CN114354810B - Method for detecting impurity N in clindamycin phosphate and method for separating impurity - Google Patents
Method for detecting impurity N in clindamycin phosphate and method for separating impurity Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 197
- UFUVLHLTWXBHGZ-MGZQPHGTSA-N [(2r,3r,4s,5r,6r)-6-[(1s,2s)-2-chloro-1-[[(2s,4r)-1-methyl-4-propylpyrrolidine-2-carbonyl]amino]propyl]-4,5-dihydroxy-2-methylsulfanyloxan-3-yl] dihydrogen phosphate Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](OP(O)(O)=O)[C@@H](SC)O1 UFUVLHLTWXBHGZ-MGZQPHGTSA-N 0.000 title claims abstract description 86
- 229960002291 clindamycin phosphate Drugs 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000000243 solution Substances 0.000 claims abstract description 107
- 238000001514 detection method Methods 0.000 claims abstract description 82
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000012488 sample solution Substances 0.000 claims abstract description 33
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 28
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical group OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000003814 drug Substances 0.000 claims abstract description 15
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 239000008363 phosphate buffer Substances 0.000 claims description 24
- 238000002347 injection Methods 0.000 claims description 22
- 239000007924 injection Substances 0.000 claims description 22
- 239000012085 test solution Substances 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 10
- 239000000523 sample Substances 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 38
- 239000008055 phosphate buffer solution Substances 0.000 abstract description 6
- 239000002904 solvent Substances 0.000 description 46
- 239000007788 liquid Substances 0.000 description 45
- 239000013558 reference substance Substances 0.000 description 33
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 32
- 238000012360 testing method Methods 0.000 description 27
- 239000000126 substance Substances 0.000 description 23
- 239000000463 material Substances 0.000 description 20
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 16
- 238000007865 diluting Methods 0.000 description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 14
- 239000000047 product Substances 0.000 description 12
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 11
- 235000011114 ammonium hydroxide Nutrition 0.000 description 11
- 239000012452 mother liquor Substances 0.000 description 10
- 239000012467 final product Substances 0.000 description 9
- 239000011550 stock solution Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
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- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical class CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 229960002227 clindamycin Drugs 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000012088 reference solution Substances 0.000 description 4
- 238000001612 separation test Methods 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000010812 external standard method Methods 0.000 description 3
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- 101000777134 Homo sapiens Ubiquitin carboxyl-terminal hydrolase 43 Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 102100031311 Ubiquitin carboxyl-terminal hydrolase 43 Human genes 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
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- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical group CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 description 2
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
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- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 150000003462 sulfoxides Chemical class 0.000 description 2
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- XGRUMIVSCDLWCM-SFYZADRCSA-N (2s,4r)-1-methyl-4-propylpyrrolidine-2-carboxamide Chemical compound CCC[C@@H]1C[C@@H](C(N)=O)N(C)C1 XGRUMIVSCDLWCM-SFYZADRCSA-N 0.000 description 1
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- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a detection method of impurity N in clindamycin phosphate and a separation method of impurities. The method for detecting the impurity N in clindamycin phosphate comprises the following steps: detecting a sample solution containing clindamycin phosphate drug by adopting high performance liquid chromatography; the mobile phase of the high performance liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is phosphate buffer solution, and the pH value of the mobile phase A is 5.0-6.0; the mobile phase B is acetonitrile-methanol solution, and the volume ratio of acetonitrile to methanol is 90:10. the detection method can realize the effective separation of the impurity N in clindamycin phosphate, and has good peak shape and separation degree; the separation method can realize the separation of various impurities in the clindamycin phosphate, the separation degree between adjacent impurity peaks meets the requirement, and the detection of the impurities in the clindamycin phosphate can be satisfied.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a detection method of impurity N in clindamycin phosphate and a separation method of impurities.
Background
The clindamycin phosphate is a chemical semisynthetic clindamycin derivative, has no antibacterial activity in vitro, can be rapidly hydrolyzed into clindamycin after entering the body, can play a pharmacological role, has strong antibacterial activity on gram-positive cocci and anaerobic bacteria, and is mainly used as a tablet, a suppository, a gel and an injection at home and abroad.
Clindamycin phosphate is known to contain impurities A, B, C, E, F, G, L, I, N, O, the specific structure of which is as follows:
Wherein, the impurity N is degradation impurity, the control limit is 0.2%, and the quality standard/control strategy is adopted. Although clindamycin phosphate was collected in ChP2020, BP2020, EP10.0, USP43 and JP17, the substances related to the injection USP43 and JP17 were not controlled, and the detection method (HPLC inspection method) of the substances related to the standard collection of the quality control standard in the clindamycin phosphate bulk drug in BP2020, chP2020 was difficult to separate impurity N.
Therefore, there is a need to optimize the detection method of clindamycin phosphate related substances and develop a separation detection method capable of realizing the separation of target impurities in clindamycin phosphate.
Disclosure of Invention
The invention aims to overcome the defect that the detection method of clindamycin phosphate related substances in the prior art is difficult to realize separation and detection of target impurities; and provides a detection method of impurity N in clindamycin phosphate and a separation method of the impurity. The detection method can realize effective separation of impurity N in clindamycin phosphate, and has good peak shape and separation degree; the separation method can realize the separation of various impurities in the clindamycin phosphate, the separation degree between adjacent impurity peaks meets the requirement, and the detection of the impurities in the clindamycin phosphate can be satisfied.
