CN111912917A - Separation method and application of oxytocin and at least ten impurities - Google Patents

Separation method and application of oxytocin and at least ten impurities Download PDF

Info

Publication number
CN111912917A
CN111912917A CN202010679399.6A CN202010679399A CN111912917A CN 111912917 A CN111912917 A CN 111912917A CN 202010679399 A CN202010679399 A CN 202010679399A CN 111912917 A CN111912917 A CN 111912917A
Authority
CN
China
Prior art keywords
impurity
oxytocin
impurities
mobile phase
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010679399.6A
Other languages
Chinese (zh)
Other versions
CN111912917B (en
Inventor
王振平
贾存宇
李晓倩
丁金国
黄臻辉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Shangyao First Biochemical Pharmaceutical Co ltd
Original Assignee
Shanghai Shangyao First Biochemical Pharmaceutical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Shangyao First Biochemical Pharmaceutical Co ltd filed Critical Shanghai Shangyao First Biochemical Pharmaceutical Co ltd
Priority to CN202010679399.6A priority Critical patent/CN111912917B/en
Publication of CN111912917A publication Critical patent/CN111912917A/en
Application granted granted Critical
Publication of CN111912917B publication Critical patent/CN111912917B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed

Landscapes

  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Physics & Mathematics (AREA)
  • Library & Information Science (AREA)
  • Engineering & Computer Science (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a separation method and application of oxytocin and at least ten 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 5.0-6.2; 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 the oxytocin and ten impurities thereof at the same time, has the characteristics of strong specificity, good separation degree, high analysis speed, high sensitivity and the like, can be used for detecting related substances of oxytocin raw materials and preparations, and solves the technical problem that no method for controlling the specific impurities of the oxytocin is available in pharmacopoeia and documents at home and abroad at present.

