Detection method of zoledronic acid residual solvent

Abstract

The invention discloses a method for detecting a zoledronic acid residual solvent. The method comprises the following steps: (1) respectively preparing a blank solution, a reference solution and a test solution; (2) sample injection detection; an instrument is a gas chromatograph with a hydrogen flame ionization detector, nitrogen is used as carrier gas, and the carrier gas is injected into the gas chromatograph by adopting headspace sample injection; and respectively carrying out sample injection detection to obtain a chromatogram of the reference substance solution and a chromatogram of the test solution, and carrying out detection analysis by adopting an external standard method to complete the determination of the zoledronic acid residual solvent. According to the method, the gas chromatograph is adopted to detect the residues of methanol, tetrahydrofuran, isopropanol and triethylamine in zoledronic acid, the detection chromatographic conditions are optimized in a targeted manner, and under the condition of ensuring good theoretical plate and separation degree, a plurality of residual solvents are placed in the same system for detection, so that the detection efficiency is greatly improved; and meanwhile, the detection chromatographic conditions are specifically optimized, so that the organic solvents are completely separated, and the linear relation is good in the investigated range.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting residual solvents of zoledronic acid. Background Art

[0002] Zoledronic acid is a bisphosphonate drug developed by Novartis. Its chemical name is 1-hydroxy-2-(imidazol-1-yl)-ethylidene-1,1-diphosphonic acid, and its structural formula is as follows: .

[0003] Zoledronic acid can be used in combination with standard anti-tumor drugs to treat bone metastases of solid tumors and bone damage caused by multiple myeloma; it is also used to treat hypercalcemia (HCM) caused by malignant tumors, osteoporosis in postmenopausal women, osteoporosis in men, and osteitis deformans (Paget's disease).

[0004] Residual solvents in drugs refer to organic volatile compounds produced or used in the production of raw materials or excipients, as well as in the preparation of preparations. Since residual solvents not only have no therapeutic effect, but may also increase the toxic side effects of drugs and affect the stability of drugs, all organic solvents should be removed as much as possible. In order to protect patients from the harm of residual organic solvents in drugs, it is necessary to measure the residual amount of organic solvents introduced into drugs during the production process.

[0005] Depending on the different preparation processes of zoledronic acid, different solvents are involved.

[0006] For example, in the "A Method for Preparing Zoledronic Acid" disclosed in Chinese patent document CN202111570123.5, the organic solvent used for crystallization is one or more of methanol, ethanol, and isopropanol.

[0007] Chinese patent document CN201010610896.7 discloses a method for preparing zoledronic acid and its sodium salt using a phase transfer catalyst, and the organic solvents used include methanol, 1,4-dioxane, tetrahydrofuran, n-butyl ether or dimethyl sulfoxide.

[0008] Chinese patent document CN201910929468 discloses a "zoledronic acid intermediate compound", which involves the use of acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethylformamide, dimethyl sulfoxide and triethylamine.

[0009] According to the ICH guidelines and the requirements of my country's Pharmacopoeia: the residual limits of methanol, tetrahydrofuran, isopropanol and triethylamine are 0.3%, 0.072%, 0.5% and 0.5% respectively.

[0010] Therefore, the determination of the residual solvents methanol, tetrahydrofuran, isopropanol and triethylamine in zoledronic acid is of great significance to the drug quality of zoledronic acid and the health of patients. Currently, there is no record of the content detection method of the residual solvents in zoledronic acid.

[0011] Chinese patent document CN201611207400 discloses a method for detecting bisphosphonates, which pretreats a sample containing bisphosphonates to obtain a pretreated sample solution, wherein the bisphosphonates include sodium pamidronate, alendronic acid, sodium risedronate and zoledronic acid; and the sample solution is detected by liquid chromatography-tandem mass spectrometry to obtain the content of bisphosphonates in the sample.

[0012] The "Method for Detecting Related Substances in Zoledronic Acid Injection by High Performance Liquid Chromatography" disclosed in Chinese patent document CN201610794684 adopts the chromatographic conditions of the invention to more comprehensively detect the related substances in zoledronic acid injection, especially to achieve a separation degree of 1.5 or more between related substance A and other peaks in the injection, thereby achieving accurate detection of related substance A and better controlling the product quality of zoledronic acid injection.

[0013] The above detection method detects zoledronic acid and its related impurities and is not suitable for the detection of residual solvents. Summary of the invention

[0014] The technical problem to be solved by the present invention is to provide a method for detecting residual solvent of zoledronic acid with high sensitivity and accuracy.

[0015] The technical solution for achieving the purpose of the present invention is a method for detecting residual solvent of zoledronic acid, comprising the following steps: ① Prepare blank solution, reference solution and test solution respectively.

[0016] The blank solution is a diluent, and the diluent is water or a sodium hydroxide solution.

[0017] The reference substance solution is a mixed solution of triethylamine, methanol, tetrahydrofuran, isopropanol and a diluent.

[0018] The test solution is obtained by dissolving zoledronic acid to be tested in a diluent.

[0019] ② Sample injection test.

[0020] The chromatographic conditions were as follows: the instrument was a gas chromatograph with a hydrogen flame ionization detector, nitrogen was used as the carrier gas, headspace injection was used to inject into the gas chromatograph, the injection port temperature was 180°C to 220°C, the column temperature was ramped, the hydrogen flame ionization detector temperature was 260°C, and the injection volume was 1 to 1.5 mL.

[0021] The chromatograms of the reference solution and the test solution were obtained by injection testing respectively, and the external standard method was used for detection and analysis to complete the determination of the residual solvent of zoledronic acid.

[0022] When preparing the reference substance solution in the above step ①, first prepare a reference stock solution, and then dilute the reference stock solution with a diluent as the reference substance solution; the preparation method of the reference stock solution is: accurately weigh triethylamine, methanol, tetrahydrofuran and isopropanol respectively, transfer them into a volumetric flask containing an appropriate amount of dimethyl sulfoxide, and then dilute to the scale with dimethyl sulfoxide, shake well, and use it as the reference stock solution.

[0023] Alternatively, when preparing the reference substance solution in step ① above, first use water as a solvent to prepare a tetrahydrofuran stock solution and a methanol-isopropanol-triethylamine stock solution respectively; then measure the tetrahydrofuran stock solution and the methanol-isopropanol-triethylamine stock solution into a volumetric flask respectively, and dilute to the scale with water.

[0024] When preparing the test solution in step ① above, accurately weigh the zoledronic acid raw material, place it in an empty bottle, add the diluent to dissolve it, and seal it with a cover to serve as the test solution.

[0025] In the above step ②, the chromatographic column is Agilent DB-5 or Agilent DB-264; the specifications are 30m*0.53mm, 1.0μm or 30m*0.53mm, 3μm.

[0026] In the above step ②, when the temperature is increased gradually, the starting temperature is 40°C, maintained for 3 to 8 minutes, and then increased to 180 to 240°C at a rate of 10 to 30°C per minute, and maintained for 6 to 10 minutes.

[0027] During the detection, the injection order of each solution into the gas chromatograph for detection is 1 injection of blank solution, 5 injections of reference solution, and 2 injections of test solution.

[0028] The detection method of the invention can simultaneously detect the residual amounts of triethylamine, methanol, tetrahydrofuran and isopropanol in zoledronic acid.

[0029] The present invention has positive effects: The invention adopts a gas chromatograph to detect the residues of methanol, tetrahydrofuran, isopropanol and triethylamine in zoledronic acid, optimizes the detection chromatographic conditions in a targeted manner, and places a plurality of residual solvents in the same system for detection while ensuring that the theoretical plates and the separation degree are both good, thereby greatly improving the detection efficiency; meanwhile, the detection chromatographic conditions are optimized in a targeted manner, so that the organic solvents are completely separated, and the linear relationship is good within the investigated range.

[0030] The detection method of the invention has sensitivity and accuracy that meet the detection requirements for residual organic solvents, and is suitable for detecting residual solvents such as methanol, tetrahydrofuran, isopropanol and triethylamine in zoledronic acid raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the gas chromatogram of the blank solution in Example 1.

[0032] Figure 2 It is the gas chromatogram of the reference substance solution in Example 1.

[0033] Figure 3 It is the gas chromatogram of the test solution in Example 1.

[0034] Figure 4 It is the gas chromatogram of the blank solution in Example 2.

[0035] Figure 5 It is the gas chromatogram of the reference substance solution in Example 2.

[0036] Figure 6 It is the gas chromatogram of the test solution in Example 2. DETAILED DESCRIPTION

[0037] (Example 1) The detection method of the zoledronic acid residual solvent of the present embodiment comprises the following steps: ① Prepare blank solution, control stock solution, reference substance solution, and sample solution containing zoledronic acid.

[0038] The blank solution is a diluent, and the diluent is obtained by dissolving 20 g of sodium hydroxide in 1000 mL of water.

[0039] The preparation method of the control stock solution is as follows: accurately weigh 500 mg, 300 mg, 72 mg, and 500 mg of triethylamine, methanol, tetrahydrofuran, and isopropanol, respectively, transfer them into a 50 mL volumetric flask containing an appropriate amount of dimethyl sulfoxide, dilute to the scale with dimethyl sulfoxide, shake well, and use the result as the control stock solution.

[0040] The preparation method of the reference solution is as follows: accurately measure 1 mL of the reference stock solution into a 100 mL volumetric flask, dilute to the mark with a diluent, shake well, and use the solution as the reference solution; accurately transfer 5 mL of the solution into a headspace bottle, cover and seal, and repeat 5 times.

[0041] The preparation method of the sample solution containing zoledronic acid is as follows: accurately weigh 0.1 g of zoledronic acid raw material, place it in an empty bottle, accurately add 5 mL of diluent to dissolve it, cover and seal it, use it as the test solution, and prepare 2 portions in parallel.

[0042] The synthetic route of the zoledronic acid raw material to be tested is referred to WO2007109542A2, and the specific reagent selection and route are as follows: imidazole and halogenated acetic acid ester are used as raw materials, triethylamine and tetrahydrofuran are used as solvents, a nucleophilic substitution reaction is first carried out to generate imidazole acetate, and then a hydrolysis reaction is carried out, and the intermediate imidazole acetic acid is obtained by crystallization with isopropanol after treatment; imidazole acetic acid is reacted under the catalysis of phosphorous acid, phosphorus trichloride and acid, and methanol is used for crystallization after treatment to obtain a crude zoledronic acid product; and finally, zoledronic acid is obtained by purification and refinement.

[0043] ② Sample injection test.

[0044] The chromatographic conditions were as follows: Instrument used: Gas chromatograph with hydrogen flame ionization detector, Agilent-7890B gas chromatograph.

[0045] Headspace sampler: Agilent-7697A.

[0046] Chromatographic column: Agilent DB-5, specification: 30m*0.53mm, 1.0μm.

[0047] Nitrogen is used as the carrier gas, and the carrier gas flow rate is 2.0 ml / min to 3.0 ml / min, and is 2.5 mL / min in this embodiment; the split ratio of the split direct injection mode is 3:1 to 7:1.

[0048] Headspace injection was adopted, the injection port temperature was 180°C to 220°C (200°C in the present embodiment), the column temperature gradient was set, the starting temperature was 40°C, maintained for 3 to 8 (4 in the present embodiment) minutes, the temperature was increased to 180 to 240°C (220°C in the present embodiment) at a rate of 10°C to 30°C (10°C in the present embodiment) per minute, and maintained for 6 to 10 (10 in the present embodiment) minutes; the hydrogen flame ionization detector temperature was 260°C, and the injection volume was 1 to 1.5 mL (1 mL in the present embodiment).

[0049] Accurately pipette 1 mL of blank solution, reference solution, and test solution, and inject into the gas chromatograph, including 2 injections of blank solution, 5 injections of reference solution, and 2 injections of test solution, and record the chromatogram. According to the chromatogram, calculate the residual amount of methanol, tetrahydrofuran, isopropanol, and triethylamine in zoledronic acid by external standard method.

[0050] See Figure 1 , Figure 1 is the gas chromatogram of the blank solution; Figure 1 It can be seen that the blank solution does not interfere with the detection of the solvent peak.

[0051] See Figure 2 , Figure 2It is the gas chromatogram of the reference solution; in the reference solution, the separation degree of the solvent peak is greater than 1.5, which meets the requirements, the peak shape is good, the peak purity is good, and the theoretical plate number is greater than 3000, which meets the standard.

[0052] See Figure 3 , Figure 3 It is a gas chromatogram of the test solution; the contents of triethylamine, methanol, tetrahydrofuran and isopropanol in the test sample are calculated to be 0.0011%, 0.0085%, 0.0001% and 0.0003%, respectively, which are all lower than the limits of ICH guidelines and Chinese Pharmacopoeia, and it is confirmed that the method of the present invention is a detection method with high sensitivity and accuracy.

[0053] (Example 2) The detection method of the zoledronic acid residual solvent of the present embodiment comprises the following steps: ① Prepare blank solution, control stock solution, reference substance solution, and sample solution containing zoledronic acid.

[0054] The blank solution is a diluent, and the diluent is water.

[0055] The preparation method of the control stock solution is: Accurately weigh 36 mg of tetrahydrofuran into a 100 mL volumetric flask, dilute to the mark with water, and mix well.

[0056] In addition, accurately weigh 30 mg of methanol, 53 mg of isopropanol, and 56 mg of triethylamine into another 100 mL volumetric flask, dilute to the mark with water, and mix well. These are used as control stock solutions.

[0057] The preparation method of the reference solution is as follows: accurately measure 5 mL of the above tetrahydrofuran stock solution and methanol-isopropanol-triethylamine stock solution into a 100 mL volumetric flask, dilute to the scale with water, mix well, and use as the reference solution.

[0058] The preparation method of the sample solution containing zoledronic acid is as follows: accurately weigh 0.1 g of zoledronic acid raw material, place it in an empty bottle, accurately add 5 mL of diluent to dissolve it, cover and seal it, use it as the test solution, and prepare 2 portions in parallel.

[0059] The synthesis method of the above-mentioned zoledronic acid raw material to be tested is also referred to WO2007109542A2, and the specific reagent selection and route are as follows (not the same batch as the raw material in Example 1): using imidazole and halogenated acetic acid ester as raw materials, triethylamine and tetrahydrofuran as solvents, first undergoing a nucleophilic substitution reaction to generate imidazole acetate, then undergoing a hydrolysis reaction, and then post-treating with isopropanol to crystallize to obtain the intermediate imidazole acetic acid; imidazole acetic acid reacts under the catalysis of phosphorous acid, phosphorus trichloride and acid, and post-treating with methanol to crystallize to obtain a crude zoledronic acid; finally, zoledronic acid is purified and refined.

[0060] ② Sample injection test.

[0061] The chromatographic conditions were as follows: Instrument used: Agilent-7890B gas chromatograph.

[0062] Headspace sampler: Agilent-7697A.

[0063] Chromatographic column: Agilent DB-264, specification: 30m*0.53mm, 3μm.

[0064] Nitrogen was used as the carrier gas with a flow rate of 2.5 mL / min.

[0065] Headspace injection was used with an injection port temperature of 180°C. The column temperature was set to a gradient temperature increase, with the starting temperature at 40°C, maintained for 8 minutes, then increased to 180°C at a rate of 20°C per minute and maintained for 6 minutes; the detector temperature was 260°C and the injection volume was 1 mL.

[0066] Accurately pipette 1 mL of blank solution, reference solution, and test solution, and inject into the gas chromatograph, including 2 injections of blank solution, 5 injections of reference solution, and 2 injections of test solution, and record the chromatogram. According to the chromatogram, calculate the residual amount of methanol, tetrahydrofuran, isopropanol, and triethylamine in zoledronic acid by external standard method.

[0067] See Figure 4 , Figure 4 is the gas chromatogram of the blank solution; Figure 4 It can be seen that the blank solution does not interfere with the detection of the solvent peak.

[0068] See Figure 5 , Figure 5 It is the gas chromatogram of the reference solution; in the reference solution, the separation degree of the solvent peak is greater than 1.5, which meets the requirements, the peak shape is good, the peak purity is good, and the theoretical plate number is greater than 3000, which meets the standard.

[0069] See Figure 6 , Figure 6 It is the gas chromatogram of the test solution; the contents of triethylamine, methanol, tetrahydrofuran and isopropanol in the test sample are calculated to be 0.002%, 0.005%, not detected and 0.0002%, respectively, which are all lower than the limits of ICH guidelines and Chinese Pharmacopoeia; tetrahydrofuran is not detected, indicating that tetrahydrofuran is removed during the purification step.

Claims

1. A method for detecting residual solvent of zoledronic acid, characterized in that The following steps are involved: ① Prepare blank solution, reference solution and test solution respectively; The blank solution is a diluent, and the diluent is water or a sodium hydroxide solution; The reference substance solution is a mixed solution of triethylamine, methanol, tetrahydrofuran, isopropanol and a diluent; The test solution is obtained by dissolving zoledronic acid to be tested in a diluent; ② Sampling test; The chromatographic conditions were as follows: the instrument was a gas chromatograph with a hydrogen flame ionization detector, nitrogen was used as the carrier gas, headspace injection was used to inject into the gas chromatograph, the injection port temperature was 180°C to 220°C, the column temperature was ramped up, the hydrogen flame ionization detector temperature was 260°C, and the injection volume was 1 to 1.5 mL; The chromatograms of the reference solution and the test solution were obtained by injection testing respectively, and the external standard method was used for detection and analysis to complete the determination of the residual solvent of zoledronic acid.

2. The method for detecting residual solvent of zoledronic acid according to claim 1, characterized in that: Step ① When preparing the reference solution, first prepare a reference stock solution, and then dilute the reference stock solution with a diluent as the reference solution; The preparation method of the control stock solution is: accurately weigh triethylamine, methanol, tetrahydrofuran and isopropanol respectively, transfer them into a volumetric flask containing an appropriate amount of dimethyl sulfoxide, dilute to the scale with dimethyl sulfoxide, shake well, and use the solution as the control stock solution.

3. The method for detecting residual solvent of zoledronic acid according to claim 1, characterized in that: Step ① When preparing the reference solution, first use water as a solvent to prepare a tetrahydrofuran stock solution and a methanol-isopropanol-triethylamine stock solution respectively; then measure the tetrahydrofuran stock solution and the methanol-isopropanol-triethylamine stock solution into a volumetric flask respectively, and dilute to the scale with water.

4. The method for detecting residual solvent of zoledronic acid according to claim 1, characterized in that: Step ① When preparing the test solution, accurately weigh the zoledronic acid raw material, place it in an empty bottle, add the diluent to dissolve it, cover and seal it, and use it as the test solution.

5. The method for detecting residual solvent of zoledronic acid according to claim 1, characterized in that: In step ②, the chromatographic column is Agilent DB-5 or Agilent DB-264; the specifications are 30m*0.53mm, 1.0μm or 30m*0.53mm, 3μm.

6. The method for detecting residual solvent of zoledronic acid according to claim 1, characterized in that: In step ②, during the gradient heating, the starting temperature is 40°C, maintained for 3 to 8 minutes, and then heated to 180 to 240°C at a rate of 10 to 30°C per minute, and maintained for 6 to 10 minutes.

7. The method for detecting residual solvent of zoledronic acid according to claim 1, characterized in that: The injection order of each solution into the gas chromatograph for detection is 1 injection of blank solution, 5 injections of reference solution, and 2 injections of test solution.

8. The method for detecting residual solvent of zoledronic acid according to claim 1, characterized in that: The residual amounts of triethylamine, methanol, tetrahydrofuran and isopropanol in zoledronic acid can be detected simultaneously.

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