CN111896642B - Separation method and application of oxytocin and three kinds of deamidation impurities - Google Patents

Separation method and application of oxytocin and three kinds of deamidation impurities Download PDF

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CN111896642B
CN111896642B CN202010679703.7A CN202010679703A CN111896642B CN 111896642 B CN111896642 B CN 111896642B CN 202010679703 A CN202010679703 A CN 202010679703A CN 111896642 B CN111896642 B CN 111896642B
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oxytocin
impurities
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CN111896642A (en
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贾存宇
王振平
李妍哲
丁金国
黄臻辉
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Sph No1 Biochemical & Pharmaceutical Co ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The invention discloses a separation method and application of oxytocin and three kinds of deamidated impurities. Which comprises the following steps: detecting the sample solution by high performance liquid chromatography; the test solution is an aqueous solution containing oxytocin; in the high performance liquid chromatography detection, a gradient elution mode is adopted for elution, a mobile phase A is a mixed solution of a water phase and acetonitrile, the water phase is a 10-150mmol/L sodium dihydrogen phosphate solution, and the pH value is 4.8-6.5; the volume ratio of the water phase to the acetonitrile is 95: 5-85: 15; mobile phase B was acetonitrile. The detection method can effectively separate oxytocin and 3 kinds of deamidated impurities thereof, has the characteristics of strong specificity, good separation degree, high analysis speed, high sensitivity and the like, can be used for detecting the oxytocin raw materials and related substances of preparations, realizes the accurate control of main process impurities and potential degradation impurities in the oxytocin production process, and improves the quality of oxytocin products.

Description

Separation method and application of oxytocin and three kinds of deamidation impurities
Technical Field
The invention relates to the field of drug detection, and in particular relates to a method for detecting oxytocin and three deamidation impurities.
Background
Oxytocin, also known as oxytocin, is a nonapeptide drug consisting of 8 amino acids, the molecular formula: c43H66N12O12S2Molecular weight 1007.2, structural formula: Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond). It can stimulate uterine contraction and milk discharge, and is mainly used for inducing and promoting parturition, shortening third stage of labor, and controlling postpartum hemorrhageAnd the like.
Oxytocin is sensitive to temperature and pH, and 3 kinds of oxytocin deamidation impurities may be generated in preparation and storage due to changes of ambient temperature or pH, as shown in table 1:
TABLE 1
Figure GDA0003409191500000011
Different kinds of impurities are introduced into a finished medicine product, so that different medication safety problems can be caused, and the content of the 3 kinds of oxytocin deamidation impurities needs to be strictly controlled in both the synthesis stage and the preparation stage of the oxytocin raw material. However, the 3 kinds of oxytocin deamidated impurities have structures similar to oxytocin, and are difficult to be effectively separated by a related substance detection method given in oxytocin raw material medicines and injection quality standards thereof, which are carried in pharmacopoeias at home and abroad (the chinese pharmacopoeia (2020 edition), the european pharmacopoeia (EP9.8), the united states pharmacopoeia (USP41) and the japanese pharmacopoeia (JP17 edition)).
Therefore, a method capable of effectively separating oxytocin and [ Glu ] at the same time is established4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]The high performance liquid chromatography of oxytocin has very important significance for the accurate quality control of oxytocin.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defect that oxytocin and [ Glu ] cannot be effectively separated simultaneously in the prior art4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Defects of oxytocin provide a detection method capable of simultaneously separating oxytocin and 3 deamidated impurities thereof. The method has the characteristics of good separation degree, high analysis speed, high sensitivity and the like, and can be used for process quality control and final product quality evaluation in a oxytocin raw material medicine synthesis process and a preparation process.
The invention solves the technical problems through the following technical scheme.
The invention provides a separation method of oxytocin and 3 deamidation impurities, which comprises the following steps: detecting the sample solution by high performance liquid chromatography;
wherein the test solution is an aqueous solution containing oxytocin; in the high performance liquid chromatography detection, a gradient elution mode is adopted for elution, a mobile phase A is a mixed solution of a water phase and acetonitrile, the water phase is a 10-150mmol/L sodium dihydrogen phosphate solution, and the pH value is 4.8-6.5; the volume ratio of the aqueous phase to the acetonitrile is (95:5) to (85: 15); the mobile phase B is acetonitrile;
the gradient elution conditions for mobile phase a and mobile phase B are as described in table 2:
TABLE 2 gradient elution conditions
Time/min Mobile phase A% Mobile phase B%
0 88-98 2-12
7 84-92 8-16
10 80-90 10-20
12 70-80 20-30
13 70-80 20-30
13.1 90-98 2-10
16 90-98 2-10
The above percentages are the ratio of the volume of mobile phase A or mobile phase B to the total volume of mobile phase A and mobile phase B;
the 3 deamidating impurities are [ Glu4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Oxytocin.
In the present invention, [ Glu ] is as defined above4Asp5]The structural formula of oxytocin is Cys-Tyr-Ile-Glu-Asp-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond).
In the present invention, [ Glu ] is as defined above4]The structural formula of oxytocin is Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond).
In the present invention, [ Asp ]5]The structural formula of oxytocin is Cys-Tyr-Ile-Gln-Asp-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond).
In the test solution, the concentration of oxytocin can be 0.002-0.2 mg/mL, preferably 0.01-0.1 mg/mL, such as 0.02 mg/mL.