In order to achieve the above object, the present invention provides the following technical solutions:
One of the technical schemes of the invention is as follows: the method for detecting impurity N in clindamycin phosphate comprises the following steps: detecting a sample solution containing clindamycin phosphate drug by adopting a first high performance liquid chromatography;
the elution mode of the first high performance liquid chromatography is as follows:
wherein,
The above% refers to volume percent;
mobile phase a is phosphate buffer; the pH value of the mobile phase A is 5.0-6.0;
mobile phase B is acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol is 90:10.
In the invention, the clindamycin phosphate drug generally refers to clindamycin phosphate bulk drug and/or clindamycin phosphate injection.
In the invention, the impurity N is C 18H34ClN2O9 PS, the molecular weight is 520.96, and the chemical name is: (2 s,4 r) -6- (((1 s,2 s) -2-chloro-1- ((2 s,4 r) -1-methyl-4-propylpyrrolidine-2-carboxamide) propyl) -4, 5-dihydroxy-2- (methylsulfinyl) tetrahydro-2H-pyran-3-yl dihydrogen phosphate, the structural formula of which is shown in formula (I):
in the present invention, in the first high performance liquid chromatography, the chromatographic column used may be a C18 chromatographic column.
Wherein, the specification of the C18 chromatographic column is preferably 150mm in column length multiplied by 4.6mm in inner diameter. The packing particle size of the C18 chromatographic column is preferably 5 μm.
For example: the model of the C18 chromatographic column is Kromasil C18 or Ultimate C18, and the specification is 150 multiplied by 4.6mm and 5 mu m.
In the present invention, the detection wavelength in the first high performance liquid chromatography may be 212 to 216nm, for example, 214nm.
In the present invention, the flow rate of the mobile phase in the first high performance liquid chromatography may be conventional in the art, and may be generally 1 to 1.5mL/min, for example, 1.1 to 1.3mL/min, and further, for example, 1.2mL/min.
In the present invention, the sample injection volume in the first hplc may be conventional in the art, for example, 50 μl.
In the present invention, in the first high performance liquid chromatography, the column temperature of the chromatographic column may be conventional in the art, for example, 35 to 45 ℃, and still more for example, 40 ℃.
In the invention, the pH value of the mobile phase A is preferably 5.3-5.7; for example 5.5.
In the invention, the mobile phase A is prepared by the following method: 3.5mL of phosphoric acid is taken, 1000mL of water is added, 2.5mL of ammonia water is added, and the pH value is adjusted to 5.5 by using the ammonia water.
In the present invention, in the first high performance liquid chromatography, the mass concentration of clindamycin phosphate in the sample solution may be conventional in the art, preferably is 1.80-3.57 mg/mL, for example 3mg/mL, where mg/mL refers to the ratio of the mass of clindamycin phosphate to the volume of the sample solution.
The sample solution can be prepared according to a conventional preparation method in the art, for example, 35.7mg clindamycin phosphate is placed in a 10mL measuring flask, a pH5.5 phosphate buffer solution-acetonitrile methanol solution (the volume ratio of the pH5.5 phosphate buffer solution to the acetonitrile methanol solution is 83:17; the volume ratio of acetonitrile to methanol is 90:10) is added, dissolved and diluted to a scale, and the scale is shaken uniformly to obtain the sample solution; for another example, 1mL clindamycin phosphate injection is placed in a 100mL measuring flask, pH5.5 phosphate buffer solution-90% acetonitrile methanol solution (the volume ratio of the pH5.5 phosphate buffer solution to the acetonitrile methanol solution is 83:17; the volume ratio of acetonitrile to methanol is 90:10) is added, dissolved and diluted to scale, and shaking is carried out uniformly, thus obtaining the clindamycin phosphate injection.
In the present invention, the elution mode of the first hplc is more preferably as follows:
wherein,
The above% refers to volume percent;
mobile phase a is phosphate buffer; the pH value of the mobile phase A is 5.7;
mobile phase B is acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol is 90:10.
The second technical scheme of the invention is as follows: a method for separating impurities in clindamycin phosphate, which comprises the following steps: detecting a sample solution containing clindamycin phosphate drug by adopting a second high performance liquid chromatography;
The chromatographic column adopted by the second high performance liquid chromatography is an XDB-C8 chromatographic column or Ulimate XB-C8 chromatographic column;
the elution mode of the second high performance liquid chromatography is as follows:
wherein,
The above% refers to volume percent;
Mobile phase C is phosphate buffer solution-acetonitrile methanol solution; the volume ratio of the phosphate buffer to the acetonitrile methanol solution is 92:8, 8;
Mobile phase D is phosphate buffer solution-acetonitrile methanol solution; the volume ratio of the phosphoric acid buffer solution to the acetonitrile methanol solution is 52:48;
In acetonitrile methanol solution of the mobile phase C and the mobile phase D, the volume ratio of acetonitrile to methanol is 90:10;
in the mobile phase C and the mobile phase D, the pH value of the phosphate buffer solution is 3.8-4.0.
In the present invention, in the second high performance liquid chromatography, the chromatographic column used is preferably an XDB-C8 chromatographic column.
The XDB-C8 chromatographic column or Ulimate XB-C8 chromatographic column is preferably 250mm long by 4.6mm inner diameter.
