Method for detecting specific impurities in LH-1801 sugar fragment

Abstract

The invention relates to the technical field of detection, and particularly provides a method for detecting specific impurities in an LH-1801 sugar fragment, which is used for detecting a methyl-alpha-D-glucopyranoside test solution by utilizing HPLC-RID (High Performance Liquid Chromatography-Resin Identifier). According to the liquid chromatography method, a sulfonated styrene-divinyl benzene polymer filled resin column is selected, the liquid chromatography method is suitable for a chromatographic column under a high water phase condition, and a mobile phase is a phosphate buffer solution. A differential detector is adopted in the liquid chromatography. The method is high in response value, glycoside products and impurities can be effectively separated, detection operation is simple, product purity and accuracy are high, system applicability, specificity, repeatability, sample introduction precision, linearity and range all meet requirements, and the problems of substance purity detection and impurity control are solved.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for detecting specific impurities in LH-1801 sugar fragments. Background Art

[0002] The key intermediate sugar fragment of the first-class innovative drug LH-1801 of Jiangsu Lianhuan Pharmaceutical Co., Ltd. has the chemical name: (3R, 4S, 5R, 6R)-3,4,5-tri(benzyloxy)-tetrahydro-6-methylpyran-2-one, CAS number: 1461750-25-3, and the structural formula is as follows:

[0003]

[0004] The current process route for the key intermediate sugar fragment of LH-1801 is as follows:

[0005]

[0006] From the control strategy of API impurity research, possible impurities in the LH-1801 sugar fragment can be traced back to the key starting material methyl-α-D-pyranoglucoside used.

[0007] Methyl-α-D-pyranoglucoside is a non-reducing glucose derivative and a tetrahydroxy polyol with a unique cyclic structure. Combined with the synthetic route of methyl-α-D-pyranoglucoside, there may be isomeric impurities such as methyl-α-D-pyranoglucoside and methyl-β-D-pyranoglucoside. The presence of isomeric impurities will affect the quality of the product, and its quality also determines the quality of the modified drug. In particular, the content of certain specific impurities will have a significant impact on the purity of subsequent products. The existing technology does not have a means to detect specific impurities in methyl-α-D-pyranoglucoside raw materials. Therefore, in order to study the control of specific impurities in this type of substance, it is necessary to study the detection of specific impurities in methyl-α-D-pyranoglucoside. Summary of the invention

[0008] In view of this, the present invention proposes a method for detecting specific impurities in the intermediate sugar fragment of LH-1801, aiming to overcome the problem that there is currently no exclusive detection method and means to detect and control its specific impurities.

[0009] The technical solution of the present invention is implemented as follows: The present invention provides a method for detecting specific impurities in LH-1801 sugar fragments, wherein the specific impurities are at least one of methyl-α-D-pyranoside and methyl-β-D-pyranoside, and the detection method adopts liquid chromatography, and the detection conditions are:

[0010] Chromatographic column: Sulfonated styrene-divinylbenzene polymer packed resin column;

[0011] Mobile phase: aqueous solution containing 0.02 mol / L sodium dihydrogen phosphate;

[0012] Column temperature: 50-80℃;

[0013] Detection temperature: 50-70℃;

[0014] Mobile phase flow rate: 0.8-1.2ml / min;

[0015] Elution mode: isocratic elution;

[0016] Injection volume: 10-20 μl;

[0017] Detector: differential refractive index detector;

[0018] The detection method steps are as follows:

[0019] Step 1, drawing standard curves of methyl-α-D-pyranoglucoside, methyl-α-D-pyranoside and methyl-β-D-pyranoside respectively;

[0020] Step 2: preparing the test solution;

[0021] Step 3: Perform liquid chromatography analysis on the sample solution according to the chromatographic conditions and the peak area in the analysis spectrum to calculate the content of the specific impurity.

[0022] In some embodiments, in step 1, the method of drawing a standard curve comprises:

[0023] Weigh 10 mg each of methyl-α-D-pyranoglucoside, methyl-α-D-pyranoside and methyl-β-D-pyranoside into a 100 ml volumetric flask, dissolve to volume with mobile phase to prepare a single standard solution of 0.1 mg / ml;

[0024] Use a pipette to accurately pipette 1.0ml, 2.0ml, 4.0ml, 5.0ml, 6.0ml, 8.0ml, 10.0ml into a 50ml volumetric flask, and then dilute to the mark with mobile phase to obtain 2.0μg / ml, 4.0μg / ml, 8.0μg / ml, 10.0μg / ml, 12.0μg / ml, 16.0μg / ml, 20.0μg / ml standard curve solutions;

[0025] The standard curve solution was taken for high performance liquid chromatography analysis. The chromatographic conditions were as follows: an Aminex HPX-87C column was used as the HPLC column with a specification of 7.8×300mm×9μm, a phosphate buffer was used as the mobile phase, a flow rate of 1.0ml / min, and a column temperature of 65°C; a differential refractometer was used as the detector, and a standard curve was drawn according to the relationship between concentration and chromatographic peak area.

