CN112858512A - Method for determining impurities of hypoglycemic drug - Google Patents

Abstract

The invention relates to the technical field of drug impurity determination, in particular to a method for determining the determination of impurities of a hypoglycemic drug, which comprises the following steps: s1, preparing reagents and equipment; s2, respectively weighing standard substances; s3, preparing a sample to be detected; s4, detecting the result; by utilizing an advanced SPE solid phase extraction sample processing technology and combining the advantages of high performance liquid chromatography tandem mass spectrometry, 13 hypoglycemic drugs of tolbutamide, tolazamide, glibenclamide, glipizide, gliclazide, gliquidone, glimepiride, phenformin hydrochloride, metformin, rosiglitazone, pioglitazone, repaglinide and nateglinide are selected as target objects, the condition of the chromatographic mass spectrometry is optimized, the sample pretreatment has a good impurity removal effect through SPE, and the detection is more accurate by adopting an MRM mode, so that the application requirements of various industries are met.

Description

Method for determining impurities of hypoglycemic drug

Technical Field

The invention relates to the technical field of drug impurity determination, in particular to a method for determining the determination of impurities of a hypoglycemic drug.

Background

The high-incidence market of diabetes mellitus has more medicine components for reducing blood sugar of western medicines, the harm of excessive medicine taking to human bodies is serious, the accurate determination of the content of the medicines not only plays an important role in the treatment of doctors, but also can control the condition of abuse of the western medicines in the market, and therefore, the method for determining the impurities of the blood sugar-reducing medicines is provided.

Disclosure of Invention

The present invention is directed to a method for confirming the determination of impurities in hypoglycemic agents, which solves the problems of the background art mentioned above.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for confirming the determination of impurities in hypoglycemic drugs comprises the following steps:

s1, preparation of reagents and equipment: water: laboratory first-grade water; methanol: sigma brand chromatographic purity; acetonitrile: sigma brand chromatographic purity; 0.1% formic acid solution: taking 1mL of formic acid, and adding water to a constant volume of 1L; 0.1% acetic acid solution: taking 1mL of acetic acid, and diluting to 1L with water; 0.1% phosphoric acid solution: taking 1mL of phosphoric acid, and adding water to a constant volume of 1L; 5mmol of the active carbon; ammonium acetate solution: weighing 0.39g of ammonium acetate, and fixing the volume to 1L by using water; 2% aqueous formic acid solution: taking 2mL of formic acid, and diluting to 100mL with water; ammonia water: tianjin Kemi European brand chromatogram; methanol/acetonitrile ═ 1: 1: methanol and acetonitrile are mixed and prepared in the same volume; 5% ammoniated methanol/acetonitrile ═ 1: 1: taking 5mL of ammonia water, and mixing the ammonia water with methanol and acetonitrile in the same volume to prepare a solution with constant volume of 100 mL; solid phase extraction column: agilent brand Bond-Elut-Plexa-PCX200mg6 mL; a centrifuge: sigma3K15 high speed refrigerated centrifuge; and (3) vortex: IKA-MS3-basic vortex mixer; ultrasonic: shumei KQ-600DV ultrasonic cleaner; high performance liquid chromatography tandem mass spectrometer: liquid chromatograph of agent-1260 + AB-Science-API-4000 mass spectrometer;

s2, weighing standard tolbutamide 0.0176g, tolazamide 0.0165g, glibenclamide 0.0202g, glipizide 0.0300g, gliclazide 0.0750g, gliquidone 0.0097g, glimepiride 0.0099g, phenformin hydrochloride 0.0140g, metformin 0.0157g, rosiglitazone 0.0083g, pioglitazone 0.0105g, repaglinide 0.0107g and nateglinide 0.0100g respectively;

using chromatographic pure methanol to fix the weighed 13 standard substances in a volumetric flask with 100mL of volume to prepare standard stock solutions with the concentrations of 176 mu g/mL of tolbutamide, 165 mu g/mL of tolazamide, 202 mu g/mL of glibenclamide, 300 mu g/mL of glipizide, 750ug/mL of gliclazide, 97 mu g/mL of gliquidone, 99ug/mL of glimepiride, 140 mu g/mL of phenformin hydrochloride, 157 mu g/mL of metformin, 83 mu g/mL of rosiglitazone, 105 mu g/mL of pioglitazone, 107 mu g/mL of repaglinide and 100 mu g/mL of nateglinide respectively;