The packing material of the XDB-C8 chromatographic column or Ulimate XB-C8 chromatographic column is preferably octyl silane bonded silica gel. The packing particle size of the XDB-C8 chromatography column or Ulimate XB-C8 chromatography column is preferably 5. Mu.m.
In the present invention, the detection wavelength in the second high performance liquid chromatography may be 210 to 218nm, for example 212 to 216nm, and further for example 214nm.
In the present invention, the flow rate of the mobile phase in the second high performance liquid chromatography may be conventional in the art, and may be generally 1.15 to 1.25mL/min, for example, 1.2mL/min.
In the present invention, the sample injection volume in the second high performance liquid chromatography may be conventional in the art, for example, 25 μl.
In the present invention, in the second high performance liquid chromatography, the column temperature of the chromatographic column may be conventional in the art, for example, 38 to 42 ℃, and still more for example, 40 ℃.
In the present invention, the pH of the phosphate buffer is preferably 3.9 in the mobile phase C and the mobile phase D.
In the invention, the mass concentration of clindamycin phosphate in the test sample solution can be conventional in the art, and is preferably 2.5-3.5 mg/mL, more preferably 3mg/mL; wherein, mg/mL refers to the ratio of the mass of clindamycin phosphate to the volume of the test solution.
The sample solution may be prepared according to a conventional preparation method in the art, for example, a solvent may be added to clindamycin phosphate drug; the solvent is phosphate buffer solution-acetonitrile methanol solution.
In the solvent, the volume ratio of the phosphate buffer solution to the acetonitrile methanol solution may be 80:20.
In the solvent, the pH of the phosphate buffer is preferably 3.9. The phosphate buffer may be conventional in the art and is preferably prepared by the following method: 3.5mL of phosphoric acid was taken, 1000mL of water and 2.5mL of concentrated ammonia solution were added, and if necessary, the pH was adjusted to 3.9.+ -. 0.05.
In the solvent, in the acetonitrile methanol solution, the volume ratio of acetonitrile to methanol is 90:10.
In the present invention, the elution mode of the second high performance liquid chromatography is more preferably as follows:
wherein,
The above% refers to volume percent;
Mobile phase C is phosphate buffer solution-acetonitrile methanol solution; the volume ratio of the phosphate buffer to the acetonitrile methanol solution is 92:8, 8;
Mobile phase D is phosphate buffer solution-acetonitrile methanol solution; the volume ratio of the phosphoric acid buffer solution to the acetonitrile methanol solution is 52:48;
In acetonitrile methanol solution of the mobile phase C and the mobile phase D, the volume ratio of acetonitrile to methanol is 90:10;
in the mobile phase C and the mobile phase D, the pH value of the phosphate buffer solution is 3.9.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The application has the positive progress effects that: the detection method can realize effective separation of the impurities in the clindamycin phosphate medicine, has good peak shape and separation degree, and can meet the detection of the impurities in the clindamycin phosphate.
Drawings
Fig. 1-1 shows high performance liquid chromatograms of the sample solution, the blank auxiliary material solution, the impurity N reference solution, the impurity O reference positioning solution, the benzaldehyde positioning solution and the blank solution in effect example 1.
FIGS. 1-2 are high performance liquid chromatograms of the control mixed solution of other impurities (A, B, C, E, F, G, L, I, N, O) in effect example 1.
FIG. 2 is a high performance liquid chromatogram of a solution of clindamycin phosphate mixed with other impurity (A, B, D, E, F, G, H, I, J, L, M) controls in effect example 2.
FIG. 3 is a high performance liquid chromatogram obtained by the detection method of example 1.
FIG. 4 is a high performance liquid chromatogram obtained by the detection method of example 2.
FIG. 5 is a high performance liquid chromatogram obtained by the detection method of example 3.
FIG. 6 is a high performance liquid chromatogram obtained by the detection method of example 4.
FIG. 7 is a high performance liquid chromatogram obtained by the detection method of example 5.
FIG. 8 is a high performance liquid chromatogram obtained by the detection method of example 6.
FIG. 9 is a high performance liquid chromatogram obtained by the detection method of example 7.
FIG. 10 is a high performance liquid chromatogram obtained by the detection method of example 8.
FIG. 11 is a high performance liquid chromatogram obtained by the detection method of example 9.
FIG. 12 is a high performance liquid chromatogram obtained by the detection method of example 10.
FIG. 13 is a high performance liquid chromatogram obtained by the detection method of example 11.
FIG. 14 is a high performance liquid chromatogram obtained by the detection method of example 12.
FIG. 15-1 is a high performance liquid chromatogram (detection wavelength 210 nm) obtained by the detection method of comparative example 1.
FIG. 15-2 is a high performance liquid chromatogram (detection wavelength 214 nm) obtained by the detection method of comparative example 1.
FIG. 16 is a high performance liquid chromatogram obtained by the detection method of comparative example 2.
FIG. 17 is a graph showing the relationship between the concentration of impurity N and the peak area in effect example 1.
FIG. 18 is a high performance liquid chromatogram obtained by the separation method of example 13.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
In the following examples and comparative examples, a commercial source of clindamycin phosphate drug substance is Tian Fang pharmaceutical Co.
