Method for measuring contents of formaldehyde and acetaldehyde in polyethylene glycol and preparation thereof

Abstract

The invention belongs to the field of medicines, and particularly provides a method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and a preparation thereof by using a high performance liquid chromatography, and the specific chromatographic conditions are as follows: water and acetonitrile are used as mobile phases, and the flow velocity is 0.8-1.2 ml / min; and the detector is an ultraviolet detector. The method comprises the following steps: deriving trace formaldehyde and acetaldehyde in polyethylene glycol into substances with ultraviolet absorption by adopting a pre-column derivation method, then detecting through an ultraviolet detector, and calculating the content of formaldehyde and acetaldehyde in polyethylene glycol and a preparation thereof through an external standard method. The determination method has the advantages of high specificity, simplicity, convenience and rapidness in operation, high sensitivity and the like.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparation. Background Art

[0002] Polyethylene glycol (PEG) is a high molecular polymer obtained by the polymerization of ethylene oxide and water acetal. It has a linear chain structure composed of ethylene oxide as repeating units and is often used as a cosolvent, stabilizer, and emulsifier in pharmaceutical preparations. Polyethylene glycol and its analogs will undergo self-oxidation reaction when exposed to light or catalyzed by metal ions to produce substances such as formaldehyde and acetaldehyde. Formaldehyde is recognized as a Class 1 carcinogen and can easily cause gene mutations, chromosome damage, etc. Therefore, it is necessary to control the formaldehyde and acetaldehyde in a polyethylene glycol and its preparations. At present, the determination of formaldehyde content in a polyethylene glycol and its preparations included in the 2020 edition of the "Chinese Pharmacopoeia" is mainly carried out by limit determination by ultraviolet spectrophotometry. This method is easily interfered by other impurities and has low specificity.

[0003] Therefore, 2,4-dinitrophenylhydrazine is used for pre-column derivatization and then determined by high performance liquid chromatography. This method has high sensitivity, simple operation and good accuracy. Summary of the invention

[0004] The purpose of the present application is to provide a method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparations, wherein the method has strong specificity and high sensitivity.

[0005] The polyethylene glycol and its analogs include polyethylene glycol 400, polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 3350, polyethylene glycol 4000, polyethylene glycol 6000, etc.

[0006] Furthermore, the formaldehyde and acetaldehyde contents in the polyethylene glycol and its preparation are determined by high performance liquid chromatography, and the specific chromatographic conditions are: using octadecylsilane bonded silica gel as a filler.

[0007] Use water as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution: preferably, from 0 to 14 min, mobile phase A changes from 70% to 20%, and continues for 5 min; from 19 to 25 min, mobile phase A changes from 70% to 0%, and continues for 5 min.

[0008] The column temperature in the method is 25-35°C, preferably 30°C.

[0009] The detection wavelength in the method is 360 nm

[0010] The injection volume in the method is 5 μl

[0011] The chromatographic column is an octadecylsilane bonded silica gel chromatographic column, and more preferably a cation chromatographic column with a specification of 4.6×250 mm and a 5 um chromatographic column, which can meet the system applicability requirements.

[0012] Preferably, the injection volume in the method is 5 μl.

[0013] In the method, the concentration of the 2,4-dinitrophenylhydrazine derivatization reagent is 5 mg / ml.

[0014] In the method, the concentrations of the formaldehyde 2,4-dinitrophenylhydrazone reference solution and the acetaldehyde 2,4-dinitrophenylhydrazone reference solution are 20 μg and 40 μg per ml, respectively.

[0015] In the method, the derivatization time is 10 to 30 minutes, preferably 15 minutes.

[0016] In the method, the amount of the derivatization reagent used is 1.5-2.5 ml, preferably 2.0 ml.

[0017] The derivatization conditions in the method are standing reaction at room temperature for 10 to 30 minutes, preferably reacting at room temperature for 15 minutes.