respectively sucking 0.568mL of tolbutamide, 0.606mL of tolazamide, 0.495mL of glibenclamide, 0.333mL of glipizide, 0.133mL of gliclazide, 1.031mL of gliquidone, 1.010mL of glimepiride, 0.714mL of phenformin hydrochloride, 0.636mL of metformin, 1.204mL of rosiglitazone, 0.952mL of pioglitazone, 0.934mL of repaglinide and 1mL to 100mL of nateglinide from the stock solution, and diluting the volume with methanol to obtain a mixed standard intermediate solution with the concentration of each standard substance of 1 ug/mL;

s3, preparation of a sample to be tested: selecting an extraction solvent; selection of an extraction mode: selecting a purification mode; establishing a pretreatment method;

and S4, detecting the result.

Preferably, the chromatographic column is an Agilent-ZORBAX-Eclipse-Plus-C18(150mmx4.6mm, 5 μm) chromatographic column; selecting a mobile phase, wherein an organic phase of the mobile phase is acetonitrile, and an acetic acid solution with an inorganic phase of 0.1% is adjusted to pH3.5 by ammonia water; flow rate: adjusting the optimal flow rate to be 0.4 mL/min; column temperature: selecting the optimal column temperature of 35 ℃; sample introduction amount: 5.0. mu.L.

Preferably, the S2 uses chromatographic pure methanol to fix the weighed 13 standard substances in a 100mL volumetric flask, and the prepared standard stock solutions have the concentrations of 176 μ g/mL of tolbutamide, 165 μ g/mL of tolazamide, 202 μ g/mL of glyburide, 300 μ g/mL of glipizide, 750ug/mL of gliclazide, 97 μ g/mL of gliquidone, 99ug/mL of glimepiride, 140 μ g/mL of phenformin hydrochloride, 157 μ g/mL of metformin, 83 μ g/mL of rosiglitazone, 105 μ g/mL of pioglitazone, 107 μ g/mL of repaglinide and 100 μ g/mL of nateglinide, respectively;

0.568mL of tolbutamide, 0.606mL of tolazamide, 0.495mL of glibenclamide, 0.333mL of glipizide, 0.133mL of gliclazide, 1.031mL of gliquidone, 1.010mL of glimepiride, 0.714mL of phenformin hydrochloride, 0.636mL of metformin, 1.204mL of rosiglitazone, 0.952mL of pioglitazone, 0.934mL of repaglinide and 1mL to 100mL of nateglinide volumetric flasks are respectively sucked from the stock solution to be subjected to constant volume by using methanol, so that the mixed standard intermediate solution with the concentration of each standard substance of 1ug/mL is obtained.

Preferably, the S3 extraction solvent selects a solid phase extraction method to remove impurities in the extract.

Preferably, the S3 extraction mode is a mode of firstly whirling and then ultrasonic, so that the extraction is firstly comprehensive and then fine, and 2-3 times of extraction and uniform mixing are added in the ultrasonic process.

Preferably, the S3 purification mode is an SPE solid phase extraction mode.

Preferably, the S3 preprocessing method establishes: accurately weighing 5.0g of sample, placing the sample in a 50mL centrifuge tube, adding 30mL of methanol, performing vortex for 2min, performing ultrasonic extraction for 15min, taking out the sample and uniformly mixing the sample, performing centrifugation for 5min at 8000r/min, transferring supernatant into a 100mL centrifuge tube, adding 20mL of methanol, repeating the extraction steps, combining uniformly mixed extracting solutions in a 100mL centrifuge tube, extracting from 50mL, accurately absorbing 10mL of the extracting solution, and adjusting the pH value to 5.0 by using 2% phosphoric acid to serve as a sample solution to be purified;

selection of the BondElutPlexaPCX200mg6mL solid phase extraction cartridge: after activating the column with 5mL of methanol and 5mL of water, the sample to be purified was added, and the column was purified using 5mL of 2% formic acid water and 5mL of methanol/acetonitrile 1: 1 eluting the column, and finally eluting the column with 5% ammoniated methanol/acetonitrile 1: and (3) collecting eluent after elution of 15mL, blowing nitrogen to be dry, using 1mL of mobile phase to fix volume, and then using a liquid chromatogram tandem mass spectrometer to carry out machine detection.