Example 1 detection of related substances in clindamycin phosphate impurity N
The chromatographic conditions for the impurity N examination in clindamycin phosphate were used as follows, and the method was established as follows:
(1) Impurity N detection chromatographic conditions
Chromatographic column: ultimate C18, 4.6X105 mm,5 μm
Mobile phase a: phosphate buffer (pH 5.5.3.5 ml phosphate, 1000ml water, 2.5ml ammonia water, pH5.5 with ammonia water)
Mobile phase B: acetonitrile-methanol (90:10)
Flow rate: 1.2mL/min
Detection wavelength: 214nm
Sample injection amount: 50 mu L
Column temperature: 40 DEG C
Gradient elution mode:
the high performance liquid chromatogram obtained by the detection method of example 1 is shown in fig. 3, and the specific data are shown in table 1 below, and the degree of separation is 1.7.
TABLE 1
Example 2 detection of related substances in clindamycin phosphate impurity N
In example 2, the mobile phase A was a phosphate buffer (3.5 ml of phosphoric acid, 1000ml of water, 2.5ml of ammonia water, and pH5.3 was adjusted with ammonia water) having a pH of 5.3, in the same manner as in example 1.
The high performance liquid chromatogram obtained by the detection method of example 2 is shown in fig. 4, and the specific data are shown in table 2 below, and the degree of separation is 4.6.
TABLE 2
Example 3 detection of related substances in clindamycin phosphate impurity N
In example 3, the mobile phase A was a phosphate buffer (3.5 ml of phosphoric acid, 1000ml of water, 2.5ml of ammonia water, and pH5.7 was adjusted with ammonia water) having a pH of 5.7, in the same manner as in example 1.
The high performance liquid chromatogram obtained by the detection method of example 3 is shown in fig. 5, and the specific data are shown in table 3 below, and the degree of separation is 7.2.
TABLE 3 Table 3
EXAMPLE 4 detection of related substances in clindamycin phosphate impurity N
The gradient elution table in example 4 is shown below, except that example 1 is followed.
The high performance liquid chromatogram obtained by the detection method of example 4 is shown in fig. 6, and the specific data is shown in table 4 below, with a degree of separation of 5.2.
TABLE 4 Table 4
EXAMPLE 5 detection of related substances in clindamycin phosphate impurity N
The gradient elution table in example 5 is shown below, except that example 1 is followed.
The high performance liquid chromatogram obtained by the detection method of example 5 is shown in fig. 7, and the specific data is shown in table 5 below, with a degree of separation of 1.4.
TABLE 5
EXAMPLE 6 detection of related substances in clindamycin phosphate impurity N
The flow rate in example 6 was 1.1ml/min, and the same as in example 1 was followed.
The high performance liquid chromatogram obtained by the detection method of example 6 is shown in fig. 8, and the specific data is shown in table 6 below, with a degree of separation of 2.6.
TABLE 6
EXAMPLE 7 detection of related substances in clindamycin phosphate impurity N
The flow rate in example 7 was 1.3ml/min, except that the flow rate was the same as in example 1.
The high performance liquid chromatogram obtained by the detection method of example 7 is shown in fig. 9, and the specific data is shown in table 7 below, with a degree of separation of 2.6.
TABLE 7
Example 8 detection of related substances in clindamycin phosphate impurity N
The column temperature in example 8 was 35℃and the same as in example 1.
The high performance liquid chromatogram obtained by the detection method of example 8 is shown in fig. 10, and the specific data is shown in table 8 below, with a degree of separation of 4.5.
TABLE 8
Example 9 detection of related substances in clindamycin phosphate impurity N
The column temperature in example 9 was 45℃and the same as in example 1.
The high performance liquid chromatogram obtained by the detection method of example 9 is shown in FIG. 11, and the specific data is shown in Table 9 below, with a degree of separation of 6.6.
TABLE 9
EXAMPLE 10 detection of related substances in clindamycin phosphate impurity N
The detection wavelength in example 10 was 212nm, and the same as in example 1 was repeated.
The high performance liquid chromatogram obtained by the detection method of example 10 is shown in FIG. 12, and the specific data is shown in Table 10 below, with a degree of separation of 4.9.
Table 10
EXAMPLE 11 detection of related substances in clindamycin phosphate impurity N
The detection wavelength in example 11 was 216nm, and the same as in example 1 was repeated.
The high performance liquid chromatogram obtained by the detection method of example 11 is shown in FIG. 13, and the specific data is shown in Table 11 below, with a degree of separation of 5.0.
TABLE 11
EXAMPLE 12 detection of related substances in clindamycin phosphate impurity N
The column in example 12 was a Kromasil column, and the procedure of example 1 was followed.
The high performance liquid chromatogram obtained by the detection method of example 12 is shown in fig. 14, and the specific data is shown in table 12 below, with a degree of separation of 5.0.
Table 12
Comparative example 1
The high performance liquid chromatograms obtained by the detection method of related substances in clindamycin phosphate injection on page 548 of four ChP2020 are shown in figure 15-1 (detection wavelength is 210 nm) and figure 15-2 (detection wavelength is 214 nm).
As can be seen from FIGS. 15-1 and 15-2, impurity J and impurity I in clindamycin phosphate injection coincide, and the separation degree of the impurity B peak and the impurity N (sulfoxide) peak is 0.42.
Comparative example 2
The high performance liquid chromatogram obtained by the detection method of related substances in clindamycin phosphate injection in BP2020 is shown in figure 16, and the specific data is shown in table 13, wherein the separation degree is 0.11.