[0026] In some embodiments, in the liquid chromatography detection conditions of the specific impurity detection method, the column temperature is 65°C.

[0027] In some embodiments, in the liquid chromatography detection conditions of the specific impurity detection method, the detection temperature is 60°C.

[0028] In some embodiments, in the liquid chromatography detection conditions of the specific impurity detection method, the flow rate of the mobile phase is 1 ml / min.

[0029] In some embodiments, in the liquid chromatography detection conditions of the specific impurity detection method, the injection volume is 10 μl.

[0030] In some embodiments, in the liquid chromatography detection conditions of the specific impurity detection method, the chromatographic column is an Aminex HPX-87C chromatographic column with a specification of 7.8×300 mm×9 μm.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The instrument provided by the present invention has low operating cost, simple and convenient detection process, good repeatability, and can be used to evaluate product quality. The present invention meets the requirements in terms of system applicability and specificity, linearity and range. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in 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 these drawings without paying creative work.

[0034] Figure 1 This is a chromatogram of the methyl-α-D-pyranoglucoside test solution in Example 1 of the present invention;

[0035] Figure 2 This is a chromatogram of the sensitivity solution of methyl-α-D-pyranoglucoside in Example 2 of the present invention;

[0036] Figure 3This is a linear relationship diagram of methyl-α-D-pyranoglucoside in Example 3 of the present invention;

[0037] Figure 4 This is a linear relationship diagram of methyl-β-D-pyranoglucoside in Example 3 of the present invention;

[0038] Figure 5 This is a linear relationship diagram of methyl-α-D-mannopyranoside in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] 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 described embodiments 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 creative work are within the scope of protection of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the 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.

[0041] Unless otherwise specified, the methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0042] 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 range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "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 end values ​​and all integers and fractions within the range. In the present specification and claims, range definitions can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0043] The instruments and reagents used in the following examples are: liquid chromatograph (Agilent 1260, Agilent); electronic balance (BS124S, Sartorius); Aminex HPX-87C chromatographic column (7.8×300 mm×9 μm, Bio-Rad, USA); phosphate was of chromatographic grade.

[0044] Example 1

[0045] This example provides a high performance liquid chromatography detection scheme for a methyl-α-D-pyranoglucoside test sample:

[0046] HPLC analysis conditions:

[0047] Chromatographic column: Aminex HPX-87C chromatographic column (7.8×300mm×9μm, Bio-Rad, USA)

[0048] The mobile phase was phosphate buffered saline;

[0049] The column temperature of the liquid chromatography method was 65°C and the detection temperature was 60°C;

[0050] The mobile phase flow rate was 1.0 ml / min, and the elution mode was isocratic;

[0051] The injection volume for liquid chromatography was 10 μl.

[0052] Solution preparation:

[0053] Preparation of mobile phase: Dissolve 2.4 g of sodium dihydrogen phosphate in 1 L of water to obtain phosphate buffer.

[0054] Preparation of test solution: Weigh 100 mg of methyl-α-D-pyranoglucoside test sample, accurately weigh it into a 10 ml volumetric flask, add the mobile phase to dissolve it and dilute it to the scale, shake well, and you will get a test solution with a concentration of 10 mg / ml.

[0055] Blank solution: mobile phase.

[0056] Determination:

[0057] HPLC-RID was used for detection, and the detection conditions were as described above.

[0058] Take blank and test solution and inject them into liquid chromatograph, record the chromatogram, the injection sequence is as follows:

[0059] name Number of injection needles blank ≥2, until the two needles completely overlap without interference Test solution 1

[0060] Record the chromatogram as Figure 1 shown.

[0061] Example 2

[0062] This embodiment is used to verify the specificity of the detection method.

[0063] HPLC analysis conditions:

[0064] Chromatographic column: Aminex HPX-87C chromatographic column (7.8×300mm×9μm, Bio-Rad, USA)

[0065] The mobile phase was phosphate buffered saline;

[0066] The column temperature of the liquid chromatography method was 65°C and the detection temperature was 60°C;

[0067] The mobile phase flow rate was 1.0 ml / min, and the elution mode was isocratic;

[0068] The injection volume for liquid chromatography was 10 μl.