Compared with the prior art, the invention has the beneficial effects that:

in the invention, by utilizing an advanced SPE solid phase extraction sample processing technology and combining the advantages of high performance liquid chromatography tandem mass spectrometry, 13 hypoglycemic drugs such as tolbutamide, tolazamide, glibenclamide, glipizide, gliquidone, glimepiride, phenformin hydrochloride, metformin, rosiglitazone, pioglitazone, repaglinide and nateglinide are selected as target objects, the condition of the chromatographic mass spectrometry is optimized, and the sample pretreatment has good impurity removal effect through SPE and is more accurate in detection by adopting an MRM mode, thereby meeting the application requirements of various industries.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative work based on the embodiments of the present invention belong to the protection scope of the present invention.

The invention provides a technical scheme that:

a method for confirming the determination of impurities in hypoglycemic drugs comprises the following steps:

s1, preparation of reagents and equipment: water: laboratory first-grade water; methanol: sigma brand chromatographic purity; acetonitrile: sigma brand chromatographic purity; 0.1% formic acid solution: taking 1mL of formic acid, and adding water to a constant volume of 1L; 0.1% acetic acid solution: taking 1mL of acetic acid, and diluting to 1L with water; 0.1% phosphoric acid solution: taking 1mL of phosphoric acid, and adding water to a constant volume of 1L; 5mmol of the active carbon; ammonium acetate solution: weighing 0.39g of ammonium acetate, and fixing the volume to 1L by using water; 2% aqueous formic acid solution: taking 2mL of formic acid, and diluting to 100mL with water; ammonia water: tianjin Kemi European brand chromatogram; methanol/acetonitrile ═ 1: 1: methanol and acetonitrile are mixed and prepared in the same volume; 5% ammoniated methanol/acetonitrile ═ 1: 1: taking 5mL of ammonia water, and mixing the ammonia water with methanol and acetonitrile in the same volume to prepare a solution with constant volume of 100 mL; solid phase extraction column: agilent brand Bond-Elut-Plexa-PCX200mg6 mL; a centrifuge: sigma3K15 high speed refrigerated centrifuge; and (3) vortex: IKA-MS3-basic vortex mixer; ultrasonic: shumei KQ-600DV ultrasonic cleaner; high performance liquid chromatography tandem mass spectrometer: liquid chromatograph of agent-1260 + AB-Science-API-4000 mass spectrometer;

s2, weighing standard tolbutamide 0.0176g, tolazamide 0.0165g, glibenclamide 0.0202g, glipizide 0.0300g, gliclazide 0.0750g, gliquidone 0.0097g, glimepiride 0.0099g, phenformin hydrochloride 0.0140g, metformin 0.0157g, rosiglitazone 0.0083g, pioglitazone 0.0105g, repaglinide 0.0107g and nateglinide 0.0100g respectively;

using chromatographic pure methanol to fix the weighed 13 standard substances in a volumetric flask with 100mL of volume to prepare standard stock solutions with the concentrations of 176 mu g/mL of tolbutamide, 165 mu g/mL of tolazamide, 202 mu g/mL of glibenclamide, 300 mu g/mL of glipizide, 750ug/mL of gliclazide, 97 mu g/mL of gliquidone, 99ug/mL of glimepiride, 140 mu g/mL of phenformin hydrochloride, 157 mu g/mL of metformin, 83 mu g/mL of rosiglitazone, 105 mu g/mL of pioglitazone, 107 mu g/mL of repaglinide and 100 mu g/mL of nateglinide respectively;

respectively sucking 0.568mL of tolbutamide, 0.606mL of tolazamide, 0.495mL of glibenclamide, 0.333mL of glipizide, 0.133mL of gliclazide, 1.031mL of gliquidone, 1.010mL of glimepiride, 0.714mL of phenformin hydrochloride, 0.636mL of metformin, 1.204mL of rosiglitazone, 0.952mL of pioglitazone, 0.934mL of repaglinide and 1mL to 100mL of nateglinide from the stock solution, and diluting the volume with methanol to obtain a mixed standard intermediate solution with the concentration of each standard substance of 1 ug/mL;

s3, preparation of a sample to be tested: selecting an extraction solvent; selection of an extraction mode: selecting a purification mode; establishing a pretreatment method;

and S4, detecting the result.