Description

Separation method and application of oxytocin and at least ten impurities
Technical Field
The invention relates to the field of drug detection, in particular to a method for detecting oxytocin and at least ten 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, controlling postpartum hemorrhage, etc.
Oxytocin is sensitive to temperature and pH, impurities such as deamidation impurities, acetylation impurities and trisulfide are likely to be generated in preparation and storage due to changes of environmental temperature or pH, and oxytocin dimer is likely to be generated in concentrated solution in the oxytocin purification process. The main process impurities and potential degradation impurities in the oxytocin production process are shown in table 1.
Different kinds of impurities are introduced into the finished medicine product, which can cause different medication safety problems. Therefore, the content of the related substances needs to be strictly controlled in the synthesis stage and the preparation stage of the oxytocin raw material. However, in the quality standards of oxytocin raw material drugs and injections thereof collected in the domestic and foreign pharmacopoeias (chinese pharmacopoeia (2020 edition), european pharmacopoeia (EP9.8), united states pharmacopoeia (USP41) and japanese pharmacopoeia (JP17 edition)), there are only requirements on the amounts of single impurity and total impurity, and no specific impurity is involved, that is, no specific impurity detection method or detection limit requirement in table 1 is involved in any of the above pharmacopoeias.
TABLE 1 potentially 12 related substances in oxytocin
Figure BDA0002585219830000011
Figure BDA0002585219830000021
In the literature relating to oxytocin-related substances, Andrea Hawe et al (Andrea Hawe, Robert Poole, Stefan Romeijn, Piotr Kasper, Rob van der Heijden, and Wim Jiskoot]Pharmaceutical Research,2009,26(7):1679-4]Oxytocin (impurity 2) and [ Gly9OH]Oxytocin (impurity 4) and [ Asp ]5]Oxytocin (impurity 5) is separated, and three impurities cannot be separated.
Ashenafi et al (Ashenafi D, Van Hemelrijck E, Chotra S, et al, liquid chromatography analysis of oxytocin and its related substructures [ J ]. J Pharm Biomed Anal,2009,51(1):24-29) examined the separation of carbetocin, acetylated oxytocin and dimer under a gradient mobile phase system of potassium dihydrogen phosphate buffer (pH 4.4) -acetonitrile (involving separation of oxytocin only from Ac-oxytocin (impurity 9), [ Cis-dimer ] oxytocin (impurity 11) and [ Trans-dimer ] oxytocin (impurity 12)).
The methods reported in the above documents are difficult to separate multiple related substances in oxytocin effectively and simultaneously. Therefore, establishing a high performance liquid chromatography which can effectively separate and detect related substances in oxytocin as much as possible has great significance for the accurate quality control of oxytocin.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect that a plurality of related substances in oxytocin are difficult to be simultaneously and effectively separated in the prior art, and provides a separation method and application capable of simultaneously separating oxytocin and ten impurities thereof. The method has the characteristics of strong specificity, 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 at least ten 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 5.0-6.2; 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 the mobile phase a and the mobile phase B are as follows:
time/min Mobile phase A% Mobile phase B%
0 90-98 2-10
7 85-92 8-15
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 ten impurities are "impurity 1, impurity 2, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11 and impurity 12" or "the impurity 1, the impurity 2, impurity 3, impurity 4, the impurity 7, the impurity 8, the impurity 9, the impurity 10, the impurity 11 and the impurity 12";
wherein the impurity 1 is [ Glu4Asp5]Oxytocin, the impurity 2 is [ Glu4]Oxytocin, the impurity 3 is [ iso-Asp ]5]Oxytocin, the impurity 4 is [ Gly9OH]Oxytocin, the impurity 5 is [ Asp ]5]Oxytocin, the impurity 6 is [ + Gly10]Oxytocin, the impurity 7 is [ -Gly9]Oxytocin, the impurity 8 is oxytocin-Ac, the impurity 9 is Ac-oxytocin, and the impurity 10 is Trisulfide]Oxytocin, the impurity 11 is [ Cis-dimer [ ]]Oxytocin and the impurity 12 are [ Trans-dimer ]]Oxytocin.
In the present invention, the impurity 1 is [ Glu ]4Asp5]Oxytocin, structureThe formula is Cys-Tyr-Ile-Glu-Asp-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond).
The impurity 2 is [ Glu4]Oxytocin has a structural formula of Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond).
The impurity 3 is [ iso-Asp ]5]Oxytocin has a structural formula of Cys-Tyr-Ile-Gln-iso-Asp-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond).
The impurity 4 is [ Gly9OH]Oxytocin has a structural formula of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (1,6 disulfide bond).
Said impurity 5 is [ Asp ]5]Oxytocin has a structural formula of Cys-Tyr-Ile-Gln-Asp-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond).
The impurity 6 is [ + Gly10]Oxytocin has a structural formula of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-Gly-NH2(1,6 disulfide bond).
The impurity 7 is [ -Gly9]Oxytocin with the structural formula of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-NH2(1,6 disulfide bond).
The impurity 8 is oxytocin-Ac, and the structural formula is Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH-Ac (1,6 disulfide bond).
The impurity 9 is Ac-oxytocin with a structural formula of Ac-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2(1,6 disulfide bond).
The impurity 10 is [ Trisulfide ]]Oxytocin has a structural formula of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2(1, 6-trisulfide bond).
The impurity 11 is [ Cis-dimer [ ]]Oxytocin has a structural formula of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2(1,1 disulfide/6, 6 disulfide).
The impurity 12 is [ Trans-dimer [ ]]Oxytocin has a structural formula of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2(1,6 disulfide/1, 6 disulfide).
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 high performance liquid chromatography detection, the chromatographic column is preferably an octadecylsilane chemically bonded silica chromatographic column, such as Waters Xbridge C18A chromatographic column. The size of the octadecylsilane bonded silica chromatographic column is preferably 150mm × 4.6mm, 5 μm.
In the present invention, preferably, the gradient elution conditions of the mobile phase a and the mobile phase B are as follows:
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 follows:
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
。
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 invention, the pH value of the water phase is preferably 5.2-6, such as 5.5.
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 28-40 ℃; preferably 30-38 ℃; more preferably 35 deg.c.
In the present invention, the total flow rate of the mobile phase A and the mobile phase B in the HPLC test is preferably 0.5-2.0mL/min, more 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 210-230nm, and is 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, a method for separating oxytocin from 12 impurities is provided, wherein the 12 impurities include the impurity 1, the impurity 2, the impurity 3, the impurity 4, the impurity 5, the impurity 6, the impurity 7, the impurity 8, the impurity 9, the impurity 10, the impurity 11 and the impurity 12;
the separation method comprises the following steps:
a. weighing a proper amount of oxytocin, the impurity 1, the impurity 2, the impurity 3, the impurity 4, the impurity 5, the impurity 6, the impurity 7, the impurity 8, the impurity 10, the impurity 11 and the impurity 12 respectively, adding water to dissolve and dilute the weighed materials to prepare 12 parts of solution with the concentration of about 0.02 mg/mL; weighing a proper amount of the impurity 9, adding 10% acetonitrile aqueous solution to dissolve and dilute the impurity to prepare a 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 30-35 ℃, and the pH value is 5.5-6.0;
c. preparing a oxytocin mixed solution, wherein the concentration of oxytocin is 0.02mg/mL, and the concentrations of impurities 1-12 are 1 mug/mL respectively;
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 an application of the separation method of the oxytocin and at least ten impurities in the analysis detection or content determination of the oxytocin and at least ten impurities.
In the invention, the oxytocin and at least ten impurities are analyzed and detected or the content of the oxytocin is measured by utilizing 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 high performance liquid chromatography detection method can effectively separate oxytocin and ten 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, solves the technical problem that no method for controlling the oxytocin specific impurities exists in pharmacopoeia and documents at home and abroad at present, realizes the accurate control of main process impurities and potential degradation impurities in the oxytocin production process, and improves the quality of oxytocin products.
Under the preferable conditions, the invention can simultaneously separate oxytocin and 12 impurities thereof.
Drawings
Fig. 1 and 2 are a complete HPLC diagram and a partially enlarged HPLC diagram, respectively, of the oxytocin mixed solution detection of comparative example 1.
Fig. 3 and 4 are a full view and a partial enlarged view of HPLC in the detection of the oxytocin mixed solution of comparative example 2, respectively.
Fig. 5 is an HPLC chart of the detection of the oxytocin mixed solution of comparative example 3.
Fig. 6 and 7 are a full view and a partial enlarged view of HPLC in the detection of the oxytocin mixed solution of comparative example 4, respectively.
Fig. 8 is a complete HPLC diagram of the detection of the oxytocin mixed solution of comparative example 5.
Fig. 9 and 10 are a full view and a partial enlarged view of HPLC in the detection of the oxytocin mixed solution of comparative example 6, respectively.
Fig. 11 and 12 are a full view and a partial enlarged view of HPLC in the detection of the oxytocin mixed solution in example 1, respectively.
Fig. 13 and 14 are a full view and a partial enlarged view of HPLC in the detection of the oxytocin mixed solution in example 2, respectively.
Fig. 15 and 16 are a full view and a partial enlarged view of HPLC in the detection of the oxytocin mixed solution in example 3, respectively.
Fig. 17 and 18 are a full view and a partial enlarged view of HPLC in the detection of the oxytocin mixed solution in example 4, respectively.
Fig. 19 and 20 are a full view and a partial enlarged view of HPLC in the detection of the oxytocin mixed solution in example 5, respectively.
Fig. 21 and 22 are a full and partial HPLC chart of the oxytocin formulation assay of example 6, respectively.
Fig. 23 and 24 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin drug substance detection in example 7, respectively.
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;
the anhydrous sodium dihydrogen phosphate, the sodium hydroxide and the hydrochloric acid are all AR grade and purchased from chemical reagents of national drug group, Inc.;
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.
Impurity 1 is obtained by refining according to the refining method of oxytocin [4-Glu,5-Asp ] impurity in Chinese patent document CN 110078796A.
Impurity 2 is obtained by refining oxytocin [4-Glu ] impurity according to Chinese patent document CN 110078797A.
The impurity 3 and the impurity 5 are obtained by refining according to a refining method of oxytocin [5-Asp ] impurity in Chinese patent document CN 110041405A.
Impurity 4 is oxytocin [ -NH ] according to Chinese patent document CN 110028556A2]The impurities are obtained by refining with a refining method.
Impurity 6 (run: OP021919YH-018), impurity 7 (run: OP021919YH-017), impurity 8 (run: OP021919YH-010), impurity 9 (run: OP021919YH-009), impurity 10 (run: OP021919YH-021), impurity 11 (run: OP021919YH-019) and impurity 12 (run: OP021919YH-020) were purchased from Zhejiang ang On Tulai biotechnologies, Inc.
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 12 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 2:
table 2 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: weighing a proper amount of oxytocin raw material, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 10, impurity 11 and impurity 12 respectively, adding water to dissolve and dilute the raw material and the impurity to prepare 12 parts of solution with the concentration of about 0.02 mg/mL; an appropriate amount of impurity 9 was weighed out, dissolved in 10% acetonitrile in water and diluted to a 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 according to retention time of oxytocin and each impurity, a chromatographic peak with retention time of 8.736min in the diagram is an oxytocin peak, and the rest 8 are impurity peaks, and the attribution is shown in table 3. The chromatographic peaks of the impurity 2, the impurity 4 and the impurity 6 are coincided; the impurity 8 and the impurity 10 are partially overlapped, and the separation degree is 0.5; the retention time of the impurity 1 and the impurity 5 is similar to that of oxytocin, and the chromatographic peak is coincided with that of oxytocin and is not detected. The chromatographic method of the comparative example has long analysis time of 45min, and can detect 8 oxytocin related substances (8 chromatographic peaks in a mixed solution chromatogram correspond to the chromatographic peaks of the impurities injected separately, which are called as 8 impurities which can be detected).
