Method for determining content of phenol and m-cresol in insulin preparation based on GC-MS / MS

Abstract

The invention relates to the field of pharmaceutical analysis, in particular to a method for determining the content of phenol and m-cresol in an insulin preparation based on GC-MS / MS (Gas Chromatography-Mass Spectrometry / Mass Spectrometry). The method comprises the following steps: (1) precisely measuring human insulin injection or insulin glargine injection, putting into a measuring flask, diluting by 100 times with a methanol aqueous solution, uniformly shaking, precisely measuring, putting into the measuring flask, diluting by 10 times with the methanol aqueous solution, and taking as a test solution; (2) a quartz capillary column is adopted, the column temperature is kept at 200 DEG C for 5 min, the sample size is 1 microliter, the split ratio is 10: 1, and carrier gas is inert gas; (3) an ionization source is an electron bombardment ion source, the energy is 70 eV, the temperature of the ion source is 200 DEG C, the interface temperature is 250 DEG C, the solvent delay is 1 min, the mass scanning range is 45-500 m / z, the electron multiplication voltage is 3.0 kV, the collision gas is inert gas, and multi-reaction monitoring MRM and full scanning Scan are collected at the same time. The specificity, the linear range, the accuracy, the repeatability, the stability and the durability of the method are superior to those of the prior art.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for determining the contents of phenol and meta-cresol in insulin preparations based on GC-MS / MS. Background Art

[0002] Insulin preparations contain phenol or m-cresol, which mainly acts as an antibacterial agent. At the same time, phenol also has the function of maintaining the stability of the spatial structure of insulin. After subcutaneous injection of insulin, phenol diffuses rapidly, which is conducive to the aggregation of insulin into dimers and monomers, making it easier to enter the blood circulation to play a role. Too much or too little phenol and m-cresol are not good. Too much will be toxic to the veins, and a small amount will not play an antibacterial role. Phenol can directly poison the human body through contact with the skin and mucous membranes. It can react chemically with proteins in the cell protoplasm to cause cell inactivation. It also has strong irritation and corrosion effects. Excessive concentration may cause adverse reactions such as skin allergies. Excessive m-cresol content can cause protein aggregation, which is harmful to the human body. It can be seen from this that the content of phenol and m-cresol must be strictly controlled. The statutory standards for phenol and m-cresol in the three insulin preparations of the 2020 edition of the "Chinese Pharmacopoeia" are titration and UV-visible spectrophotometry, respectively, and it is not possible to measure both at the same time. The titration method for determining the content of phenol-containing preparations is complicated and time-consuming, and it is difficult to judge the end point color change; the reagents used in the UV-visible spectrophotometry method are cumbersome and time-consuming to prepare, have high requirements for reagents, and auxiliary materials will interfere with the detection of meta-cresol content.

[0003] GC-MS / MS technology has the characteristics of high sensitivity, strong separation ability, rapid and accurate method, and has been widely used in drug component analysis and quality evaluation. This study established a GC-MS / MS method for the simultaneous determination of phenol and meta-cresol content, and verified the method's specificity, linear range, accuracy, repeatability, stability, and durability. This method has the advantages of high separation efficiency, low environmental pollution, diverse separation modes, high automation, low sample and reagent consumption, low sample purity requirements, and the ability to determine two components at the same time, which provides technical guidance for further controlling the quality of the preparation and ensuring its safety and effectiveness.

[0004] Currently, there is no report on a method for determining the content of phenol and m-cresol in insulin preparations based on GC-MS / MS. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a method for determining the content of phenol and m-cresol in insulin preparations based on GC-MS / MS.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for determining the content of phenol and meta-cresol in an insulin preparation based on GC-MS / MS, the method comprising the following steps:

[0008] (1) Accurately measure human insulin injection or glargine insulin injection into a volumetric bottle, dilute it 100 times with methanol-water solution, shake well, accurately measure again into a volumetric bottle, dilute it 10 times with methanol-water solution as the test solution;

[0009] (2) A quartz capillary column was used, the column temperature was maintained at 200°C for 5 min, the injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was an inert gas;

[0010] (3) The ionization source was an electron bombardment ion source with an energy of 70 eV, an ion source temperature of 200 °C, an interface temperature of 250 °C, a solvent delay of 1 min, a mass scan range of 45-500 m / z, an electron multiplier voltage of 3.0 kV, an inert gas as the collision gas, and multiple reaction monitoring (MRM) and full scan were collected simultaneously.

