A detection method for determining racemic-3-n-butylphthalide related substances by HPLC

Abstract

The invention provides a detection method for determining racemic 3-n-butylphthalide related substances by high performance liquid chromatography (HPLC), wherein the method uses an octadecylsilane bonded silica gel chromatographic column, a perchloric acid aqueous solution, methanol and acetonitrile as mobile phases for linear gradient elution, the column temperature is 15°C-30°C, and the detection wavelength is 225-230nm. The detection method provided by the invention has good specificity, sensitivity, separation of each absorption peak, accuracy and stability. The detection method provided by the present invention can effectively solve the separation problem of racemic 3-n-butylphthalide and 2-propyl-3-butyl-1-indanone, 2-valerylbenzoic acid, 3-butyl-3-hydroxyphthalide, propylphthalide, butylphthalide, toluphthalide, ethylphthalide, pentylphthalide, isopropylphthalide, 3-isobutylphthalide, 3-butylisoindolin-1-one, 2-propyl-3-hydroxy-1-indanone, o-pentylbenzoic acid and 3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone, and successfully avoids the influence of chloroform solvent peak on the detection of preparation-related substances.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and in particular relates to a detection method for determining racemic-3-n-butylphthalide raw material medicine or racemic-3-n-butylphthalide preparation related substances by high performance liquid chromatography (HPLC). Background Art

[0002] Racemic-3-n-butylphthalide (also known as butylphthalide) is a drug independently researched and developed in my country for the treatment of mild and moderate acute ischemic stroke. The structural formula of racemic-3-n-butylphthalide is Phthalate compounds are unstable and are easily affected by factors such as light, temperature, acid, and alkali, causing changes and mutual conversion. Related substances, i.e. impurities, refer to any substance that affects the purity of the drug, mainly starting materials, intermediates, polymers, side reaction products introduced during the production process, and degradation products during production and storage. In addition to being related to the pharmacological activity of the drug itself, the adverse reactions of drugs in clinical use are sometimes closely related to the impurities present in the drug. Therefore, developing appropriate analytical methods and accurately distinguishing and measuring the content of impurities is a key task in controlling drug quality.

[0003] The difficulty in detecting butylphthalide related substances lies in the difficulty in separating the various impurities produced during the synthesis and storage process from the main components. Although a variety of detection methods for racemic-3-n-butylphthalide related substances have been reported at home and abroad, these methods all have defects of one kind or another, especially the inability to effectively separate and detect the various impurities present in the production and storage process of butylphthalide. For example, the national registered standard WS 1 -(X-124)-2005Z contains the method for detecting butylphthalide-related substances: it is unable to solve the separation problem of phthalic acid, intermediate I, intermediate II, propylphthalide, butylenephthalide, dibutylphthalide and racemic-3-n-butylphthalide, and it is even more unable to solve the effective separation and inspection of more impurities. The above defects are proved in the Chinese patent application CN201610395891.4. Although the application uses a high performance liquid chromatograph, octadecylsilane bonded silica gel as a filler, 0.2% disodium hydrogen phosphate and acetonitrile as a mobile phase for gradient elution, and the detection wavelength is 226-230nm, the detection conditions solve the problem of phthalic acid, intermediate I, intermediate II, propylphthalide, butylenephthalide, dibutylphthalide impurities and the main component are difficult to separate, but the detection method described in the patent application still cannot effectively separate and detect other phthalide derivative impurities other than the above six impurities.

[0004] Chinese patent application CN201910466352.9 has developed a method for simultaneously detecting 13 butylphthalide-related substances. However, the inventors conducted a specific investigation on the potential process impurities and degradation impurities AN (see Table 1 in the specification of this application) in the butylphthalide preparation process currently used by the applicant according to the method recorded in the patent application and found that the peaks of racemic-3-n-butylphthalide and impurity N (3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone) overlap, racemic-3-n-butylphthalide and impurity J (3-isobutylphthalide) have poor separation, and impurity K (3-butylisoindolin-1-one) and impurity I (isopropylphthalide) have poor separation (see Example 1 in the specification of this application for specific experimental results).

[0005] In addition, the national registered standard WS 1 -(X-122)-2005Z, the preparation method of the test solution of butylphthalide soft capsules is to take an appropriate amount of the content under the filling amount difference item (approximately equivalent to 50 mg of butylphthalide), accurately weigh it, put it in a 100mL volumetric flask, add 1mL of chloroform to dissolve it, add methanol to dilute to the scale, shake it well, and use it as the test solution. However, the study found that chloroform has ultraviolet absorption in liquid phase inspection, and its solvent peak may affect the inspection of related substances in soft capsules.

