Method for simultaneously detecting six GLP-1RA drugs

Abstract

The invention relates to the technical field of medicines, and discloses a method for simultaneously detecting six GLP-1RA medicines, which comprises the steps of sample solution preparation, standard solution preparation, liquid chromatography condition, mass spectrum condition, qualitative determination and quantitative determination. According to the method disclosed by the invention, the six GLP-1RA medicine components such as the semeglutide in the sample are simultaneously determined by a high performance liquid chromatography-triple quadrupole mass spectrometry method for the first time, the six components can be rapidly and effectively separated and detected by single-time sample introduction analysis for 30 minutes, and the method is simple in pretreatment, strong in specificity, good in accuracy and repeatability, high in sensitivity and high in sensitivity. The method can be used for accurately and efficiently qualitatively and quantitatively detecting the components of the semeglutide, the lisinitide, the beraglutide, the exenatide, the liraglutide and the tilpoitide added in the sample, fills the blank of detection methods and detection standards in the related detection fields, and provides technical support for supervision departments to fight against illegal behaviors of illegally adding medicine components. The health and safety of people are guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for simultaneously detecting six GLP-1RA drugs. Background Art

[0002] The new glucose-lowering drug glucagon-like peptide-1 receptor agonist (GLP-1RA) has been widely recognized by relevant treatment guidelines at home and abroad for its glucose-lowering efficacy and metabolic benefits. At present, there are more than 10 GLP-1RA varieties on the market worldwide, and domestic drugs on the market include semaglutide, lixisenatide, belaglutide, exenatide, liraglutide and telpotide. In June 2021, the U.S. FDA approved the 2.4 mg strength of semaglutide for the treatment of overweight and obesity in adults, becoming the first GLP-1RA preparation for weight management. Clinical data show that semaglutide has a significant weight loss effect, but the research subjects are within the approved indication range, that is, adults with a body mass index (BMI) ≥ 27kg·m -2 With at least one complication of obesity or BMI>30 kg·m -2 Simple obesity. Studies have shown that semaglutide can directly stimulate the hypothalamus and hindbrain, reduce appetite through indirect activation of the vagus nerve, delay gastric emptying, increase satiety, and achieve weight loss by reducing energy intake. Common adverse reactions include gastrointestinal discomfort, acute pancreatitis, etc., and there may be risks of depression, acute kidney damage and thyroid tumors. Therefore, this type of drug is used as a hypoglycemic and weight-loss drug with clear indications and contraindications, and should be used strictly according to prescriptions.

[0003] With the publicity of the "weight loss" effect of GLP-1RA drugs such as semaglutide on the Internet, "weight loss" products containing semaglutide and other GLP-1RA drug ingredients have appeared in the market, including oral preparations, lyophilized preparations and injections. Therefore, it is necessary to establish accurate and efficient qualitative and quantitative methods for simultaneous determination of semaglutide, lixisenatide, belaglutide, exenatide, liraglutide and telpotide in samples, so as to effectively combat illegal production and provide technical support for market supervision and protect people's health and safety. Summary of the invention

[0004] The present invention provides a method for simultaneously detecting six GLP-1RA drugs, which can realize accurate and efficient qualitative and quantitative detection of semaglutide, lixisenatide, belaglutide, exenatide, liraglutide and telpotide components added to samples, filling the gaps in detection methods and inspection standards in the relevant inspection field.

[0005] The present application embodiment provides a method for simultaneously detecting 6 GLP-1RA drugs, comprising the following steps:

[0006] S110, sample solution preparation: take an appropriate amount of sample, add solvent to dissolve or dilute, filter through a 0.22 μm filter membrane, and take the filtrate as the sample solution;

[0007] S120, preparation of standard solution: weigh appropriate amount of semaglutide, lixisenatide, belaglutide, exenatide, liraglutide and telportide reference substances, add solvent to dissolve and prepare standard stock solution; accurately measure appropriate amount of standard stock solution, dilute with solvent to prepare standard solution or standard series solution;

[0008] S130, Liquid chromatography conditions: The chromatographic column used was Kinetex C18 (4.6 mm × 150 mm, 2.6 μm), or an equivalent chromatographic column; the mobile phase A was an aqueous solution containing 0.1% formic acid, and the mobile phase B was a solution of methanol-acetonitrile (1:1) containing 0.1% formic acid, with a gradient elution procedure;