[0069] Solution preparation:

[0070] Preparation of mobile phase: Dissolve 2.4 g of sodium dihydrogen phosphate in 1 L of water to obtain phosphate buffer.

[0071] Blank: Mobile phase

[0072] Specific impurity solution: Take 10 mg of methyl-α-D-pyranoside and methyl-β-D-pyranoside into a 100 ml volumetric flask and dissolve them to the mark with mobile phase.

[0073] Sensitivity solution: Take 100 mg of methyl-α-D-pyranoglucoside into a 10 ml volumetric flask, then transfer 0.2 ml of the specific impurity solution into the same volumetric flask, add the mobile phase to dissolve it and dilute to the scale.

[0074] Test solution: Take 100 mg of methyl-α-D-pyranoglucoside, accurately weigh it, put it into a 10 ml volumetric flask, add the mobile phase to dissolve it and dilute it to the scale, shake well, and you will get a methyl-α-D-pyranoglucoside solution with a concentration of 10 mg / ml.

[0075] Determination method:

[0076] Inject blank, system suitability solution and test solution in the following order and record the chromatogram.

[0077] Injection order:

[0078] name Number of injection needles blank ≥2, until the two needles completely overlap without interference Sensitivity solution 1 Test solution 1

[0079] Chromatogram Figure 2 As shown, the blank has no interference, the peak separation of each component in the sensitivity solution is ≥1.5, and the signal-to-noise ratio S / N of specific impurities is greater than 10.

[0080] The peak time, relative retention time, signal-to-noise ratio S / N and separation of each component are shown in the following table:

[0081]

[0082] Example 3

[0083] This example is used to verify the linearity and range of the standard curve.

[0084] HPLC analysis conditions:

[0085] Chromatographic column: Aminex HPX-87C chromatographic column (7.8×300mm×9μm, Bio-Rad, USA)

[0086] The mobile phase was phosphate buffered saline;

[0087] The column temperature of the liquid chromatography method was 65°C and the detection temperature was 60°C;

[0088] The mobile phase flow rate was 1.0 ml / min, and the elution mode was isocratic;

[0089] The injection volume for liquid chromatography was 10 μl.

[0090] Solution preparation:

[0091] Stock solution: Accurately weigh 10 mg each of methyl-α-D-pyranoglucoside, methyl-α-D-pyranoside and methyl-β-D-pyranoside into a 100 ml volumetric flask, dissolve to volume with mobile phase to prepare a single standard solution of 0.1 mg / ml;

[0092] Use a pipette to accurately pipette 1.0ml, 2.0ml, 4.0ml, 5.0ml, 6.0ml, 8.0ml, 10.0ml into a 50ml volumetric flask, and then dilute to the scale with mobile phase to obtain 2.0μg / ml, 4.0μg / ml, 8.0μg / ml, 10.0μg / ml, 12.0μg / ml, 16.0μg / ml, 20.0μg / ml standard curve solutions.

[0093] Linear solution ① (2.0 μg / ml, 20%): Accurately measure 1.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0094] Linear solution ② (4.0 μg / ml, 40%): Accurately measure 2.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0095] Linear solution ③ (8.0 μg / ml, 80%): Accurately measure 4.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0096] Linear solution ④ (10.0 μg / ml, 100%): Accurately measure 5.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0097] Linear solution ⑤ (12.0 μg / ml, 120%): Accurately measure 6.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0098] Linear solution ⑥ (16.0 μg / ml, 160%): Accurately measure 8.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0099] Linear solution ⑦ (20.0 μg / ml, 200%): Accurately measure 10.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0100] Determination method:

[0101] Take linear solution ① to linear solution ⑦, inject 2 times each, and record the chromatogram. Perform linear regression based on the concentration and peak area of ​​the linear solution to draw a standard curve.

[0102] result:

[0103] The linear data of methyl-α-D-pyranoglucoside are as follows:

[0104] Limit concentration ratio / % Concentration / μg / ml Peak area / nRIU 20 2.01 2849.61 40 4.01 5784.79 80 8.03 12124.31 100 10.03 14936.21 120 12.04 17148.66 160 16.05 23457.17 200 20.07 29473.86

[0105] The linear relationship diagram of methyl-α-D-pyranoglucoside is as follows Figure 3 The linear data of methyl-β-D-pyranoglucoside are as follows:

[0106] Limit concentration ratio / % Concentration / μg / ml Peak area / nRIU 20 2.05 2569.20 40 4.11 5178.43 80 8.22 10497.25 100 10.27 12931.46 120 12.32 15267.91 160 16.43 20897.34 200 20.54 25649.57

[0107] The linear relationship diagram of methyl-β-D-pyranoglucoside is as follows Figure 4 The linear data of methyl-α-D-pyranoside are shown in the following table:

[0108] Limit concentration ratio / % Concentration / μg / ml Peak area / nRIU 20 2.04 2219.76 40 4.08 4636.85 80 8.15 8371.54 100 10.19 10766.18 120 12.23 12984.53 160 16.30 17705.18 200 20.38 22543.80

[0109] The linear relationship diagram of methyl-α-D-pyranoside is shown in Figure 5 shown.

