Method for detecting initiator and degradation product thereof in polymer

Abstract

The invention discloses a method for detecting an initiator and a degradation product thereof in a polymer, a chromatographic column is an octadecyl bonded silica gel column, a gradient elution mode is adopted, and a detector is a diode array detector (DAD). The result shows that under the chromatographic condition, various impurities generated by degradation of the sevelamer carbonate catalyst can be well separated; the precision, repeatability and stability results are good. The method is simple and convenient to operate, high in repeatability and stable and reliable in result, and can be used for detecting the sevelamer carbonate initiator and the degradation product thereof.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a method for detecting an initiator in a polymer and its degradation products. Background Art

[0002] Sevelamer carbonate, chemical name: poly(propyleneamine-co-N,N'-diallyl-1,3-diamino-2-hydroxypropane) carbonate. Sevelamer carbonate is obtained by polymerizing allylamine hydrochloride with an initiator under certain conditions to obtain polyallylamine, cross-linking polypropyleneamine with epichlorohydrin, and finally acidifying with carbonic acid to obtain sevelamer carbonate. In the first step of allylamine hydrochloride polymerization, an initiator is usually added so that the reaction can be polymerized at room temperature. The addition of the initiator directly leads to the introduction of impurities that are not related to the efficacy of the drug, further increasing the risk of side effects during drug treatment.

[0003] Azobis(isobutylimidazoline) hydrochloride (VA-044) is a white solid with good solubility. It is soluble in some organic solvents, such as alcohols and ketones, but has low solubility in water. VA-044 is often used as a free radical initiator. Its molecular structure contains two azo groups, which can be decomposed by heat or light under appropriate conditions to generate free radicals, which can initiate free radical polymerization reactions and promote the formation of polymers. Therefore, VA-044 is widely used as an initiator in polymerization reactions, especially in polymer synthesis and coating preparation. Secondly, VA-044 is also used as a polymer modifier. By introducing VA-044 into the polymer system, cross-linking and modification of the material can be achieved, and the physical properties and chemical stability of the polymer can be improved. This makes VA-044 have application potential in the fields of plastics, rubbers and adhesives.

[0004] At present, there is no relevant report or effective means to accurately and efficiently detect azobisisobutylimidazoline hydrochloride (VA-044) and isopropylimidazoline. The instability of azobisisobutylimidazoline hydrochloride (VA-044) also further increases the difficulty of the detection method. Summary of the invention

[0005] In order to overcome the above defects, the present invention provides a method for detecting initiators and their degradation products in polymers, which can simultaneously and accurately and quantitatively determine azobisisobutylimidazoline hydrochloride (VA-044) and isopropylimidazoline, and has the characteristics of high sensitivity, strong accuracy, good reproducibility, and stable method.

[0006] The invention tries various types of chromatographic columns, and finally determines the determination method of azobisisobutylimidazoline hydrochloride (VA-044) and isopropylimidazoline by changing the mobile phase ratio, flow rate, chromatographic column temperature, test sample extraction method and the like.

[0007] A method for detecting an initiator and its degradation products in a polymer comprises the following steps:

[0008] 1) Prepare blank solution, reference solution and test solution respectively;

[0009] 2) using an octadecylsilane bonded silica gel chromatographic column, diluting the reference solution and the test solution with a diluent added with an organic acid, respectively, and injecting the solution, and performing retention separation by gradient elution;

[0010] 3) Calculate the content of initiator and its degradation products by peak area using the external standard method of reference substance.

[0011] The blank solution is a 0.01-1.0% TFA acetonitrile aqueous solution, the reference solution is a 0.02-0.10 mg / ml azobisisobutylimidazoline hydrochloride or isopropylimidazoline solution, and the test solution is a 300-700 mg / ml polymer that may contain an initiator and its degradation products.