The chromatographic column Agilent-ZORBAX-Eclipse-Plus-C18(150mmx4.6mm, 5 μm) chromatographic column; selecting a mobile phase, wherein an organic phase of the mobile phase is acetonitrile, and an acetic acid solution with an inorganic phase of 0.1% is adjusted to pH3.5 by ammonia water; flow rate: adjusting the optimal flow rate to be 0.4 mL/min; column temperature: selecting the optimal column temperature of 35 ℃; sample introduction amount: 5.0. mu.L.

The S2 uses chromatographic pure methanol to fix the volume of 13 weighed standard substances in a 100mL volumetric flask, and the concentrations of prepared standard stock solutions are 176 mu g/mL of tolbutamide, 165 mu g/mL of tolazamide, 202 mu g/mL of glibenclamide, 300 mu g/mL of glipizide, 750ug/mL of gliclazide, 97 mu g/mL of gliquidone, 99ug/mL of glimepiride, 140 mu g/mL of phenformin hydrochloride, 157 mu g/mL of metformin, 83 mu g/mL of rosiglitazone, 105 mu g/mL of pioglitazone, 107 mu g/mL of repaglinide and 100 mu g/mL of nateglinide respectively;

0.568mL of tolbutamide, 0.606mL of tolazamide, 0.495mL of glibenclamide, 0.333mL of glipizide, 0.133mL of gliclazide, 1.031mL of gliquidone, 1.010mL of glimepiride, 0.714mL of phenformin hydrochloride, 0.636mL of metformin, 1.204mL of rosiglitazone, 0.952mL of pioglitazone, 0.934mL of repaglinide and 1mL to 100mL of nateglinide volumetric flasks are respectively sucked from the stock solution to be subjected to constant volume by using methanol, so that the mixed standard intermediate solution with the concentration of each standard substance of 1ug/mL is obtained.

And the S3 extraction solution selects a solid phase extraction mode to remove impurities in the extracting solution.

The S3 extraction mode is a mode of firstly whirling and then ultrasonic, so that the extraction is firstly comprehensive and then fine, and 2-3 times of extraction and uniform mixing are added in the ultrasonic process.

And the S3 purification mode selects an SPE solid phase extraction mode.

The S3 preprocessing method establishes: accurately weighing 5.0g of sample, placing the sample in a 50mL centrifuge tube, adding 30mL of methanol, performing vortex for 2min, performing ultrasonic extraction for 15min, taking out the sample and uniformly mixing the sample, performing centrifugation for 5min at 8000r/min, transferring supernatant into a 100mL centrifuge tube, adding 20mL of methanol, repeating the extraction steps, combining uniformly mixed extracting solutions in a 100mL centrifuge tube, extracting from 50mL, accurately absorbing 10mL of the extracting solution, and adjusting the pH value to 5.0 by using 2% phosphoric acid to serve as a sample solution to be purified;

selection of the BondElutPlexaPCX200mg6mL solid phase extraction cartridge: after activating the column with 5mL of methanol and 5mL of water, the sample to be purified was added, and the column was purified using 5mL of 2% formic acid water and 5mL of methanol/acetonitrile 1: 1 eluting the column, and finally eluting the column with 5% ammoniated methanol/acetonitrile 1: and (3) collecting eluent after elution of 15mL, blowing nitrogen to be dry, using 1mL of mobile phase to fix volume, and then using a liquid chromatogram tandem mass spectrometer to carry out machine detection.

