Dalbavancin B 0 > 21.807 21.812 11.3 <![CDATA[Dalbavancin B 1 > 22.947 22.959 3.0 <![CDATA[Dalbavancin B 2 > 23.589 23.593 1.7 Impurity 3 24.833 24.835 3.2

[0095] The results showed that the blank solution and blank excipient solution had no interference with the test sample detection; in the spiked test sample solution, impurities 1, A 0 , A 1 、Impurity 2、B 0 , B 1 , B 2 , impurity 3 has the same retention time as the corresponding positioning solution, A 0 , A 1 , B 0 , B 1 , B 2 The separation between adjacent peaks is greater than 1.5. The method of the present invention has good specificity.

[0096] 2. Repeatability test

[0097] The reference solution was prepared in the same manner as in Verification Example 1, and 2 portions were prepared in parallel.

[0098] The test solution was prepared in the same manner as in Verification Example 1, and 6 portions were prepared in parallel.

[0099] Take 10 μl of each of the above prepared solutions, inject them into high performance liquid chromatograph, detect them according to the conditions described in Example 1, and record the chromatogram. Calculate the content of dalbavancin in the test solution. The test results are shown in Table 5.

[0100] 3. Intermediate precision test

[0101] Another analyst replaced a different chromatographic column on a different date and repeated the test to verify Example 2. The test results are shown in Table 5.

[0102] Table 5 Repeatability and intermediate precision test results

[0103]

[0104] As can be seen from the table above, the RSDs of the dalbavancin content in the 6 samples were 0.59% and 0.91%, respectively, less than 2.0%; the RSD of the dalbavancin content in the 12 samples was 0.83%, less than 3.0%, both meeting the requirements, indicating that the method of the present invention has good precision.

[0105] 4. Linearity test of each component

[0106] (1) Dalbavancin B 0 Linear solution

[0107] Dalbavancin B 0 Stock solution: Take 66 mg of dalbavancin hydrochloride working reference (containing approximately 0 50mg), accurately weighed, placed in a 25ml volumetric flask, added with diluent to dissolve and dilute to scale, shaken well, to make a 1ml dalbavancin B 0 2 mg solution as dalbavancin B 0 Stock solution.

[0108] B 0 Linear solution 1: Accurately measure 5 ml of B 0 Place linear solution 3 in a 10 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0109] B 0 Linearity solution 2: Precisely measure dalbavancin B 0 Place 1 ml of the stock solution in a 25 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0110] B 0 Linearity solution 3: Precisely measure dalbavancin B 0 Place 1 ml of the stock solution in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0111] B 0 Linearity solution 4: Precisely measure dalbavancin B 0 Place 3 ml of the stock solution in a 50 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0112] B 0 Linearity solution 5: Precisely measure dalbavancin B 0 Place 2 ml of the stock solution in a 25 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0113] (2) Dalbavancin A 0 Linear solution

[0114] Dalbavancin A 0 Stock solution: weigh dalbavancin A0 Reference substance 5.6mg (containing approximately A 0 4 mg) was placed in a 20 ml volumetric flask, and the diluent was added to dissolve and dilute to the mark, and shaken to make a volume containing about dalbavancin A per 1 ml. 0 0.2 mg solution as dalbavancin A 0 Stock solution.

[0115] A 0 Linear solution 1: Precision measurement 1 ml A 0 Place linear solution 4 in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0116] A 0 Linear solution 2: Precisely measure dalbavancin A 0 Place 1 ml of the stock solution in a 200 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0117] A 0 Linearity solution 3: Precisely measure dalbavancin A 0 Place 3 ml of the stock solution in a 200 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0118] A 0 Linearity solution 4: Precisely measure dalbavancin A 0 Place 3 ml of the stock solution in a 100 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0119] A 0 Linear solution 5: Precisely measure dalbavancin A 0 Place 1 ml of the stock solution in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0120] (3) Dalbavancin A 1 Linear solution

[0121] Dalbavancin A 1 Stock solution: weigh dalbavancin hydrochloride A 1 Reference substance 4.9mg (containing approximately A 1 4 mg) was placed in a 20 ml volumetric flask, and the diluent was added to dissolve and dilute to the mark, and shaken to make a volume containing about dalbavancin A per 1 ml. 1 0.2 mg solution as dalbavancin A 1 Stock solution.