[0011] Preferably, the volume concentration of methanol in the methanol aqueous solution in step (1) is 50%.

[0012] Preferably, the quartz capillary column in step (2) has a specification of 30 m×0.25 mm, 0.50 μm Agilent DB-WAXETR.

[0013] Preferably, in step (2), the column temperature is programmed to start at 150°C, maintained for 10 min, and finally increased to 200°C at a rate of 10°C / min and maintained for 5 min.

[0014] Preferably, in step (2), the inert gas is nitrogen with a purity of 99.99%.

[0015] Preferably, in step (3), the inert gas is argon with a purity of 99.99%.

[0016] Preferably, the acquisition parameters of MRM in step (3) are: the quantitative ion pair of phenol is m / z94→66 (CE: 15eV), and the qualitative ion pair is m / z94→55 (CE: 18eV); the quantitative ion pair of m-cresol is m / z108→79 (CE: 15eV), and the qualitative ion pair is m / z108→77 (CE: 24eV).

[0017] Compared with the prior art, the method of the present invention has the following advantages:

[0018] 1. The present invention establishes a method for simultaneously determining phenol and m-cresol in insulin preparations using GC-MS / MS technology, and the specificity, linear range, accuracy, repeatability, stability and durability of the method are superior to those of the prior art.

[0019] 2. The present invention establishes a method for simultaneously determining phenol and meta-cresol in insulin preparations using GC-MS / MS technology. This method has the advantages of high separation efficiency, low environmental pollution, diverse separation modes, high degree of automation, low sample and reagent consumption, low sample purity requirements, and the ability to simultaneously determine two components. This method provides technical guidance for further controlling the quality of the preparation and ensuring its safety and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the total ion current chromatogram of m-cresol reference solution.

[0021] Figure 2 This is the total ion current chromatogram of the phenol reference solution.

[0022] Figure 3 The total ion current chromatogram of the mixed reference solution.

[0023] Figure 4 is the total ion current chromatogram of the blank solvent.

[0024] Figure 5 This is the total ion current chromatogram of the human insulin injection sample produced by manufacturer A.

[0025] Figure 6 This is the total ion current chromatogram of the human insulin injection sample produced by manufacturer B.

[0026] Figure 7 This is the total ion current chromatogram of the insulin glargine injection sample produced by manufacturer C.

[0027] Figure 8 This is the MRM diagram of the human insulin injection sample produced by manufacturer A.

[0028] Fig. 9 This is the MRM diagram of the human insulin injection sample produced by manufacturer B.

[0029] Fig.10 This is the MRM diagram of the insulin glargine injection sample produced by manufacturer C. DETAILED DESCRIPTION

[0030] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0031] Example 1

[0032] 1. Experimental Materials

[0033] 1.1 Instruments and Equipment

[0034] Shimadzu Nexis TQ8040 series GC-MS / MS coupling instrument (Shimadzu Corporation, Japan); XPR205DU electronic balance (d=0.01 mg, METTLERTOLEDO Company).

[0035] 1.2 Reagents and test drugs

[0036] Reference substances: phenol (China Food and Drug Inspection Institute, batch number: 1-F-18-2, content 99.1%), m-cresol (China Food and Drug Inspection Institute, batch number: 101153-201602, 99.3%). Methanol (Merck, Germany, HPLC ≥ 99.9, batch number F22MAJ201), water is ultrapure water.

[0037] Human insulin injection (the company is Company A, batch numbers are 202208B018, 202304B010, and 202310B008; the company is Company B, batch numbers are 202303016, 202304017, and 20231008), and glargine insulin injection (the company is Company C, batch numbers are GP202206008, GP202307001, and GP202310002).