In the invention, the concentration of a single impurity contained in the sample solution can be 0.00002-0.2 mg/mL, preferably 0.002-0.1 mg/mL, for example 0.001 mg/mL.
In the present invention, in the HPLC assay, the chromatographic column is preferably an octadecylsilane bonded silica gel chromatographic column, such as Waters Xbridge C18A chromatographic column. The octadecylsilane bonded silica gel chromatography column preferably has a specification of 150mm x 4.6mm,5μm。
in the present invention, preferably, the gradient elution conditions of the mobile phase a and the mobile phase B are as follows:
TABLE 3 gradient elution conditions
Time/min Mobile phase A% Mobile phase B%
0 94-96 4-6
7 86-90 10-14
10 84-86 14-16
12 73-77 23-27
13 73-77 23-27
13.1 94-96 4-6
16 94-96 4-6
。
More preferably, the gradient elution conditions are as shown in table 4:
TABLE 4 gradient elution conditions
Figure GDA0003409191500000031
Figure GDA0003409191500000041
In the invention, the water phase is preferably 80-120 mmol/L sodium dihydrogen phosphate solution, and more preferably 100mmol/L sodium dihydrogen phosphate solution.
In the present invention, the pH of the aqueous phase is preferably 5.0 to 6.0, for example 5.4.
In the present invention, the volume fractions of the aqueous phase and the organic phase in the mobile phase a total 100 parts. The volume ratio of the aqueous phase to the acetonitrile is preferably (92:8) to (88:12), more preferably 90: 10.
In the invention, in the high performance liquid chromatography detection, the temperature of a chromatographic column can be 20-45 ℃; preferably from 25 ℃ to 45 ℃ for example 32 ℃.
In the invention, in the high performance liquid chromatography detection, the total flow rate of the mobile phase A and the mobile phase B can be 0.5-2.0 mL/min, and preferably 1.5 mL/min.
In the invention, in the high performance liquid chromatography detection, the sample volume of the test solution can be 10-100 μ L, preferably 100 μ L.
In the invention, in the high performance liquid chromatography detection, the detection wavelength can be 210-230 nm, and preferably 220 nm.
In the invention, in the high performance liquid chromatography detection, the detector used can be an ultraviolet detector or a photodiode array detector.
In a preferred embodiment of the present invention, the method for determining oxytocin and 3 kinds of deamidation impurities thereof by using high performance liquid chromatography specifically comprises the following steps:
a. respectively taking appropriate amount of oxytocin and the [ Glu4Asp5]Oxytocin, the [ Glu4]Oxytocin and said [ Asp ]5]Weighing oxytocin, adding water to dissolve and dilute the oxytocin to prepare 4 parts of solution with the concentration of about 0.02 mg/mL;
b. taking 100 mu L of each solution, detecting according to the high performance liquid chromatography, and recording the retention time of oxytocin and each impurity; in the detection process of the high performance liquid chromatography, the column temperature is 32-45 ℃, and the pH value is 4.8-5.4;
c. preparing oxytocin mixed solution, wherein the oxytocin concentration is 0.02mg/mL, [ Glu4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]The oxytocin concentrations are respectively 1 mug/mL;
d. and (c) sampling 100 mu L of the oxytocin mixed solution, detecting according to the high performance liquid chromatography, and observing the oxytocin, the impurities and the separation condition of the impurities in the oxytocin mixed solution according to the retention time of the oxytocin and the impurities in the step b.
The invention also provides application of the separation method of the oxytocin and the 3 deamidated impurities in analysis detection or content determination of the oxytocin and the 3 deamidated impurities.
In the invention, the oxytocin and the 3 deamidated impurities are analyzed and detected or the content of the oxytocin and the 3 deamidated impurities is measured by using the detection method of the high performance liquid chromatography.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the high performance liquid chromatography detection method can effectively separate oxytocin and 3 kinds of deamidated impurities thereof, has the characteristics of strong specificity, good separation degree, high analysis speed, high sensitivity and the like, can be used for detecting oxytocin raw materials and related substances of preparations, solves the technical problem that no method for controlling the specific impurities of oxytocin is available in pharmacopoeia at home and abroad and documents at present, realizes the accurate control of main process impurities and potential degradation impurities in an oxytocin production process, and improves the quality of oxytocin products.
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FIG. 1 is a complete HPLC chart of the detection of the oxytocin mixed solution in comparative example 1.
FIG. 2 is an enlarged view of a portion of HPLC showing the detection of the oxytocin mixed solution in comparative example 1.
FIG. 3 is a complete HPLC chart of the detection of the oxytocin mixed solution in comparative example 2.
FIG. 4 is an enlarged view of a portion of HPLC showing the detection of the oxytocin mixed solution in comparative example 2.
FIG. 5 is a complete HPLC chart of the detection of the oxytocin mixed solution in comparative example 3.
FIG. 6 is a complete HPLC chart of the detection of the oxytocin mixed solution in comparative example 4.
FIG. 7 is an enlarged view of a portion of HPLC showing the detection of the oxytocin mixed solution in comparative example 4.
FIG. 8 is a complete HPLC chart of the detection of the oxytocin mixed solution in comparative example 5.
FIG. 9 is a complete HPLC chart of the detection of the oxytocin mixed solution in comparative example 6.
FIG. 10 is a complete HPLC chart of the detection of the oxytocin mixed solution in example 1.
FIG. 11 is an enlarged view of a portion of HPLC showing the detection of oxytocin mixture solution in example 1.