TABLE 13
The figure shows that impurities F and A in clindamycin phosphate injection are not completely separated, and impurities B and N (sulfoxide), impurities H and G, impurities I and J, and impurities L and clindamycin phosphate coincide pairwise.
Effect example 1
1. Specificity test
(1) Solution preparation
Blank solvent: phosphate buffer-90% acetonitrile in methanol (83:17) at pH 5.5.
Blank auxiliary material solution (sodium hydroxide, edetate sodium, benzyl alcohol, water for injection): taking 1mL of blank auxiliary material solution, placing the blank auxiliary material solution into a 100mL measuring flask, adding a solvent to dilute the blank auxiliary material solution to a scale, and shaking the blank auxiliary material solution uniformly to obtain the finished product.
Test solution: taking 1mL of a sample solution, placing the sample solution into a 100mL measuring flask, adding a pH 5.5 phosphate buffer solution-90% acetonitrile methanol solution (83:17), diluting to a scale, and shaking uniformly to obtain the final product.
Impurity N control solution: taking 1mg of impurity N reference substance, placing into a 10mL measuring flask, adding a pH 5.5 phosphate buffer solution-90% acetonitrile methanol solution (83:17), dissolving and diluting to scale, and shaking uniformly to obtain impurity N reference substance mother liquor; measuring impurity N reference mother liquor 0.3mL, placing into a 10mL measuring flask, adding solvent to dilute to scale, and shaking to obtain the final product.
Impurity O control positioning solution: taking 1mg of impurity O reference substance, putting into a 10mL measuring flask, adding a solvent to dissolve and dilute to scale, and shaking uniformly to obtain impurity O reference substance mother liquor; precisely measuring 0.3mL of impurity O reference mother liquor, placing into a 10mL measuring flask, adding solvent to dilute to scale, and shaking uniformly to obtain the final product.
Benzaldehyde positioning solution: taking 1mg of benzaldehyde reference substance, placing into a 10mL measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking uniformly to obtain a benzaldehyde reference substance mother solution; precisely measuring 0.3mL of benzaldehyde reference mother liquor, placing into a 10mL measuring flask, adding solvent to dilute to scale, and shaking to obtain the final product.
Adopts the under-term atlas of' raw material medicine internal control impurity N related substance verification-specificity
(2) Measuring 50 mu L of each of the blank solvent, the blank auxiliary material solution, the sample solution, the impurity N reference substance solution, the impurity O reference substance positioning solution and the benzaldehyde positioning solution, respectively injecting the sample solution, the impurity N reference substance solution, the impurity O reference substance positioning solution and the benzaldehyde positioning solution into a liquid chromatograph according to the impurity N detection chromatographic conditions (same as in example 1), and recording chromatograms as shown in fig. 1 (the sample solution, the blank auxiliary material solution, the impurity N reference substance solution, the impurity O reference substance positioning solution, the benzaldehyde positioning solution and the blank solution chromatograms are sequentially arranged from top to bottom).
2. Separation test of other impurities
Other impurity control mixed solution: taking the impurity A, B, C, E, F, G, L, I, N, O reference substance and clindamycin phosphate crude drug with proper amounts respectively, dissolving with a solvent, diluting to prepare a solution containing 3.6 mug of each impurity and 0.72mg of clindamycin phosphate in 1mL, and shaking uniformly to obtain the medicine.
Measuring 50 μl of the mixed solution of other impurity reference substances, respectively injecting into a liquid chromatograph according to the detection chromatographic conditions of impurity N, and recording the chromatogram, as shown in FIG. 3, wherein 9.9min is the peak position of impurity N.
The results of the specificity-separation test are shown in Table 14 below.
TABLE 14
3. Conclusion(s)
The blank auxiliary material solution, the impurity O positioning solution, the benzaldehyde positioning solution and other impurity reference substance solutions do not interfere with the detection of impurity N, and the method can be used for detecting the impurity N in the clindamycin phosphate injection.
4. Linear relationship of concentration versus peak area for different test solutions:
Impurity N control stock: weighing impurity N reference substance about 2.2mg, precisely weighing, placing into 25mL measuring flask, adding blank solvent, dissolving, diluting to scale, and shaking.
Impurity N control solution: precisely measuring 1mL of impurity N reference stock solution, placing into a 25mL measuring flask, adding solvent to dilute to scale, and shaking to obtain the final product. (concentration: 3.5. Mu.g/mL) a proper amount of a linear test stock solution (i.e., the above-mentioned "impurity N reference stock solution") was taken, and diluted with a blank solvent to a solution of a quantitative limit concentration (quantitative limit concentration is shown in Table 15 below) as an impurity N linear solution 1; and precisely measuring 0.5mL, 1.0mL, 2.0mL, 3.0mL and 5.0mL of the linear test stock solution respectively, placing the linear test stock solution into 25mL measuring flasks respectively, diluting to the scale with a solvent, shaking the linear test stock solution uniformly to obtain linear test solution serving as impurity N linear solutions 2, 3,4, 5 and 6.
According to the method for detecting impurity N, which is related to the detection of substances in clindamycin phosphate (same as in example 1), 50. Mu.L each of the above-mentioned linear solutions 1 to 6 of impurity N (labeled as samples 1 to 6, respectively) was precisely measured, injected into a liquid chromatograph, and the chromatogram was recorded to determine the peak area of impurity N. And (3) carrying out linear regression by taking the concentration (mug/mL) as an abscissa and the peak area as an ordinate to obtain a linear regression equation. The results of the linearity and range tests are shown in Table 15 below.