[0122] A 1 Linear solution 1: Accurately measure 1 ml of A 1 Place linear solution 5 in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0123] A1 Linear solution 2: Precisely measure dalbavancin A 1 Place 1 ml of the stock solution in a 200 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0124] A 1 Linearity solution 3: Precisely measure dalbavancin A 1 Place 3 ml of the stock solution in a 200 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0125] A 1 Linearity solution 4: Precisely measure dalbavancin A 1 Place 3 ml of the stock solution in a 100 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0126] A 1 Linear solution 5: Precisely measure dalbavancin A 1 Place 1 ml of the stock solution in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0127] (4) Dalbavancin B 1 Linear solution

[0128] Dalbavancin B 1 Stock solution: weigh dalbavancin B 1 Reference substance 4.9mg (containing approximately B 1 4 mg) was placed in a 20 ml volumetric flask, and the diluent was added to dissolve and dilute to the mark, and shaken to make a volume containing about dalbavancin B per 1 ml. 1 0.2 mg solution as dalbavancin B 1 Stock solution.

[0129] B 1 Linearity solution 1: Accurately measure dalbavancin B 1 Place 1 ml of the stock solution in a 200 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0130] B 1 Linear solution 2 :Precisely measure dalbavancin B 1 Place 3 ml of the stock solution in a 200 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0131] B 1 Linearity solution 3: Precisely measure dalbavancin B 1 Place 3 ml of the stock solution in a 100 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0132] B 1 Linear solution 4 :Precisely measure dalbavancin B 1Place 1 ml of the stock solution in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0133] B 1 Linearity solution 5: Precisely measure dalbavancin B 1 Place 2 ml of the stock solution in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0134] (5) Dalbavancin B 2 Linear solution

[0135] Dalbavancin B 2 Stock solution: weigh dalbavancin B 2 Reference substance 5.1mg (containing approximately B 2 4 mg) was placed in a 20 ml volumetric flask, and the diluent was added to dissolve and dilute to the mark, and shaken to make a volume containing about dalbavancin B per 1 ml. 2 0.2 mg solution as dalbavancin B 2 Stock solution.

[0136] B 2 Linearity solution 1: Accurately measure dalbavancin B 2 Place 1 ml of the stock solution in a 200 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0137] B 2 Linearity solution 2: Precisely measure dalbavancin B 2 Place 3 ml of the stock solution in a 200 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0138] B 2 Linearity solution 3: Precisely measure dalbavancin B 2 Place 3 ml of the stock solution in a 100 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0139] B 2 Linearity solution 4: Precisely measure dalbavancin B 2 Place 1 ml of the stock solution in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0140] B 2 Linearity solution 5: Precisely measure dalbavancin B 2 Place 2 ml of the stock solution in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0141] 10 μl of each of the above prepared solutions were taken and injected into a high performance liquid chromatograph, and the detection was performed according to the conditions described in Example 1, and the chromatogram was recorded. The linearity results of each component are shown in Table 6.

[0142] Table 6 Linearity results of each component

[0143]

[0144] It can be seen from the above table that the correlation coefficients of each component are all 0.9999, the correction factors are all between 0.8 and 1.2, and the linear relationship between each component is good.

[0145] 5. Durability test

[0146] The column temperature was set to 35°C, and other chromatographic conditions were the same as those described in Example 1. The test results are shown in Table 7.

[0147] The column temperature was set to 45°C, and other chromatographic conditions were the same as those described in Example 1. The test results are shown in Table 7.

[0148] The flow rate was set to 0.45 mi / min, and other chromatographic conditions were the same as those described in Example 1. The test results are shown in Table 7.

[0149] The flow rate was set to 0.55 mi / min, and other chromatographic conditions were the same as those described in Example 1. The test results are shown in Table 7.

[0150] The mobile phase buffer salt pH was adjusted to 5.8, and other chromatographic conditions were the same as those described in Example 1. The test results are shown in Table 7.

[0151] The mobile phase buffer salt pH was adjusted to 6.2, and other chromatographic conditions were the same as those described in Example 1. The test results are shown in Table 7.

[0152] The test results under normal conditions are the same as those of the repeatability test.

[0153] The test results of different chromatographic columns are the same as those of the intermediate precision test.