FIG. 12 is a complete HPLC chart of the detection of the oxytocin mixed solution in example 2.
FIG. 13 is an enlarged view of a portion of HPLC showing the detection of oxytocin mixture solution in example 2.
FIG. 14 is a complete HPLC chart of the detection of the oxytocin mixed solution in example 3.
FIG. 15 is an enlarged view of a portion of HPLC showing the detection of oxytocin mixture solution in example 3.
FIG. 16 is a complete HPLC chart of the detection of the oxytocin mixed solution in example 4.
FIG. 17 is an enlarged view of a portion of HPLC showing the detection of oxytocin mixture solution in example 4.
FIG. 18 is a complete HPLC chart of the detection of the oxytocin mixed solution in example 5.
FIG. 19 is an enlarged view of a portion of HPLC showing the detection of oxytocin mixture solution in example 5.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the examples, the sources and models of the products are as follows:
acetonitrile was HPLC grade, purchased from Fisher corporation;
anhydrous sodium dihydrogen phosphate was purchased from national pharmaceutical group chemical agents ltd;
oxytocin preparation Pitocin (oxocin Injection, Usp), lot No. 323838; available from Parr Pharmaceutical Inc. (Par Pharmaceutical Inc).
The oxytocin raw material is obtained by refining according to a refining method of oxytocin in Chinese patent document CN 110016072A.
[Glu4Asp5]Oxytocin is oxytocin [4-Glu,5-Asp ] according to Chinese patent document CN 110078796A]The impurities are obtained by refining with a refining method. [ Glu4]Oxytocin is oxytocin [4-Glu ] according to Chinese patent document CN 110078797A]The impurities are obtained by refining with a refining method. [ Asp ]5]Oxytocin is oxytocin [5-Asp ] according to Chinese patent document CN 110041405A]The impurities are obtained by refining with a refining method.
In the examples, the instrument models and sources used were as follows:
1260 definition high performance liquid chromatograph (Agilent, usa); xbridge C18 column (5 μm, 4.6X 150 mm); XS205DU analytical balance (Mettler-Toledo, Switzerland); AL204 analytical balance (Mettler-Toledo, switzerland); FiveEasy pH meter (Mettler-Toledo, Switzerland).
In an embodiment, the data processing software used is the Chemstation chemical workstation.
Comparative example 1
The comparative example adopts a method of Chinese pharmacopoeia (2020 edition)/European pharmacopoeia (EP9.8) to detect the separation condition of oxytocin and 3 kinds of deamidation impurities thereof in an oxytocin mixed solution.
The experimental conditions are shown below. In the experiment, the column temperature and the Xbridge chromatographic column are selected by self (the column temperature and the type of the chromatographic column are not mentioned in Chinese pharmacopoeia, and the chromatographic column length is 125mm specified in European pharmacopoeia), and other experimental conditions are carried out according to the Chinese pharmacopoeia or the European pharmacopoeia.
A chromatographic column: xbridge C18Chromatographic column (5 μm, 4.6 mm. times.150 mm)
Column temperature: 30 deg.C
Mobile phase: the mobile phase A is 100mmol/L sodium dihydrogen phosphate solution, and the mobile phase B is acetonitrile in volume ratio: water 50:50 mixed solution. In the Chinese pharmacopoeia or the European pharmacopoeia, the pH value of the mobile phase A is not adjusted, and only 100mmol/L sodium dihydrogen phosphate solution is used as the mobile phase A, and the pH value is 4.4.
Flow rate: 1.0 mL/min.
Detection wavelength: 220nm
The gradient is shown in table 5:
table 5 elution procedure of comparative example 1
Time/min Mobile phase A% Mobile phase B%
0 70 30
30 40 60
30.1 70 30
45 70 30
Sample introduction amount: 100 μ L.
A detector: ultraviolet detector
1. Sample preparation and detection: respectively taking appropriate amount of oxytocin and [ Glu4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Oxytocin is weighed, dissolved by adding water and diluted to prepare 4 parts of solution with the concentration of about 0.02 mg/mL.
Taking 100 mu L of each solution, detecting by high performance liquid chromatography according to the experimental conditions of the comparative example 1, and recording the retention time of oxytocin and each impurity.
2. Taking 20mg of oxytocin raw material, placing the oxytocin raw material in a 50mL volumetric flask, dissolving the oxytocin raw material in water and fixing the volume to obtain 0.4mg/mL oxytocin solution; and (3) measuring 1mL of each 0.4mg/mL oxytocin solution and 0.02mg/mL of each impurity solution prepared in the step (1), putting the solutions in the same 20mL volumetric flask, diluting with water, fixing the volume, and shaking up to obtain a solution containing about 0.02mg of oxytocin and 1 mu g of each impurity per 1mL, namely an oxytocin mixed solution.
Taking 100 mu L of oxytocin mixed solution, detecting by high performance liquid chromatography according to the experimental conditions of comparative example 1 to obtain chromatogram 1, and the enlarged view of which is shown in figure 2.
The experimental results are as follows: FIG. 1 and FIG. 2 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin mixed solution detection, respectively, and a chromatographic peak with a retention time of 8.826min is a shrinkage chart according to the retention time of oxytocin and each impurityGongsu peak, [ Glu4Asp5]Oxytocin and [ Asp ]5]The retention time of oxytocin is the same as that of oxytocin, and chromatographic peaks coincide. Chromatographic peak with retention time of 8.478min [ Glu4]Oxytocin, which is partially coincided with oxytocin chromatographic peak. The chromatographic method of the comparative example has long analysis time of 45min, and cannot effectively separate oxytocin from 3 kinds of deamidation impurities.