Example (b): preparing reagents and equipment: water: laboratory first-grade water; methanol: sigma brand chromatographic purity; acetonitrile: sigma brand chromatographic purity; 0.1% formic acid solution: taking 1mL of formic acid, and adding water to a constant volume of 1L; 0.1% acetic acid solution: taking 1mL of acetic acid, and diluting to 1L with water; 0.1% phosphoric acid solution: taking 1mL of phosphoric acid, and adding water to a constant volume of 1L; 5mmol of the active carbon; ammonium acetate solution: weighing 0.39g of ammonium acetate, and fixing the volume to 1L by using water; 2% aqueous formic acid solution: taking 2mL of formic acid, and diluting to 100mL with water; ammonia water: tianjin Kemi European brand chromatogram; methanol/acetonitrile ═ 1: 1: methanol and acetonitrile are mixed and prepared in the same volume; 5% ammoniated methanol/acetonitrile ═ 1: 1: taking 5mL of ammonia water, and mixing the ammonia water with methanol and acetonitrile in the same volume to prepare a solution with constant volume of 100 mL; solid phase extraction column: agilent brand Bond-Elut-Plexa-PCX200mg6 mL; a centrifuge: sigma3K15 high speed refrigerated centrifuge; and (3) vortex: IKA-MS3-basic vortex mixer; ultrasonic: shumei KQ-600DV ultrasonic cleaner; high performance liquid chromatography tandem mass spectrometer: liquid chromatograph of agent-1260 + AB-Science-API-4000 mass spectrometer; the column was an Agilent-ZORBAX-Eclipse-Plus-C18(150mmx4.6mm, 5 μm) column; selecting a mobile phase, wherein an organic phase of the mobile phase is acetonitrile, and an acetic acid solution with an inorganic phase of 0.1% is adjusted to pH3.5 by ammonia water; gradient elution: after the time and the proportion of the organic-inorganic mobile phase are repeatedly adjusted, the characteristic ion peaks of the 13 medicines can be efficiently presented within 7min by using the following gradient elution mode, and the next injection sample is injected into the equilibrium column environment in the last three minutes:

0-1min 0.1% acetic acid solution of 50% acetonitrile and 50% ammonia water with pH 3.5;

50-85% acetonitrile and 50-15% ammonia water for 1-4min to adjust the pH value to be 0.1% acetic acid solution with 3.5;

4-7min 0.1% acetic acid solution containing 85% acetonitrile and 15% ammonia water at pH 3.5;

85-50% acetonitrile for 7-10min, and 0.1% acetic acid solution with pH3.5 adjusted by 15-50% ammonia water;

flow rate: adjusting the optimal flow rate to be 0.4 mL/min; column temperature: selecting the optimal column temperature of 35 ℃; sample introduction amount: 5.0 mu L; respectively weighing 0.0176g of standard tolbutamide, 0.0165g of tolazamide, 0.0202g of glibenclamide, 0.0300g of glipizide, 0.0750g of gliclazide, 0.0097g of gliquidone, 0.0099g of glimepiride, 0.0140g of phenformin hydrochloride, 0.0157g of metformin, 0.0083g of rosiglitazone, 0.0105g of pioglitazone, 0.0107g of repaglinide and 0.0100g of nateglinide; using chromatographic pure methanol to fix the weighed 13 standard substances in a volumetric flask with 100mL of volume to prepare standard stock solutions with the concentrations of 176 mu g/mL of tolbutamide, 165 mu g/mL of tolazamide, 202 mu g/mL of glibenclamide, 300 mu g/mL of glipizide, 750ug/mL of gliclazide, 97 mu g/mL of gliquidone, 99ug/mL of glimepiride, 140 mu g/mL of phenformin hydrochloride, 157 mu g/mL of metformin, 83 mu g/mL of rosiglitazone, 105 mu g/mL of pioglitazone, 107 mu g/mL of repaglinide and 100 mu g/mL of nateglinide respectively; respectively sucking 0.568mL of tolbutamide, 0.606mL of tolazamide, 0.495mL of glibenclamide, 0.333mL of glipizide, 0.133mL of gliclazide, 1.031mL of gliquidone, 1.010mL of glimepiride, 0.714mL of phenformin hydrochloride, 0.636mL of metformin, 1.204mL of rosiglitazone, 0.952mL of pioglitazone, 0.934mL of repaglinide and 1mL to 100mL of nateglinide from the stock solution, and diluting the volume with methanol to obtain a mixed standard intermediate solution with the concentration of each standard substance of 1 ug/mL; preparation of a sample to be tested: selecting an extraction solvent; selection of an extraction mode: selecting a purification mode; establishing a pretreatment method; selecting a mode of firstly whirling and then ultrasonically, enabling the extraction to be complete and fine, taking out and uniformly mixing for 2-3 times in the ultrasonic process, selecting an SPE solid phase extraction mode in a purification mode, accurately weighing 5.0g of sample, placing the sample in a 50mL centrifuge tube, adding 30mL of methanol, whirling for 2min, ultrasonically extracting for 15min, taking out and uniformly mixing in the process, centrifuging for 5min at 8000r/min, transferring the supernatant into a 100mL centrifuge tube, adding 20mL of methanol, repeating the extraction step, combining uniformly mixed extracting solutions in the 100mL centrifuge tube, extracting from 50mL, accurately absorbing 10mL of the solution, adjusting the pH value to 5.0 by using 2% phosphoric acid to serve as a sample solution to be purified; selection of the BondElutPlexaPCX200mg6mL solid phase extraction cartridge: after activating the column with 5mL of methanol and 5mL of water, the sample to be purified was added, and the column was purified using 5mL of 2% formic acid water and 5mL of methanol/acetonitrile 1: 1 eluting the column, and finally eluting the column with 5% ammoniated methanol/acetonitrile 1: 15mL of eluent is collected after elution, nitrogen is blown to be dry, after 1mL of mobile phase constant volume is used, a liquid chromatography tandem mass spectrometer is used for detecting on a computer, 3 medicines and food eaten by 3 diabetic patients are extracted from the market, the detection is carried out by the method, the obtained results show that metformin is detected in the hypoglycemic medicines, some of the hypoglycemic medicines also contain phenformin or glyburide, and the food eaten by the diabetic patients is basically not detected, so that the method can be applied to the detection of 13 hypoglycemic medicines in different matrixes.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (7)