[0154] Table 7 Durability results under different conditions

[0155]

[0156] As can be seen from the above table, under different column temperatures (35°C, 45°C); different mobile phase pH values ​​(5.8, 6.2); different flow rates (0.45ml / min, 0.55ml / min); chromatographic columns of the same brand but different batches (Agilent InfinityLab Poroshell120EC-C18 4.6×100mm, 2.7μm), the system applicability meets the requirements; the absolute difference between the dalbavancin content measured under each condition and the dalbavancin content measured under normal conditions is less than 2.0%, which proves that the method of the present invention has good durability.

[0157] In summary, the method used in the present invention, A 0 , A 1 , B 0 , B 1 , B2 The five components can be well separated, and have the advantages of strong specificity, high accuracy, good precision and durability. This method can not only detect the total content of dalbavancin hydrochloride for injection, but also achieve A 0 , A 1 , B 0 , B 1 , B 2 Determination of the content of each component.

[0158] Comparative Example 1

[0159] Take one bottle of dalbavancin hydrochloride for injection (500 mg), add 20% acetonitrile to dissolve the contents and dilute to make a solution containing about 0.2 mg of dalbavancin per 1 ml, which is used as the test solution.

[0160] Chromatographic conditions

[0161] Chromatographic column: YMC Triart-C18, 4.6 mm × 250 mm, 3 μm;

[0162] Detection wavelength: 210nm;

[0163] Column temperature: 40°C;

[0164] Flow rate: 0.5 ml / min;

[0165] Mobile Phase:

[0166] Mobile phase A: 0.025 mol / L ammonium dihydrogen phosphate buffer solution, pH = 5.8-6.2;

[0167] Mobile phase B: acetonitrile;

[0168] The elution procedure is as follows:

[0169] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 5 80 20 10 67 33 45 67 33 46 80 20 50 80 20

[0170] Accurately measure 10 μl of blank solution and test solution and inject them into HPLC, and record the chromatogram. Figure 6 shown.

[0171] The results showed that in the chromatogram of the test solution, A 0 , A 1 , B 0 , B 1 , B 2 The peaks appeared in sequence, with retention times of 23.789min, 24.971min, 32.681min, 34.521min, and 36.207min, respectively. The separation between the five components was greater than 1.5. However, after zooming in on the spectrum, it was found that the main peak B 0 There is an impurity peak in the front that is not completely separated.

[0172] Comparative Example 2

[0173] The test solution was prepared in the same manner as in Comparative Example 1.

[0174] Chromatographic conditions

[0175] Chromatographic column: YMC Triart-C18, 4.6 mm × 250 mm, 3 μm;

[0176] Detection wavelength: 210nm;

[0177] Column temperature: 45°C;

[0178] Flow rate: 0.4 ml / min;

[0179] Mobile Phase:

[0180] Mobile phase A: 0.025 mol / L ammonium dihydrogen phosphate buffer solution, pH = 5.8-6.2;

[0181] Mobile phase B: acetonitrile;

[0182] The elution procedure is as follows:

[0183] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 5 80 20 10 67 33 45 67 33 46 80 20 50 80 20

[0184] Accurately measure 10 μl of blank solution and test solution and inject them into HPLC, and record the chromatogram. Figure 7 shown.

[0185] The results showed that in the chromatogram of the test solution, A 0 , A 1 , B 0 , B 1 , B 2 The peaks appeared in sequence, with retention times of 27.180min, 28.552min, 37.623min, 39.763min, and 42.112min, respectively. The separation between the five components was greater than 1.5. However, after zooming in on the spectrum, it was found that the main peak B 0 There is an impurity peak in the front that is not completely separated.

[0186] Comparative Example 3

[0187] The test solution was prepared in the same manner as in Comparative Example 1.

[0188] Chromatographic conditions

[0189] Chromatographic column: YMC Triart-C18, 4.6 mm × 250 mm, 3 μm;

[0190] Detection wavelength: 210nm;

[0191] Column temperature: 40°C;

[0192] Flow rate: 0.6 ml / min;

[0193] Mobile Phase:

[0194] Mobile phase A: 0.025 mol / L ammonium dihydrogen phosphate buffer solution, pH = 5.8-6.2;

[0195] Mobile phase B: acetonitrile;

[0196] The elution procedure is as follows:

[0197] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 5 80 20 10 67 33 45 67 33 46 80 20 50 80 20

[0198] Accurately measure 10 μl of blank solution and test solution and inject them into HPLC, and record the chromatogram. Figure 8 shown.