[0006] It can be seen that the currently available detection methods for related substances of racemic-3-n-butylphthalide raw materials or racemic-3-n-butylphthalide preparations still cannot meet the needs of effectively separating and detecting related substances of butylphthalide in the butylphthalide preparation process currently used by the applicant. Summary of the invention

[0007] In order to overcome the defects of the prior art and to meet the actual needs of the applicant in the production process of racemic-3-n-butylphthalide raw materials and their preparations, the present invention provides a high performance liquid chromatography method for efficiently separating process impurities and degradation impurities generated during the production and storage of racemic-3-n-butylphthalide. From the physical and chemical properties of racemic-3-n-butylphthalide, it can be seen that process impurities are mainly reaction byproducts during the production of raw materials, and such impurities coexist in raw materials and their preparations. Degradation impurities are mainly certain functional groups or molecular structures that change due to external reasons during production or storage. Such impurities are related to the molecular structure and the environment. The impurity AN listed by us includes process impurities and degradation impurities of racemic-3-n-butylphthalide and its preparations, and can be well separated in this method. Therefore, this method is also suitable for the inspection of impurities in the production and storage of racemic-3-n-butylphthalide related preparations. This method has the advantages of good specificity, high sensitivity, good repeatability, etc.

[0008] Specifically, the present invention is realized through the following technical solutions:

[0009] The invention provides a detection method for determining racemic-3-n-butylphthalide related substances by HPLC. The method adopts an octadecylsilane bonded silica gel chromatographic column, the detector is an ultraviolet detector, the detection wavelength is 225-230nm, the flow rate is 0.9mL / min-1.1mL / min, the column temperature is 15℃-30℃, the injection volume is 10-50μL, and the mobile phase is composed of phase A, phase B and phase C, wherein phase A is a perchloric acid aqueous solution, phase B is methanol and phase C is acetonitrile;

[0010] The related substances are selected from one or more of the following: 2-propyl-3-butyl-1-indanone, 2-pentanoylbenzoic acid, 3-butyl-3-hydroxyphthalide, propylphthalide, butylphthalide, toluphthalide, ethylphthalide, pentylphthalide, isopropylphthalide, 3-isobutylphthalide, 3-butylisoindolin-1-one, 2-propyl-3-hydroxy-1-indanone, o-pentylbenzoic acid and 3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone;

[0011] The linear gradient elution conditions were as follows:

[0012]

[0013] Preferably, the racemic-3-n-butylphthalide is a racemic-3-n-butylphthalide bulk drug or a racemic-3-n-butylphthalide preparation, and more preferably, the racemic-3-n-butylphthalide preparation is a soft capsule or an injection.

[0014] Furthermore, for the detection method of related substances of racemic-3-n-butylphthalide preparations, the method also includes the step of preparing a preparation test solution: taking an appropriate amount of the preparation content, accurately weighing, placing it in a volumetric bottle, adding an appropriate amount of acetone to dissolve it, then diluting it to the scale with methanol, and shaking well, and the preparation step does not use chloroform.

[0015] In one embodiment, the octadecylsilane bonded silica gel chromatography column has a column length of 150 mm×4.6 mm and a filler particle size of 3 μm.

[0016] Preferably, the octadecylsilane bonded silica gel chromatographic column is Discovery HS-C18.

[0017] In another embodiment, the flow rate is 1.0 mL / min, the injection volume is 10 μL, the detection wavelength is 228 nm, the column temperature is 18°C-25°C, or the column temperature is 18°C, 20°C, 22°C or 25°C.

[0018] In yet another embodiment, mobile phase A is a 0.001%-0.025% perchloric acid aqueous solution.

[0019] Preferably, 0.005%-0.015% perchloric acid aqueous solution.

[0020] In a preferred embodiment, the related substances are composed of: 2-propyl-3-butyl-1-indanone, 2-pentanoylbenzoic acid, 3-butyl-3-hydroxyphthalide, propylphthalide, butylphthalide, toluphthalide, ethylphthalide, pentylphthalide, isopropylphthalide, 3-isobutylphthalide, 3-butylisoindolin-1-one, 2-propyl-3-hydroxy-1-indanone, o-pentylbenzoic acid and 3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone.

[0021] In a more preferred embodiment, the linear elution conditions are as follows:

[0022]

[0023] In another embodiment, the diluent is used to prepare the test solution or the reference solution. For the detection method of related substances of racemic-3-n-butylphthalide API, the diluent is 0.01% perchloric acid aqueous solution-methanol in a volume ratio of 50:50. In addition, methanol, ethanol, acetone, and acetonitrile can also be used as diluents.

[0024] In another more preferred embodiment, the separation degree of racemic-3-n-butylphthalide from 3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone, the separation degree of racemic-3-n-butylphthalide from impurity J (3-isobutylphthalide), and the separation degree of impurity K (3-butylisoindolin-1-one) from impurity I (isopropylphthalide) are all greater than 1.5.