TABLE 3 oxytocin mixture solution chromatographic peak attribution
Chromatographic peak Retention time/min Impurity assignment
1 6.915 Impurity 3
2 8.375 Impurities 2, 4, 6
3 8.736 Oxytocin, impurities 1, 5
4 9.093 Impurity 7
5 11.048 Impurity 8
6 11.203 Impurities 10
7 13.318 Impurity 11
8 13.640 Impurity 9
9 15.407 Impurities 12
Comparative example 2
The comparative example uses the method of United states pharmacopoeia (USP41) to detect the separation of oxytocin and 12 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 column temperature is mentioned in the United states pharmacopoeia)Using room temperature, chromatographic column C18Chromatography column (5 μm, 4.6 mm. times.120 mm), flow rate of 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 4:
table 4 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. 13 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, a chromatographic peak with a retention time of 7.909min in the diagram is an oxytocin peak, and the rest 7 peaks are impurity peaks, and the attribution is shown in table 5. The chromatographic peaks of the impurity 4 and the impurity 6 are coincided; the chromatographic peaks of the impurity 8 and the impurity 10 are coincided; the retention time of the impurity 1, the impurity 2 and the impurity 5 is similar to that of oxytocin, and the chromatographic peak is coincided with the oxytocin and is not detected. The chromatographic method of the comparative example has the analysis time of 40min, and can detect 7 oxytocin related substances (only 7 impurity peaks are detected in a mixed solution).
TABLE 5 oxytocin Mixed solution chromatogram Peak assignment
Figure BDA0002585219830000101
Figure BDA0002585219830000111
Comparative example 3
In this comparative example, the separation of oxytocin from a mixed oxytocin solution and 12 related substances 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 phases disclosed in chinese, european or united states pharmacopeia, as in 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: 35 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. 13 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 oxytocin mixed solution detection, and oxytocin and 12 related substances are not detected. Under the chromatographic conditions of the comparative example, oxytocin and 12 related substances 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 from the oxytocin mixture solution and 12 related substances 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 13 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, figure 6 is obtained, and the enlarged view is shown in figure 7.
The experimental results are as follows: fig. 6 and 7 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, a chromatographic peak with a retention time of 6.012min in the diagram is an oxytocin peak, and the rest 7 peaks are impurity peaks, and the attributions are shown in table 6. Only 7 chromatographic peaks of the 12 oxytocin related substances are detected, and the chromatographic peaks of the impurity 4 and the impurity 6 are coincided; the chromatographic peaks of the impurity 8 and the impurity 10 are coincided; the retention time of the impurity 1, the impurity 2 and the impurity 5 is similar to that of oxytocin, and the chromatographic peak is coincided with the oxytocin and is not detected. In the chromatographic method of the comparative example, the pH of the mobile phase A water phase is 4.4, and other conditions are the same as those in example 1, and potential 7 related substances in oxytocin can be detected.
TABLE 6 chromatographic peak assignment of oxytocin mixed solution
Figure BDA0002585219830000121
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 5.5): the separation of oxytocin from the oxytocin mixture solution and 12 related substances 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 5.5): the other conditions were the same as in example 1 except that the acetonitrile was 80: 20.
1. The preparation method of 13 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 comparative example 5 to obtain figure 8.
The experimental results are as follows: FIG. 8 is a complete HPLC chart of the detection of the oxytocin mixed solution, and according to the retention time of oxytocin and each impurity, a chromatographic peak with the retention time of 1.888min in the chart is an oxytocin peak, and the other 3 are impurity peaks. Because oxytocin and 12 related substances are eluted from the chromatographic column too early, the retention time is similar, and the oxytocin and the 12 related substances cannot be effectively separated from each other.
Comparative example 6
In this comparative example, the separation of oxytocin and 12 related substances in a mixed oxytocin solution was examined as in example 1 except for the elution procedure and the operation and conditions other than that of 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 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 13 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 μ L of oxytocin mixed solution, detecting by high performance liquid chromatography according to the experimental conditions of comparative example 6, figure 9 is obtained, and the enlarged view is shown in figure 10.
The experimental results are as follows: fig. 9 and fig. 10 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, in which a chromatographic peak with the retention time of 2.865min is an oxytocin peak, and the remaining 5 chromatographic peaks marked with the retention time are impurity peaks, and the elution procedure in the chromatographic method of the comparative example is not within the scope of claim 1 of the present invention, and the rest is the same as in example 1, and 5 related substances potential in oxytocin can be detected.
Example 1
This example is an example of the separation of oxytocin and 12 related substances, and the experimental conditions are as follows:
a chromatographic column: xbridge C18Chromatographic column (5 μm, 4.6X 150mm)
Column temperature: 35 deg.C
Mobile phase: mobile phase a was 100mmol/L sodium dihydrogen phosphate solution (pH 5.5): 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 13 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, and a spectrum 11 is obtained, and the enlarged view is shown in FIG. 12.