Comparative example 2
The comparative example uses the method of United states pharmacopoeia (USP41) to detect the separation of oxytocin and 3 kinds of deamidated impurities in the oxytocin mixed solution.
The experimental conditions are shown below. Wherein, the experimental column temperature and the Xbridge chromatographic column are self-selected and are not mentioned in the United states pharmacopoeia (the United states pharmacopoeia mentions that the column temperature is room temperature, and the chromatographic column C is used18Chromatography column (5 μm, 4.6 mm. times.120 mm), flow rate about 1.5mL/min, adjustable), other experimental conditions were performed according to the United states Pharmacopeia.
A chromatographic column: xbridge C18 column (5 μm, 4.6 mm. times.150 mm)
Column temperature: 25 deg.C
Mobile phase: the mobile phase A is 100mmol/L sodium dihydrogen phosphate solution, and the mobile phase B is acetonitrile in volume ratio: water 50:50 mixed solution. In the United states Pharmacopeia, the pH value of the mobile phase A is not adjusted, and only 100mmol/L sodium dihydrogen phosphate solution is used as the mobile phase A, and the pH value is 4.4.
Flow rate: 1.0 mL/min.
The gradient is shown in table 6:
table 6 elution procedure of comparative example 2
Time/min Mobile phase A% Mobile phase B%
0 70 30
20 50 50
25 0 100
30 0 100
30.1 70 30
40 70 30
Sample introduction amount: 100 μ L.
A detector: ultraviolet detector
Detection wavelength: 220nm
1. 4 parts of each solution having a concentration of about 0.02mg/ml were prepared in the same manner as in step 1 of comparative example 1.
Taking 100 mu L of each solution, detecting by high performance liquid chromatography according to the experimental conditions of comparative example 2, and recording the retention time of oxytocin and each impurity.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
Taking 100 μ L of oxytocin mixed solution, detecting by high performance liquid chromatography according to experimental conditions of comparative example 2 to obtain chromatogram 3, and an enlarged view of the chromatogram is shown in FIG. 4.
The experimental results are as follows: FIG. 3 and FIG. 4 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin mixed solution detection, respectively, and according to the retention time of oxytocin and each impurity, the chromatographic peak with the retention time of 7.934min in the diagram is an oxytocin peak, [ Glu [ -Glu ] ]4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]The retention time of oxytocin is the same as that of oxytocin, and the oxytocin is not detected. The chromatographic method of the comparative example has the analysis time of 40min, and oxytocin and 3 deamidation impurities cannot be separated.
Comparative example 3
In this comparative example, the separation of oxytocin and 3 deamidating impurities thereof in an oxytocin mixed solution was examined by the same elution procedure as in example 1, except that the mobile phase a and the mobile phase B were the mobile phase disclosed in chinese, european or united states pharmacopeia, as compared with example 1, and the operation and conditions were the same as in example 1. The experimental conditions were as follows:
a chromatographic column: xbridge C18Chromatographic column (5 μm, 4.6 mm. times.150 mm)
Column temperature: 32 deg.C
Detection wavelength: 220nm
Mobile phase: the mobile phase A is 100mmol/L sodium dihydrogen phosphate solution, and the pH value is 4.4; the mobile phase B is acetonitrile in volume ratio: water 50:50 mixed solution.
Flow rate: 1.5 ml/min.
Gradient elution procedure as in example 1
1. 4 parts of each solution having a concentration of about 0.02mg/ml were prepared in the same manner as in step 1 of comparative example 1.
Taking 100 mu L of each solution, detecting by high performance liquid chromatography according to the experimental conditions of comparative example 3, and recording the retention time of oxytocin and each impurity.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
And (3) taking 100 mu L of oxytocin mixed solution, and detecting by using a high performance liquid chromatography according to the experimental conditions of the comparative example 3 to obtain a chromatogram 5.
The experimental results are as follows:
FIG. 5 is an HPLC chart of the detection of oxytocin mixed solution, oxytocin and 3 deamidation impurities are not detected. Under the chromatographic conditions of the comparative example, oxytocin and 3 kinds of deamidated impurities thereof are difficult to elute from the chromatographic column.
Comparative example 4
This comparative example is compared to example 1 except that mobile phase a is a 100mmol/L sodium dihydrogen phosphate solution (pH 4.4): the separation of oxytocin and 3 kinds of deamidated impurities thereof from the oxytocin mixed solution was examined in the same manner as in example 1 except that the acetonitrile was changed to 90: 10. The experimental conditions were as follows:
chromatographic conditions are as follows: mobile phase a was 100mmol/L sodium dihydrogen phosphate solution (pH 4.4): the other conditions were the same as in example 1 except that the acetonitrile was changed to a 90:10 mixed solution.
1. The preparation method of 4 parts of oxytocin solutions with the concentration of about 0.02mg/ml and each impurity solution is the same as that of the step 1 of the comparative example 1.
Taking 100 mu L of each solution, detecting by high performance liquid chromatography according to the experimental conditions of comparative example 4, and recording the retention time of oxytocin and each impurity.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
Taking 100 μ L of oxytocin mixed solution, detecting by high performance liquid chromatography according to the experimental conditions of comparative example 4 to obtain a spectrum 6, wherein the enlarged view is shown in figure 7.