TABLE 15
Conclusion: the concentration of the impurity N is within the concentration range of 0.16 mug/mL (quantitative limit) to 16.32 mug/mL, the linear equation of the concentration and the peak area is y= 5862.4424x-1591.3248, the correlation coefficient R 2 is 0.9995, and the linear relation meets the requirements.
5. Quantitative limit detection limit test results
Quantitative limit: taking the solution (1) of the impurity N linear solution in the step 4, gradually diluting the solution into a solution with a series of concentration by using a blank solvent, precisely measuring 50 mu L of the solution according to the chromatographic conditions of the impurity N detection method in the embodiment 1, injecting the solution into a liquid chromatograph, recording the chromatogram, and ensuring that the signal to noise ratio is not lower than 10:1 is determined as a quantitative limit.
Limit of detection: precisely measuring 3mL of quantitative limiting solution, placing in a10 mL measuring flask, diluting to a scale with a solvent, shaking uniformly, precisely measuring 50 mu L of the quantitative limiting solution respectively, injecting into a liquid chromatograph, recording a chromatogram, and determining the signal-to-noise ratio of 3:1 as a detection limit. The results are shown in Table 16 below.
Table 16
Conclusion: the quantitative limit concentration of the impurity N is 0.16 mug/ml, which is equivalent to 0.005% of the concentration of the solution of the test sample; the detection limit concentration is 0.05 mug/ml, which is equivalent to 0.001% of the concentration of the solution of the test sample, and can meet the detection requirement.
6. Recovery test results
Impurity N control stock: weighing about 5mg of impurity N reference substance, precisely weighing, placing into 50mL measuring flask, adding blank solvent, dissolving, diluting to scale, and shaking.
Impurity N control solution: precisely measuring 3mL of impurity N reference stock solution, placing into a 100mL measuring flask, diluting to scale with blank solvent, and mixing uniformly.
Test solution: precisely measuring 1mL of clindamycin phosphate injection, placing into a 100mL measuring flask, adding a blank solvent for dissolution, diluting to a scale, and shaking uniformly to obtain the clindamycin phosphate injection.
Recovery rate 50%,100%,150% test sample solution: 1mL of the product is precisely measured respectively, the product is placed in a 100mL measuring flask, 1.5mL,3mL and 4.5mL of the reference stock solution are respectively added, the solvent is added for dilution to the scale, and the product is obtained after shaking uniformly (3 parts of the product are prepared in parallel).
Precisely measuring 50 mu L of impurity N reference substance solution, 50 mu L of impurity N sample solution and 50 mu L of impurity N recovery rate sample solution respectively according to the chromatographic conditions of the impurity N detection method in the embodiment 1, respectively injecting into a liquid chromatograph, and recording a chromatogram; the measured amount of impurity N was calculated as the peak area by the external standard method, and the recovery rate was calculated from the measured amount and the addition amount. The test results are shown in Table 17 below.
TABLE 17
Conclusion: the average recovery rate of 9 samples of impurity N is 97.99% -102.96%, RSD is 1.4%, and the accuracy of the method is good.
7. Results of the repeatability test
Solvent: pH5.5 phosphate buffer-90% acetonitrile in methanol (83:17)
Test solution: and (3) preparing the medium-concentration test sample solution in the test of 6 recovery rate.
Control solution: taking about 2.5mg of impurity N reference substance, placing into a25 mL measuring flask, adding solvent to dissolve and dilute to scale, shaking uniformly, precisely measuring 3mL, placing into a 100mL measuring flask, adding solvent to dilute to scale, and shaking uniformly to obtain the product.
According to the chromatographic conditions of the impurity N detection method in the embodiment 1, precisely measuring 50 mu L of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording a chromatogram; the content of impurity N was calculated as peak area according to the external standard method. The test results are shown in Table 18 below.
TABLE 18
| Sample number | 1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Impurity N content (%) | 0.22 | 0.22 | 0.22 | 0.23 | 0.22 | 0.22 | 0.22 | 1.8 |
Test results and analysis: the RSD value of the impurity N content measured by 6 samples is 1.8%, and the precision of the method is good.
8. Results of intermediate precision test
The "reproducibility" test was repeated by another analyst at different times using different instruments. RSD values were calculated for the measurements of 12 samples obtained by two analysts. The test results are shown in Table 19 below.
TABLE 19
Conclusion: the RSD of the same impurity content measured in 12 samples is less than 12%, and the method has good precision.
9. Test results of stability of test sample solution
Test solution: taking 1mL of clindamycin phosphate injection, placing the injection into a 100mL measuring flask, adding a solvent to dissolve and dilute the injection to a scale, and shaking the injection uniformly to obtain the clindamycin phosphate injection.
The sample solutions were allowed to stand at room temperature, and 50. Mu.L of each of the sample solutions was precisely measured at 0, 4, 8, 12 and 24 hours after the preparation, and the sample solutions were injected into a liquid chromatograph under the chromatographic conditions of the impurity N detection method in example 1, and the stability of the sample solutions at room temperature was examined over 24 hours. The test results are shown in Table 20 below.