[0199] The results showed that in the chromatogram of the test solution, A 0 , A 1 , B 0 , B 1 , B 2 The peaks appeared in sequence, with retention times of 21.048min, 21.988min, 28.112min, 29.586min, and 30.913min, respectively. The separation between the five components was greater than 1.5. However, after zooming in on the spectrum, it was found that the main peak B 0 There is an impurity peak in the front that is not completely separated.

[0200] Comparative Example 4

[0201] Take one bottle of dalbavancin hydrochloride for injection (500 mg), add 20% acetonitrile to dissolve the contents and dilute to make a solution containing approximately 0.1 mg of dalbavancin per 1 ml, which is used as the test solution.

[0202] Chromatographic conditions

[0203] Column: Agilent InfinityLab Poroshell 120EC-C18, 4.6 mm × 100 mm, 2.7 μm;

[0204] Detection wavelength: 210nm;

[0205] Column temperature: 40°C;

[0206] Flow rate: 0.5 ml / min;

[0207] Mobile Phase:

[0208] Mobile phase A: 0.025 mol / L ammonium dihydrogen phosphate buffer solution-acetonitrile (80:20), pH = 5.8-6.2;

[0209] Mobile phase B: 0.025 mol / L ammonium dihydrogen phosphate buffer solution-acetonitrile (66:34), pH = 5.8-6.2;

[0210] The elution procedure is as follows:

[0211] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 4 5 95 22 5 95 22.1 95 5 30 95 5

[0212] Accurately measure 10 μl of blank solution and test solution and inject them into HPLC, and record the chromatogram. Fig. 9 shown.

[0213] Comparative Example 4 The elution procedure is obtained by using the method converter for Example 1. The results show that in the chromatogram of the test solution, A 0 , A 1 , B 0 , B 1 , B 2 The peaks appeared in sequence, with retention times of 10.081min, 10.468min, 13.121min, 13.745min and 14.195min respectively. The separation degrees between the five components were all greater than 1.5. However, compared with Comparative Example 1 or Example 1, the peaks of the five components were more concentrated and the baseline fluctuation was more obvious.

[0214] Comparative Example 5

[0215] The test solution was prepared in the same manner as in Comparative Example 4.

[0216] Chromatographic conditions

[0217] Column: Agilent InfinityLab Poroshell 120EC-C18, 4.6 mm × 100 mm, 2.7 μm;

[0218] Detection wavelength: 210nm;

[0219] Column temperature: 40°C;

[0220] Flow rate: 0.5 ml / min;

[0221] Mobile Phase:

[0222] Mobile phase A: 0.025 mol / L ammonium dihydrogen phosphate buffer solution-acetonitrile (80:20), pH = 5.8-6.2;

[0223] Mobile phase B: 0.025 mol / L ammonium dihydrogen phosphate buffer solution-acetonitrile (66:34), pH = 5.8-6.2;

[0224] The elution procedure is as follows:

[0225]

[0226]

[0227] Accurately measure 20 μl of blank solution and test solution and inject them into the HPLC instrument. Record the chromatogram. Fig.10 shown.

[0228] The results showed that in the chromatogram of the test solution, A 0 , A 1 , B 0 , B 1 , B 2 The peaks appeared in sequence, with retention times of 13.729min, 14.434min, 19.099min, 20.049min, and 20.755min, respectively. The separation between the five components was greater than 1.5. However, after zooming in on the spectrum, it was found that the main peak B 0 There is an impurity peak in the front that is not completely separated.

[0229] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An analytical method for detecting the content of dalbavancin, characterized in that: The method comprises detecting dalbavancin by reversed-phase high performance liquid chromatography, wherein the mobile phase is ammonium dihydrogen phosphate solution-acetonitrile and the elution procedure is gradient elution.

2. The analytical method for detecting the content of dalbavancin according to claim 1, characterized in that: The mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is a diammonium phosphate solution-acetonitrile in a volume ratio of 80:20, and the mobile phase B is a diammonium phosphate solution-acetonitrile in a volume ratio of (60-70):(30-40); preferably, the mobile phase B is a diammonium phosphate solution-acetonitrile in a volume ratio of 66:34; preferably, the concentration of the diammonium phosphate solution in the mobile phase A and the mobile phase B is 0.025 mol / L, and the pH is 5.8-6.

2.