[0025] In another most preferred embodiment, the quantification limit of racemic-3-n-butylphthalide and related substances is in the range of 0.90 ng-2.46 ng; and the detection limit is in the range of 0.27-0.74 ng.

[0026] In another embodiment, the detection method for related substances of racemic-3-n-butylphthalide raw materials can also be used for the detection of related substances of racemic-3-n-butylphthalide injection, which effectively solves the separation problem between the related substances and the related substances and racemic-3-n-butylphthalide, and the blank excipient solution in this detection method does not interfere with the detection of related substances.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] According to the actual production needs, the applicant has targeted the butylphthalide-related substances in the butylphthalide preparation process currently used by the applicant, mainly the process impurities and degradation impurities AN, and has obtained a high performance liquid chromatography method for efficiently separating the process impurities and degradation impurities AN generated in the production and storage of racemic-3-n-butylphthalide. The detection method can effectively solve the separation problem of racemic-3-n-butylphthalide and 2-propyl-3-butyl-1-indanone, 2-pentanoylbenzoic acid, 3-butyl-3-hydroxyphthalide, propylphthalide, butylphthalide, toluphthalide, ethylphthalide, pentylphthalide, isopropylphthalide, 3-isobutylphthalide, 3-butylisoindolin-1-one, 2-propyl-3-hydroxy-1-indanone, o-pentylbenzoic acid and 3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone.

[0029] In addition, for the detection method of related substances in butylphthalide soft capsule preparations, the applicant has also improved the preparation method of butylphthalide soft capsule test solution, successfully avoiding the influence of chloroform solvent peak on the detection of related substances in soft capsules. Moreover, this method can also be used to check related substances in racemic-3-n-butylphthalide-related preparations (such as injections, self-emulsifying capsules, etc.), and the detection method can efficiently separate process impurities and degradation impurities generated during the production and storage of these preparations.

[0030] The detection method of the invention has the advantages of good specificity, high sensitivity, good repeatability and the like, and can be used for simultaneous monitoring of multiple related substances in the industrial production, preparation and storage of racemic-3-n-butylphthalide. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : HPLC spectrum of impurity AN of the present invention detected using CN201910466352.9.

[0032] Figure 2 : HPLC spectrum of impurity separation in the method of embodiment 2 of the present invention.

[0033] Figure 3 : HPLC spectrum of impurity separation in the method of embodiment 3 of the present invention.

[0034] Figure 4 : HPLC spectrum of impurity separation in the method of embodiment 4 of the present invention.

[0035] Figure 5 : HPLC spectrum of impurity separation in the method of embodiment 5 of the present invention.

[0036] Figure 6 : HPLC spectrum of impurity separation in the method of embodiment 6 of the present invention.

[0037] Figure 7 : HPLC spectrum of impurity separation in the method of embodiment 7 of the present invention.

[0038] Figure 8 : HPLC spectrum of impurity separation in the method of Example 8 of the present invention.

[0039] Fig. 9 : HPLC spectrum of impurity separation in the method of embodiment 9 of the present invention.

[0040] Fig.10 : HPLC spectrum of blank solution in the method of embodiment 10 of the present invention.

[0041] Fig.11 : HPLC spectrum of impurity separation in the method of embodiment 10 of the present invention.

[0042] Fig.12 : HPLC spectrum of blank solution in the method of embodiment 11 of the present invention.

[0043] Fig.13 : HPLC spectrum of impurity separation in the method of embodiment 11 of the present invention. DETAILED DESCRIPTION

[0044] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0045] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0047] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0048] Table 1 below provides a list of information on process impurities and degradation impurities AN of the present invention.

[0049] Table 1: Process impurities and degradation impurities AN of the present invention

[0050]

[0051] Embodiment 1:

[0052] With reference to the method disclosed in Chinese patent application CN201910466352.9, based on the butylphthalide preparation process adopted by the applicant and the relevant impurities in Chinese patent application CN201910466352.9, the potential process impurities and degradation impurities AN in the currently used butylphthalide preparation process and potassium 2-(α-hydroxypentyl)benzoate in Chinese patent application CN201910466352.9 were specifically investigated.

[0053] The specific chromatographic conditions are as follows:

[0054] Column: Ultimate XB-Phenyl 250mm×4.6mm, 5μm

[0055] Mobile phase A: 0.1% acetic acid in water

[0056] Mobile phase B: methanol-acetonitrile (50:50)

[0057] Flow rate: 1.0mL / min

[0058] Column temperature: 30°C

[0059] Detection wavelength: 227nm (UV detector)

[0060] Injection volume: 20 μL

[0061] Gradient elution was performed as shown in Table 2 below.