Table 20
Note that:
test results and analysis: the sample solution is placed at room temperature for 24 hours, the change rate of impurity N is less than 20%, and the solution is stable.
Example 13
The separation method of the impurity A, B, C, E, F, G, L, N, O in clindamycin phosphate comprises the following steps:
solvent: phosphate buffer (pH 3.9) (3.5 mL of phosphoric acid, 1000mL of water and 2.5mL of concentrated ammonia solution are added, and the pH is adjusted to 3.9.+ -. 0.05) -90% acetonitrile in methanol (80:20) with concentrated ammonia solution if necessary.
Test solution: the product is precisely measured and diluted quantitatively with solvent to prepare solution containing clindamycin 3mg in each lmL.
Control solution: the solution of the test sample is precisely measured and diluted quantitatively with a solvent to prepare a solution containing about 30 mug of clindamycin per 1 mL.
Sensitivity solution: a proper amount of control solution is precisely measured, and the control solution is quantitatively diluted by a solvent to prepare a solution containing about 0.6 mug of clindamycin in each 1 mL.
Control solution: respectively precisely weighing the respective proper amounts of the reference substances of the impurity A and the impurity E, dissolving the reference substances by using a reference solution, and quantitatively diluting to prepare a mixed solution containing about 6 mug of the impurity A and 30 mug of the impurity E in each 1 mL.
Blank auxiliary material solution (sodium hydroxide, edetate sodium, benzyl alcohol, water for injection): precisely measuring 1mL of blank auxiliary materials with prescription amount, placing into a 50mL measuring flask, adding diluent for dissolution, diluting to scale, and shaking uniformly to obtain the final product.
System applicability solution: and (3) dissolving and diluting the impurity A, the impurity B, the impurity C, the impurity E, the impurity F, the impurity G, the impurity L, the impurity N, the impurity O and the clindamycin phosphate reference by adding a solvent to prepare a solution containing about 6 mug of the impurity A, 24 mug of the impurity B, 9 mug of the impurity C, 45 mug of the impurity E, 90 mug of the impurity F, 6 mug of the impurity G, 24 mug of the impurity L, 6 mug of the impurity N, 24 mug of the impurity O and 3.57mg of clindamycin phosphate in 1 mL.
Chromatographic conditions: octyl silane bonded silica gel as filler (XDB-C8, 4.6 mm. Times.250 mm,5 μm or column with comparable performance); linear gradient elution was performed using phosphate buffer (pH 3.9) -90% acetonitrile in methanol (92:8) as mobile phase C and phosphate buffer (pH 3.9) -90% acetonitrile in methanol (52:48) as mobile phase D, as follows; the flow rate is 1.2mL/min; column temperature is 40 ℃; the detection wavelength is 214nm; sample injection volume 25. Mu.L;
The gradient elution recipe was as follows:
System applicability requirements: in the system applicability solution chromatogram, the peak-out sequence is sequentially an impurity G, an impurity F, an impurity A, an impurity O, an impurity B (the relative retention time of the impurity N and the impurity B is consistent), an impurity L, clindamycin phosphate, an impurity C and an impurity E; the separation degree between the impurity F peak and the impurity A peak, between the clindamycin phosphate peak and the impurity E peak, and between the impurity L peak and the clindamycin phosphate peak should be more than 2.0, 6.0 and 1.5 respectively. In the sensitivity solution chromatograms, the signal to noise ratio of the peak to peak height of the principal component should be greater than 10.
Assay: precisely measuring the sample solution, the reference substance solution, the reference solution and the blank auxiliary material solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
Limit: in the chromatogram of the sample solution, except for blank auxiliary material peaks, impurity peaks exist, the impurity A and the impurity E are calculated according to an external standard method by peak areas, the impurity A is not more than 0.2% of the marked amount, and the impurity E is not more than 1.5% of the marked amount; the area of the impurity F peak is not more than 3 times (3.0%) of the area of the main peak of the control solution, the area of the impurity O peak is not more than 0.5 times (0.5%) of the area of the main peak of the control solution, the areas of the impurity C and the impurity G are calculated according to the corrected peak areas (multiplied by correction factors 1.2 and 0.5 respectively), the sum of the areas of the impurity B and the impurity N peak is not more than 0.2 times (0.5%) of the area of the main peak of the control solution, the area of the impurity L peak is not more than 0.2 times (0.2%) of the area of the main peak of the control solution, and the sum of the areas of the individual impurities (excluding the impurity A and the impurity E peak) is not more than 6.0%. The peak of the chromatogram of the sample solution, which is smaller than the main peak area of the sensitive solution, is ignored (0.02%).
The high performance liquid chromatogram obtained by the separation method of example 13 is shown in FIG. 18, and the specific data are shown in Table 21 below.
Table 21
Effect example 2
1. Specificity test
1.1 Blank interference and separation test
(1) The separation of impurity N from other impurities (under the term of "raw material medicine internal control impurity N related substance verification-specificity")
Impurity N control solution: dissolving impurity N reference substance in diluent, diluting to obtain solution containing 3.5mg per 1ml, and shaking.
Other impurities and main medicine mixed solution: taking the impurity A, B, D, E, F, G, H, I, J, L, M reference substance and clindamycin phosphate in proper amounts, dissolving in a solvent, diluting to obtain a solution containing 3.6 mug of each impurity and 0.72mg of clindamycin phosphate in 1mL, and shaking uniformly. The chromatographic conditions were checked according to impurity N, and the mixture was injected into a liquid chromatograph, and the chromatogram was recorded as shown in FIG. 2.