3. The analytical method for detecting the content of dalbavancin according to claim 2, characterized in that: The procedure for mobile phase gradient elution was as follows: 。 4. The analytical method for detecting the content of dalbavancin according to claim 1, characterized in that: The chromatographic column of the high performance liquid chromatography method is a chromatographic column filled with octadecyl bonded silica gel; preferably, the chromatographic column has a specification of 4.6 mm×100 mm, 2.7 μm or 4.6 mm×250 mm, 3 μm; further preferably, the chromatographic column has a specification of 4.6 mm×100 mm, 2.7 μm.

5. The analytical method for detecting the content of dalbavancin according to claim 1, characterized in that: The column temperature of the high performance liquid chromatography is 35-45°C; more preferably, the column temperature is 40°C; Preferably, the flow rate of the HPLC method is 0.4 to 0.6 ml / min; further preferably, the flow rate is 0.5 ml / min; Preferably, the injection volume of the HPLC method is 10 to 20 μl; further preferably, the injection volume is 10 μl; Preferably, the detection wavelength of the HPLC is 200-220 nm; further preferably, the detection wavelength is 210 nm.

6. The analytical method for detecting the content of dalbavancin according to claim 1, characterized in that: The analytical method comprises measuring the test solution and the reference solution, respectively injecting them into a liquid chromatograph, recording the chromatogram, and calculating the peak area according to the external standard curve method; Preferably, a standard curve is drawn using the peak area obtained by linear solution detection, and the contents of components A0, A1, B0, B1, and B2 in the test sample are calculated using the standard curve; Preferably, the dalbavancin sample is dissolved in a diluent to obtain a test solution; further preferably, the test solution contains about 0.1 mg of dalbavancin per 1 ml; Preferably, the diluent is a mixed solution of acetonitrile and water; further preferably, the diluent is acetonitrile-water (20:80).

7. The analytical method for detecting the content of dalbavancin according to claim 1, characterized in that: The high performance liquid chromatography method can achieve a separation degree greater than 1.5 between the five components of dalbavancin, namely, A0, A1, B0, B1, and B2; Preferably, the high performance liquid chromatography method can achieve separation and detection of dalbavancin components A0, A1, B0, B1, B2 and impurities 1, impurity 2, and impurity 3; Preferably, the HPLC method can achieve the quantitative detection concentration range and linear relationship of dalbavancin components A0, A1, B0, B1, and B2, see Table 6 of the specification.

8. A reagent combination for detecting the content of dalbavancin, characterized in that: The reagent combination comprises a mobile phase A and a mobile phase B; the mobile phase A is a diammonium phosphate solution-acetonitrile in a volume ratio of 80:20, and the mobile phase B is a diammonium phosphate solution-acetonitrile in a volume ratio of (60-70):(30-40); further preferably, the mobile phase B is a diammonium phosphate solution-acetonitrile in a volume ratio of 66:34; Preferably, the reagent combination further comprises a diluent; the diluent is a mixed solution of acetonitrile and water, and more preferably, the diluent is acetonitrile-water (20:80); Preferably, the reagent combination further comprises stock solutions of dalbavancin components A0, A1, B0, B1 and B2; further preferably, it further comprises stock solutions of impurity 1, impurity 2 and impurity 3; preferably, the concentration of the stock solution is 0.1-0.5 mg / ml; Preferably, the reagent combination further comprises a localization solution of dalbavancin components A0, A1, B0, B1 and B2; preferably, the concentration of the localization solution is 1-10 μg / ml; Preferably, the reagent combination further comprises a localization solution of impurity 1, impurity 2 and impurity 3; preferably, the concentration of the localization solution is 1-10 μg / ml; Preferably, the reagent combination further comprises a linear solution of dalbavancin components A0, A1, B0, B1 and B2; Preferably, the B0 linear solution includes a series of linear gradient B0 solutions with a concentration in the range of 0.01g / ml-2mg / ml; the B2 linear solution or B1 linear solution includes a series of linear gradient B2 solutions or B1 solutions with a concentration in the range of 0.0005mg / ml-0.025mg / ml; the A0 linear solution includes a series of linear gradient A0 solutions with a concentration in the range of 0.0002mg / ml-0.02mg / ml; the A1 linear solution includes a series of linear gradient A1 solutions with a concentration in the range of 0.0005mg / ml-0.02mg / ml.

9. A reagent combination for detecting the content of dalbavancin according to claim 8, characterized in that: The stock solution, positioning solution and linearity solution are all prepared based on diluent.

10. Use of the analytical method according to any one of claims 1 to 7 and the reagent combination according to claim 8 or 9 in the detection and quality control of dalbavancin hydrochloride preparations for injection.

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