[0062] Table 2: Gradient elution conditions of Example 1

[0063]

[0064] Preparation of test solution: Take appropriate amounts of impurity B, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity K, impurity L, impurity M, impurity N, potassium 2-(α-hydroxypentyl)benzoate and butylphthalide, dissolve and dilute with solvent [0.1% acetic acid aqueous solution-methanol (50:50)] to make a solution containing approximately 10 μg each of impurity B, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, impurity N, potassium 2-(α-hydroxypentyl)benzoate and 1 mg of butylphthalide per 1 mL.

[0065] The test results are shown in Table 3 and Figure 1 shown.

[0066] Table 3: Results of testing the impurity AN of the present invention using the CN201910466352.9 method

[0067] name Retention time (min) Separation Impurity F 10.110 - Impurity L 12.763 6.03 Potassium 2-(α-hydroxypentyl)benzoate 14.694 4.03 Impurity G 15.385 1.30 Impurity B 18.990 6.11 Impurity I 19.965 1.54 Impurity K 20.385 0.65 Impurity D 22.009 2.66 Impurity C 23.030 1.64 Impurity J 29.203 9.77 Butylphthalide 30.132 1.38 Impurity N 30.132 Coincident with the main peak Impurity H 37.348 10.42 Impurity M 38.744 2.25

[0068] The results show that the peaks of racemic-3-n-butylphthalide and impurity N (3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone) overlap, the separation degree of racemic-3-n-butylphthalide and impurity J (3-isobutylphthalide) is poor, and the separation degree of impurity K (3-butylisoindolin-1-one) and impurity I (isopropylphthalide) is poor. It can be seen that the detection method described in CN201910466352.9 cannot meet the needs of effective separation and detection of butylphthalide-related substances in the butylphthalide preparation process currently used by the applicant.

[0069] Embodiment 2:

[0070] The chromatographic conditions used were as follows:

[0071] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0072] Column temperature: 20℃

[0073] Flow rate: 1.0mL / min

[0074] Detection wavelength: 228nm (UV detector)

[0075] Injection volume: 10 μL

[0076] Mobile phase A: 0.01% perchloric acid in water

[0077] Mobile phase B: methanol

[0078] Mobile phase C: acetonitrile

[0079] Gradient elution was performed as shown in Table 4 below.

[0080] Table 4: Gradient elution conditions of Example 2

[0081]

[0082] Test solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity K, impurity L, impurity M, impurity N and racemic-3-n-butylphthalide, dissolve and dilute with solvent [0.01% perchloric acid aqueous solution-methanol (50:50)] to make a solution containing about 10 μg each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, impurity N and 1 mg of racemic-3-n-butylphthalide per 1 mL;

[0083] Take 10 μL of the test solution for inspection. The results show that the separation between each impurity and between each impurity and racemic-3-n-butylphthalide is greater than 1.5 (see Figure 2 ).

[0084] The results showed that under this chromatographic condition, the separation degree of 15 substances including impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity K, impurity L, impurity M, impurity N and racemic-3-n-butylphthalide was greater than 1.5 (see Figure 2 ), compared with the chromatographic conditions in CN201910466352.9, racemic-3-n-butylphthalide and impurity N, racemic-3-n-butylphthalide and impurity J, impurity K and impurity I can be effectively separated, and the separation degrees are greater than 1.5, which can meet the requirements for accurately checking racemic-3-n-butylphthalide related substances.

[0085] Embodiment 3:

[0086] The chromatographic conditions used were as follows:

[0087] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0088] Column temperature: 18°C

[0089] Flow rate: 1.0mL / min

[0090] Detection wavelength: 228nm (UV detector)

[0091] Injection volume: 10 μL

[0092] Mobile phase A: 0.01% perchloric acid in water

[0093] Mobile phase B: methanol

[0094] Mobile phase C: acetonitrile

[0095] Gradient elution was performed as shown in Table 5 below.

[0096] Table 5: Example 3 Gradient elution conditions

[0097]

[0098] Take 10 μL of the test solution for inspection. The results are shown in Figure 3 .

[0099] The results showed that under the chromatographic conditions, the separation degree between racemic-3-n-butylphthalide and impurity J was 1.595, and the separation degree between racemic-3-n-butylphthalide and impurity N was 3.946. Both separation degrees were greater than 1.5, which could meet the requirements for accurate inspection of racemic-3-n-butylphthalide related substances.