[0012] Preferably, the blank solution is 0.1% TFA acetonitrile aqueous solution, the reference solution is 0.05 mg / ml azobisisobutylimidazoline hydrochloride, isopropylimidazoline solution, and the test solution is a polymer that may contain an initiator and its degradation products, and the test solution is 500 mg / ml.

[0013] The present invention takes sevelamer carbonate as an example, and the test solution is a 500 mg / ml sevelamer carbonate extract.

[0014] The reference substance solution is 0.05 mg / ml azobisisobutylimidazoline hydrochloride (VA-044) (ADAMASP2721803-1) and 0.05 mg / ml isopropylimidazoline (Y152319, Shandong Walderson Biotechnology Co., Ltd.).

[0015] The organic acid includes but is not limited to phosphoric acid, formic acid, trifluoroacetic acid and the like.

[0016] The amount of organic acid added to the diluent is 0.05-0.2%, and more preferably, the amount added is 0.1%.

[0017] The diluent is preferably a diluent with trifluoroacetic acid added; more preferably, the diluent is TFA:acetonitrile:water=0.1:

[0018] 10:90(v:v:v).

[0019] The high performance liquid chromatography column is an octadecylsilane bonded silica gel (Agilent Zorbax SB-Aq 4.6mm*250mm5μm or a column with equivalent polarity).

[0020] The gradient elution, mobile phase A is acetonitrile, mobile phase B is 0.1% TFA aqueous solution, the gradient program is as follows:

[0021]

[0022] The HPLC conditions used a diode array detector (DAD) as the detector, a temperature of 40° C., and a wavelength of 210 nm.

[0023] The chromatographic column temperature is 40°C.

[0024] The injection volume is 10 μl.

[0025] The method for preparing the test sample solution extract is as follows: weigh 5 g of the test sample and add 10 ml of a diluent (TFA: acetonitrile: water = 0.1:10:90), sonicate for 15 minutes, vortex for 1 minute, shake once every 5 minutes for a total of 60 minutes, centrifuge for 30 minutes, and filter the supernatant using a 0.22 μm organic membrane.

[0026] The external standard method calculation formula is:

[0027]

[0028] A sam : Peak area of ​​target solvent in sample solution;

[0029] A std : Average peak area of ​​target solvent in reference solution 2;

[0030] C std : The concentration of the target solvent in reference solution 2, mg / mL;

[0031] W s : Sample weight, mg;

[0032] V s : dilution volume of sample solution, mL;

[0033] LOD: loss on drying of sample, %.

[0034] The chromatographic conditions were as follows:

[0035] HPLC parameters:

[0036] Chromatographic column: Agilent Zorbax SB-Aq 4.6mm*250mm 5μm or equivalent Column temperature: 40℃, flow rate: 1.0ml / min, wavelength: 220nm, run time: 10min, injection volume: 10μl;

[0037] Mobile phase A: acetonitrile, mobile phase B: 0.1% TFA aqueous solution; isocratic elution.

[0038] Solution preparation method

[0039] Blank solution / diluent: TFA:acetonitrile:water=0.1:10:90 (v:v:v).

[0040] Reference solution: Accurately weigh 20 mg of azobisisobutylimidazoline hydrochloride (VA-044) reference substance and 20 mg of isopropylimidazoline reference substance into the same 20 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1.0 ml of the previous solution into a 20 ml volumetric flask, add diluent to the scale to obtain 0.05 mg / ml azobisisobutylimidazoline hydrochloride reference substance solution and 0.05 mg / ml isopropylimidazoline reference substance solution.

[0041] Test solution: weigh 5 g of the test sample and add 10 ml of diluent (TFA: acetonitrile: water = 0.1:10:90). Ultrasonicate for 15 minutes, then vortex for 1 minute. Shake once every 5 minutes for a total of 60 minutes. Centrifuge for 30 minutes, and filter the supernatant using a 0.22 μm organic membrane.

[0042] System suitability solution: weigh 5 g of the test sample and add 10 ml of the reference solution into the same centrifuge tube. Ultrasonicate for 15 minutes, then vortex for 1 minute. Shake once every 5 minutes for a total of 60 minutes. Centrifuge for 30 minutes and filter the supernatant using a 0.22 μm organic membrane.

[0043] System suitability: Take the system suitability solution and inject it directly, record the chromatogram, the blank has no interference, and the resolution of each peak is not less than 1.5.

[0044] Limit of Quantitation

[0045] Accurately weigh azobisisobutylimidazoline hydrochloride (VA-044) and isopropylimidazoline, and dilute them to low concentrations with a diluent. When azobisisobutylimidazoline hydrochloride (VA-044) is diluted to 0.00025 mg / ml (relative to the test sample concentration of 0.00025%) and isopropylimidazoline is diluted to 0.000125 mg / ml (relative to the test sample concentration of 0.000125%), the measured S / N is in the range of 10-15, which is taken as the quantitative limit concentration.

[0046] Linearity and range

[0047] Accurately measure the reference substance stock solution, take multiple portions (≥5) according to the gradient, place them in volumetric flasks of the same volume, make up to volume with diluent, inject them separately and record the chromatograms, calculate the linear regression equation and correlation coefficient based on the concentration and peak area.

[0048] Accuracy

[0049] The recovery rates of 6 spiked impurities with 100% impurity limit concentration (0.05 mg / ml) of azobisisobutylimidazoline hydrochloride (VA-044) added to the test sample were 104.88% to 106.77%, and the recovery rates of 6 spiked impurities with 100% impurity limit concentration (0.05 mg / ml) of isopropylimidazoline added to the test sample were 99.69% to 105.80%.

[0050] A reference solution and a separation solution were prepared and sequentially injected at different time intervals. The results showed that the solution of the present invention was stable for at least 43 hours.

[0051] Beneficial effects:

[0052] The advantages of the present invention are that a conventional high performance liquid chromatograph is used for detection, the method is simple and easy to operate, and has the characteristics of high sensitivity, strong accuracy, good reproducibility, and method stability.

[0053] Since azobisisobutylimidazoline hydrochloride (VA-044) and isopropylimidazoline will decompose after being dissolved in water, and are insoluble in conventional organic phases such as methanol and acetonitrile, the detection is difficult, the reproducibility is poor, and the sample is completely decomposed before injection. In order to overcome the difficulties, the present invention combines multiple methods such as oscillation, ultrasound, vortex, centrifugation, etc. to perform sample pretreatment, and adds a certain amount of trifluoroacetic acid (about 0.1%), etc. so that the method can keep the sample stable under long-term conditions, and can accurately and effectively detect the initiator azobisisobutylimidazoline hydrochloride (VA-044) and its degradation product isopropylimidazoline. The highest detection capability can reach 2.5ppm.

[0054] The detection method using different chromatographic columns, different rates and different gradient programs can effectively separate different impurities of different polarities. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Example 3 Experimental chromatogram;

[0056] Figure 2 Example 4 separation chromatogram;

[0057] Figure 3 Example 4 test sample chromatogram;

[0058] Figure 4 Example 4 Accuracy impurity addition chromatogram. DETAILED DESCRIPTION

[0059] Azobisisobutylimidazoline hydrochloride VA-044 was purchased from ADAMAS P2721803-1;

[0060] Isopropyl imidazoline was purchased from Shandong Walderson Biotechnology Co., Ltd. Y152319;

[0061] Example 1

[0062] HPLC method:

[0063] Chromatographic column: Agilent Zorbax SB-C18 4.6mm*250mm 5μm;

[0064] Column temperature: 40°C, flow rate: 1.0 ml / min, wavelength: 220 nm, run time: 10 min, injection volume: 10 μl;

[0065] Mobile phase A: acetonitrile, mobile phase B: 0.1% TFA aqueous solution;

[0066] The elution procedure is as follows:

[0067]

[0068] Blank solution / diluent: acetonitrile: water = 10:90.

[0069] Reference solution: Accurately weigh 20 mg of azobisisobutylimidazoline hydrochloride (VA-044) (ADAMAS P2721803-1) reference substance and 20 mg of isopropylimidazoline (Y152319, Shandong Wodeson Biotechnology Co., Ltd.) reference substance into the same 20 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1.0 ml of the previous solution into a 20 ml volumetric flask, add diluent to the scale to obtain 0.05 mg / ml azobisisobutylimidazoline hydrochloride reference solution and 0.05 mg / ml isopropylimidazoline reference solution.

[0070] Test solution: weigh 5 g of the test sample (SC-030-2210401, Jiangsu Hengsheng Pharmaceutical Co., Ltd.) and add 10 ml of diluent. Ultrasonicate for 15 minutes, then vortex for 1 minute. Shake once every 5 minutes for a total of 60 minutes. Centrifuge for 30 minutes, and filter the supernatant with a 0.22 μm organic membrane.

[0071] The experimental results showed that the reference solution did not produce any peak.

[0072] Example 2

[0073] HPLC method:

[0074] Chromatographic column: Agilent Zorbax Aq-C18 4.6mm*250mm 5μm;

[0075] Column temperature: 40°C, flow rate: 1.0 ml / min, wavelength: 220 nm, run time: 10 min, injection volume: 10 μl;

[0076] Mobile phase A: acetonitrile, mobile phase B: 0.1% TFA aqueous solution;

[0077] The elution procedure is as follows:

[0078]

[0079] Blank solution / diluent: acetonitrile: water = 10:90.

[0080] Reference solution: Accurately weigh 20 mg of azobisisobutylimidazoline hydrochloride (VA-044) (ADAMAS P2721803-1) reference substance and 20 mg of isopropylimidazoline (Y152319, Shandong Wodeson Biotechnology Co., Ltd.) reference substance into the same 20 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1.0 ml of the previous solution into a 20 ml volumetric flask, add diluent to the scale to obtain 0.05 mg / ml azobisisobutylimidazoline hydrochloride reference solution and 0.05 mg / ml isopropylimidazoline reference solution.

[0081] Test solution: weigh 5 g of the test sample (SC-030-2210401, Jiangsu Hengsheng Pharmaceutical Co., Ltd.) and add 10 ml of diluent. Ultrasonicate for 15 minutes, then vortex for 1 minute. Shake once every 5 minutes for a total of 60 minutes. Centrifuge for 30 minutes, and filter the supernatant with a 0.22 μm organic membrane.

[0082] The experimental results show that the reference solution has a peak, the separation meets the requirements, and the peak shape is poor.

[0083] Example 3

[0084] HPLC method:

[0085] Chromatographic column: Agilent Zorbax Aq-C18 4.6mm*250mm 5μm;

[0086] Column temperature: 40°C, flow rate: 1.0 ml / min, wavelength: 220 nm, run time: 10 min, injection volume: 10 μl;

[0087] Mobile phase A: acetonitrile, mobile phase B: 0.1% TFA aqueous solution;

[0088] The elution procedure is as follows:

[0089]

[0090] Blank solution / diluent: acetonitrile: water = 10:90.

[0091] Reference solution: Accurately weigh 20 mg of azobisisobutylimidazoline hydrochloride (VA-044) (ADAMAS P2721803-1) reference substance and 20 mg of isopropylimidazoline (Y152319, Shandong Wodeson Biotechnology Co., Ltd.) reference substance into the same 20 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1.0 ml of the previous solution into a 20 ml volumetric flask, add diluent to the scale to obtain 0.05 mg / ml azobisisobutylimidazoline hydrochloride reference solution and 0.05 mg / ml isopropylimidazoline reference solution.

[0092] Test solution: weigh 5 g of the test sample (SC-030-2210401, Jiangsu Hengsheng Pharmaceutical Co., Ltd.) and add 10 ml of diluent. Ultrasonicate for 15 minutes, then vortex for 1 minute. Shake once every 5 minutes for a total of 60 minutes. Centrifuge for 30 minutes, and filter the supernatant with a 0.22 μm organic membrane.

[0093] The experimental results show that the reference solution has a peak, the separation meets the requirements, but the system applicability cannot meet the requirements, the peak area is greatly reduced, and the peak shape is poor.

[0094] Example 4

[0095] HPLC method:

[0096] Chromatographic column: Agilent Zorbax Aq-C18 4.6mm*250mm 5μm;

[0097] Column temperature: 40°C, flow rate: 1.0 ml / min, wavelength: 220 nm, run time: 10 min, injection volume: 10 μl;

[0098] Mobile phase A: acetonitrile, mobile phase B: 0.1% TFA aqueous solution;

[0099] The elution procedure is as follows:

[0100]

[0101] Blank solution / diluent: TFA:acetonitrile:water=0.05:10:90 (v:v:v).

[0102] Reference solution: Accurately weigh 20 mg of azobisisobutylimidazoline hydrochloride (VA-044) (ADAMAS P2721803-1) reference substance and 20 mg of isopropylimidazoline (Y152319, Shandong Wodeson Biotechnology Co., Ltd.) reference substance into the same 20 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1.0 ml of the previous solution into a 20 ml volumetric flask, add diluent to the scale to obtain 0.05 mg / ml azobisisobutylimidazoline hydrochloride reference solution and 0.05 mg / ml isopropylimidazoline reference solution.

[0103] Test solution: weigh 5 g of the test sample (SC-030-2210401, Jiangsu Hengsheng Pharmaceutical Co., Ltd.) and add 10 ml of diluent. Ultrasonicate for 15 minutes, then vortex for 1 minute. Shake once every 5 minutes for a total of 60 minutes. Centrifuge for 30 minutes, and filter the supernatant with a 0.22 μm organic membrane.

[0104] The experimental results show that the reference solution has a peak, the separation meets the requirements, the peak area reduction is reduced, and the system applicability cannot meet the requirements.

[0105] Example 5

[0106] HPLC method:

[0107] Chromatographic column: Agilent Zorbax Aq-C18 4.6mm*250mm 5μm;

[0108] Column temperature: 40°C, flow rate: 1.0 ml / min, wavelength: 220 nm, run time: 10 min, injection volume: 10 μl;

[0109] Mobile phase A: acetonitrile, mobile phase B: 0.1% TFA aqueous solution

[0110] The elution procedure is as follows:

[0111]

[0112] Blank solution / diluent: TFA:acetonitrile:water=0.1:10:90 (v:v:v).

[0113] Reference solution: Accurately weigh 20 mg of azobisisobutylimidazoline hydrochloride (VA-044) (ADAMAS P2721803-1) reference substance and 20 mg of isopropylimidazoline (Y152319, Shandong Wodeson Biotechnology Co., Ltd.) reference substance into the same 20 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1.0 ml of the previous solution into a 20 ml volumetric flask, add diluent to the scale to obtain 0.05 mg / ml azobisisobutylimidazoline hydrochloride reference solution and 0.05 mg / ml isopropylimidazoline reference solution.

[0114] Test solution: weigh 5 g of the test sample (SC-030-2210401, Jiangsu Hengsheng Pharmaceutical Co., Ltd.) and add 10 ml of diluent. Ultrasonicate for 15 minutes, then vortex for 1 minute. Shake once every 5 minutes for a total of 60 minutes. Centrifuge for 30 minutes, and filter the supernatant with a 0.22 μm organic membrane.

[0115] The experimental results show that the reference solution has a peak, the separation degree meets the requirements, the system applicability meets the requirements, and meets the detection needs.

[0116] Detection limit

[0117] The concentration solution obtained by diluting the reference solution several times to obtain a S / N ≥ 3 is the LOD solution.

[0118] Experimental results:

[0119] When the concentration of the reference solution was 1 ppm, the S / N of azobisisobutylimidazoline hydrochloride (VA-044) was 15.48 and that of isopropylimidazoline was 6.31.

[0120] Accuracy

[0121] Prepare reference solution and test solution.

[0122] 100% limit concentration solution: weigh about 500 mg of the test sample, place it in a 10 ml centrifuge tube, accurately add 5.0 ml of the reference solution, seal it with a stopper, and vortex for 30 seconds. After centrifugation at 4500 rpm for 10 minutes, take the supernatant and filter it through 0.22 μm. Prepare 6 copies in parallel.

[0123] Experimental results:

[0124] Azobis(isobutylimidazoline) hydrochloride (VA-044)

[0125]

[0126]

[0127] The recovery rates of 6 spiked impurities with 100% impurity limit concentration (0.05 mg / ml) of azobisisobutylimidazoline hydrochloride (VA-044) added to the test sample were 104.88% to 106.77%, and the RSD of the recovery rates of 6 spiked impurities was 0.68%.

[0128] Isopropyl imidazoline

[0129]

[0130] The recovery rates of 6 spiked impurities with 100% impurity limit concentration (0.05 mg / ml) of isopropyl imidazoline added to the test sample were 99.69% to 105.80%, and the RSD of the recovery rates of 6 spiked impurities was 2.74%.

[0131] stability

[0132] Prepare reference solution and resolution solution, and perform sequential injections at different time intervals.

[0133] Experimental results:

[0134]

[0135] The solution is stable for at least 43 hours.

[0136] Although the embodiments of the present invention have been described in detail, it should be understood that the various changes, substitutions, and alterations could be made hereto without departing from the spirit and scope of the invention.

Claims

1. A method for detecting an initiator and its degradation products in a polymer, characterized in that: The following steps are involved: 1) Prepare blank solution, reference solution and test solution respectively; 2) using an octadecylsilane bonded silica gel chromatographic column, diluting the reference solution and the test solution with a diluent added with an organic acid, respectively, and injecting the solution, and performing retention separation by gradient elution; 3) Calculate the content of initiator and its degradation products by peak area according to the external standard method of reference substance; The initiator is azobisisobutylimidazoline hydrochloride, and the degradation product is isopropylimidazoline.

2. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The blank solution is a 0.01-1.0% TFA acetonitrile aqueous solution, the reference solution is a 0.02-0.10 mg / ml azobisisobutylimidazoline hydrochloride or isopropylimidazoline solution, and the test solution is a 300-700 mg / ml polymer that may contain an initiator and its degradation products.

3. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The organic acid includes but is not limited to phosphoric acid, formic acid, and trifluoroacetic acid.

4. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The octadecylsilane bonded silica gel chromatographic column is Agilent Zorbax SB-Aq or a chromatographic column with equivalent polarity.

5. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The gradient elution, mobile phase A is acetonitrile, mobile phase B is 0.1% TFA, the gradient program is as follows:

6. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The HPLC conditions used a diode array detector as the detector, the temperature of the detector was 40° C., and the wavelength was 210 nm.

7. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The temperature of the chromatographic column is 40°C.

8. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The chromatographic injection volume was 10 μl.

9. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The preparation method of the test solution is as follows: weigh 5 g of the test sample and add 10 ml of the diluent, sonicate for 15 minutes, vortex for 1 minute, shake once every 5 minutes for a total of 60 minutes, centrifuge for 30 minutes, and filter the supernatant with a 0.22 μm organic membrane.

10. The method for detecting an initiator and its degradation products in a polymer according to claim 1, characterized in that: The external standard method calculation formula is: A sam : Peak area of ​​target solvent in sample solution; A std : Average peak area of ​​target solvent in reference solution 2; C std : The concentration of the target solvent in reference solution 2, mg / mL; W s : Sample weight, mg; V s : dilution volume of sample solution, mL; LOD: loss on drying of sample, %.

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