The experimental results are as follows: FIG. 6 and FIG. 7 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin mixed solution detection, respectively, wherein a chromatographic peak with a retention time of 6.142min is an oxytocin peak according to the retention time of oxytocin and each impurity, and [ Glu ] is a peak of oxytocin4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]The retention time of oxytocin is the same as that of oxytocin, and the oxytocin is not detected. In the chromatographic method of the comparative example, the pH of the water phase of the mobile phase A is 4.4, other conditions are the same as those in example 1, and oxytocin and 3 deamidation impurities cannot be separated.
Comparative example 5
This comparative example is compared to example 1 except that mobile phase a is a 100mmol/L sodium dihydrogen phosphate solution (pH 5.4): the separation of oxytocin and 3 kinds of deamidated impurities thereof from the oxytocin mixed solution was examined in the same manner as in example 1 except that the acetonitrile was 80: 20. The experimental conditions were as follows:
chromatographic conditions are as follows: mobile phase a was 100mmol/L sodium dihydrogen phosphate solution (pH 5.4): the other conditions were the same as in example 1 except that the acetonitrile was 80: 20.
1. The preparation method of 4 parts of oxytocin solutions with the concentration of about 0.02mg/ml and each impurity solution is the same as that of the step 1 of the comparative example 1.
Taking 100 mu L of each solution, detecting by high performance liquid chromatography according to the experimental conditions of the comparative example 5, and recording the retention time of oxytocin and each impurity.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
Taking 100 mu L of oxytocin mixed solution, detecting by high performance liquid chromatography according to the experimental conditions of the comparative example 5 to obtain a spectrum 8.
The experimental results are as follows: FIG. 8 is a complete HPLC chart of oxytocin solution detection, according to the retention time of oxytocin and each impurity, the chromatographic peak with retention time of 1.875min in the chart is the oxytocin peak, and the chromatographic peak with retention time of 1.521min is [ Glu [4]Oxytocin Peak, chromatographic Peak with a Retention time of 1.641min, [ Asp ]5]Oxytocin peak, [ Glu4Asp5]Oxytocin elutes from the column prematurely, coinciding with the solvent peak. The comparative chromatographic procedure was carried out in 100mmol/L sodium dihydrogen phosphate solution (pH 5.4) except for mobile phase a: as for the mixed solution of acetonitrile 80:20, oxytocin and [ Glu ] may be mixed in the same manner as in example 14]Oxytocin and [ Asp ]5]Oxytocin isolation ([ Glu)4]Oxytocin and [ Asp ]5]Separation of oxytocin 1.2, [ Asp ]5]Oxytocin-oxytocin separation degree 2.1), but [ Glu4Asp5]Oxytocin is coincident with a solvent peak (oxytocin used is an acetate salt, acetic acid exists in oxytocin solution, and the solvent peak is referred to as an acetic acid peak) and cannot be effectively detected.
Comparative example 6
In this comparative example, the separation of oxytocin and 3 kinds of deamidated impurities thereof in a mixed oxytocin solution was examined in the same manner as in example 1 except for the elution procedure as in example 1. The experimental conditions were as follows:
chromatographic conditions are as follows: the elution procedure is as in Table 7, with the other conditions being the same as in example 1.
Table 7 elution procedure of comparative example 6
Time/min Mobile phase A% Mobile phase B%
0 85 15
7 84 16
10 82 18
12 75 25
13 75 25
13.1 95 5
16 95 5
1. The preparation method of 4 parts of oxytocin solutions with the concentration of about 0.02mg/ml and each impurity solution is the same as that of the step 1 of the comparative example 1.
Taking 100 mu L of each solution, detecting by high performance liquid chromatography according to the experimental conditions of comparative example 6, and recording the retention time of oxytocin and each impurity.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
Taking 100 mu L of oxytocin mixed solution, detecting by high performance liquid chromatography according to the experimental conditions of the comparative example 6 to obtain a map 9.
The experimental results are as follows: FIG. 9 is a complete HPLC chart of oxytocin solution detection, according to the retention time of oxytocin and various impurities, the chromatographic peak with retention time of 2.011min in the chart is the oxytocin peak, and the chromatographic peak with retention time of 1.586min is [ Glu [4]Oxytocin peak, chromatographic peak with retention time of 1.728min is [ Asp ]5]Oxytocin peak, [ Glu4Asp5]Oxytocin elutes from the column prematurely, coinciding with the solvent peak.
The elution procedure in the comparative example chromatographic method is out of the scope of claim 1 of the present invention, and oxytocin and [ Glu ] can be mixed in the same manner as in example 14]Oxytocin and [ Asp ]5]Oxytocin isolation ([ Glu)4]Oxytocin and [ Asp ]5]Separation of oxytocin 1.3, [ Asp ]5]Oxytocin-oxytocin separation degree 2.4), but [ Glu4Asp5]Oxytocin coincided with a solvent peak (oxytocin used is an acetate salt, acetic acid exists in oxytocin solution, and the solvent peak refers to the acetic acid peak), and thus the oxytocin could not be effectively detected.
Example 1
This example is an example of separation of oxytocin and 3 kinds of deamidation impurities thereof, and experimental conditions are as follows:
a chromatographic column: xbridge C18Chromatographic column(5μm，4.6×150mm)
Column temperature: 32 deg.C
Mobile phase: mobile phase a was 100mmol/L sodium dihydrogen phosphate solution (pH 5.4): the acetonitrile is a 90:10 mixed solution, and the mobile phase B is acetonitrile.