[0110] Linear regression was performed with concentration (μg / ml) as the horizontal axis and the corresponding peak area as the vertical axis. The linear results and linear equation are shown in Figure 3 As shown, the results show that methyl-α-D-pyranoglucoside, methyl-α-D-pyranoglucoside and methyl-β-D-pyranoglucoside are detected according to the chromatographic conditions described in Example 1. Methyl-α-D-pyranoglucoside has a good linear relationship in the concentration range of 2.01 μg / ml to 20.07 μg / ml, and the linear equation is y=1464x-7.794, R 2 =0.999; methyl-α-D-pyranoside has a good linear relationship in the concentration range of 2.04μg / ml to 20.38μg / ml, and the linear equation is y=1099x-203.4, R 2 =0.998; methyl-β-D-pyranoglucoside has a good linear relationship in the concentration range of 2.05μg / ml to 20.54μg / ml, and the linear equation is y=1252x-49.46, R 2 =0.999.

[0111] 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 protection scope of the present invention.

Claims

1. A method for detecting specific impurities in LH-1801 sugar fragments, It is characterized in that The specific impurity is at least one of methyl-α-D-pyranoglucoside and methyl-β-D-pyranoglucoside, and the detection method adopts liquid chromatography, and the detection conditions are: Chromatographic column: Sulfonated styrene-divinylbenzene polymer packed resin column; Mobile phase: aqueous solution containing 0.02 mol / L sodium dihydrogen phosphate; Column temperature: 50-80℃; Detection temperature: 50-70℃; Mobile phase flow rate: 0.8-1.2ml / min; Elution mode: isocratic elution; Injection volume: 10-20 μl; Detector: differential refractometer; The detection method steps are as follows: Step 1, drawing standard curves of methyl-α-D-pyranoglucoside, methyl-α-D-pyranoside and methyl-β-D-pyranoside respectively; Step 2: preparing the test solution; Step 3: Perform liquid chromatography analysis on the sample solution according to the chromatographic conditions and the peak area in the analysis spectrum to calculate the content of the specific impurity.

2. The method for detecting specific impurities in LH-1801 sugar fragments according to claim 1, It is characterized in that In step 1, the method of drawing a standard curve includes: Weigh 10 mg each of methyl-α-D-pyranoglucoside, methyl-α-D-pyranoside and methyl-β-D-pyranoside into a 100 ml volumetric flask, dissolve to volume with mobile phase to prepare a single standard solution of 0.1 mg / ml; Use a pipette to accurately pipette 1.0ml, 2.0ml, 4.0ml, 5.0ml, 6.0ml, 8.0ml, 10.0ml into a 50ml volumetric flask, and then dilute to the mark with mobile phase to obtain 2.0μg / ml, 4.0μg / ml, 8.0μg / ml, 10.0μg / ml, 12.0μg / ml, 16.0μg / ml, 20.0μg / ml standard curve solutions; The standard curve solution was taken for high performance liquid chromatography analysis. The chromatographic conditions were as follows: Aminex HPX-87C column was used as the HPLC column with a specification of 7.8×300mm×9μm, the mobile phase was phosphate buffer, the flow rate was 1.0ml / min, and the column temperature was 65°C; a differential refractometer was used as the detector, and a standard curve was drawn according to the relationship between concentration and chromatographic peak area.

3. The method for detecting specific impurities in LH-1801 sugar fragments according to claim 1, It is characterized in that The column temperature was 65°C.

4. The method for detecting specific impurities in LH-1801 sugar fragments according to claim 1, It is characterized in that The detection temperature is 60°C.

5. The method for detecting specific impurities in LH-1801 sugar fragments according to claim 1, It is characterized in that The flow rate of the mobile phase was 1 ml / min.

6. The method for detecting specific impurities in LH-1801 sugar fragments according to claim 1, It is characterized in that The injection volume was 10 μl.

7. The method for detecting specific impurities in LH-1801 sugar fragments according to claim 1, It is characterized in that The chromatographic column is an Aminex HPX-87C chromatographic column with specifications of 7.8×300mm×9μm.

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