Method for detecting impurity hydrazine hydrate in candesartan cilexetil intermediate

Abstract

The invention belongs to the field of pharmaceutical analysis, and aims to provide a high performance liquid chromatography detection method for detecting hydrazine hydrate impurities doped in candesartan cilexetil intermediates (candesartan cyclization compound (C6) and candesartan (C8)). The method can be used for monitoring the content of the impurity hydrazine hydrate in the candesartan cilexetil intermediate, is high in chromatographic peak separation degree of the impurity hydrazine hydrate in the candesartan cilexetil intermediate, has relatively high system applicability, and has the characteristics that specificity, repeatability, quantitation limit, detection limit, accuracy and durability all meet standards and the like. According to the present invention, the vacancy of the existing impurity hydrazine hydrate detection method is filled, the detection method has characteristics of simple operation, short time and low cost, and the quality controllability of the impurity hydrazine hydrate in candesartan cilexetil intermediates (candesartan cyclization compound (C6) and candesartan (C8)) is ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting hydrazine hydrate, an impurity in candesartan cilexetil intermediate. Background Art

[0002] Candesartan cilexetil has the chemical name: 2-Ethoxy-1-[[2-(1H-tetrazol-5-yl)[1,1-biphenyl]-4-yl]methyl]-1H-benzimidazole-7-carboxylic acid 1-[[(cyclohexyloxy)carbonyl]oxy]ethyl ester, CAS: 145040-37-5, and its structural formula is as follows: , which is a prodrug of candesartan. In the gastrointestinal tract, it can be rapidly and completely hydrolyzed into candesartan. Candesartan is an angiotensin II AT1 receptor antagonist, which antagonizes the vasoconstrictive effect of angiotensin II by binding to the vascular smooth muscle AT1 receptor, thereby reducing the peripheral vascular resistance. It is a long-acting receptor antagonist. Candesartan cilexetil is a prodrug of candesartan, with a potency 10 times that of losartan, good selectivity of action (the affinity for the AT1 receptor is more than 10,000 times higher than that for the AT2 receptor), and a long action time (taking the drug once a day). Since it is a prodrug and the release of its active effect in the body after oral administration is relatively gentle, it is an ideal drug for the treatment of hypertension.

[0003] Candesartan cyclic compound (C6), CAS: 139481-41-7, and its structural formula is as follows: Candesartan (C8), CAS: 139481-59-7, and its structural formula is as follows: , both of which are important intermediates in the synthesis of candesartan cilexetil, and hydrazine hydrate , also known as hydrazine hydrate, is a harmful impurity produced by the mixing of hydrazine (N 2 H 4 ) with water during the synthesis of the above intermediates. Hydrazine hydrate not only endangers the life and health of production personnel, but can also be carried into the active pharmaceutical ingredient or preparation along with the subsequent process flow, which will directly affect the quality, safety, and effectiveness of the final product. There is no detection method for this single impurity disclosed in the existing literature technology. Therefore, an effective, stable, and reliable detection method for the above hydrazine hydrate impurity needs to be found. Summary of the Invention

[0004] The purpose of the present invention is to provide an effective, stable, and reliable high-performance liquid chromatography detection method for the above harmful impurity hydrazine hydrate in view of the blank in the detection of the above harmful impurity hydrazine hydrate in the existing detection methods.

[0005] The present invention provides a method for detecting hydrazine hydrate, an impurity in candesartan cilexetil intermediate, and the technical solution is as follows: (1) Preparation of solutions: Dilute hydrochloric acid solution, benzaldehyde - methanol solution, blank solution, hydrazine hydrate stock solution, reference solution, test solution (C6), test solution (C8); the blank solution includes dilute hydrochloric acid solution, benzaldehyde solution and n - heptane; the hydrazine hydrate stock solution includes hydrazine sulfate solution and dilute hydrochloric acid solution; the reference solution includes hydrazine sulfate solution, dilute hydrochloric acid solution, benzaldehyde solution and n - heptane; the test solution (C6) includes candesartan cyclic compound sample solution, dilute hydrochloric acid solution, benzaldehyde solution and n - heptane; the test solution (C8) includes candesartan sample solution, dilute hydrochloric acid solution, benzaldehyde solution and n - heptane; (2) Determination method: After the system is stable, inject 1 needle of the blank solution, 5 needles of the reference solution, and 1 needle of each test solution, and record the chromatogram. The chromatographic conditions are as follows: Chromatographic column: Octadecylsilyl silica gel bonded phase is used as the filler; Flow rate: 1.0 ml / min; Detection wavelength: 305 nm; Column temperature: 30 °C; Injection volume: 20 μl; Mobile phase A: 0.3 g / L disodium ethylenediaminetetraacetate solution; Mobile phase B: Acetonitrile, isocratic elution; Furthermore, the preparation of the dilute hydrochloric acid solution: Take an appropriate amount of concentrated hydrochloric acid, place it in a 100 - ml volumetric flask, add water to dilute to the scale, and shake well; Furthermore, the preparation of the benzaldehyde - methanol solution: Take a certain amount of benzaldehyde, place it in a 100 - ml volumetric flask, add a certain amount of formaldehyde, shake, and then make up the volume to the scale with water and shake well; Furthermore, the preparation of the blank solution: Accurately measure a certain amount of dilute hydrochloric acid solution, place it in a 5 - ml centrifuge tube, accurately add a certain amount of benzaldehyde, vortex - shake, then accurately add a certain amount of n - heptane, vortex - shake, centrifuge, let stand, and inject the supernatant (n - heptane layer); Furthermore, the preparation of the hydrazine stock solution: Weigh accurately a certain amount of hydrazine sulfate, place it in a 100 - ml volumetric flask, dissolve it with water and dilute to the scale, shake well, then accurately measure 1 ml and place it in a 100 - ml volumetric flask, dilute to the scale with dilute hydrochloric acid, and shake well; Furthermore, the preparation of the reference solution: Accurately measure a certain amount of the hydrazine stock solution, place it in a 5 - ml centrifuge tube, accurately add a certain amount of benzaldehyde solution, vortex - shake, then accurately add a certain amount of n - heptane, vortex - shake, centrifuge, let stand, and inject the supernatant (n - heptane layer); Furthermore, the preparation of the test solution (C6): Accurately measure a certain amount of candesartan cyclic compound sample, place it in a 5 - ml centrifuge tube, accurately add a certain amount of dilute hydrochloric acid solution and benzaldehyde solution, vortex - shake, then accurately add a certain amount of n - heptane, vortex - shake, centrifuge, let stand, and inject the supernatant (n - heptane layer); Further, the preparation of the test solution (C8): Accurately measure a certain amount of candesartan sample and place it in a 5-ml centrifuge tube. Then, accurately add a certain amount of dilute hydrochloric acid solution and benzaldehyde solution, vortex and oscillate. Next, accurately add a certain amount of n-heptane, vortex and oscillate again, centrifuge, and let it stand. Take the supernatant (n-heptane layer) for injection; (3)

[0006] Wherein, Ru: the peak area of hydrazine impurity in the test solution; Rs: the average peak area of hydrazine impurity in 5 needles of the reference solution; Cs: the concentration of hydrazine impurity in the reference solution (μg / ml); Cu: the concentration of the test solution (g / ml).

[0007] The beneficial effects of the present invention are as follows: The object of the present invention is to provide a high-performance liquid chromatography detection method for hydrazine hydrate impurity in candesartan cilexetil intermediate. The detection method has a high chromatographic peak resolution for hydrazine impurity in candesartan cilexetil intermediate, has high system suitability, and meets the standards in terms of specificity, precision, quantitative limit, detection limit, and method durability.

[0008] The detection method for the impurity C further includes method verification before detection. The determination results of this analytical method verification are shown in Table 1 below: Description of the Drawings

[0009] Figure 1 Chromatogram of the reference solution in Example 2 Figure 2 Chromatogram of the blank solution in Example 2 Figure 3 Chromatogram of the hydrazine localization solution in Example 3 Figure 4 Chromatogram of the selective solution (C6) in Example 3 Figure 5 Chromatogram of the selective solution (C8) in Example 3 Figure 6 Chromatogram of the test solution (C6) in Example 4 Figure 7 Chromatogram of the test solution (C8) in Example 4 Figure 8 Chromatogram of the LOQ solution in Example 5 Figure 9 Chromatogram of the LOD solution in Example 5 Embodiments

[0010] The present invention will be further described below in conjunction with the drawings in the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Examples

[0011] (1) Experimental materials and instrument conditions Experimental materials: Hydrazine sulfate, manufacturer: Guangzhou Chemical Reagent Factory; Benzaldehyde, manufacturer: Tianjin Kemiou Chemical Reagent Co., Ltd.; Disodium ethylenediaminetetraacetate, manufacturer: Tianjin Damao Chemical Reagent Factory; Concentrated hydrochloric acid, manufacturer: Zhuhai Huachengda Chemical Co., Ltd.; Methanol, manufacturer: Xilong Scientific Co., Ltd.; Acetonitrile, manufacturer: Xilong Scientific Co., Ltd.; Ultra-pure water, manufacturer: Zhuhai Runde Pharmaceutical Co., Ltd.; n-Heptane, manufacturer: Tianjin Kemiou Chemical Reagent Co., Ltd.; Candesartan cyclic compound, manufacturer: Zhuhai Runde Pharmaceutical Co., Ltd.; Candesartan, manufacturer: Zhuhai Runde Pharmaceutical Co., Ltd.

[0012] Instruments: Liquid chromatograph: Agilent Technologie Agilent 1260; Electronic analytical balance: METTLERTOLED XSE205DU; Chromatographic column: Grace Alltima TM C18 4.6×250mm, 5μm; Centrifuge: Shanghai Anting Scientific Instrument Factory TGL-16C; Oscillator: Dalong Xingchuang Experimental Instrument Co., Ltd. MX-S.

[0013] Determination method: Inject the blank solution, reference solution, and test solution into the liquid chromatograph respectively, and record the chromatogram. The chromatographic conditions are as follows: Chromatographic column: Octadecylsilane chemically bonded silica gel as the filler; Flow rate: 1.0 ml / min; Detection wavelength: 305 nm; Column temperature: 30 °C; Injection volume: 20 μl; Mobile phase A: 0.3 g / L disodium ethylenediaminetetraacetate solution; Mobile phase B: Acetonitrile, isocratic elution, for 25 min.

[0014] (2) Experimental procedures: Preparation of dilute hydrochloric acid solution: Take 23.4 ml of concentrated hydrochloric acid, place it in a 100 ml volumetric flask, add water to dilute to the mark, and shake well; Preparation of benzaldehyde-methanol solution: Take 6.0 ml of benzaldehyde (equivalent to 6 g of benzaldehyde), place it in a 100 ml volumetric flask, add 50 ml of formaldehyde, shake, and then add water to volume to the mark and shake well; (Concentration of benzaldehyde-methanol solution: 60 g / L) Preparation of blank solution: Accurately measure 1.0 ml of dilute hydrochloric acid solution, place it in a 5 ml centrifuge tube, accurately add 1.0 ml of benzaldehyde solution, vortex for 90 s, then accurately add 2.0 ml of n-heptane, vortex for 120 s, centrifuge at 3500 rpm for 4 min, let stand, and inject the supernatant (n-heptane layer); Preparation of hydrazine hydrate stock solution: Weigh accurately 38 mg of hydrazine sulfate, place it in a 100-ml volumetric flask, dissolve it with water and dilute to the mark, shake well. Then accurately measure 1.0 ml of this diluted solution, place it in a 100-ml volumetric flask, and dilute to the mark with dilute hydrochloric acid, shake well; (Concentration of hydrazine sulfate: 3.8 μg / ml, equivalent to hydrazine 0.94 μg / ml) Preparation of reference solution: Accurately measure 1.0 ml of hydrazine stock solution, place it in a 5-ml centrifuge tube, accurately add 1.0 ml of benzaldehyde solution, vortex for 90 s, then accurately add 2.0 ml of n-heptane, vortex for 120 s, centrifuge at 3500 rpm for 4 min, let it stand, and inject the supernatant (n-heptane layer); (Concentration of hydrazine sulfate: 1.9 μg / ml, equivalent to hydrazine 0.47 μg / ml) Preparation of test solution (C6): Accurately weigh 20 mg of candesartan cyclic compound sample, place it in a 5-ml centrifuge tube, accurately add 1.0 ml of dilute hydrochloric acid solution and 1.0 ml of benzaldehyde solution, vortex for 90 s, then accurately add 2.0 ml of n-heptane, vortex for 120 s, centrifuge at 3500 rpm for 4 min, let it stand, and inject the supernatant (n-heptane layer); (Concentration: 10 mg / l) Preparation of test solution (C8): Accurately weigh 20 mg of candesartan sample, place it in a 5-ml centrifuge tube, accurately add 1.0 ml of dilute hydrochloric acid solution and 1.0 ml of benzaldehyde solution, vortex for 90 s, then accurately add 2.0 ml of n-heptane, vortex for 120 s, centrifuge at 3500 rpm for 4 min, let it stand, and inject the supernatant (n-heptane layer); (Concentration: 10 mg / l) Preparation of hydrazine hydrate localization solution: Accurately measure 1.0 ml of hydrazine hydrate stock solution, place it in a 5-ml centrifuge tube, accurately add 1.0 ml of benzaldehyde solution, vortex for 90 s, then accurately add 2.0 ml of n-heptane, vortex for 120 s, centrifuge at 3500 rpm for 4 min, let it stand, and inject the supernatant (n-heptane layer); Preparation of selectivity solution (C6): Accurately weigh 19.97 mg of candesartan cyclic compound sample, place it in a 5-ml centrifuge tube, accurately add 1.0 ml of hydrazine stock solution and 1.0 ml of benzaldehyde solution, vortex for 90 s, then accurately add 2.0 ml of n-heptane, vortex for 120 s, centrifuge at 3500 rpm for 4 min, let it stand, and inject the supernatant (n-heptane layer); Preparation of selectivity solution (C8): Accurately weigh 20.03 mg of candesartan sample, place it in a 5-ml centrifuge tube, accurately add 1.0 ml of hydrazine stock solution and 1.0 ml of benzaldehyde solution, vortex for 90 s, then accurately add 2.0 ml of n-heptane, vortex for 120 s, centrifuge at 3500 rpm for 4 min, let it stand, and inject the supernatant (n-heptane layer); Preparation of LOQ solution: Accurately pipette 1.0 ml of hydrazine hydrate stock solution into a 5-ml centrifuge tube, accurately add 1.0 ml of benzaldehyde solution, vortex for 90 s, then accurately add 2.0 ml of n-heptane, vortex for 120 s, centrifuge at 3500 rpm for 4 s, let stand, and inject the supernatant (n-heptane layer) for analysis; Preparation of LOD solution: Accurately pipette 3.0 ml of LOQ solution into a 10-ml volumetric flask, dilute to the mark with water, and mix well; After the system is stable, inject 1 needle of blank solution, 5 needles of reference solution, and 1 needle of each test solution, and record the chromatogram. , where Ru: peak area of hydrazine hydrate in the test solution; Rs: average peak area of hydrazine hydrate in 5 needles of reference solution; Cs: concentration of hydrazine hydrate in the reference solution (μg / ml); Cu: concentration of the test solution (g / ml).

[0015] Example 2 System suitability test of the detection method described in the present invention System suitability is achieved by measuring the RSD of the peak areas of hydrazine in 5 needles of reference solution, and it is required that the RSD of the peak areas of hydrazine in 5 needles of reference solution ≤ 10.0%.

[0016] Prepare the blank solution and reference solution as in Example 1. Under the chromatographic conditions described in Example 1, inject 1 needle of blank solution and 5 needles of reference solution, and record the chromatogram. As Figure 1 、 Figure 2 shown, the conversion results according to the formula are shown in Table 2 below:

[0017] Example 3 Specificity test of the detection method described in the present invention The specificity of the method is achieved by measuring whether the blank solution interferes with the detection of hydrazine hydrate and the resolution between hydrazine hydrate and adjacent peaks in the selectivity solution. It is required that the blank solution should have no interference with the detection of hydrazine hydrate, and the resolution between hydrazine hydrate and adjacent impurity peaks in the selectivity solution should be ≥ 1.5.

[0018] Prepare the blank solution, localization solution and selectivity solution as described in Example 1. After the system is balanced, inject 1 needle of blank solution, 1 needle of localization solution, 1 needle of selectivity solution (C6) and 1 needle of selectivity solution (C8), and record the chromatogram. As Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 shown, the selectivity test results are as described in Table 3 below:

[0019] Example 4 Precision test of the detection method described in the present invention The precision is achieved by determining the RSD of the hydrazine hydrate determination results of 6 test solutions, and it is required that the RSD of the hydrazine hydrate determination results of the 6 test solutions should be ≤ 10.0%.

[0020] Prepare the blank solution, reference solution, test solution (C6), and test solution (C8) as described in Example 1. After the system is balanced, inject 1 needle of the blank solution and 6 needles of the test solution (C6), and record the chromatogram. As Figure 1 , Figure 6 , then after the system is balanced again, inject 1 needle of the blank solution and 6 needles of the test solution (C8), and record the chromatogram. As Figure 1 , Figure 7 , and then calculate the results according to the formula, as shown in Table 4 and Table 5 below:

[0021]

[0022] Example 5 Detection Limit and Quantitation Limit Tests of the Detection Method of the Present Invention The lowest detection limit is obtained by detecting the ratio of its response signal to noise (S / N) of 3:1, and the lowest quantitation limit is obtained by detecting the ratio of its response signal to noise of 10:1. At the quantitation limit concentration level, 6 LOQ solutions are repeatedly examined. It is required that the RSD of the hydrazine peak areas in the 6 LOQ solutions should be ≤ 10.0% to confirm that the quantitation limit test results have a certain precision. It is required that LOQ 肼 ≤ 47 ppm, LOD = LOQ.

[0023] Prepare the blank solution, LOQ solution, and LOD solution as described in Example 1. After the system is balanced, inject 1 needle of the blank solution, 1 needle of each of the 6 LOQ solutions, and 1 needle of the LOD solution, and record the chromatogram. As Figure 1 , Figure 8 , Figure 9 , and the quantitation limit, detection limit test results, and LOQ repeatability results are shown in Tables 6 and 7 below:

[0024]

[0025] Example 6 Robustness Test of the Detection Method of the Present Invention Examine the rule of the detection results changing with time when the reference solution and the test solution are injected after being placed at room temperature for 0 day, 1 day, and 2 days, so as to provide a basis for the placement time of the reference solution and the test solution during detection: It is required that, compared with 0 day, the recovery rate of hydrazine in the reference solution should be between 90.0% and 110.0% after being placed at room temperature for 2 days, and the change value of the hydrazine determination result in the test solution should be within 20% of the limit within 2 days of being placed at room temperature, then the solution is stable.

[0026] Prepare the reference solution and the test solution according to Example 1, and place them at room temperature for 0 day, 1 day, and 2 days respectively. After the system is stable, inject one needle of the reference solution and the test solution with different placement times, record the chromatogram, and obtain the results in Table 8 as follows:

[0027] Matters not covered in this invention are well-known technologies.

[0028] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention. The purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for detecting hydrazine hydrate, an impurity in candesartan cilexetil intermediate, characterized in that, the detection method comprises the following steps: (1) Preparation of solutions: dilute hydrochloric acid solution, benzaldehyde-methanol solution, blank solution, hydrazine hydrate stock solution, reference solution, C6 test solution, C8 test solution; the blank solution comprises dilute hydrochloric acid solution, benzaldehyde solution and n-heptane; the hydrazine hydrate stock solution comprises hydrazine sulfate solution and dilute hydrochloric acid solution; the reference solution comprises hydrazine sulfate solution, dilute hydrochloric acid solution, benzaldehyde solution and n-heptane; the C6 test solution comprises candesartan cyclization product sample solution, dilute hydrochloric acid solution, benzaldehyde solution and n-heptane; the C8 test solution comprises candesartan sample solution, dilute hydrochloric acid solution, benzaldehyde solution and n-heptane; (2) Determination method: After the system is stable, inject 1 needle of the blank solution, 5 needles of the reference solution, and 1 needle of each test solution, and record the chromatogram. The chromatographic conditions are as follows: Chromatographic column: packed with octadecylsilane chemically bonded silica gel; Flow rate: 1.0 ml / min; Detection wavelength: 305 nm; Column temperature: 30 °C; Injection volume: 20 μl; Mobile phase A: 0.3 g / L disodium ethylenediaminetetraacetate solution; Mobile phase B: acetonitrile solution, isocratic elution.

2. The detection method according to claim 1, characterized in that: the mobile phase B is an acetonitrile solution with a volume ratio of acetonitrile to water of 35:65; Preparation of the blank solution: Precise measure a certain amount of dilute hydrochloric acid solution, place it in a 5 ml centrifuge tube, precisely add a certain amount of benzaldehyde, vortex, then precisely add a certain amount of n-heptane, vortex, centrifuge, let stand, and inject the supernatant n-heptane layer; Preparation of the hydrazine hydrate stock solution: Weigh precisely a certain amount of hydrazine sulfate, place it in a 100 ml volumetric flask, dissolve it with water and dilute to the mark, shake well, then precisely measure 1 ml, place it in a 100 ml volumetric flask, and dilute to the mark with dilute hydrochloric acid, shake well; Preparation of the reference solution: Precise measure a certain amount of the hydrazine hydrate stock solution, place it in a 5 ml centrifuge tube, precisely add a certain amount of benzaldehyde solution, vortex, then precisely add a certain amount of n-heptane, vortex, centrifuge, let stand, and inject the supernatant n-heptane layer; Preparation of the C6 test solution: Precise measure a certain amount of candesartan cyclization product (C6) sample, place it in a 5 ml centrifuge tube, precisely add a certain amount of dilute hydrochloric acid solution and benzaldehyde solution, vortex, then precisely add a certain amount of n-heptane, vortex, centrifuge, let stand, and inject the supernatant n-heptane layer; Preparation of the C8 test solution: Precise measure a certain amount of candesartan (C8) sample, place it in a 5 ml centrifuge tube, precisely add a certain amount of dilute hydrochloric acid solution and benzaldehyde solution, vortex, then precisely add a certain amount of n-heptane, vortex, centrifuge, let stand, and inject the supernatant n-heptane layer.

Images