1. A method for confirming the determination of impurities in hypoglycemic drugs is characterized by comprising the following steps:

s1, preparation of reagents and equipment: water: laboratory first-grade water; methanol: sigma brand chromatographic purity; acetonitrile: sigma brand chromatographic purity; 0.1% formic acid solution: taking 1mL of formic acid, and adding water to a constant volume of 1L; 0.1% acetic acid solution: taking 1mL of acetic acid, and diluting to 1L with water; 0.1% phosphoric acid solution: taking 1mL of phosphoric acid, and adding water to a constant volume of 1L; 5mmol of the active carbon; ammonium acetate solution: weighing 0.39g of ammonium acetate, and fixing the volume to 1L by using water; 2% aqueous formic acid solution: taking 2mL of formic acid, and diluting to 100mL with water; ammonia water: tianjin Kemi European brand chromatogram; methanol/acetonitrile ═ 1: 1: methanol and acetonitrile are mixed and prepared in the same volume; 5% ammoniated methanol/acetonitrile ═ 1: 1: taking 5mL of ammonia water, and mixing the ammonia water with methanol and acetonitrile in the same volume to prepare a solution with constant volume of 100 mL; solid phase extraction column: agilent brand Bond-Elut-Plexa-PCX200mg6 mL; a centrifuge: sigma3K15 high speed refrigerated centrifuge; and (3) vortex: IKA-MS3-basic vortex mixer; ultrasonic: shumei KQ-600DV ultrasonic cleaner; high performance liquid chromatography tandem mass spectrometer: liquid chromatograph of agent-1260 + AB-Science-API-4000 mass spectrometer;

s2, weighing standard tolbutamide 0.0176g, tolazamide 0.0165g, glibenclamide 0.0202g, glipizide 0.0300g, gliclazide 0.0750g, gliquidone 0.0097g, glimepiride 0.0099g, phenformin hydrochloride 0.0140g, metformin 0.0157g, rosiglitazone 0.0083g, pioglitazone 0.0105g, repaglinide 0.0107g and nateglinide 0.0100g respectively;

using chromatographic pure methanol to fix the weighed 13 standard substances in a volumetric flask with 100mL of volume to prepare standard stock solutions with the concentrations of 176 mu g/mL of tolbutamide, 165 mu g/mL of tolazamide, 202 mu g/mL of glibenclamide, 300 mu g/mL of glipizide, 750ug/mL of gliclazide, 97 mu g/mL of gliquidone, 99ug/mL of glimepiride, 140 mu g/mL of phenformin hydrochloride, 157 mu g/mL of metformin, 83 mu g/mL of rosiglitazone, 105 mu g/mL of pioglitazone, 107 mu g/mL of repaglinide and 100 mu g/mL of nateglinide respectively;

respectively sucking 0.568mL of tolbutamide, 0.606mL of tolazamide, 0.495mL of glibenclamide, 0.333mL of glipizide, 0.133mL of gliclazide, 1.031mL of gliquidone, 1.010mL of glimepiride, 0.714mL of phenformin hydrochloride, 0.636mL of metformin, 1.204mL of rosiglitazone, 0.952mL of pioglitazone, 0.934mL of repaglinide and 1mL to 100mL of nateglinide from the stock solution, and diluting the volume with methanol to obtain a mixed standard intermediate solution with the concentration of each standard substance of 1 ug/mL;

s3, preparation of a sample to be tested: selecting an extraction solvent; selection of an extraction mode: selecting a purification mode; establishing a pretreatment method;

and S4, detecting the result.