Flow rate: 1.5 mL/min.
The gradient is shown in table 8:
table 8 elution procedure of example 1
Time/min Mobile phase A% Mobile phase B%
0 95 5
7 88 12
10 85 15
12 75 25
13 75 25
13.1 95 5
16 95 5
Sample introduction amount: 100 μ L
A detector: ultraviolet detector
Detection wavelength: 220nm
1. The preparation method of 4 parts of oxytocin solutions with the concentration of about 0.02mg/ml and each impurity solution is the same as that of the step 1 of the comparative example 1.
mu.L of each solution was taken, and the retention time of oxytocin and each impurity was recorded by HPLC according to the experimental conditions of example 1.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
100 μ L of oxytocin mixed solution is detected by high performance liquid chromatography according to the experimental conditions of example 1 to obtain a spectrum 10, and the enlarged view is shown in FIG. 11.
The experimental results are as follows: FIG. 10 and FIG. 11 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin mixed solution detection, respectively, wherein a chromatographic peak with a retention time of 7.128min is an oxytocin peak according to the retention time of oxytocin and each impurity, and the other 3 chromatographic peaks are [ Glu ] respectively4Asp5]Oxytocin (retention time 4.455min), [ Glu4]Oxytocin (retention time 5.590min) and [ Asp ]5]Oxytocin (retention time 6.278 min). The chromatographic method of the embodiment has short analysis time of 16min, and can be used for mixing oxytocin with [ Glu4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Oxytocin can be effectively separated, 3 deamidated impurities can be effectively separated, and the minimum separation degree is more than 2.0.
Example 2
The present example is an example of separation of oxytocin and 3 kinds of deamidated impurities thereof at a column temperature of 45 ℃, and experimental conditions are as follows:
chromatographic conditions are as follows: the column temperature was 45 ℃ and other conditions were the same as in example 1.
1. The preparation method of 4 parts of oxytocin solutions with the concentration of about 0.02mg/ml and each impurity solution is the same as that of the step 1 of the comparative example 1.
Taking 100 mu L of each solution, detecting by high performance liquid chromatography according to the experimental conditions of the example 2, and recording the retention time of oxytocin and each impurity.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
100 μ L of oxytocin mixed solution is detected by high performance liquid chromatography according to the experimental conditions of example 2, and a spectrum 12 is obtained, and the enlarged view is shown in FIG. 13.
The experimental results are as follows: FIG. 12 and FIG. 13 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin mixed solution detection, respectively, wherein a chromatographic peak with a retention time of 7.127min is an oxytocin peak according to the retention time of oxytocin and each impurity, and the other 3 chromatographic peaks are [ Glu ] respectively4Asp5]Oxytocin (retention time 4.334min), [ Glu4]Oxytocin (Retention time 5.521min) and [ Asp ]5]Oxytocin (retention time 6.116 min).
In the chromatographic method of the embodiment, the temperature of the chromatographic column is 45 ℃, and oxytocin and [ Glu ] can be mixed under the same other conditions as in the embodiment 14Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Oxytocin can be effectively separated, 3 deamidated impurities can be effectively separated, and the minimum separation degree is more than 2.0.
Example 3
This example is an example of separation of oxytocin and its 3 deamidating impurities at pH 4.8 of mobile phase a, with the following experimental conditions:
chromatographic conditions are as follows: mobile phase a was 100mmol/L sodium dihydrogen phosphate solution (pH 4.8): the other conditions were the same as in example 1 except that the acetonitrile was changed to a 90:10 mixed solution.
1. The preparation method of 4 parts of oxytocin solutions with the concentration of about 0.02mg/ml and each impurity solution is the same as that of the step 1 of the comparative example 1.
mu.L of each solution was taken, and the detection was performed by high performance liquid chromatography according to the experimental conditions of example 4, and the retention time of oxytocin and each impurity was recorded.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
100 mu L of oxytocin mixed solution is detected by high performance liquid chromatography according to the experimental conditions of example 4, and a spectrum 14 is obtained, and the enlarged view of the spectrum is shown in figure 15.
The experimental results are as follows: FIG. 14 and FIG. 15 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin mixed solution detection, respectively, wherein a chromatographic peak with a retention time of 6.378min is an oxytocin peak according to the retention time of oxytocin and each impurity, and the other 3 chromatographic peaks are [ Glu ] respectively4Asp5]Oxytocin (retention time 5.270min), [ Glu4]Oxytocin (retention time 5.590min) and [ Asp ]5]Oxytocin (retention time 6.027 min).
In this example, the pH of mobile phase a was 4.8 and oxytocin and [ Glu ] were reacted under the same conditions as in example 14Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Oxytocin can be effectively separated, 3 deamidated impurities can be effectively separated, and the minimum separation degree is more than 2.0.
Example 4
In this example, the separation of oxytocin and 3 kinds of deamidation impurities thereof in a mixed oxytocin solution was examined in the same manner as in example 1 except for the elution procedure in the present example. The experimental conditions were as follows:
chromatographic conditions are as follows: the elution procedure is as in Table 9, with the other conditions being the same as in example 1.