The experimental results are as follows: fig. 11 and fig. 12 are a complete HPLC diagram and a partially enlarged HPLC diagram of the detection of the oxytocin mixed solution, respectively, and according to the retention time of oxytocin and each impurity, a chromatographic peak with a retention time of 7.173min in the diagram is an oxytocin peak, and the rest 12 peaks are impurity peaks, and the attributions thereof are shown in table 9.12 oxytocin related substances are detected, oxytocin can be effectively separated from impurities, and the minimum separation degree is 1.1. The chromatographic method of the embodiment has short analysis time of 16min, and can detect 12 potential related substances in oxytocin.
TABLE 9 chromatographic peak assignment of oxytocin mixed solution
Chromatographic peak Retention time/min Impurity assignment
1 4.324 Impurity 1
2 5.541 Impurity 2
3 5.705 Impurity 3
4 5.875 Impurity 4
5 6.217 Impurity 5
6 6.761 Impurity 6
7 7.173 Oxytocin
8 7.622 Impurity 7
9 8.704 Impurity 8
10 9.008 Impurity 9
11 9.601 Impurities 10
12 10.920 Impurity 11
13 12.459 Impurities 12
Example 2
This example is an example of separation of oxytocin and 12 related substances thereof at a column temperature of 30 ℃, and the experimental conditions are as follows:
chromatographic conditions are as follows: the column temperature was 30 ℃ and other conditions were the same as in example 1.
1. The preparation method of 13 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 13 is obtained, and the enlarged view is shown in FIG. 14.
The experimental results are as follows: fig. 13 and 14 are a complete HPLC diagram and a partially enlarged HPLC diagram of the detection of the oxytocin mixed solution, respectively, where the chromatographic peak with the retention time of 7.158min is the oxytocin peak and the remaining 12 are impurity peaks according to the retention time of oxytocin and each impurity, and the attribution is shown in table 10. 12 oxytocin related substances are detected, wherein the separation degree of the impurity 2 and the impurity 3 is the minimum and is 0.7; oxytocin can be effectively separated from other impurities and other impurities. This example can detect potentially 12 related substances in oxytocin.
TABLE 10 chromatographic peak assignment of oxytocin mixed solution
Chromatographic peak Retention time/min Impurity assignment
1 4.337 Impurity 1
2 5.551 Impurity 2
3 5.672 Impurity 3
4 5.978 Impurity 4
5 6.257 Impurity 5
6 6.760 Impurity 6
7 7.158 Oxytocin
8 7.578 Impurity 7
9 8.676 Impurity 8
10 9.132 Impurity 9
11 9.501 Impurities 10
12 10.717 Impurity 11
13 12.344 Impurities 12
Example 3
This example is an example of separation of oxytocin and 12 related substances thereof at a column temperature of 40 ℃, and the experimental conditions are as follows:
chromatographic conditions are as follows: the column temperature was 40 ℃ and other conditions were the same as in example 1.
1. The preparation method of 13 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 3, 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 the example 3, and a spectrum 15 is obtained, and the enlarged view of the spectrum is shown in a figure 16.
The experimental results are as follows: fig. 15 and fig. 16 are a complete HPLC diagram and a partially enlarged HPLC diagram of the detection of the oxytocin mixed solution, respectively, and according to the retention time of oxytocin and each impurity, the chromatographic peak with the retention time of 7.187min in the diagram is an oxytocin peak, and the rest 11 peaks are impurity peaks, and the attributions are shown in table 11. 11 oxytocin related substances can be detected from 12 oxytocin related substances, wherein the impurity 3 is coincided with the impurity 4, and the separation degree of the impurity 8 and the impurity 9 is the minimum and is 0.8; oxytocin can be effectively separated from other impurities and other impurities.
The chromatographic method of the embodiment has the column temperature of 40 ℃, other conditions are the same as the conditions of the embodiment 1, and potential 11 related substances in oxytocin can be detected.
TABLE 11 chromatographic peak assignment of oxytocin mixed solution
Chromatographic peak Retention time/min Impurity assignment
1 4.294 Impurity 1
2 5.544 Impurity 2
3 5.769 Impurities 3 and 4
4 6.188 Impurity 5
5 6.767 Impurity 6
6 7.187 Oxytocin
7 7.660 Impurity 7
8 8.741 Impurity 8
9 8.891 Impurity 9
10 9.694 Impurities 10
11 11.103 Impurity 11
12 12.548 Impurities 12
Example 4
This example is the case of separation of oxytocin and its 12 related substances under the condition of mobile phase a pH 5.0, and the experimental conditions are as follows:
chromatographic conditions are as follows: mobile phase a was 100mmol/L sodium dihydrogen phosphate solution (pH 5.0): 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 13 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 17 is obtained, and the enlarged view is shown in FIG. 18.
The experimental results are as follows: fig. 17 and 18 are a complete HPLC diagram and a partially enlarged HPLC diagram of the detection of the oxytocin mixed solution, respectively, in which a chromatographic peak with a retention time of 6.416min is an oxytocin peak and the remaining 11 are impurity peaks according to the retention time of oxytocin and each impurity, and the attributions are shown in table 12. 11 oxytocin related substances can be detected from 12 oxytocin related substances, wherein the impurity 5 is coincided with the impurity 6, and the separation degree of the impurity 4 and the impurity 5 is the minimum and is 0.8; oxytocin can be effectively separated from other impurities and other impurities.
In the chromatographic method of the embodiment, the pH of the mobile phase A and the water phase is 5.0, and other conditions are the same as those in the embodiment 1, and potential 11 related substances in oxytocin can be detected.
TABLE 12 chromatographic peak assignment of oxytocin mixed solution
Chromatographic peak Retention time/min Impurity assignment
1 4.858 Impurity 1
2 5.098 Impurity 2
3 5.392 Impurity 3
4 5.748 Impurity 4
5 5.923 Impurities 5, 6
6 6.416 Oxytocin
7 6.743 Impurity 7
8 7.905 Impurity 8
9 8.392 Impurity 9
10 9.040 Impurities 10
11 9.671 Impurity 11
12 11.088 Impurities 12
Example 5
This example is the case of separation of oxytocin and 12 related substances under the condition that the mobile phase a has pH of 6.0, and the experimental conditions are as follows:
chromatographic conditions are as follows: mobile phase a was 100mmol/L sodium dihydrogen phosphate solution (pH 6.0): 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 13 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 test was performed by HPLC under the conditions of example 5, 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 5, and a map 19 is obtained, and the enlarged view is shown in fig. 20.