[0009] Mass spectrometry conditions: electrospray ion source (ESI source) was used; the ion source mode was positive ion multiple reaction monitoring mode (MRM), the ion source temperature (TEM) was 350°C; the electrospray voltage (IS) was 5500V; the curtain gas (CUR) was 30psi; the spray gas (GS1) was 50psi; the auxiliary heating gas (GS2) was 50psi;

[0010] S140, Qualitative determination: Take the sample solution and the standard solution and measure them under the same analytical conditions. If the sample presents chromatographic peaks of quantitative ion pairs and qualitative ion pairs, the retention time of the characteristic ion peak of the measured component is consistent with the retention time of the mixed reference solution for qualitative detection, and the maximum allowable deviation of the relative abundance ratio of the selected monitoring ion pair and the relative abundance ratio of the monitoring ion pair of the mixed reference solution for screening with equivalent concentration does not exceed the provisions of the following table, then it can be determined that the corresponding component exists in the sample; the maximum allowable deviation of the relative ion abundance ratio during qualitative confirmation is:

[0011]

[0012]

[0013] Furthermore, it also includes step S140, quantitative determination: taking sample solutions and standard series solutions for sampling and analysis, taking the series concentrations of the components to be measured as the horizontal axis and the peak area of ​​the components to be measured as the vertical axis, performing linear regression, and drawing a standard curve, whose linear correlation coefficient should be greater than 0.99, substituting the corresponding quantitative ion pair chromatographic peak area in the sample into the standard curve to calculate the concentration of the components to be measured in the sample, and then calculating the content.

[0014] Furthermore, the specific content of S110 is:

[0015] S111, preparation of solvent: weigh 1.2g of sodium dihydrogen phosphate, add 900ml of water to dissolve, adjust the pH value to 7.5±0.1 with 1mol / L sodium hydroxide solution, dilute with water to 1000ml, and mix well;

[0016] S112. Sample processing:

[0017] a. When the sample is a liquid sample: take 1.0 ml of the sample solution, place it in a 5 ml volumetric flask, dilute it with solvent and make up to the mark, and mix well;

[0018] b. When the sample is a freeze-dried solid sample: If the sample is provided with a diluent, use the diluent to dissolve the freeze-dried solid sample to prepare a solution. Take 1.0 ml of the solution and place it in a 5 ml volumetric flask. Dilute it with a solvent and make it up to the mark, and mix well. If the sample is not provided with a diluent, use 10 ml of the solvent to dissolve the freeze-dried solid sample to prepare a solution.

[0019] c. When the sample is a raw material drug sample: weigh about 5 mg of the sample, place it in a 25 ml volumetric flask, dissolve it with solvent and dilute to the mark, and mix well;

[0020] d. When the sample is other solid samples: take a piece or a pellet of other solid samples, place it in a 100ml volumetric flask, add 75ml of solvent, shake to dissolve, dilute to scale with solvent, shake well, centrifuge at 5000r / min for 10min, and take the supernatant;

[0021] S113. The prepared solution is filtered through a 0.22 μm filter membrane, and the filtrate is taken as the sample solution.

[0022] Furthermore, in step S130, the monitoring ion pairs of each component and the related parameter settings are shown in the following table:

[0023]

[0024]

[0025] Note: * indicates quantitative ion.

[0026] Furthermore, in step S130, the flow rate is 0.8 μl / min; the column temperature is 40° C.; the injection volume is 2 μl;

[0027] Further, the gradient elution program is shown in the following table:

[0028]

[0029] Furthermore, in step S120, the concentration of the prepared standard solution is qualitatively determined to be a mixed reference solution containing 0.2 mg / ml or 0.3 mg / ml of each component.

[0030] Furthermore, in step S120, the standard series solutions prepared for quantitative determination are mixed reference solutions containing 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, and 0.5 mg / ml of each component.

[0031] Furthermore, in S113, the concentration of the sample solution is 0.2 mg / ml-0.3 mg / ml.