[0100] Embodiment 4:

[0101] The chromatographic conditions used were as follows:

[0102] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0103] Column temperature: 22°C

[0104] Flow rate: 1.0mL / min

[0105] Detection wavelength: 228nm (UV detector)

[0106] Injection volume: 10 μL

[0107] Mobile phase A: 0.01% perchloric acid in water

[0108] Mobile phase B: methanol

[0109] Mobile phase C: acetonitrile

[0110] Gradient elution was performed as shown in Table 6 below.

[0111] Table 6: Example 4 Gradient elution conditions

[0112]

[0113]

[0114] Take 10 μL of the test solution for inspection. The results are shown in Figure 4 .

[0115] The results showed that under the chromatographic conditions, the separation degree between racemic-3-n-butylphthalide and impurity J was 1.546, and the separation degree between racemic-3-n-butylphthalide and impurity N was 4.087. Both separation degrees were greater than 1.5, which could meet the requirements for accurate inspection of racemic-3-n-butylphthalide related substances.

[0116] Embodiment 5:

[0117] The chromatographic conditions used were as follows:

[0118] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0119] Column temperature: 20℃

[0120] Flow rate: 1.0mL / min

[0121] Detection wavelength: 228nm (UV detector)

[0122] Injection volume: 10 μL

[0123] Mobile phase A: 0.005% perchloric acid in water

[0124] Mobile phase B: methanol

[0125] Mobile phase C: acetonitrile

[0126] Gradient elution was performed as shown in Table 7 below.

[0127] Table 7: Example 5 Gradient elution conditions

[0128]

[0129] Take 10 μL of the test solution for inspection. The results are shown in Figure 5 .

[0130] The results showed that under the chromatographic conditions, the separation degree between racemic-3-n-butylphthalide and impurity J was 1.587, and the separation degree between racemic-3-n-butylphthalide and impurity N was 4.016. Both separation degrees were greater than 1.5, which could meet the requirements for accurate inspection of racemic-3-n-butylphthalide related substances.

[0131] Embodiment 6:

[0132] The chromatographic conditions used were as follows:

[0133] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0134] Column temperature: 20℃

[0135] Flow rate: 1.0mL / min

[0136] Detection wavelength: 228nm (UV detector)

[0137] Injection volume: 10 μL

[0138] Mobile phase A: 0.015% perchloric acid in water

[0139] Mobile phase B: methanol

[0140] Mobile phase C: acetonitrile

[0141] Gradient elution was performed as shown in Table 8 below.

[0142] Table 8: Example 6 Gradient elution conditions

[0143]

[0144] Take 10 μL of the test solution for inspection. The results are shown in Figure 6 .

[0145] The results showed that under the chromatographic conditions, the separation degree between racemic-3-n-butylphthalide and impurity J was 1.602, and the separation degree between racemic-3-n-butylphthalide and impurity N was 4.017. Both separation degrees were greater than 1.5, which could meet the requirements for accurate inspection of racemic-3-n-butylphthalide related substances.

[0146] Embodiment 7:

[0147] The chromatographic conditions used were as follows:

[0148] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0149] Column temperature: 25°C

[0150] Flow rate: 1.0mL / min

[0151] Detection wavelength: 228nm (UV detector)

[0152] Injection volume: 10 μL

[0153] Mobile phase A: 0.01% perchloric acid in water

[0154] Mobile phase B: methanol

[0155] Mobile phase C: acetonitrile

[0156] Gradient elution was performed as shown in Table 9 below.

[0157] Table 9: Example 7 Gradient elution conditions

[0158]

[0159] Take 10 μL of the test solution for inspection. The results are shown in Figure 7 .

[0160] The results showed that under the chromatographic conditions, the separation degree between racemic-3-n-butylphthalide and impurity J was 1.567, and the separation degree between racemic-3-n-butylphthalide and impurity N was 5.450. Both separation degrees were greater than 1.5, which could meet the requirements for accurate inspection of racemic-3-n-butylphthalide related substances.

[0161] Embodiment 8:

[0162] The chromatographic conditions used were as follows:

[0163] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0164] Column temperature: 25°C

[0165] Flow rate: 1.0mL / min

[0166] Detection wavelength: 228nm (UV detector)

[0167] Injection volume: 10 μL

[0168] Mobile phase A: 0.01% perchloric acid in water

[0169] Mobile phase B: methanol

[0170] Mobile phase C: acetonitrile

[0171] Gradient elution was performed as shown in Table 10 below.

[0172] Table 10: Example 8 gradient elution conditions

[0173]

[0174] Take 10 μL of the test solution for inspection. The results are shown in Figure 8 .

[0175] The results showed that under the chromatographic conditions, the separation degree between racemic-3-n-butylphthalide and impurity J was 1.545, and the separation degree between racemic-3-n-butylphthalide and impurity N was 3.465. The separation degrees were both greater than 1.5, which could meet the requirements for accurate inspection of racemic-3-n-butylphthalide related substances.

