Method for detecting content of polysorbate 80 in complex preparation

Abstract

The invention discloses a method for detecting the content of polysorbate 80 in a complex preparation. The complex preparation contains a liposome component prepared from DOPC. The method comprises the following steps: 1, determining the DOPC content in a preparation by adopting a high performance liquid chromatography; 2, adding a strong alkali solution into the preparation, hydrolyzing the DOPC and the polysorbate 80 to generate oleic acid, and determining the content of the oleic acid by adopting a high performance liquid chromatography; and 3, calculating the amount of oleic acid generated by hydrolysis through DOPC, and then calculating the content of polysorbate 80 in the preparation. The method provided by the invention can eliminate the interference of the DOPC component in the preparation on the determination of the content of the polysorbate 80, has strong specificity and good stability, and can be used for accurately determining the content of the polysorbate 80 in a complex preparation containing DOPC interference.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting the content of polysorbate 80 in a complex preparation. Background Art

[0002] Polysorbate 80 is a hydrophilic nonionic surfactant and one of the commonly used pharmaceutical excipients in pharmaceutical preparations. It is a light yellow to orange-yellow viscous liquid that is easily soluble in water, soluble in ethanol, vegetable oil, ethyl acetate, methanol, toluene, and insoluble in mineral oil. Polysorbate 80 has multiple functions and effects. When added to biological preparations, it can enhance the stability of proteins or antigens in biological preparations. As a pharmaceutical excipient, excessive use of polysorbate 80 can lead to adverse consequences such as allergies and hemolysis. Therefore, it is crucial to strictly control the amount of polysorbate 80 used in biological preparations.

[0003] The determination of polysorbate 80 content is divided into two methods: spectral method and chromatography. The 2020 edition of the "Chinese Pharmacopoeia" uses spectral colorimetry for the determination of polysorbate 80 content. This method has cumbersome pre-treatment steps and weak anti-interference ability. Polysorbate 80 does not contain chromophores in its molecular structure. Chromatographic analysis requires that polysorbate 80 be hydrolyzed into oleic acid first. The molecular structures of polysorbate 80 and oleic acid are as follows:

[0004]

[0005] When the biological preparation contains liposome components prepared by DOPC, the polysorbate 80 content in the preparation measured by the hydrolysis spectral method will be greatly interfered. This is because DOPC can also produce two oleic acids after alkaline hydrolysis. The molecular structure of DOPC is as follows:

[0006]

[0007] Therefore, for this complex preparation, a new detection method needs to be developed to determine the content of polysorbate 80 therein. Summary of the invention

[0008] The object of the present invention is to provide a method for detecting the content of polysorbate 80 in a complex preparation, so as to solve the problem of detecting the content of polysorbate 80 in a complex preparation, especially in a liposome preparation prepared by DOPC, in the prior art.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a method for detecting the content of polysorbate 80 in a complex preparation, wherein the complex preparation contains a liposome component prepared from DOPC, comprising the following steps:

[0011] (1) diluting the preparation sample with anhydrous ethanol, centrifuging and collecting the supernatant, and detecting the DOPC content therein by high performance liquid chromatography;

[0012] (2) diluting the preparation sample with anhydrous ethanol, adding a strong alkaline solution, hydrolyzing DOPC and polysorbate 80 at high temperature, and detecting the oleic acid content in the sample by high performance liquid chromatography;

[0013] (3) Calculate the amount of oleic acid produced by the hydrolysis of DOPC, calculate the content of oleic acid produced by the complete hydrolysis of polysorbate 80, and calculate the content of polysorbate 80 based on the content of oleic acid produced by the complete hydrolysis of polysorbate 80. The content of polysorbate 80 is 1.517 times the content of oleic acid produced by the complete hydrolysis of polysorbate 80.

[0014] Furthermore, the chromatographic columns used for the detection of DOPC and oleic acid are the same, namely, C18 columns with dimensions of 4.6×250 mm and a particle size of 5 μm.

[0015] Furthermore, the strong alkaline solution is a NaOH or KOH solution with a concentration of 0.2M-0.5M.

[0016] Furthermore, during the hydrolysis, the water bath temperature is 50-80° C., and the hydrolysis time is 16-24 hours.

[0017] Furthermore, the mobile phase for the high performance liquid chromatography detection is acetonitrile-20 mM potassium dihydrogen phosphate buffer.

[0018] Furthermore, the HPLC detection was performed at a flow rate of 1 ml / min, a detection wavelength of 195 nm, a column temperature of 30° C., an injection volume of 20 μl, and a running time of 30 min.

[0019] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects:

[0020] The present invention provides a method for detecting the content of polysorbate 80 in a complex preparation. The method can eliminate the interference of the DOPC component in the preparation on the determination of the content of polysorbate 80, has strong specificity and good stability, and can be used for the accurate determination of the content of polysorbate 80 in a complex preparation containing DOPC interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0022] Figure 1is the chromatogram of the reference substance DOPC of Example 1 of the present invention;

[0023] Figure 2 It is the chromatogram of oleic acid, the reference substance of Example 2 of the present invention. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] DOPC content determination

[0031] 1.1 Sample pretreatment

[0032] Accurately pipette 1 ml of the preparation sample, dilute it with anhydrous ethanol to make 5 ml, mix well, centrifuge at 12000 rpm for 10 min, and take the supernatant sample for later use.

[0033] 1.2 Preparation of reference solution

[0034] Weigh a certain amount of DOPC sample, dissolve it in anhydrous ethanol and make up to volume, mix it evenly and use it as the reference stock solution. Dilute the stock solution to the use concentration when necessary.

[0035] 1.3 High performance liquid chromatography detection

[0036] The high performance liquid chromatograph was turned on, and the chromatographic analysis column was connected, the flow rate was set to 1 ml / min, the detection wavelength was 205 nm, the column temperature was 30°C, the injection volume was 20 μl, and the running time was 30 min. The mobile phase was acetonitrile-20 mM potassium dihydrogen phosphate buffer (80:20).

[0037] 1.4 Result calculation

[0038] The peak areas of the reference substances and test samples with different concentrations were integrated and the average value was calculated. The content of DOPC in the test samples was calculated using the external standard method.

[0039] Example 2

[0040] 2.1 Preparation of test samples

[0041] Accurately measure 0.6 ml of the preparation sample, add 0.6 ml of 300 mM NaOH, mix, centrifuge at 3000 g for 1 min, incubate at 60 ° C for 18 h, equilibrate to room temperature, centrifuge at 3000 g for 1 min, add 0.30 ml of 5 M NaOH and 1.20 ml of acetonitrile, mix, ultrasonicate for 5 min, mix, centrifuge at 3000 g for 5 min, let stand for 30 min, take 0.60 ml of the upper organic phase, add 0.15 ml of 20 mM anhydrous potassium dihydrogen phosphate, mix, and analyze using high performance liquid chromatography.

[0042] 2.2 Preparation of reference solution

[0043] Accurately measure a certain amount of oleic acid and dilute it to a series of concentrations with 20 mM potassium dihydrogen phosphate solution. The oleic acid standard solution is prepared and used immediately.

[0044] 2.3 High performance liquid chromatography detection

[0045] The high performance liquid chromatograph was turned on, and the chromatographic analysis column was connected, the flow rate was set to 1 ml / min, the detection wavelength was 195 nm, the column temperature was 30°C, the injection volume was 20 μl, and the running time was 30 min. The mobile phase was acetonitrile-20 mM potassium dihydrogen phosphate buffer (80:20).

[0046] 2.4 Result calculation

[0047] The peak areas of the reference substances and test samples of different concentrations were integrated and the average value was obtained. The content of oleic acid in the test sample was calculated by the external standard method. According to the content of DOPC measured in 1.4, the content of oleic acid produced after its complete hydrolysis was calculated. The content of oleic acid produced by the complete hydrolysis of polysorbate 80 is the content of oleic acid in the test sample measured minus the calculated content of DOPC after complete hydrolysis. The content of polysorbate 80 is 1.517 multiplied by the content of oleic acid produced by the hydrolysis of polysorbate 80 (the molar concentrations of polysorbate 80 and oleic acid are the same. When converting the mass concentration, the molecular weight of polysorbate 80 is divided by the molecular weight of oleic acid, and then multiplied by the mass concentration of oleic acid to obtain the mass concentration of polysorbate 80).

[0048] Finally, it should be noted that:

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the content of polysorbate 80 in a complex preparation, wherein the complex preparation contains a liposome component prepared from DOPC, characterized in that: The following steps are involved: (1) Dilute the preparation sample with anhydrous ethanol, centrifuge and take the supernatant, and detect the DOPC content therein by high performance liquid chromatography; (2) Dilute the preparation sample with anhydrous ethanol, add a strong alkaline solution, hydrolyze DOPC and polysorbate 80 at high temperature, and detect the oleic acid content in the sample by high performance liquid chromatography; (3) Calculate the amount of oleic acid produced by the hydrolysis of DOPC, calculate the content of oleic acid produced by the complete hydrolysis of polysorbate 80, and calculate the content of polysorbate 80 based on the content of oleic acid produced by the complete hydrolysis of polysorbate 80. The content of polysorbate 80 is 1.517 times the content of oleic acid produced by the complete hydrolysis of polysorbate 80.

2. The method for detecting the content of polysorbate 80 in a complex preparation according to claim 1, characterized in that: The chromatographic columns used for the detection of DOPC and oleic acid are the same, that is, C18 columns, with dimensions of 4.6×250 mm and a particle size of 5 μm.

3. The method for detecting the content of polysorbate 80 in a complex preparation according to claim 1, characterized in that: The strong alkaline solution is NaOH or KOH solution with a concentration of 0.2M~0.5M.

4. The method for detecting the content of polysorbate 80 in a complex preparation according to claim 1, characterized in that: The water bath temperature during the hydrolysis is 50-80° C., and the hydrolysis time is 16-24 hours.

5. The method for detecting the content of polysorbate 80 in a complex preparation according to claim 1, characterized in that: The mobile phase during the high performance liquid chromatography detection is acetonitrile-20 mM potassium dihydrogen phosphate buffer at v / v 80:

20.

6. The method for detecting the content of polysorbate 80 in a complex preparation according to claim 1, characterized in that: The high performance liquid chromatography detection was performed with a flow rate of 1 ml / min, a detection wavelength of 195 nm, a column temperature of 30° C., an injection volume of 20 μl, and a running time of 30 min.

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