[0032] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0033] The method of the present invention is the first to simultaneously determine six GLP-1RA drug components such as semaglutide in samples by high performance liquid chromatography-triple quadrupole mass spectrometry. The six components can be quickly and effectively separated and detected in a single sample analysis of 30 minutes. The method has simple pretreatment, strong specificity, good accuracy and repeatability, and can achieve accurate and efficient qualitative and quantitative detection of semaglutide, lixisenatide, belaglutide, exenatide, liraglutide and telpotide components added to the sample, filling the gaps in detection methods and inspection standards in related inspection fields, providing technical support for regulatory authorities to crack down on illegal acts of illegally adding drug ingredients, and protecting people's health and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0035] Figure 1 It is the MRM total ion current chromatogram of the standard solution;

[0036] Figure 2 This is the extracted ion chromatogram of lixisenatide in the standard solution;

[0037] Figure 3 This is the extracted ion chromatogram of belaglutide in the standard solution;

[0038] Figure 4 This is the extracted ion chromatogram of exenatide in the standard solution;

[0039] Figure 5 This is the extracted ion chromatogram of semaglutide in the standard solution;

[0040] Figure 6 This is the extracted ion chromatogram of liraglutide in the standard solution;

[0041] Figure 7 This is the extracted ion chromatogram of tepoxetine in standard solution. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0043] like Figure 1-Figure 7 As shown, the present invention provides a method for simultaneously detecting 6 GLP-1RA drugs, comprising the following steps:

[0044] 1. Reagents and Materials

[0045] Methanol, mass spectrometry grade; acetonitrile, mass spectrometry grade; formic acid, mass spectrometry grade; sodium dihydrogen phosphate, analytical grade; sodium hydroxide, analytical grade. Unless otherwise specified, all reagents used in this method are of analytical grade or above, and purified water is homemade. The reference substance information of lixisenatide, belaglutide, exenatide, semaglutide, liraglutide and telportide is detailed in the table below:

[0046]

[0047] 2. Instruments and equipment

[0048] High performance liquid chromatography-triple quadrupole mass spectrometer (AB Sciex Triple Quad5500); analytical balance: sensitivity 0.0001g and 0.00001; acid-base detector; high-speed centrifuge.

[0049] 3. Analysis steps

[0050] 3.1 Blank solvent

[0051] Take the solvent (weigh 1.20 g of sodium dihydrogen phosphate, add 900 ml of water to dissolve, adjust the pH value to 7.5±0.1 with 1 mol / L sodium hydroxide solution, dilute with water to 1000 ml, and mix well) as the blank solvent.

[0052] 3.2 Standard stock solution

[0053] Weigh 5 mg each of lixisenatide, belaglutide, exenatide, semaglutide, liraglutide and telpotide, accurately weigh, place in a 5 ml volumetric flask, dissolve with solvent and dilute to scale, mix well, and prepare a mixed reference standard stock solution with a concentration of 1.0 mg / ml.

[0054] 3.3 Qualitative detection standard solution

[0055] Take an appropriate amount of the standard stock solution and dilute it with a solvent to prepare a standard solution for qualitative detection with a concentration of 0.2 mg / ml or 0.3 mg / ml.

[0056] 3.4 Quantitative detection of standard solution

[0057] When using the standard curve method for quantitative analysis, take an appropriate amount of the standard stock solution and dilute it with a solvent to prepare a series of mixed reference solutions with concentrations of 0.5 mg / ml, 0.4 mg / ml, 0.3 mg / ml, 0.2 mg / ml, and 0.1 mg / ml (the concentration range of the mixed reference solution series can be adjusted according to actual conditions).

[0058] 3.5 Sample processing

[0059] (1) Liquid sample: Take 1.0 ml of sample solution and place it in a 5 ml volumetric flask. Dilute with solvent and make up to the mark. Mix well.

[0060] (2) Freeze-dried solid samples: If the sample is provided with a diluent, take one bottle (tube) of the diluent to dissolve one bottle (tube) of freeze-dried solid sample to make a solution. Take 1.0 ml of the solution and place it in a 5 ml volumetric flask. Dilute it with solvent and make up to the mark. Mix well. If no diluent is provided, use solvent (about 10 ml) to dissolve one bottle (tube) of freeze-dried solid sample to make a solution.

[0061] (3) API sample: weigh about 5 mg of the sample, place it in a 25 ml volumetric flask, dissolve it with solvent and make up to the mark, and mix well;

[0062] (4) Other solid samples: Take one piece or one grain of other solid sample (such as tablets, capsule contents, etc.), place it in a 100 ml volumetric flask, add 75 ml of solvent, shake to dissolve, dilute to scale with solvent, shake well, centrifuge at 5000 r / min for 10 min, and take the supernatant;

[0063] The prepared solution was filtered through a 0.22 μm filter membrane, and the filtrate was taken as the sample solution (the sample solution concentration can be prepared to a range of 0.2 mg / ml-0.3 mg / ml).