Table 9 elution procedure of example 4
Time/min Mobile phaseA％ Mobile phase B%
0 88 12
7 84 16
10 82 18
12 75 25
13 75 25
13.1 95 5
16 95 5
1. The preparation method of 4 parts of oxytocin solutions with the concentration of about 0.02mg/ml and each impurity solution is the same as that of the step 1 of the comparative example 1.
mu.L of each solution was taken, and the detection was performed by high performance liquid chromatography according to the experimental conditions of example 4, and the retention time of oxytocin and each impurity was recorded.
2. The preparation method of the oxytocin mixed solution is the same as that of the step 2 of the comparative example 1.
100 μ L of oxytocin mixed solution is detected by high performance liquid chromatography according to the experimental conditions of example 4, and a map 16 is obtained, and the enlarged view is shown in FIG. 17.
The experimental results are as follows: FIG. 16 and FIG. 17 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin mixed solution detection, respectively, according to the retention time of oxytocin and each impurity, the chromatographic peak with the retention time of 3.034min in the diagram is an oxytocin peak, and the other 3 chromatographic peaks are [ Glu ] respectively4Asp5]Oxytocin (retention time 1.882min), [ Glu4]Oxytocin (Retention time 2.279min) and [ Asp ]5]Oxytocin (retention time 2.552 min).
The chromatographic method of this example was carried out in the same manner as in example 1 except that the elution procedure was different, and oxytocin and [ Glu ] were used in combination4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Oxytocin can be effectively separated, 3 deamidated impurities can be effectively separated, and the minimum separation degree is more than 2.0.
Example 5
This example is a test example of related substances of oxytocin preparation
The substances related to Oxytocin (Oxytocin Injection, Usp) were measured using the same experimental conditions as in example 1.
1. The preparation method of 4 parts of oxytocin solutions with the concentration of about 0.02mg/ml and each impurity solution is the same as that of the step 1 of the comparative example 1.
mu.L of each solution was taken, and the retention time of oxytocin and each impurity was recorded by HPLC according to the experimental conditions of example 1.
2. A100 mL volume of oxytocin preparation (batch No. 323838) was subjected to HPLC analysis under the same conditions as in example 1 to obtain a chromatogram 18, which is shown in FIG. 19 as an enlarged view.
Fig. 18 and 19 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin preparation detection, respectively. According to the retention time of oxytocin and each impurity, the chromatographic peak of the retention time 7.166min in the figure is oxytocin peak, and the chromatographic peak of the retention time 5.541min in the figure is [ Glu4]Oxytocin, retention time 5.The chromatographic peak at 930min is unknown impurity 1 and the chromatographic peak at retention time 6.239min is [ Asp ]5]Oxytocin, and the chromatographic peak with the retention time of 9.060min is the unknown impurity 2. The chromatographic peak with the retention time of 1-2 min is an acetic acid solvent peak, the chromatographic peak with the retention time of 12-14 min is chlorobutanol in the oxytocin preparation auxiliary material, and the two chromatographic peaks are not counted as an integral. The results of the contents of the related substances of the oxytocin preparation calculated by an area normalization method are shown in table 10.
TABLE 10 oxytocin formulation-related substance results
Figure GDA0003409191500000161
The percentage is the mass percentage of each component in the oxytocin preparation relative to the oxytocin.
Example 6
Sensitivity test
The experiment was carried out under the same experimental conditions as in example 1.
1. Respectively taking appropriate amount of oxytocin raw material and [ Glu4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Precisely weighing oxytocin, adding water to dissolve and dilute the oxytocin to prepare 4 parts of solution with the concentration of 0.02 mg/ml;
2. collecting oxytocin and [ Glu ] with concentration of 0.02mg/ml4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]And putting 1ml of each oxytocin solution into the same 100ml measuring flask, diluting with water to constant volume to obtain a mixed solution of oxytocin and each impurity with the concentration of 0.2 mu g/ml. The mixed solution was gradually diluted with water to give a mixed solution having concentrations of each component of 0.04. mu.g/ml, 0.02. mu.g/ml, and 0.01. mu.g/ml, respectively.
4. Detecting by high performance liquid chromatography according to experimental conditions of example 1, wherein in the obtained chromatogram, the height of an impurity peak is about 3-5 times higher than the baseline noise, and the concentration of the solution at the moment is the detection limit of the component; in the obtained chromatogram, the impurity peak height is about 10-20 times higher than the baseline noise, and the concentration of the solution at the moment is the quantitative limit of the components. The detection limits and the quantitative limits of oxytocin and 3 kinds of deamidated impurities thereof are shown in table 11, which shows that the method has high sensitivity.
TABLE 11 detection and quantitation limits for oxytocin and 3 related substances
Components Detection limit ng Limit of quantification ng
[Glu4Asp5]Oxytocin 1.2 2.3
[Glu4]Oxytocin 1.2 2.3
[Asp5]Oxytocin 1.2 2.4
Oxytocin 1.2 2.5

Claims (18)

1. A separation method of oxytocin and three deamidated impurities is characterized by comprising the following steps: detecting the sample solution by high performance liquid chromatography;
wherein the test solution is an aqueous solution containing oxytocin; in the high performance liquid chromatography detection, a gradient elution mode is adopted for elution, a mobile phase A is a mixed solution of a water phase and acetonitrile, the water phase is a 10-150mmol/L sodium dihydrogen phosphate solution, and the pH value is 4.8-6.5; the volume ratio of the water phase to the acetonitrile is (95:5) - (85: 15); the mobile phase B is acetonitrile;
the gradient elution conditions for the mobile phase a and the mobile phase B are as follows:
Figure DEST_PATH_IMAGE001
the above percentages are the ratio of the volume of mobile phase A or mobile phase B to the total volume of mobile phase A and mobile phase B;
the three deamidating impurities are [ Glu4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]Oxytocin;
the [ Glu4Asp5]The structural formula of oxytocin is Cys-Tyr-Ile-Glu-Asp-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond);
the [ Glu4]The structural formula of oxytocin is Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond);
said [ Asp5]The structural formula of oxytocin is Cys-Tyr-Ile-Gln-Asp-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond);
in the high performance liquid chromatography detection, a chromatographic column is an octadecylsilane chemically bonded silica chromatographic column.