[0018] The beneficial effects of the present invention are:

[0019] The method has the advantages of high sensitivity, high accuracy and convenience when determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparations.

[0020] It can be seen from the specificity chromatogram that the separation degree of formaldehyde 2,4-dinitrophenylhydrazone and acetaldehyde 2,4-dinitrophenylhydrazone is about 9, and the blank solvent and blank auxiliary material peaks have no interference with the determination.

[0021] It can be seen from the system suitability chromatogram that the separation degree of formaldehyde 2,4-dinitrophenylhydrazone and acetaldehyde 2,4-dinitrophenylhydrazone is about 9, and the RSDs of the peak areas of five consecutive injections are 0.2% and 0.2%, respectively, with good precision.

[0022] It can be seen from the calculation results of the accuracy chromatogram that the recoveries of formaldehyde 2,4-dinitrophenylhydrazone and acetaldehyde 2,4-dinitrophenylhydrazone are both between 98% and 101%, and the method has high accuracy.

[0023] The linear relationship between formaldehyde 2,4-dinitrophenylhydrazone and acetaldehyde 2,4-dinitrophenylhydrazone in this method is good, the linear relationship coefficient (r) is ≥0.999, and the Y-axis intercept is within 2% of the 100% response value.

[0024] Good repeatability and precision: The repeatability and precision of the method in this application are good, and the RSD values ​​are all less than 2%.

[0025] High sensitivity: The detection limits of formaldehyde and acetaldehyde in this method can reach 1.2ng / ml and 1.6ng / ml respectively; the quantitative limits can reach 3.6ng / ml and 4.7ng / ml respectively.

[0026] When the chromatographic conditions of the analytical method change slightly, the formaldehyde and acetaldehyde contents calculated by the external standard method shall be compared with the results under repeatability, and the RSD% shall be ≤2.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Specificity spectra of formaldehyde 2,4-dinitrophenylhydrazone linear and acetaldehyde 2,4-dinitrophenylhydrazone reference solutions;

[0028] Figure 2 Blank derivatization reagent spectrum

[0029] Figure 3 Linearity of formaldehyde 2,4-dinitrophenylhydrazone and quantification limit of acetaldehyde 2,4-dinitrophenylhydrazone

[0030] Figure 4 Detection limit spectrum of formaldehyde 2,4-dinitrophenylhydrazone and acetaldehyde 2,4-dinitrophenylhydrazone

[0031] Figure 5 Chromatographic spectrum of compound polyethylene glycol 3350 electrolyte oral solution sample solution

[0032] Figure 6 Formaldehyde 2,4-dinitrophenylhydrazone linear graph

[0033] Figure 7 Acetaldehyde 2,4-dinitrophenylhydrazone linear graph DETAILED DESCRIPTION

[0034] Below, the specific embodiments of the present invention are further described in conjunction with examples to more clearly describe the advantages and features of the present invention. However, these examples are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0035] 1. Specificity test

[0036] Blank interference test

[0037] Instrument: High performance liquid chromatography.

[0038] Chromatographic conditions: Use octadecylsilane bonded silica gel as filler (Welch Ultimate XB-18 250*4.6mm, 5μm or chromatographic column with equivalent performance); use water as mobile phase A, acetonitrile as mobile phase B, and gradient elution.

[0039] Preparation of blank solution: accurately measure 1 ml of acetonitrile and put it into a 10 ml volumetric flask, add 1 ml of acetonitrile and 2 ml of 2,4-dinitrophenylhydrazine derivatization solution, shake for a few seconds and then let it react for 15 minutes, then add acetonitrile to dilute to the scale and shake well to obtain

[0040] Preparation of formaldehyde-2,4-dinitrophenylhydrazone fixing solution: Accurately measure 1 ml of formaldehyde-2,4-dinitrophenylhydrazone stock solution, place it in a 50 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0041] Preparation of acetaldehyde-2,4-dinitrophenylhydrazone positioning solution: Accurately measure 5 ml of acetaldehyde-2,4-dinitrophenylhydrazone stock solution, place it in a 50 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0042] Preparation of reference solution and test solution: Accurately measure 1 ml of formaldehyde-2,4-dinitrophenylhydrazone stock solution and 5 ml of acetaldehyde-2,4-dinitrophenylhydrazone stock solution, place in a 50 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0043] Determination method: Accurately measure 5 μl of blank solution, blank auxiliary solution, 1 ml of formaldehyde-2,4-dinitrophenylhydrazone fixing solution and 5 ml of acetaldehyde-2,4-dinitrophenylhydrazone stock solution, respectively, and inject them into liquid chromatograph to record the chromatogram.

[0044] Specificity results name Retention time (min) Separation Formaldehyde-2,4-dinitrophenylhydrazone 12.387 Acetaldehyde-2,4-dinitrophenylhydrazone 13.872 8.9

[0045] 2 System suitability test

[0046] Determination method: Accurately measure 5 μl of each reference solution (reference solution 1 is injected continuously for 5 times, and reference solution 2 is injected continuously for 2 times), inject them into the liquid chromatograph respectively, and record the chromatogram.

[0047] System suitability test results

[0048] The peak separation of formaldehyde-2,4-dinitrophenylhydrazone and acetaldehyde-2,4-dinitrophenylhydrazone in the chromatogram of the reference solution should be greater than 2.0. The RSD of the peak area of ​​the reference solution injected 5 times in a row should not be greater than 2.0%. The relative recovery of two different reference solutions should be between 98.0% and 102.0%.

[0049] 3 Linear relationship and range test

[0050] Preparation of linearization solution According to the table below, accurately measure appropriate amounts of formaldehyde-2,4-dinitrophenylhydrazone and acetaldehyde-2,4-dinitrophenylhydrazone reference stock solutions, place them in corresponding volumetric bottles, add acetonitrile to dilute to the scale, shake well, and prepare corresponding linear solutions.

[0051] Determination method: Accurately measure 5 μl of blank solvent and linear solution of each concentration, inject them into liquid chromatograph respectively, and record the chromatogram.

[0052] Linear results Linear graph attached Figure 6 , Attachment Figure 7 .

[0053] Perform linear regression analysis with concentration as the horizontal axis (X) and peak area as the vertical axis (Y). The correlation coefficient (r) shall not be less than 0.998, the Y-axis intercept shall be within 2% of the 100% response value, and the relative standard deviation of the response factor shall not be greater than 2.0%.

[0054] 4. Accuracy

[0055] Preparation of Formaldehyde-2,4-Dinitrophenylhydrazone Reference Stock Solution and Acetaldehyde-2,4-Dinitrophenylhydrazone Reference Stock Solution: See the operating method.

[0056] Preparation of Accuracy Test Solution: According to the table below, accurately measure the appropriate amount of formaldehyde-2,4-dinitrophenylhydrazone and acetaldehyde-2,4-dinitrophenylhydrazone reference substance stock solutions, place them in the corresponding volumetric bottles, add acetonitrile to dilute to the scale, shake well, and prepare the accuracy test solution of the corresponding concentration. Prepare three copies of each concentration in the same way.

[0057] Determination method: Accurately measure 5 μl of blank solvent, reference solution (reference solution 1 is injected continuously 5 times, reference solution 2 is injected continuously 2 times), accuracy test solution of each concentration and reference solution 1, respectively, and inject them into liquid chromatograph to record the chromatogram.

[0058] Accuracy results

[0059] The average recovery rate at each concentration should be between 98.0% and 102.0%, and the RSD% of the 9 recovery rate data should not be greater than 2.0%.

[0060] 5. Precision test

[0061] Repeatability test

[0062] Test solution: Take 1 ml of the product into a 10 ml volumetric flask, add 1 ml of acetonitrile and 2 ml of 2,4-dinitrophenylhydrazine derivatization solution, shake for a few seconds and then leave to react for 15 minutes, then add acetonitrile to dilute to the mark, shake well, and the solution is ready. Prepare 6 portions in the same way. ⑤ Determination method: Accurately measure 5 μl of blank solvent, reference solution (reference solution 1 is injected continuously 5 times, reference solution 2 is injected continuously 2 times), each test solution and reference solution 1, respectively, and inject them into liquid chromatograph to record the chromatogram.

[0063] Reproducible results

[0064] Calculate the formaldehyde content and acetaldehyde content in 6 test solution, calculate the RSD% value of the 6 contents, RSD% should be ≤5.0%.

[0065] 6. Limit of detection and limit of quantification

[0066] Detection limit solution: Accurately measure appropriate amount of formaldehyde-2,4-dinitrophenylhydrazone 20% linear solution and acetaldehyde-2,4-dinitrophenylhydrazone 20% linear solution, add acetonitrile to dilute to a certain concentration, so that the concentration when the S / N of the formaldehyde-2,4-dinitrophenylhydrazone peak and the acetaldehyde-2,4-dinitrophenylhydrazone peak is ≈ 3 is used as the detection limit concentration.

[0067] Quantitation limit solution: Accurately measure appropriate amount of formaldehyde-2,4-dinitrophenylhydrazone 20% linear solution and acetaldehyde-2,4-dinitrophenylhydrazone 20% linear solution, add acetonitrile to dilute to a certain concentration, so that the concentration when the S / N of the formaldehyde-2,4-dinitrophenylhydrazone peak and the acetaldehyde-2,4-dinitrophenylhydrazone peak is ≈ 10 is used as the detection limit concentration.

[0068] Determination method: Accurately measure 5 μl of blank solvent and each detection limit concentration solution (injected 3 times continuously), inject them into liquid chromatograph respectively, and record the chromatogram.

[0069] Limit of Detection and Limit of Quantitation Results

[0070] The RSD% of the peak area of ​​the detection limit solution for three consecutive injections should be ≤20.0%; the RSD% of the peak area of ​​the quantification limit solution for six consecutive injections should be ≤10.0%.

[0071] The above content shows that this method has high sensitivity for the determination of formaldehyde and acetaldehyde in polyethylene glycol and its preparations, and the method has strong specificity and high accuracy.

Claims

1. A method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparations, characterized in that: The determination was carried out using high performance liquid chromatography, and the specific chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as filler; water was used as mobile phase A, acetonitrile was used as mobile phase B, and elution was performed according to a gradient; the column temperature was 30°C; the detection wavelength was 360nm, and the injection volume was 5ul.

2. The method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparation according to any one of claim 1, characterized in that: The column temperature was 30°C.

3. The method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparation according to any one of claim 1, characterized in that: The detection wavelength is 360 nm.

4. According to claim 1, the method for determining the formaldehyde and acetaldehyde contents in polyethylene glycol and its preparations, the elution gradient is that the mobile phase A changes from 70% to 20% from 0 to 14 min, and lasts for 5 min; and the mobile phase A changes from 70% to 0% from 19 to 25 min, and lasts for 5 min.

5. A method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparations according to claims 2 and 7, using 2,4-dinitrophenylhydrazine for derivatization, characterized in that The concentration of 2,4-dinitrophenylhydrazine was 5 mg / ml.

6. A method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparations according to claims 2 and 7, wherein 2,4-dinitrophenylhydrazine is used for derivatization, and the amount of the derivatization test solution used is 1.5 to 2.5 ml.

7. A method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparations according to claims 2 and 7, wherein 2,4-dinitrophenylhydrazine is used for derivatization, and the derivatization temperature is 10-40°C.

8. A method for determining the content of formaldehyde and acetaldehyde in polyethylene glycol and its preparations according to claims 2 and 7, wherein 2,4-dinitrophenylhydrazine is used for derivatization, and the derivatization condition is to allow the reaction to stand for 10 to 20 minutes.

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