The experimental results are as follows: fig. 19 and 20 are a complete HPLC diagram and a partially enlarged HPLC diagram of the detection of the oxytocin mixed solution, respectively, in which a chromatographic peak with a retention time of 7.758min is an oxytocin peak and the remaining 12 are impurity peaks according to the retention time of oxytocin and each impurity, and the attributions are shown in table 13. 12 oxytocin related substances are detected, and the separation degree of impurities 3 and 4 is minimum and is 0.9; oxytocin can be effectively separated from other impurities and other impurities.
In the chromatographic method of the embodiment, the pH of the mobile phase A and the water phase is 6.0, and other conditions are the same as those in the embodiment 1, and potential 12 related substances in oxytocin can be detected.
TABLE 13 chromatographic peak assignment of oxytocin mixed solution
Chromatographic peak Retention time/min Impurity assignment
1 4.424 Impurity 1
2 5.838 Impurity 2
3 6.066 Impurity 3
4 6.225 Impurity 4
5 6.617 Impurity 5
6 7.323 Impurity 6
7 7.758 Oxytocin
8 8.277 Impurity 7
9 8.996 Impurity 8
10 9.321 Impurity 9
11 10.304 Impurities 10
12 11.770 Impurity 11
13 12.829 Impurities 12
Example 6
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 13 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. Taking 100 μ L of oxytocin preparation (batch No. 323838) (commercial oxytocin preparation is 10IU/ml, the mass concentration is about 0.02mg/ml, and the oxytocin preparation can be directly used as a sample to be detected), detecting by high performance liquid chromatography according to the experimental conditions of example 1 to obtain a map 21, wherein the enlarged view is shown in figure 22.
Fig. 21 and 22 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 in step 1 of example 1, the chromatographic peak with retention time of 7.149min in the figure is oxytocin peak, the chromatographic peak with retention time of 5.528min is impurity 2, the chromatographic peak with retention time of 5.911min is impurity 4, the chromatographic peak with retention time of 6.218min is impurity 5, and the chromatographic peak with retention time of 9.043min is impurity 9. The chromatographic peak with the retention time of 1-2 min is a solvent peak (acetic acid), the chromatographic peak with the retention time of 12-14 min is chlorobutanol (an auxiliary material and an antiseptic in the oxytocin preparation), and the two chromatographic peaks are not counted as integration. The results of the contents of the related substances of the oxytocin preparation calculated by an area normalization method are shown in table 14.
TABLE 14 oxytocin formulation-related substance results
Batch number Impurity 2 (%) Impurity 4 (%) Impurity 5 (%) Impurity 9 (%) Total impurities (%)
323838 3.2 3.4 0.7 0.6 7.9
The percentage is the mass percentage of each component in the oxytocin preparation relative to the oxytocin.
Example 7
This example is an example of detecting substances related to oxytocin raw materials.
The substances related to oxytocin raw materials were measured under the same experimental conditions as in example 1.
1. The preparation method of 13 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. 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. Measuring 1mL of oxytocin solution, placing the oxytocin solution in a 20mL volumetric flask, diluting with water, fixing the volume, shaking up to obtain a solution containing about 0.02mg of oxytocin per 1mL, namely the oxytocin solution, and detecting by high performance liquid chromatography according to the experimental conditions of example 1 to obtain a map 23, wherein the enlarged view is shown in figure 24.
Fig. 23 and 24 are a complete HPLC diagram and a partially enlarged HPLC diagram of oxytocin drug substance detection, respectively. According to the chromatographic retention time of oxytocin and various impurities, a chromatographic peak with the retention time of 7.102min in the figure is an oxytocin peak, a chromatographic peak with the retention time of 9.129min is impurity 9, a chromatographic peak with the retention time of 9.438min is impurity 10, a chromatographic peak with the retention time of 12.318min is impurity 12, and a chromatographic peak with a peak area smaller than one thousandth of the area of the oxytocin peak is not counted as an integral. The results of the contents of the oxytocin raw material drugs calculated by the area normalization method are shown in table 15.
TABLE 15 results for oxytocin formulations related substances
/ Impurity 9 (%) Impurity 10 (%) Impurity 12 (%) Total impurities (%)
Oxytocin raw material 0.3 0.2 0.2 0.7
The percentage is the mass percentage of each component in the oxytocin raw material relative to the oxytocin.
Example 8
Sensitivity test
The experiment was carried out under the same experimental conditions as in example 1.
1. Respectively precisely weighing a proper amount of oxytocin raw material, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 10, impurity 11 and impurity 12, adding water to dissolve and dilute the materials to prepare 12 parts of solution with the concentration of about 0.02 mg/ml;
2. precisely weighing a proper amount of impurity 9, adding 10% acetonitrile water solution for dissolving and diluting to prepare a solution with the concentration of about 0.02 mg/mL;
3. taking 1mL of oxytocin raw material with the concentration of 0.02mg/mL, 1 impurity, 2 impurity, 3 impurity, 4 impurity, 5 impurity, 6 impurity, 7 impurity, 8 impurity, 9 impurity, 10 impurity, 11 impurity and 12 impurity solution respectively, putting the raw material, the impurity, the 6 impurity, the 7 impurity, the 8 impurity, the 9 impurity, the 10 impurity, the 11 impurity and the 12 impurity solution into the same 100mL measuring flask, diluting with water to constant volume, and obtaining mixed solution with oxytocin and each impurity 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 limit and the quantitative limit of oxytocin and 12 related substances thereof are shown in table 16, which shows that the method has high sensitivity.
TABLE 16 detection and quantitation limits of oxytocin and its 12 related substances
Components Detection limit ng Limit of quantification ng
Impurity 1 1.2 2.3
Impurity 2 1.2 2.3
Impurity 3 1.0 1.9
Impurity 4 0.8 1.6
Impurity 5 1.2 2.4
Impurity 6 1.3 2.5
Oxytocin 1.2 2.5
Impurity 7 1.0 1.9
Impurity 8 0.9 1.9
Impurity 9 1.3 2.5
Impurities 10 0.8 1.5
Impurity 11 2.0 4.0
Impurities 12 3.3 6.5