[0064] 3.6 Instrument reference conditions

[0065] 3.6.1 Chromatographic conditions

[0066] Chromatographic column: Kinetex C18 (4.6 mm × 150 mm, 2.6 μm), or equivalent;

[0067] Mobile phase: A is an aqueous solution containing 0.1% formic acid, B is a solution of methanol-acetonitrile (1:1) containing 0.1% formic acid. Flow rate: 0.8 ml / min; column temperature: 40°C; injection volume: 2 μl. The gradient elution program is shown in the table below:

[0068] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 15 30 70 20 30 70 25 70 30 30 70 30

[0069] 3.6.2 Mass spectrometry conditions

[0070] Ion source: electrospray ion source (ESI source); ion source mode: positive ion multiple reaction monitoring mode (MRM); ion source temperature (TEM): 350°C; electrospray voltage (IS): 5500V; curtain gas (CUR): 30psi; spray gas (GS1): 50psi; auxiliary heating gas (GS2): 50psi. The monitoring ion pairs and related parameter settings are shown in the following table:

[0071]

[0072]

[0073] Note: 1. * indicates the recommended quantitative ion; 2. When using different mass spectrometers, the instrument parameters may be different. The mass spectrometer parameters should be optimized to the best before measurement.

[0074] 3.7 Qualitative judgment

[0075] Take the sample solution and the standard solution and measure them under the same analytical conditions. If the sample presents chromatographic peaks of quantitative ion pairs and qualitative ion pairs, the retention time of the characteristic ion peak of the measured component is consistent with the retention time of the mixed reference solution for qualitative detection, and the maximum allowable deviation of the relative abundance ratio of the selected monitoring ion pair and the relative abundance ratio of the monitoring ion pair of the mixed reference solution for screening with equivalent concentration does not exceed the provisions of the following table, then it can be determined that the corresponding component exists in the sample; the maximum allowable deviation of the relative ion abundance ratio for qualitative confirmation is:

[0076]

[0077] 3.8 Quantitative determination

[0078] Take sample solution and standard series solution for analysis, use the series concentration of the component to be measured as the horizontal axis and the peak area of ​​the component to be measured as the vertical axis, perform linear regression, and draw a standard curve. The linear correlation coefficient should be greater than 0.99. Substitute the corresponding quantitative ion pair chromatographic peak area in the sample into the standard curve to calculate the concentration of the component to be measured in the sample, and then calculate the content.

[0079] 3.9 Parallel testing

[0080] According to the above steps, parallel experiments were carried out on the same sample.

[0081] 3.10 Blank test

[0082] Take blank solvent and perform determination according to the above determination conditions and steps.

[0083] 4. System suitability

[0084] The signal-to-noise ratio of each component peak in the chromatogram of the standard solution should be greater than 10.

[0085] 5. Limit of detection and limit of quantification

[0086] The detection limits, quantification limits, detection concentrations and minimum quantification concentrations of lixisenatide, belaglutide, exenatide, semaglutide, liraglutide and telportide in this method are shown in the following table:

[0087]

[0088] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting 6 GLP-1RA drugs. It is characterized in that The following steps are involved: S110, sample solution preparation: take an appropriate amount of sample, add solvent to dissolve or dilute, filter through a 0.22 μm filter membrane, and take the filtrate as the sample solution; S120, preparation of standard solution: weigh appropriate amount of semaglutide, lixisenatide, belaglutide, exenatide, liraglutide and telportide reference substances, add solvent to dissolve and prepare standard stock solution; accurately measure appropriate amount of standard stock solution, dilute with solvent to prepare standard solution or standard series solution; S130, Liquid chromatography conditions: The chromatographic column used was Kinetex C18 (4.6 mm × 150 mm, 2.6 μm), or an equivalent chromatographic column; the mobile phase A was an aqueous solution containing 0.1% formic acid, and the mobile phase B was a solution of methanol-acetonitrile (1:1) containing 0.1% formic acid, with a gradient elution procedure; Mass spectrometry conditions: electrospray ion source (ESI source) was used; the ion source mode was positive ion multiple reaction monitoring mode (MRM), the ion source temperature (TEM) was 350°C; the electrospray voltage (IS) was 5500V; the curtain gas (CUR) was 30psi; the spray gas (GS1) was 50psi; the auxiliary heating gas (GS2) was 50psi; S140, Qualitative determination: Take the sample solution and the standard solution and measure them under the same analytical conditions. If the sample presents chromatographic peaks of quantitative ion pairs and qualitative ion pairs, the retention time of the characteristic ion peak of the measured component is consistent with the retention time of the mixed reference solution for qualitative detection, and the maximum allowable deviation of the relative abundance ratio of the selected monitoring ion pair and the relative abundance ratio of the monitoring ion pair of the mixed reference solution for screening with equivalent concentration does not exceed the provisions of the following table, then it can be determined that the corresponding component exists in the sample; the maximum allowable deviation of the relative ion abundance ratio during qualitative confirmation is:

2. The method for simultaneously detecting 6 GLP-1RA drugs according to claim 1, It is characterized in that The method also includes step S140, quantitative determination: taking sample solutions and standard series solutions for analysis, taking the series concentrations of the components to be measured as the horizontal coordinates and the peak areas of the components to be measured as the vertical coordinates, performing linear regression, and drawing a standard curve, whose linear correlation coefficient should be greater than 0.99, substituting the corresponding quantitative ion pair chromatographic peak area in the sample into the standard curve to calculate the concentration of the components to be measured in the sample, and then calculating the content.

3. The method for simultaneously detecting 6 GLP-1RA drugs according to claim 1, It is characterized in that The specific content of S110 is: S111, preparation of solvent: weigh 1.2g of sodium dihydrogen phosphate, add 900ml of water to dissolve, adjust the pH value to 7.5±0.1 with 1mol / L sodium hydroxide solution, dilute with water to 1000ml, and mix well; S112. Sample processing: a. When the sample is a liquid sample: take 1.0 ml of the sample solution, place it in a 5 ml volumetric flask, dilute it with solvent and make up to the mark, and mix well; b. When the sample is a freeze-dried solid sample: If the sample is provided with a diluent, use the diluent to dissolve the freeze-dried solid sample to prepare a solution. Take 1.0 ml of the solution and place it in a 5 ml volumetric flask. Dilute it with a solvent and make it up to the mark, and mix well. If the sample is not provided with a diluent, use 10 ml of the solvent to dissolve the freeze-dried solid sample to prepare a solution. c. When the sample is a raw material drug sample: weigh about 5 mg of the sample, place it in a 25 ml volumetric flask, dissolve it with solvent and dilute to the mark, and mix well; d. When the sample is other solid samples: take a piece or a pellet of other solid samples, place it in a 100ml volumetric flask, add 75ml of solvent, shake to dissolve, dilute to scale with solvent, shake well, centrifuge at 5000r / min for 10min, and take the supernatant; S113. The prepared solution is filtered through a 0.22 μm filter membrane, and the filtrate is taken as the sample solution.

4. The method for simultaneously detecting 6 GLP-1RA drugs according to claim 1, It is characterized in that In step S130, the monitoring ion pairs of each component and the related parameter settings are shown in the following table: Note: * indicates quantitative ion.

5. The method for simultaneously detecting 6 GLP-1RA drugs according to claim 1, It is characterized in that In the step S130, the flow rate is 0.8 μl / min; the column temperature is 40° C.; and the injection volume is 2 μl.

6. The method for simultaneously detecting 6 GLP-1RA drugs according to claim 1, It is characterized in that The gradient elution program is shown in the following table: 。 7. The method for simultaneously detecting 6 GLP-1RA drugs according to claim 1, It is characterized in that In the step S120, the concentration of the prepared standard solution is qualitatively determined to be a mixed reference solution containing 0.2 mg / ml or 0.3 mg / ml of each component.

8. The method for simultaneously detecting 6 GLP-1RA drugs according to claim 1, It is characterized in that In step S120, the standard series solutions prepared for quantitative determination are mixed reference solutions containing 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, and 0.5 mg / ml of each component.

9. The method for simultaneously detecting 6 GLP-1RA drugs according to claim 3, It is characterized in that In S113, the concentration of the sample solution is 0.2 mg / ml-0.3 mg / ml.

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