2. The method for separating oxytocin and three deamidated impurities according to claim 1, wherein the concentration of oxytocin in the test solution is 0.002-0.2 mg/mL;
and/or the concentration of a single impurity contained in the test solution is 0.00002-0.2 mg/mL;
and/or in the high performance liquid chromatography detection, the chromatographic column is Waters Xbridge C18A chromatographic column.
3. The method for separating oxytocin and three deamidated impurities according to claim 2, wherein the concentration of oxytocin in the test solution is 0.01-0.1 mg/mL.
4. The method of claim 3, wherein the concentration of oxytocin in the test solution is 0.02 mg/mL.
5. The method for separating oxytocin and three deamidated impurities according to claim 2, wherein the concentration of a single impurity contained in the test solution is 0.002-0.1 mg/mL.
6. The method of claim 2, wherein the sample solution contains a single impurity at a concentration of 0.001 mg/mL.
7. The method of claim 1, wherein the octadecylsilane chemically bonded silica chromatographic column has a size of 150mm x 4.6mm, 5 μm.
8. The method of claim 1, wherein the gradient elution conditions of mobile phase a and mobile phase B are as follows:
Figure DEST_PATH_IMAGE002
。
9. the method of claim 1, wherein the gradient elution conditions are as follows:
Figure DEST_PATH_IMAGE003
。
10. the separation method of oxytocin and three deamidation impurities as claimed in claim 1, characterized in that the water phase is 80-120 mmol/L sodium dihydrogen phosphate solution;
and/or the pH value of the water phase is 5.0-6.0;
and/or the volume ratio of the aqueous phase to the acetonitrile is (92:8) - (88: 12);
and/or in the high performance liquid chromatography detection, the temperature of a chromatographic column is 20-45 ℃.
11. The method of claim 10, wherein the aqueous phase is a 100mmol/L sodium dihydrogen phosphate solution;
and/or the pH of the aqueous phase is 5.4;
and/or the volume ratio of the aqueous phase to the acetonitrile is 90: 10;
and/or in the high performance liquid chromatography detection, the temperature of a chromatographic column is 25-45 ℃.
12. The method of claim 11, wherein the column temperature of the column is 32 ℃ in the HPLC assay.
13. The separation method of oxytocin and three deamidated impurities according to claim 1, wherein in the high performance liquid chromatography detection, the total flow rate of the mobile phase A and the mobile phase B is 0.5-2.0 mL/min;
and/or in the high performance liquid chromatography detection, the sample volume of the sample solution is 10-100 muL;
and/or in the high performance liquid chromatography detection, the detection wavelength is 210-230 nm;
and/or in the high performance liquid chromatography detection, the detector is an ultraviolet detector.
14. The method for separating oxytocin and three deamidated impurities according to claim 1, wherein the detector used in the high performance liquid chromatography is a photodiode array detector.
15. The method for separating oxytocin and three deamidated impurities according to claim 13 or 14, wherein the total flow rate of mobile phase a and mobile phase B is 1.5mL/min in the high performance liquid chromatography detection;
and/or the sample volume of the sample solution is 100 muL in the high performance liquid chromatography detection;
and/or in the high performance liquid chromatography detection, the detection wavelength is 220 nm.
16. The method of claim 1 for separating oxytocin and three deamidated impurities, comprising the steps of:
a. respectively taking appropriate amount of oxytocin and the [ Glu4Asp5]Oxytocin, the [ Glu4]Oxytocin and said [ Asp ]5]Weighing oxytocin, adding water to dissolve and dilute the oxytocin to prepare 4 parts of solution with the concentration of 0.02 mg/mL;
b. taking 100 mu L of each solution, detecting according to a high performance liquid chromatography, and recording the retention time of oxytocin and each impurity; in the detection process of the high performance liquid chromatography, the column temperature is 32-45 ℃, and the pH value is 4.8-5.4;
c. preparing oxytocin mixed solution, wherein the oxytocin concentration is 0.02mg/mL, [ Glu4Asp5]Oxytocin, [ Glu4]Oxytocin and [ Asp ]5]The oxytocin concentrations are respectively 1 mug/mL;
d. and (c) sampling 100 muL of the oxytocin mixed solution, detecting according to the high performance liquid chromatography, and observing the oxytocin, impurities and the separation condition of the impurities in the oxytocin mixed solution according to the retention time of the oxytocin and the impurities in the step b.
17. Use of a method for the separation of oxytocin according to any one of claims 1 to 16 and three deamidated impurities in the analytical detection of oxytocin and the three deamidated impurities.
18. Use of a method for separating oxytocin according to any one of claims 1 to 16 and three deamidated impurities in the determination of the content of oxytocin and the three deamidated impurities.
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