Claims (10)

1. A separation method of oxytocin and at least ten 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 5.0-6.2; 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 the mobile phase a and the mobile phase B are as follows:
time/min Mobile phase A% Mobile phase B% 0 90-98 2-10 7 85-92 8-15 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 ten impurities are "impurity 1, impurity 2, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11 and impurity 12" or "the impurity 1, the impurity 2, impurity 3, impurity 4, the impurity 7, the impurity 8, the impurity 9, the impurity 10, the impurity 11 and the impurity 12";
wherein the impurity 1 is [ Glu4Asp5]Oxytocin, the impurity 2 is [ Glu4]Oxytocin, the impurity 3 is [ iso-Asp ]5]Oxytocin, the impurity 4 is [ Gly9OH]Oxytocin, the impurity 5 is [ Asp ]5]Oxytocin, the impurity 6 is [ + Gly10]Oxytocin, the impurity 7 is [ -Gly9]Oxytocin, the impurity 8 is oxytocin-Ac, the impurity 9 is Ac-oxytocin, and the impurity 10 is Trisulfide]Oxytocin, the impurity 11 is [ Cis-dimer [ ]]Oxytocin and the impurity 12 are [ Trans-dimer ]]Oxytocin.
2. The method of claim 1, wherein the concentration of oxytocin in the test solution is 0.002-0.2 mg/mL, preferably 0.01-0.1 mg/mL, such as 0.02 mg/mL;
and/or the concentration of single impurity contained in the test solution is 0.00002-0.2 mg/mL, preferably 0.002-0.1 mg/mL, such as 0.001 mg/mL;
and/or, in the high performance liquid chromatography detection, the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, such as Waters Xbridge C18A chromatographic column; the size of the octadecylsilane bonded silica chromatographic column is preferably 150mm × 4.6mm, 5 μm.
3. A method of separating oxytocin according to claim 1 and at least ten impurities, characterized in that the conditions of gradient elution of the mobile phase a and the mobile phase B are as follows:
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
。
4. A method of separating oxytocin according to claim 3 and at least ten impurities, characterized in that the gradient elution conditions are as follows:
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
。
5. The method for separating oxytocin and at least ten impurities according to claim 1, characterized in that the aqueous phase is 80-120 mmol/L sodium dihydrogen phosphate solution;
and/or the pH value of the water phase is 5.2-6;
and/or the mobile phase A has a volume ratio of the water phase to the acetonitrile of (92:8) - (88: 12).
6. The process for the separation of oxytocin according to claim 5 and at least ten impurities, characterized in that the aqueous phase is a 100mmol/L sodium dihydrogen phosphate solution;
and/or the pH of the aqueous phase is 5.5;
and/or the volume ratio of the water phase to the acetonitrile in the mobile phase A is 90: 10.
7. The method for separating oxytocin according to claim 1 and at least ten impurities, characterized in that in the high performance liquid chromatography detection, the column temperature of the chromatographic column is 28 ℃ to 40 ℃;
and/or the flow rate of the total flow rate of the mobile phase A and the mobile phase B in the high performance liquid chromatography detection is 0.5-2.0 mL/min;
and/or in the high performance liquid chromatography detection, the sample volume of the test solution is 10-100 mu L;
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 or a photodiode array detector.
8. The method for separating oxytocin according to claim 7 and at least ten impurities, characterized in that in the high performance liquid chromatography detection, the column temperature of the chromatographic column is 30-38 ℃; preferably 35 ℃;
and/or the flow rate of the total flow rate of the mobile phase A and the mobile phase B in the high performance liquid chromatography detection is 1.5 mL/min;
and/or, in the high performance liquid chromatography detection, the sample volume of the test solution is 100 mu L;
and/or in the high performance liquid chromatography detection, the detection wavelength is 220 nm.
9. The method for separating oxytocin and at least ten impurities according to claim 1, characterized in that the method for separating oxytocin and 12 impurities comprises the following steps:
the 12 impurities include the impurity 1, the impurity 2, the impurity 3, the impurity 4, the impurity 5, the impurity 6, the impurity 7, the impurity 8, the impurity 9, the impurity 10, the impurity 11, and the impurity 12;
the separation method comprises the following steps:
a. weighing a proper amount of oxytocin, the impurity 1, the impurity 2, the impurity 3, the impurity 4, the impurity 5, the impurity 6, the impurity 7, the impurity 8, the impurity 10, the impurity 11 and the impurity 12 respectively, adding water to dissolve and dilute the weighed materials to prepare 12 parts of solution with the concentration of 0.02 mg/mL; weighing a proper amount of the impurity 9, adding acetonitrile water solution to dissolve and dilute the impurity to prepare a solution with the concentration of 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 30-35 ℃, and the pH value is 5.5-6.0;
c. preparing a oxytocin mixed solution, wherein the concentration of oxytocin is 0.02mg/mL, and the concentrations of impurities 1-12 are 1 mug/mL respectively;
d. and (c) sampling 100 mu L of the oxytocin mixed solution, performing separation detection according to the high performance liquid chromatography in the step b, and observing the oxytocin, each impurity and the separation condition among the impurities in the oxytocin mixed solution according to the retention time of the oxytocin and each impurity in the step b.
10. Use of a method for the separation of oxytocin according to any one of claims 1-9 and at least ten impurities for the analytical detection or content determination of oxytocin and at least the ten impurities.
CN202010679399.6A 2020-07-15 2020-07-15 Separation method and application of oxytocin and at least ten impurities Active CN111912917B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010679399.6A CN111912917B (en) 2020-07-15 2020-07-15 Separation method and application of oxytocin and at least ten impurities