[0176] Embodiment 9:

[0177] The chromatographic conditions used were as follows:

[0178] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0179] Column temperature: 30°C

[0180] Flow rate: 1.0mL / min

[0181] Detection wavelength: 228nm (UV detector)

[0182] Injection volume: 10 μL

[0183] Mobile phase A: 0.01% perchloric acid in water

[0184] Mobile phase B: methanol

[0185] Mobile phase C: acetonitrile

[0186] Gradient elution was performed as shown in Table 11 below.

[0187] Table 11: Example 8 gradient elution conditions

[0188]

[0189]

[0190] Take 10 μL of the test solution for inspection. The results are shown in Fig. 9 .

[0191] The results showed that under these chromatographic conditions, the separation degree between racemic-3-n-butylphthalide and impurity J was 1.436, and the separation degree between racemic-3-n-butylphthalide and impurity N was 3.465. The column temperature of 30°C affected the separation of racemic-3-n-butylphthalide and impurity J.

[0192] Embodiment 10:

[0193] The chromatographic conditions used were as follows:

[0194] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0195] Column temperature: 20℃

[0196] Flow rate: 1.0mL / min

[0197] Detection wavelength: 228nm (UV detector)

[0198] Injection volume: 10 μL

[0199] Mobile phase A: 0.01% perchloric acid in water

[0200] Mobile phase B: methanol

[0201] Mobile phase C: acetonitrile

[0202] Gradient elution was performed as shown in Table 12 below.

[0203] Table 12: Example 9 gradient elution conditions

[0204]

[0205] Impurity stock solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity K, impurity L, impurity M and impurity N, dissolve and dilute with methanol to make a solution containing approximately 0.1 mg each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N per 1 mL.

[0206] Test solution: Take an appropriate amount of the contents of butylphthalide soft capsules (approximately equivalent to 10 mg of racemic-3-n-butylphthalide), accurately weigh, place in a 10 mL volumetric bottle, add 2 mL of acetone to dissolve, add 1 mL of the above impurity stock solution, and then dilute to the scale with methanol, shake well, and obtain.

[0207] Blank solution: Take 2 mL of acetone, place it in a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, and obtain.

[0208] Take 10 μL of the test solution and blank solution for inspection. The results are shown in Fig.10 and Fig.11 The results show that the separation between each impurity and between each impurity and racemic-3-n-butylphthalide is greater than 1.5. In addition, acetone has no absorption under this chromatographic condition and does not interfere with the inspection of each impurity and racemic-3-n-butylphthalide related substances.

[0209] Note: Butylphthalide soft capsules are homemade products, and the main ingredients are racemic-3-n-butylphthalide and vegetable oil.

[0210] Embodiment 11:

[0211] The chromatographic conditions used were as follows:

[0212] Chromatographic column: Discovery HS-C18, 150 mm × 4.6 mm, 3 μm

[0213] Column temperature: 20℃

[0214] Flow rate: 1.0mL / min

[0215] Detection wavelength: 228nm (UV detector)

[0216] Injection volume: 10 μL

[0217] Mobile phase A: 0.01% perchloric acid in water

[0218] Mobile phase B: methanol

[0219] Mobile phase C: acetonitrile

[0220] Gradient elution was performed as shown in Table 13 below.

[0221] Table 13: Gradient elution conditions of Example 10

[0222]

[0223] Test solution: Take 1 mL of the impurity stock solution in Example 10, place it in a 10 mL volumetric flask, dilute to the scale with butylphthalide injection, shake well, and obtain.

[0224] Blank excipient solution: prepare a solution by mixing hydroxypropyl-beta-cyclodextrin, sodium chloride and water according to the prescription ratio of the self-developed product.

[0225] Take 10 μL of the test solution and blank excipient solution for inspection. The results are shown in Fig.12 and Fig.13The results showed that the separation between the impurities and between the impurities and racemic-3-n-butylphthalide was greater than 1.5, and the blank auxiliary material solution did not interfere with the detection of the impurities and racemic-3-n-butylphthalide related substances.

[0226] Note: Butylphthalide injection is a self-developed product. Its main ingredients are racemic-3-n-butylphthalide, water, hydroxypropyl-beta-cyclodextrin and sodium chloride.

[0227] Embodiment 12:

[0228] In order to prove the applicability and accuracy of this method, the quantitative limit, detection limit, linearity and range, accuracy, precision, and solution stability of this detection method were also investigated. The specific experimental results are as follows:

[0229] (1) Limit of quantification

[0230] Impurity quantification limit solution: Take appropriate amount of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, impurity N and racemic-3-n-butylphthalide, dilute to appropriate concentration with diluent, and the LOQ solution is when the S / N value is greater than 10.

[0231] The results show that the S / N of racemic-3-n-butylphthalide and its impurities are greater than 10, and the quantitative limits of racemic-3-n-butylphthalide and its impurities are in the range of 0.90ng-2.46ng, indicating that this method has high sensitivity. See Table 14 for details.

[0232] Table 14: Limit of quantitation test results

[0233] Compound Name Concentration (ng) S / N Impurity A 2.46 31.15 Impurity B 1.66 16.74 Impurity C 2.25 25.88 Impurity D 1.04 26.16 Impurity E 1.08 26.85 Impurity F 1.07 41.03 Impurity G 0.90 22.18 Impurity H 1.61 29.67 Impurity I 1.05 21.58 Impurity J 1.38 18.79 Impurity K 0.99 22.18 Impurity L 2.27 23.53 Impurity M 2.24 39.66 Impurity N 1.06 17.88 Racemic-3-n-butylphthalide 1.06 13.84

[0234] (2) Detection limit

[0235] Take an appropriate amount of the quantitative limit solution and dilute it with a diluent to an appropriate concentration. When the S / N value is about 3, it is the LOD solution. The results show that the detection limit range of racemic-3-n-butylphthalide and each impurity is 0.27-0.74ng, indicating that this method has high sensitivity. See Table 15 for details.

[0236] Table 15: Detection limit test results

[0237]

[0238]

[0239] (3) Linearity and range

[0240] Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, impurity N and racemic-3-n-butylphthalide, weigh them accurately, add solvent to dissolve and dilute to prepare a linear series solution with a concentration of LOQ-200%.

[0241] The results show that the linear correlation coefficients of each impurity with racemic-3-n-butylphthalide are all greater than 0.9990, the response factor RSD at each concentration is less than 10.0%, and the percentage of the Y-axis intercept to the 100% response value is less than 25%, indicating that racemic-3-n-butylphthalide and each impurity have a good linear relationship between concentration and peak area within the quantitative limit of -2 μg / mL. See Table 16 for details.

[0242] Table 16: Linearity and range test results

[0243] Compound Name Linear regression equation Linear correlation coefficient Response Factor RSD Impurity A y=12691x+841.3 0.9994 4.59％ Impurity B y=25687x-477.63 0.9999 2.97％ Impurity C y=18240x+428.66 0.9994 2.50％ Impurity D y=35223x+350.45 0.9999 7.27％ Impurity E y=51066x-678.31 1.0000 8.29％ Impurity F y=43091x-220.42 1.0000 2.98％ Impurity G y=43091x-220.42 1.0000 1.21％ Impurity H y=29853x+43.088 1.0000 0.25％ Impurity I y=37987x-69.513 1.0000 1.70％ Impurity J y=32338x-419.1 0.9999 1.03％ Impurity K y=35369x-929.31 0.9999 2.08％ Impurity L y=13672x-261.53 1.0000 4.14％ Impurity M y=20616x-86.224 1.0000 0.35％ Impurity N y=33299x+2.4333 1.0000 1.80％ Racemic-3-n-butylphthalide y=30777x-240.57 1.0000 1.40％

[0244] (4) Accuracy test

[0245] LOQ recovery solution: Dissolve 20 mg of 3-n-butylphthalide, weigh accurately, place in a 20 mL volumetric flask, add solvent to dissolve and dilute to scale, shake well, dissolve with LOQ solution and dilute to scale. Prepare three portions in total.

[0246] 100% recovery solution: Add 20 mg of 3-n-butylphthalide, weigh accurately, place in a 20 mL volumetric flask, and accurately measure 5 mL of 200% solution under the linearity and range items, dilute to the scale with solvent. Prepare three times in total.

[0247] 120% recovery solution: Dissolve 20 mg of 3-n-butylphthalide, weigh accurately, place in a 20 mL volumetric flask, and accurately measure 6 mL of the 200% solution under the linearity and range item, dilute to the mark with solvent. Prepare three times in total.

[0248] The results showed that the recovery rate of each impurity ranged from 91.04% to 108.67%, and the RSD was less than 10.0%, indicating that the accuracy of this method was good. See Table 17 for details.

[0249] Table 17: Accuracy test results

[0250] Compound Name Average recovery rate% RSD% Impurity A 91.04 3.27 Impurity B 103.06 2.71 Impurity C 108.67 6.06 Impurity D 94.45 6.82 Impurity E 99.08 6.52 Impurity F 100.72 0.72 Impurity G 108.14 7.07 Impurity H 100.77 7.07 Impurity I 105.38 8.55 Impurity J 91.78 7.28 Impurity K 99.19 3.77 Impurity L 97.22 3.47 Impurity M 101.84 0.93 Impurity N 105.26 6.56

[0251] (5) Precision

[0252] Test solution: Dissolve 20 mg of 3-n-butylphthalide, weigh accurately, place in a 20 mL volumetric flask, and accurately measure 5 mL of 200% solution under the linearity and range items, dilute to the scale with solvent, and shake well. Prepare 6 portions in the same way.

[0253] Six test sample solutions were prepared by different personnel using different instruments on different dates for inspection.

[0254] The results show that among the 12 data sets, the RSD of impurities with impurity mass > 0.1% is less than 10%, and the RSD of impurities with impurity mass ≤ 0.1% is less than 15%, indicating that the precision of this method is good. See Table 18 for details.

[0255] Table 18: Precision test results

[0256]

[0257]

[0258] (6) Solution stability

[0259] Test solution: weigh about 20 mg of racemic 3-n-butylphthalide into a 20 mL volumetric flask, add diluent to dissolve and dilute to scale, shake well. Place at room temperature for a period of time before injecting (0h, 4h, 8h, 12h, 31h, 48h); calculate the impurities and corresponding changes at each time point.

[0260] The results show that the RSD of the impurity changes in the test solution within 48 hours is less than 10%, indicating that the test solution is stable within 48 hours. See Table 19 for details.

[0261] Table 19: Test solution stability test results (area normalization method)

[0262] Time (h) Impurity A(%) Impurity D(%) Total impurities% 0 0.018 0.069 0.087 4 0.018 0.070 0.088 8 0.018 0.069 0.087 12 0.018 0.071 0.089 31 0.021 0.069 0.090 48 0.022 0.070 0.092 RSD% 9.57 1.17 2.18

[0263] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for determining racemic-3-n-butylphthalide related substances by HPLC, Features: The method adopts an octadecylsilane bonded silica gel chromatographic column, the filler particle size of the octadecylsilane bonded silica gel chromatographic column is 3 μm, the detector is an ultraviolet detector, the detection wavelength is 225-230 nm, the column temperature is 18°C-25°C, and the mobile phase is composed of phase A, phase B and phase C, wherein phase A is 0.001%-0.025% perchloric acid aqueous solution, phase B is methanol and phase C is acetonitrile; The related substances include: 3-isobutylphthalide, 3-butylisoindolin-1-one, isophenylpropanolphthalide, 3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone; The linear gradient elution conditions were as follows: ； The racemic-3-n-butylphthalide is a racemic-3-n-butylphthalide raw material or a racemic-3-n-butylphthalide preparation. The method for detecting related substances of the racemic-3-n-butylphthalide preparation further comprises the step of preparing a test solution for the preparation: taking an appropriate amount of the preparation content, accurately weighing it, placing it in a volumetric bottle, adding an appropriate amount of acetone to dissolve it, and then diluting it to the scale with methanol, and shaking it well to obtain, The resolution of racemic-3-n-butylphthalide and 3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone, the resolution of racemic-3-n-butylphthalide and 3-isobutylphthalide, and the resolution of racemic-3-butylisoindolin-1-one and isopropylphthalide are all greater than 1.

5.

2. The detection method according to claim 1, Features: The racemic 3-n-butylphthalide preparation is a soft capsule or an injection.

3. The detection method according to claim 1, Features: The column temperature is 18°C, 20°C, 22°C or 25°C.

4. The detection method according to claim 1, Features: Mobile phase A is 0.005%-0.015% perchloric acid aqueous solution.

5. The detection method according to claim 1, Features: The flow rate is 0.9 mL / min-1.1 mL / min, the injection volume is 10-50 μL, and the detection wavelength is 228 nm.

6. The detection method according to claim 5, Features: The flow rate was 1.0 mL / min, and the injection volume was 10 μL.

7. The detection method according to claim 1, Features: The specification of the octadecylsilane bonded silica gel chromatographic column is 150 mm×4.6 mm, and the octadecylsilane bonded silica gel chromatographic column is Discovery HS-C18.

8. The detection method according to claim 1, Features: The related substances are composed of the following: 2-propyl-3-butyl-1-indanone, 2-pentanoylbenzoic acid, 3-butyl-3-hydroxyphthalide, propylphthalide, butylphthalide, toluphthalide, ethylphthalide, pentylphthalide, isopropylphthalide, 3-isobutylphthalide, 3-butylisoindolin-1-one, 2-propyl-3-hydroxy-1-indanone, o-pentylbenzoic acid and 3-butyl-4,5,6,7-tetrahydro-1(3H)-isobenzofuranone.

9. The detection method according to claim 1, Features: The quantification limits of racemic-3-n-butylphthalide and its related substances were in the range of 0.90 ng-2.46 ng; the detection limits were in the range of 0.27-0.74 ng.

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