2. The method for confirming the determination of impurities in a hypoglycemic drug according to claim 1, wherein the chromatographic column uses an Agilent-ZORBAX-Eclipse-Plus-C18(150mmx4.6mm, 5 μm) chromatographic column; selecting a mobile phase, wherein an organic phase of the mobile phase is acetonitrile, and an acetic acid solution with an inorganic phase of 0.1% is adjusted to pH3.5 by ammonia water; flow rate: adjusting the optimal flow rate to be 0.4 mL/min; column temperature: selecting the optimal column temperature of 35 ℃; sample introduction amount: 5.0. mu.L.

3. The method for confirming the determination of impurities in hypoglycemic drugs according to claim 1, wherein said S2 uses chromatographically pure methanol to fix the weighed 13 standard substances in a 100mL volumetric flask, and the concentrations of the prepared standard stock solutions are 176 μ g/mL tolbutamide, 165 μ g/mL tolazamide, 202 μ g/mL glyburide, 300 μ g/mL glipizide, 750ug/mL gliclazide, 97 μ g/mL gliquidone, 99ug/mL glimepiride, 140 μ g/mL phenformin hydrochloride, 157 μ g/mL metformin, 83 μ g/mL rosiglitazone, 105 μ g/mL pioglitazone, 107 μ g/mL repaglinide, and 100 μ g/mL nateglinide, respectively;

0.568mL of tolbutamide, 0.606mL of tolazamide, 0.495mL of glibenclamide, 0.333mL of glipizide, 0.133mL of gliclazide, 1.031mL of gliquidone, 1.010mL of glimepiride, 0.714mL of phenformin hydrochloride, 0.636mL of metformin, 1.204mL of rosiglitazone, 0.952mL of pioglitazone, 0.934mL of repaglinide and 1mL to 100mL of nateglinide volumetric flasks are respectively sucked from the stock solution to be subjected to constant volume by using methanol, so that the mixed standard intermediate solution with the concentration of each standard substance of 1ug/mL is obtained.

4. The method for confirming the determination of impurities in hypoglycemic drugs according to claim 1, wherein the S3 extraction solution selects a solid phase extraction method to remove impurities in the extraction solution.

5. The method for determining impurities in hypoglycemic drugs according to claim 1, wherein the S3 extraction method is a vortex-followed ultrasound method, so that the extraction is complete and fine, and the extraction is performed 2-3 times during the ultrasound process.

6. The method for confirming the determination of impurities in hypoglycemic drugs according to claim 1, wherein the S3 purification mode is SPE solid phase extraction mode.

7. The method for confirming the determination of impurities in hypoglycemic drugs according to claim 1, wherein the pretreatment method of S3 is to establish: accurately weighing 5.0g of sample, placing the sample in a 50mL centrifuge tube, adding 30mL of methanol, performing vortex for 2min, performing ultrasonic extraction for 15min, taking out the sample and uniformly mixing the sample, performing centrifugation for 5min at 8000r/min, transferring supernatant into a 100mL centrifuge tube, adding 20mL of methanol, repeating the extraction steps, combining uniformly mixed extracting solutions in a 100mL centrifuge tube, extracting from 50mL, accurately absorbing 10mL of the extracting solution, and adjusting the pH value to 5.0 by using 2% phosphoric acid to serve as a sample solution to be purified;

selection of the BondElutPlexaPCX200mg6mL solid phase extraction cartridge: after activating the column with 5mL of methanol and 5mL of water, the sample to be purified was added, and the column was purified using 5mL of 2% formic acid water and 5mL of methanol/acetonitrile 1: 1 eluting the column, and finally eluting the column with 5% ammoniated methanol/acetonitrile 1: and (3) collecting eluent after elution of 15mL, blowing nitrogen to be dry, using 1mL of mobile phase to fix volume, and then using a liquid chromatogram tandem mass spectrometer to carry out machine detection.