Test solution: about 35.7mg of the product is taken, precisely weighed, placed in a 10mL measuring flask, added with a diluent for dissolution and dilution to a scale, and uniformly shaken to obtain the product.
(2) Blank solvent, blank auxiliary material, impurity O, benzaldehyde and separation condition of sample solution and impurity N
Blank solvent: pH 5.5 phosphate buffer-90% acetonitrile in methanol (83:17).
Blank auxiliary material solution: weighing 1mL of blank auxiliary material solution, placing in a 100mL measuring flask, adding solvent to dilute to a scale, and shaking uniformly to obtain the product.
Test solution: taking 1mL of the product, placing in a 100mL measuring flask, adding solvent to dilute to a scale, and shaking uniformly to obtain the product.
Impurity N control solution: taking 1mg of impurity N reference substance, placing into a 10mL measuring flask, adding solvent to dissolve and dilute to scale, and shaking uniformly to obtain mother liquor of the impurity N reference substance; measuring impurity N reference mother liquor 0.3mL, placing into a 10mL measuring flask, adding solvent to dilute to scale, and shaking to obtain the final product.
Impurity O control positioning solution: taking 1mg of impurity O reference substance, putting into a 10mL measuring flask, adding a solvent to dissolve and dilute to scale, and shaking uniformly to obtain impurity O reference substance mother liquor; precisely measuring 0.3mL of impurity O reference mother liquor, placing into a 10mL measuring flask, adding solvent to dilute to scale, and shaking uniformly to obtain the final product.
Benzaldehyde positioning solution: taking 1mg of benzaldehyde reference substance, placing into a 10mL measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking uniformly to obtain a benzaldehyde reference substance mother solution; precisely measuring 0.3mL of benzaldehyde reference mother liquor, placing into a 10mL measuring flask, adding solvent to dilute to scale, and shaking to obtain the final product.
Precisely measuring 50 μl of each of the blank solvent, the blank auxiliary material solution, the sample solution, the impurity N reference substance solution, the other impurity reference substance mixed solution, the impurity O reference substance positioning solution and the benzaldehyde positioning solution, respectively injecting the mixture into a liquid chromatograph according to chromatographic conditions of an impurity N measuring method, recording a chromatogram, and observing whether the blank solvent, the blank auxiliary material, the impurity O and the benzaldehyde positioning solution interfere with the detection of the impurity N of the sample. The results of the specificity-separation test are shown in Table 22 below.
Table 22
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Claims (7)
1. The method for detecting the impurity N in the clindamycin phosphate is characterized by comprising the following steps of: detecting a sample solution containing clindamycin phosphate drug by adopting a first high performance liquid chromatography;
The structural formula of the impurity N is shown as the formula (I): Formula (I);
the elution mode of the first high performance liquid chromatography is as follows:
wherein,
The above% refers to volume percent;
Mobile phase a is phosphate buffer; the pH value of the mobile phase A is 5.0-5.7;
mobile phase B is acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol is 90:10;
in the first high performance liquid chromatography, the adopted chromatographic column is a C18 chromatographic column; the specification of the C18 chromatographic column is 150mm in column length multiplied by 4.6mm in inner diameter; the filler particle size of the C18 chromatographic column is 5 mu m;
The column temperature of the chromatographic column is 35-45 ℃; the detection wavelength is 212-216 nm; the flow rate of the mobile phase is 1-1.5 mL/min; the sample injection volume is 50 mu L; the mass concentration of clindamycin phosphate in the sample solution is 1.80-3.57 mg/mL; wherein, mg/mL refers to the ratio of the mass of clindamycin phosphate to the volume of the test solution.
2. The method for detecting impurity N in clindamycin phosphate according to claim 1, wherein,
In the first high performance liquid chromatography, the column temperature of the chromatographic column is 40 ℃.
3. The method for detecting the impurity N in the clindamycin phosphate according to claim 1, wherein the method for detecting the impurity N in the clindamycin phosphate satisfies one or more of the following conditions i to ii:
(i) In the first high performance liquid chromatography, the detection wavelength is 214nm;
(ii) In the first high performance liquid chromatography, the flow rate of the mobile phase is 1.1-1.3 mL/min.
4. The method for detecting impurity N in clindamycin phosphate according to claim 3, wherein the flow rate of the mobile phase in the first high performance liquid chromatography is 1.2mL/min.
5. The method for detecting impurity N in clindamycin phosphate according to claim 1, wherein the pH value of the mobile phase A is 5.3-5.7;
and/or, in the first high performance liquid chromatography, the mass concentration of clindamycin phosphate in the test solution is 3mg/mL, wherein mg/mL refers to the ratio of the mass of clindamycin phosphate to the volume of the test solution.
6. The method for detecting impurity N in clindamycin phosphate according to claim 5, wherein the pH value of the mobile phase A is 5.5.
7. The method for detecting impurity N in clindamycin phosphate according to claim 1, wherein the first high performance liquid chromatography is eluted in the following manner:
wherein,
The above% refers to volume percent;
mobile phase a is phosphate buffer; the pH value of the mobile phase A is 5.7;
mobile phase B is acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol is 90:10.
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