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010679399.6A CN111912917B (en) 2020-07-15 2020-07-15 Separation method and application of oxytocin and at least ten impurities

Publications (2)

Publication Number Publication Date
CN111912917A true CN111912917A (en) 2020-11-10
CN111912917B CN111912917B (en) 2022-03-15

Family

ID=73280973

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010679399.6A Active CN111912917B (en) 2020-07-15 2020-07-15 Separation method and application of oxytocin and at least ten impurities

Country Status (1)

Country Link
CN (1) CN111912917B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113252807A (en) * 2021-04-15 2021-08-13 武汉大安制药有限公司 High performance liquid chromatography separation method for oxytocin raw material medicine related substances

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160377635A1 (en) * 2013-06-26 2016-12-29 Martin-Protean Llc Measurement of Oxytocin and Vasopressin
CN106749539A (en) * 2017-01-03 2017-05-31 上海上药第生化药业有限公司 A kind of preparation method of oxytocin deamidation impurity
CN111060635A (en) * 2019-11-27 2020-04-24 吉尔生化（上海）有限公司 Analysis and detection method of carbetocin
CN111077243A (en) * 2019-12-16 2020-04-28 成都市海通药业有限公司 Detection method for oxytocin injection

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160377635A1 (en) * 2013-06-26 2016-12-29 Martin-Protean Llc Measurement of Oxytocin and Vasopressin
CN106749539A (en) * 2017-01-03 2017-05-31 上海上药第生化药业有限公司 A kind of preparation method of oxytocin deamidation impurity
CN111060635A (en) * 2019-11-27 2020-04-24 吉尔生化（上海）有限公司 Analysis and detection method of carbetocin
CN111077243A (en) * 2019-12-16 2020-04-28 成都市海通药业有限公司 Detection method for oxytocin injection

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ANDREA HAWE等: "Towards Heat-stable Oxytocin Formulations Analysis of Degradation Kinetics and Identification of Degradation Products", 《PHARMACEUTICAL RESEARCH》 *
BRENT LARSEN等: "Reverse Phase High Pressure Liquid Chromatography for the Separation of Peptide Hormone Diastereoisomers", 《JOURNAL OF CHROMATOGRAPHIC SCIENCE》 *
BRENT LARSEN等: "THE SEPARATION OF PEPTIDE HORMONE DIASTEREOISOMERS BY REVERSE PHASE HIGH PRESSURE LIQUID CHROMATOGRAPHY", 《ZNT. J. PEPTIDE PROTEIN RES.》 *
KARINE FABIO等: "Heat-Stable Dry Powder Oxytocin Formulations for Delivery by Oral Inhalation", 《AAPS PHARMSCITECH》 *
张慧等: "高效液相色谱法分析卡贝缩宫素及其有关杂质", 《药物分析杂志》 *
王思鸿: "HPLC测定缩宫素注射液中有关物质", 《国际医药卫生导报》 *
黄青等: "缩宫素注射液的有关物质研究", 《药物分析杂志》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113252807A (en) * 2021-04-15 2021-08-13 武汉大安制药有限公司 High performance liquid chromatography separation method for oxytocin raw material medicine related substances
CN113252807B (en) * 2021-04-15 2022-05-06 远大生命科学(武汉)有限公司 High performance liquid chromatography separation method for oxytocin raw material medicine related substances

Also Published As

Publication number Publication date
CN111912917B (en) 2022-03-15

Similar Documents

Publication Publication Date Title
CN107102079A (en) Detect the application of Liquid Chromatography-Tandem Mass Spectrometry method and the clinical pharmacokinetics research of Atorvastatin and metabolin in human plasma
CN111912917B (en) Separation method and application of oxytocin and at least ten impurities
CN110478313B (en) Sodium carbazochrome injection
CN113866337B (en) Mass analysis method for separating and measuring oseltamivir phosphate isomer
Djordjevic et al. Liquid chromatographic-mass spectrometric method for the determination of fluoxetine and norfluoxetine in human plasma: application to clinical study
Wei et al. Improved HPLC method for doxorubicin quantification in rat plasma to study the pharmacokinetics of micelle‐encapsulated and liposome‐encapsulated doxorubicin formulations
CN111812240B (en) Separation method and application of oxytocin and three impurities
CN114965822B (en) Drug analysis method for efficiently determining ganglioside GM1 and impurities thereof
CN111830154B (en) Separation method and application of oxytocin and 8 epimers thereof
CN107328874B (en) Resolution reagent and separation detection method for palonosetron hydrochloride optical isomer
CN114518423B (en) Method for detecting impurities in dopamine hydrochloride injection
CN111896642B (en) Separation method and application of oxytocin and three kinds of deamidation impurities
CN113252807B (en) High performance liquid chromatography separation method for oxytocin raw material medicine related substances
CN110824074A (en) Method for detecting related substances of ticagrelor by using high performance liquid chromatography
CN104237399A (en) Method for detecting ambrisenta crud materials and content of ambrisenta active ingredients in preparations
CN1790013A (en) Method for simultaneous determination of protocatechuic acid content and 5-hydroxymethyl furfural content in pulse-activating injection
CN110412164B (en) Method for detecting related substances of mexiletine hydrochloride
CN115032284A (en) Method for separating and detecting related substances in chewable tablets
CN114224904B (en) Clindamycin phosphate and quality control method
CN108398493B (en) Quality detection method for centella asiatica and its extract and preparation
CN105136952A (en) Method for separation and determination of acitretin and genotoxic impurities thereof
CN115684445B (en) Detection method for related substances in valsartan amlodipine pharmaceutical composition
CN118501322B (en) Method for detecting prednisolone sodium succinate related substances for injection
CN115494183B (en) Method for detecting 1,2, 4-triazole in triazole medicines
CN115078576B (en) Analytical method for related substances of paracetamol and dihydrocodeine tablet

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant