Method for analyzing purity of micromolecular monodisperse polyethylene glycol

Abstract

The invention discloses a method for detecting and analyzing the purity of micromolecular monodisperse polyethylene glycol, which comprises the following steps: diluting a heptapolyethylene glycol test solution with methanol, and detecting by using HPLC-RID (High Performance Liquid Chromatography-Resin Identifier). According to the liquid chromatography method disclosed by the invention, a chromatographic column suitable for a high water phase condition is selected, and a mobile phase is a methanol-ammonium formate solution. A differential detector is adopted in the liquid chromatography. According to the method, the response value is high, the product and impurities can be effectively separated, the detection operation is simple, the product purity and accuracy are high, and the system applicability, specificity, repeatability, sample injection precision, linearity and range all meet the requirements. The problems of purity detection and impurity control of the substance are solved.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical analysis, and specifically proposes a method for analyzing the purity of small molecule monodisperse polyethylene glycol. Background Art

[0002] Polyethylene glycol (PEG) is a polymer composed of repeating -(O-CH2-CH2)- units with excellent hydrophilicity and biocompatibility. Functionalized monodisperse PEG inserted into small molecule drugs can increase drug solubility and stability, thereby extending the drug's half-life in the body. It can also reduce steric hindrance and binding interactions associated with high-molecular-weight PEG-modified polymers, thereby reducing immunogenicity, improving pharmacokinetics, increasing circulation time, and reducing toxicity.

[0003] HeptaPEG is a polymer in which seven PEG monomers, combined with other monomers or compounds, form a polymer chain. These PEG monomers are chemically linked together to form a copolymer structure with other monomers. The specific copolymer can vary depending on the other monomers used, depending on the requirements of the target application. For example, heptaPEG can be copolymerized with acrylic acid monomers to form a heptaPEG-acrylic acid copolymer, which is used to prepare hydrogels or biomaterials; or it can be coupled with drug molecules to form a polymer drug used to improve drug solubility, bioavailability, and stability. The purity of heptaPEG determines the purity of the modified drug. Impurities can significantly affect the efficacy evaluation of PEGylated drugs. Therefore, to investigate the purity testing and impurity control issues of this type of substance, a series of studies on the purity testing of heptaPEG have been conducted. Summary of the Invention

[0004] In view of this, the present invention proposes a method for analyzing the purity of small molecule monodisperse polyethylene glycol.

[0005] The technical solution of the present invention is achieved as follows: The present invention provides a method for analyzing the purity of small molecule monodisperse polyethylene glycol, comprising the following steps: Dissolve the test sample in methanol to prepare a test solution with a concentration of 0.15–0.25 mg / ml; Detection was performed using high performance liquid chromatography coupled with a differential refractive index detector; The chromatographic column is a polar modified bonded phase chromatographic column suitable for high aqueous phase conditions; The mobile phase was a mixture of methanol and ammonium formate solution; The elution mode was isocratic.

[0006] In some embodiments, the chromatographic column is a C18 chromatographic column using surface charged hybrid particle technology.

[0007] In some embodiments, the chromatographic column is a Poroshell 120 EC-C18 chromatographic column with a size of 15 cm×4.6 mm and a filler particle size of 4.0 μm.

[0008] In some embodiments, the column temperature is 50-70°C; and the differential detector temperature is 50-60°C.

[0009] In some embodiments, the mobile phase is composed of methanol and 5 mmol / L ammonium formate solution in a volume ratio of 20:80.

[0010] In some embodiments, the mobile phase flow rate is 0.4-0.6 ml / min, including but not limited to 0.4 ml / min, 0.5 ml / min, 0.6 ml / min; the injection volume is 10-20 μl, including but not limited to 10 μl, 15 μl, 20 μl.

[0011] In some embodiments, the test substance is heptapolyethylene glycol.

[0012] The structural formula of heptapolyethylene glycol is as follows:

[0013] The present invention also provides a drug quality control method, which uses the above-mentioned analytical method to detect the raw material purity and impurity content of the polyethylene glycol-modified drug.

[0014] The present invention has the following beneficial effects compared to the prior art: The instrument provided by the present invention has low operating costs, a simple and convenient detection process, and good repeatability, and can be used to evaluate product quality. The present invention meets the requirements in terms of system applicability and specificity, repeatability, injection precision, linearity and range. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 these drawings without paying any creative work.

[0016] Figure 1 This is a chromatogram of the heptapolyethylene glycol test solution in Example 1 of the present invention; Figure 2 Graph showing the linearity and range of the heptapolyethylene glycol reference solution in Example 2 of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0019] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0020] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0021] Detection of Heptapolyethylene Glycol by High Performance Liquid Chromatography Instruments and chemical experiments used in the examples include a liquid chromatograph (Agilent 1260, Agilent); an electronic balance (BS124S, Sartorius); and a Poroshell 120 EC-C18 column (15 cm × 4.6 mm, 4.0 μm, Agilent). The differential detector parameters were set according to the instrument manufacturer's recommendations, specifically a gain of 8 and positive polarity. Methanol was chromatographically grade, and ammonium formate was analytically grade.

[0022] Example 1 1. HPLC analysis conditions: Chromatographic column: Poroshell 120 EC-C18 column (15 cm × 4.6 mm, 4.0 μm, Agilent) with a methanol-ammonium formate solution as the mobile phase; The column temperature for liquid chromatography was 60°C and the detection temperature was 55°C; The mobile phase flow rate was 0.5 ml / min, and the elution mode was isocratic; The injection volume for liquid chromatography was 10 μl. Solution preparation: Mobile phase preparation: methanol: 5 mmol / L methanolic ammonium solution = 20:80 (v / v).

[0023] Preparation of test solution: Weigh 40 mg of heptapolyethylene glycol test sample accurately into a 200 ml volumetric flask, dissolve it in methanol, dilute to the scale, and shake well to obtain a heptapolyethylene glycol solution with a concentration of 0.2 mg / ml.

[0024] Blank solution: methanol.

[0025] Determination Detection was performed using HPLC-RID, and the detection conditions were as described above.

[0026] Take blank and test solution and inject them into liquid chromatograph, record the chromatogram, the injection sequence is as follows:

[0027] The chromatogram results are as follows Figure 1 shown.

[0028] Example 2 Specificity and system suitability 1.1 Solution preparation: Blank: Methanol System suitability solution: Take 10 μl of toluene, 10 μl of hexaethylene glycol, and about 40 mg of heptaethylene glycol, accurately weigh them, transfer them to the same 200 ml volumetric flask, add methanol to dissolve them, and dilute to the scale.

[0029] Test solution: Take 40 mg of the heptapolyethylene glycol test sample, accurately weigh it, and put it into a 200 ml volumetric flask. Add methanol to dissolve it and dilute it to the scale. Shake well to obtain a heptapolyethylene glycol solution with a concentration of 0.2 mg / ml.

[0030] 1.2 Assay Inject blank, system suitability solution, and test solution in the following order and record the chromatogram.

[0031] Injection order:

[0032] 1.3 Results: No interference in blank; the separation degree between the main peak and adjacent peaks in the test solution is ≥1.5.

[0033] Injection precision 2.1 Solution preparation: Take the test solution under 1.1 and inject it 6 times continuously, and record the chromatogram.

[0034] 2.2 Results:

[0035] Solution stability 3.1 Assay Take the test solution under 1.1 and inject it at 0h, 1h, 2h, 4h and 6h respectively, and record the chromatogram.

[0036] 3.2 Results:

[0037] Linearity and range 4.1 Solution preparation: Stock solution: Take about 50 mg of heptapolyethylene glycol reference substance, accurately weigh it, place it in a 50 ml volumetric flask, dilute it to the scale with methanol, shake well, and obtain it.

[0038] Linear solution ① (0.30 mg / ml, 150%): Accurately measure 3.0 ml of the stock solution, place it in a 10 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0039] Linear solution ② (0.24 mg / ml, 120%): Accurately measure 2.4 ml of the stock solution, place it in a 10 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0040] Linear solution ③ (0.20 mg / ml, 100%): Accurately measure 2.0 ml of the stock solution, place it in a 10 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0041] Linear solution ④ (0.16 mg / ml, 80%): Accurately measure 1.6 ml of the stock solution, place it in a 10 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0042] Linear solution ⑤ (0.10 mg / ml, 50%): Accurately measure 1.0 ml of the stock solution, place it in a 10 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0043] 4.2 Determination method: Take two injections of each of the linear solutions ① through ⑤ and record the chromatogram. Perform linear regression based on the concentration and peak area of ​​the linear solutions to create a standard curve.

[0044] 4.3 Results:

[0045] With the concentration (mg / ml) as the horizontal axis and the corresponding peak area as the vertical axis, linear regression is performed. The linear results and linear equation are as follows: Figure 2 As shown, the results showed that the heptapolyethylene glycol control was detected according to the chromatographic conditions described in Example 1, and had good linearity in the concentration range of 0.10~0.30mg / ml, with a linear equation of y=88.678x-239.1, R=0.999, and intercept ratio=2.8%.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for analyzing the purity of small molecule monodisperse polyethylene glycol, characterized in that , including the following steps: Dissolve the test sample in methanol to prepare a test solution with a concentration of 0.15–0.25 mg / ml; Detection was performed using high performance liquid chromatography coupled with a differential refractive index detector; The chromatographic column is a polar modified bonded phase chromatographic column suitable for high aqueous phase conditions; The mobile phase was a mixture of methanol and ammonium formate solution; The elution mode was isocratic.

2. The analysis method according to claim 1, wherein The chromatographic column is a C18 chromatographic column using surface charged hybrid particle technology.

3. The analysis method according to claim 2, wherein The chromatographic column is a Poroshell 120 EC-C18 chromatographic column with a specification of 15 cm×4.6 mm and a filler particle size of 4.0 μm.

4. The analysis method according to claim 1, wherein The column temperature was 50–70°C and the differential detector temperature was 50–60°C.

5. The analysis method according to claim 1, wherein The mobile phase is composed of methanol and 5 mmol / L ammonium formate solution in a volume ratio of 20:

80.

6. The analysis method according to claim 1, wherein The mobile phase flow rate was 0.4–0.6 ml / min and the injection volume was 10–20 μl.

7. The analysis method according to claim 1, wherein The test sample is heptapolyethylene glycol.

8. A drug quality control method, characterized in that: The analytical method according to any one of claims 1 to 7 is used to detect the raw material purity and impurity content of the polyethylene glycol-modified drug.

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