[0038] 2. Experimental Methods

[0039] 2.1 Reference solution

[0040] Take 10 mg of phenol, weigh it accurately, and place it in a 20 mL volumetric flask. Take 27 mg of m-cresol, weigh it accurately, and place it in a 50 mL volumetric flask. Dissolve it with 50% methanol aqueous solution and dilute it to the mark. Shake it well to use as a single reference substance stock solution. Accurately weigh 2.0 ml of phenol reference substance stock solution and 2.0 ml of m-cresol reference substance stock solution, place them in the same 50 mL volumetric flask, dilute them to the mark with 50% methanol aqueous solution, and shake it well to use as a mixed reference substance solution.

[0041] 2.2 Test solution and blank solution

[0042] Accurately measure 0.5 ml of human insulin injection and glargine insulin injection respectively and place them in different 50 ml volumetric bottles, dilute to the mark with 50% methanol aqueous solution, shake well, then accurately measure 1 ml and place it in a 10 ml volumetric bottle, dilute to the mark with 50% methanol aqueous solution as the test solution. Take 50% methanol aqueous solution as the blank solution.

[0043] 2.3 GC-MS / MS conditions

[0044] A 30m×0.25mm, 0.50μm Agilent DB-WAXETR quartz capillary column was used. The column temperature was raised to 150°C for 10 min, then raised to 200°C at a rate of 10°C / min for 5 min. The injection volume was 1μL, the split ratio was 10:1, and the carrier gas was 99.99% pure nitrogen.

[0045] The ionization source was an electron bombardment ion source with an energy of 70 eV, an ion source temperature of 200°C, an interface temperature of 250°C, a solvent delay of 1 min, a mass scanning range of 45-500 m / z, an electron multiplier voltage of 3.0 kV, and a collision gas of argon with a purity of 99.99%. Multiple reaction monitoring MRM and full scan Scan were collected simultaneously.

[0046] MRM acquisition parameters: the quantitative ion pair of phenol was m / z94→66 (CE: 15 eV), and the qualitative ion pair was m / z94→55 (CE: 18 eV); the quantitative ion pair of m-cresol was m / z108→79 (CE: 15 eV), and the qualitative ion pair was m / z108→77 (CE: 24 eV).

[0047] 3. Experimental Results

[0048] 3.1 System Applicability

[0049] Take the reference solution, the mixed reference solution, the test solution and the blank solution respectively and measure them according to the chromatographic conditions under "2.3" and record the chromatogram. The retention time of phenol is 13.5min and the retention time of m-cresol is 15.0min. The separation degree of the two is good, and the theoretical plate number is greater than 5000. The blank solution does not interfere with the determination. The results are shown in Figure 1 .

[0050] 3.2 Methodological validation

[0051] Linear relationship investigation: Accurately measure 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 of the mixed reference solution under "2.1" and place them in different 10ml volumetric bottles, add 50% methanol aqueous solution to dilute to the scale, as a series of standard curve solutions, and measure according to the chromatographic conditions under "2.3". Draw the standard curve with mass concentration as the horizontal axis and peak area as the vertical axis, perform linear regression, and calculate the regression equation. Results Phenol has a good linear relationship in the range of 0.11-21.97μg / ml, and the linear equation is y=1522802.9x+61332.5 (r=0.9998); meta-cresol has a good linear relationship in the range of 0.10-9.84μg / ml, and the equation is y=833321.0x+73196.5 (r=0.9997).

[0052] Precision test: Take the mixed reference solution under "2.1" and inject it 6 times continuously according to the chromatographic conditions under "2.3" to calculate the RSD of the peak areas of phenol and m-cresol. The RSD of phenol and m-cresol were 1.8% and 0.6% respectively, indicating that the instrument is stable and the method has good precision.

[0053] Repeatability test: Take the same batch of human insulin injection (Company A, batch number 202208B018, containing only meta-cresol) and prepare 6 test solutions at the same time according to the method under "2.2". According to the chromatographic conditions under "2.3", the sample was injected and measured. The RSD of the meta-cresol content was 0.7%. Take the same batch of human insulin injection (Company B, batch number 202303016, containing only phenol) and prepare 6 test solutions at the same time according to the method under "2.2". According to the chromatographic conditions under "2.3", the sample was injected and measured. The RSD of phenol was 1.1%. It shows that the repeatability of the method is good.

[0054] Sample recovery test: Accurately measure 0.5 ml of human insulin injection (Company A, batch number 202208B018) and place it in a 50 ml volumetric flask, dilute it to the mark with 50% methanol aqueous solution, shake well, and then accurately measure 0.5 ml and place it in a 10 ml volumetric flask, accurately add 1.0 ml of the mixed reference solution under "2.1", dilute it to the mark with 50% methanol, prepare 6 portions in the same way, as recovery solutions 1 to 6, and measure them according to the chromatographic conditions under "2.3", calculate the recovery and relative standard deviation (RSD), and verify the accuracy of the method. Results The average sample recovery of phenol was 98.0%, and the RSD was 1.5%; the average sample recovery of m-cresol was 97.4%, and the RSD was 1.4%.

[0055] Solution stability test: Take the recovery solution under the "addition recovery test" and place it at room temperature for 0, 2, 4, 6, 8, and 12 hours, and calculate the RSD of the peak area of ​​phenol and m-cresol to verify the stability of the method. The results showed that the RSD of the peak area of ​​phenol and m-cresol was 1.5% and 1.4%, respectively, indicating that the stability of the test solution within 12 hours was qualitatively good.

[0056] Detection limit and quantification limit The mixed reference solution under "2.1" was diluted step by step, and the lower limit of quantification of the method was determined when the signal-to-noise ratio S / N = 10, and the detection limit of the method was determined when S / N = 3 to verify the sensitivity of the method. The results showed that the detection limit of phenol was 0.14ng / ml and the quantification limit was 0.45ng / ml; the detection limit of m-cresol was 0.067ng / ml and the quantification limit was 0.22ng / ml, indicating that the method has high sensitivity.

[0057] 3.3 Sample content determination

[0058] Take samples of 9 batches of 2 varieties of human insulin injection and glargine insulin injection, prepare the test solution according to the method under "2.2", inject and measure according to the chromatographic conditions under "2.3", and calculate the contents of phenol and m-cresol. The results are shown in Table 1.

[0059] Table 1 Sample measurement results

[0060]

[0061] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for determining the content of phenol and m-cresol in insulin preparations based on GC-MS / MS, characterized in that: The method comprises the following steps: (1) Accurately measure human insulin injection or glargine insulin injection into a volumetric bottle, dilute it 100 times with methanol-water solution, shake well, accurately measure again into a volumetric bottle, dilute it 10 times with methanol-water solution as the test solution; (2) A quartz capillary column was used, the column temperature was maintained at 200°C for 5 min, the injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was an inert gas; (3) The ionization source was an electron bombardment ion source with an energy of 70 eV, an ion source temperature of 200 °C, an interface temperature of 250 °C, a solvent delay of 1 min, a mass scan range of 45-500 m / z, an electron multiplier voltage of 3.0 kV, an inert gas as the collision gas, and multiple reaction monitoring (MRM) and full scan were collected simultaneously.

2. The method according to claim 1, characterized in that The volume concentration of methanol in the methanol aqueous solution in step (1) is 50%.

3. The method according to claim 1, characterized in that The specification of the quartz capillary column in step (2) is 30m×0.25mm, 0.50μm Agilent DB-WAXETR.

4. The method according to claim 1, characterized in that In the step (2), the column temperature is programmed to start at 150°C, maintained for 10 min, and finally raised to 200°C at a rate of 10°C / min and maintained for 5 min.

5. The method according to claim 1, characterized in that In the step (2), the inert gas is nitrogen with a purity of 99.99%.

6. The method according to claim 1, characterized in that In the step (3), the inert gas is argon with a purity of 99.99%.

7. The method according to claim 1, characterized in that The MRM acquisition parameters in the step (3) are: the quantitative ion pair of phenol is m / z94→66 (CE: 15 eV), and the qualitative ion pair is m / z94→55 (CE: 18 eV); the quantitative ion pair of m-cresol is m / z108→79 (CE: 15 eV), and the qualitative ion pair is m / z108→77 (CE: 24 eV).

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