A method for separating guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and impurities by high performance liquid chromatography

Abstract

The invention discloses a high performance liquid chromatography separation method of guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and impurities, that is, using a high performance liquid chromatograph, Kromasil 100-3.5-C18 3.5μm×4.6mm×250mm as a chromatographic column, a mixed solution consisting of phosphoric acid water and acetonitrile as a mobile phase, and controlling the flow rate of the mobile phase to be 1mL / min; the chromatographic column temperature is 40°C; the injection volume is 10μL; and chromatographic separation is performed under the condition of a detection wavelength of 220nm. A separation and determination of guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and impurities is effectively achieved, and the method is simple, low-cost, highly sensitive, and has a good separation effect; and at the same time, a method can be used to simultaneously detect 2,6-dichlorophenylacetic acid and five impurities, and the quality control of guanfacine hydrochloride is completed.

Description

Technical Field

[0001] The invention relates to a method for separating guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and impurities by high performance liquid chromatography, and belongs to the field of drug analysis and detection. Background Art

[0002] 2,6-Dichlorophenylacetic acid It is a key intermediate in the synthesis of guanfacine hydrochloride. Its chemical formula is C8H6C l2 O2, English name: 2,6-Dichlorophenylacetic acid. It is mainly used to synthesize guanfacine hydrochloride. Guanfacine hydrochloride was approved for marketing by the US Food and Drug Administration in 2009, and is mainly used to treat attention deficit and hyperactivity disorder in children and adolescents aged 6 to 17. Clinical studies have found that guanfacine hydrochloride can directly bind to receptors in the prefrontal cortex, and the excitation of postsynaptic α2a-receptors may produce effects such as enhancing working memory, reducing distraction, improving attention regulation, improving behavioral inhibition and enhancing impulse control. Guanfacine hydrochloride is not a controlled substance and has no known abuse or dependence. 2,6-Dichlorophenylacetic acid is an important intermediate in the synthesis of guanfacine hydrochloride, and its purity directly affects the purity of the final synthetic product, guanfacine hydrochloride. Therefore, the research on 2,6-dichlorophenylacetic acid and its impurity separation method has important industrial value.

[0003] In the process of synthesizing the intermediate, many by-products are generated due to incomplete reaction, including five impurities, whose structural formulas are shown in Table 1.

[0004] Table 1: Structural formulas of impurities in 2,6-dichlorophenylacetic acid

[0005]

[0006] Strict quality control is required for the impurities introduced during the reaction process of synthesizing the intermediate 2,6-dichlorophenylacetic acid. Therefore, it is of great significance to use a detection method with strong specificity, high sensitivity, good accuracy and easy reproducibility to control the potential impurities in 2,6-dichlorophenylacetic acid. Summary of the invention

[0007] The purpose of the present invention is to provide a method for separating guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and impurities by high performance liquid chromatography, which has good peak shape and symmetry, high sensitivity, good separation degree, and accurate and reliable results. The guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its five impurities (2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid, 3,5-dichlorophenylacetic acid) can be effectively, quickly and sensitively detected and separated.

[0008] The invention provides a method for separating and detecting 2,6-dichlorophenylacetic acid and impurities by using high performance liquid chromatography, comprising: preparing a detection solution with a diluent from a sample of 2,6-dichlorophenylacetic acid containing impurities, using a Kromasil 100-3.5-C18 chromatographic column, 3.5 μm×4.6 mm×250 mm, using phosphoric acid water and acetonitrile as mobile phases, and performing separation by gradient elution.

[0009] The mobile phase flow rate was 1 mL / min;

[0010] The column temperature was 40°C;

[0011] The injection volume was 10 μL;

[0012] The detection wavelength is 220nm;

[0013] The mobile phase was 0.1% phosphoric acid in water and acetonitrile;

[0014] The pH value of phosphoric acid water is 2.07;

[0015] The gradient elution program was:

[0016]

[0017] The preferred chromatographic separation conditions are:

[0018] The mobile phase flow rate was 1.0 mL / min;

[0019] The column temperature was 40°C;

[0020] Injection volume: 10 μL;

[0021] The detection wavelength is 220 nm.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The method of the present invention can accurately detect 2,6-dichlorophenylacetic acid by using high performance liquid chromatography, which can not only effectively detect 2,6-dichlorophenylacetic acid, but also effectively separate and detect five impurities generated in the process.

[0024] The separation method of the invention has good peak shape and separation degree, high sensitivity, accurate and reliable results, low cost, simplicity and strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a full wavelength scan of the guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid in Example 1, and the detection wavelength is determined to be 220 nm;

[0026] Figure 2The chromatographic separation diagram of the guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and five impurities in Examples 2, 3 and 4;

[0027] Figures 3 to 8 A chromatographic separation diagram showing the location of the guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and five impurities in Example 5;

[0028] Fig. 9 This is a chromatographic separation diagram of the guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and five impurities in Example 6. DETAILED DESCRIPTION

[0029] In order to make the present invention more clearly understood, preferred embodiments are described in detail below in conjunction with the accompanying drawings. The models and manufacturers of the instruments used in each embodiment are shown in Table 2.

[0030] Table 2 Instrument models and manufacturers

[0031]

[0032]

[0033] The batch numbers and manufacturer information of the reagents used in the examples are shown in Table 3.

[0034] Table 3 Batch numbers and manufacturers of reagents used

[0035] name batch number Manufacturer water - Watsons Acetonitrile 114210907 Shanghai Xingke High Purity Solvent Co., Ltd. Methanol 212210804 Shanghai Xingke High Purity Solvent Co., Ltd. Orthophosphoric acid P1864148 General-Reagent Perchloric acid 20210903 Sinopharm Chemical Reagent Co., Ltd. Dipotassium hydrogen phosphate P1675368 General-Reagent

[0036] Example 1

[0037] The invention discloses a method for determining a full-wavelength scanning wavelength in a high-performance liquid chromatography separation method of a guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities. The method comprises the following steps: weighing 50 mg of each of 2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid and 3,5-dichlorophenylacetic acid in five 50-mL volumetric flasks, adding a diluent acetonitrile to dissolve the mixture and diluting the mixture to the scale line to obtain an impurity stock solution; then weighing 100 mg of a sample 2,6-dichlorophenylacetic acid in a 100-mL volumetric flask, transferring 50 μL of each impurity stock solution into the 100-mL volumetric flask, adding a diluent acetonitrile to dissolve the mixture and diluting the mixture to the scale line to obtain a sample to be tested. A Kromasil 100-3.5-C18 3.5 μm × 4.6 mm × 250 mm column was used as the separation column, and the mobile phase was 0.1% phosphoric acid water and acetonitrile. The separation was performed under the following separation conditions:

[0038] The gradient elution program was:

[0039]

[0040] The mobile phase flow rate was 1.0 mL / min;

[0041] The column temperature was 40°C;

[0042] The injection volume was 10 μL.

[0043] The full wavelength scan is performed and the scanning results are shown in Figure 1 Considering that a wavelength that is too low will be interfered by more background substances, 220nm was selected as the detection wavelength of 2,6-dichlorophenylacetic acid.

[0044] Example 2

[0045] A high performance liquid chromatography separation method for guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities, the steps of which are as follows: weigh 50 mg of each of 2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid and 3,5-dichlorophenylacetic acid in 5 50 mL volumetric flasks, add diluent acetonitrile to dissolve and dilute to the scale line to make a fixed volume, as an impurity stock solution; then weigh 100 mg of sample 2,6-dichlorophenylacetic acid in a 100 mL volumetric flask, transfer 50 μL of each impurity stock solution to the 100 mL volumetric flask, add diluent acetonitrile to dissolve and dilute to the scale line to make a fixed volume as a sample to be tested. That is, a high performance liquid chromatograph is used, a mixed solution consisting of phosphoric acid water and acetonitrile is used as a mobile phase, and the following chromatographic columns and separation conditions are used for separation:

[0046] The chromatographic columns are SHIMADZU VP-ODS 250L×4.6mm×5μm, Kromasil 100-3.5-C18 3.5μm×4.6mm×250mm, Agilent ZORBAX SB-C18 250mm×4.6mm×5μm, and Nanospectroscopy ChromCoreTM120 C18 250mm×4.6mm×5μm.

[0047] The mobile phase was 0.1% phosphoric acid in water: acetonitrile;

[0048] The gradient elution program was:

[0049]

[0050] The mobile phase flow rate was 1.0 mL / min;

[0051] The column temperature was 40°C;

[0052] The injection volume was 10 μL;

[0053] The detection wavelength is 220 nm.

[0054] The chromatographic separation results are shown in Table 4.

[0055] Table 4 Separation results of different chromatographic column models

[0056]

[0057] From the results in Table 4, it can be seen that the different models and manufacturers of the chromatographic columns in this embodiment have an impact on the chromatographic peak separation. Agilent ZORBAX SB-C18 250mm×4.6mm×5μm did not separate the various substances, and the SHIMADZU VP-ODS 250L×4.6mm×5μm and sodium spectrum analysis ChromCoreTM 120 C18 250mm×4.6mm×5μm chromatographic columns did not have a separation greater than 1.5 for all substances. In terms of retention time and separation, the separation of the main substance and impurities using the Kromasil 100-3.5-C18 3.5μm×4.6mm×250mm chromatographic column was greater than 1.5, and the separation was good. Finally, Kromasil 100-3.5-C18 3.5μm×4.6mm×250mm was selected as the best chromatographic column for HPLC separation of guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities; the separation results are shown in Figure 2 .

[0058] Example 3

[0059] A high performance liquid chromatography separation method for guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities, the steps of which are as follows: weigh 50 mg of each of 2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid and 3,5-dichlorophenylacetic acid in 5 50 mL volumetric flasks, add diluent acetonitrile to dissolve and dilute to the scale line to make a fixed volume, as an impurity stock solution; then weigh 100 mg of sample 2,6-dichlorophenylacetic acid in a 100 mL volumetric flask, transfer 50 μL of each impurity stock solution to the 100 mL volumetric flask, add diluent acetonitrile to dissolve and dilute to the scale line to make a fixed volume as a sample to be tested. A Kromasil 100-3.5-C18 3.5 μm×4.6 mm×250 mm column is used as a separation chromatographic column, and the following separation conditions are used for separation:

[0060] Mobile phase A: B (acetonitrile);

[0061] The gradient elution program was:

[0062]

[0063] The mobile phase flow rate was 1.0 mL / min;

[0064] The column temperature was 40°C;

[0065] The injection volume was 10 μL;

[0066] The detection wavelength is 220nm;

[0067] The chromatographic separation results are shown in Table 5.

[0068] Table 5 Separation results of different mobile phase types

[0069]

[0070]

[0071] From the results in Table 5, it can be seen that the series of mobile phase types studied in this example have an impact on the chromatographic peak separation. Except for 0.1% phosphoric acid water as the mobile phase, the separation of each substance is greater than 1.5, and the separation of other mobile phases does not meet the requirements of the Chinese Pharmacopoeia; and most C18 chromatographic columns are suitable for pH values ​​between 2.0 and 8.0. Excessive acidity will narrow the selection range of the chromatographic column; perchloric acid poses a great safety hazard when used. Considering the effects of separation, retention time, safety and pH value, 0.1% phosphoric acid water was selected as the optimal mobile phase for the HPLC separation of guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities; the separation results are shown in Figure 2 .

[0072] Example 4

[0073] A high performance liquid chromatography separation method for guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities, the steps of which are as follows: weigh 50 mg of each of 2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid and 3,5-dichlorophenylacetic acid in 5 50 mL volumetric flasks, add a diluent to dissolve acetonitrile and dilute to the scale line to make a fixed volume, as an impurity stock solution; then weigh 100 mg of sample 2,6-dichlorophenylacetic acid in a 100 mL volumetric flask, transfer 50 μL of each impurity stock solution to the 100 mL volumetric flask, add a diluent acetonitrile to dissolve and dilute to the scale line to make a fixed volume as a sample to be tested. A Kromasil 100-3.5-C18 3.5 μm×4.6 mm×250 mm column is used as a separation chromatographic column, and the following ratio separation conditions of different mobile phases are used for separation:

[0074] The mobile phase was 0.1% phosphoric acid in water: acetonitrile;

[0075] The gradient elution program is shown in Table 6.

[0076] Figure 6 Elution programs for different gradient methods

[0077]

[0078] The mobile phase flow rate was 1.0 mL / min;

[0079] The column temperature was 40°C;

[0080] The injection volume was 10 μL;

[0081] The detection wavelength is 220 nm.

[0082] The separation results are shown in Table 7.

[0083] Table 7 Separation results of different gradient elution programs

[0084]

[0085] From the results in Table 7, it can be seen that the ratio of a series of mobile phases studied in this example has an impact on the separation of chromatographic peaks. Taking into account the factors of retention time and separation, the relatively good gradient method was finally selected as method 2, that is, at 0 minutes, the ratio of 0.1% phosphoric acid water and acetonitrile was 80:20, and after 32 minutes of gradient elution, the ratio changed to 50:50; the separation results are shown in Figure 2 .

[0086] Example 5

[0087] A method for separating guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities by high performance liquid chromatography is provided for calibrating the peak positions of 2,6-dichlorophenylacetic acid and its five impurities, and the steps are as follows:

[0088] Weigh 50 mg of 2,6-dichlorophenylacetic acid into a 50 mL volumetric flask, add diluent acetonitrile to dissolve and dilute to volume; then weigh 50 mg of five impurities (2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid, 3,5-dichlorophenylacetic acid) into 5 50 mL volumetric flasks, add diluent acetonitrile to dissolve and dilute to volume to serve as the sample solution to be tested;

[0089] According to the chromatographic conditions: chromatographic column: Kromasil 100-3.5-C18 3.5μm×4.6mm×250mm; mobile phase: 0.1% phosphoric acid water: acetonitrile; gradient elution method is the gradient method 2 in the above-mentioned implementation case 4; flow rate 1.00mL / min; column temperature: 40℃; detector wavelength 220nm; injection volume 10μL, high performance liquid chromatography analysis was performed; the chromatographic separation results are shown in Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 .

[0090] from Figure 3 It can be seen that the retention time of 2,6-dichlorophenylacetic acid is 21.919 min; Figure 4 It can be seen that the retention time of 2,3-dichlorophenylacetic acid is 22.742min; Figure 5 It can be seen that the retention time of 2,5-dichlorophenylacetic acid is 23.691min; Figure 6 It can be seen that the retention time of 2,4-dichlorophenylacetic acid is 24.717min; Figure 7 It can be seen that the retention time of 3,4-dichlorophenylacetic acid is 25.404min; Figure 8 It can be seen that the retention time of 3,5-dichlorophenylacetic acid is 27.078 min.

[0091] Example 6

[0092] A high performance liquid chromatography separation method for guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities comprises the following steps: weighing 50 mg of each of 2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid and 3,5-dichlorophenylacetic acid in five 50-mL volumetric flasks, adding a diluent to dissolve acetonitrile and diluting to the scale line to make a fixed volume, as an impurity stock solution; then weighing 100 mg of sample 2,6-dichlorophenylacetic acid in a 100-mL volumetric flask, respectively taking 100 μL of each impurity stock solution into the 100-mL volumetric flask, adding a diluent acetonitrile to dissolve and diluting to the scale line to make a fixed volume as a sample to be tested.

[0093] According to the chromatographic conditions: chromatographic column: Kromasil 100-3.5-C18 3.5μm×4.6mm×250mm, mobile phase is 0.1% phosphoric acid water: acetonitrile; gradient elution method is the gradient method 2 in the above-mentioned implementation case 4; flow rate 1.00mL / min; column temperature: 40℃; UV detector wavelength 220nm; injection volume 10μL; high performance liquid chromatography analysis; the chromatogram separation results are shown in Table 8.

[0094] Table 8 Chromatographic separation results of each substance

[0095]

[0096]

[0097] Separation results are shown in Fig. 9 ,from Fig. 9It can be seen that the guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its five impurities (2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid, 3,5-dichlorophenylacetic acid) can be well separated with good separation degree, indicating that this method is suitable for the detection of the synthesis of guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its impurities.

[0098] In summary, the high performance liquid chromatography separation method of the guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and its five impurities (2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid and 3,5-dichlorophenylacetic acid) of the present invention can effectively separate the intermediate 2,6-dichlorophenylacetic acid from the five impurities, and the separation degree can reach 1.71-14.33.

Claims

1. A method for separating guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and impurities by high performance liquid chromatography, characterized in that: The impurities in the 2,6-dichlorophenylacetic acid are 2,3-dichlorophenylacetic acid, 2,4-dichlorophenylacetic acid, 2,5-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid, and 3,5-dichlorophenylacetic acid; A high performance liquid chromatograph was used, with a Kromasil column as the chromatographic column, wherein the chromatographic column was Kromasil 100-3.5-C18 3.5 μm×4.6 mm×250 mm as the chromatographic column, and a mixture of phosphoric acid water and acetonitrile as the mobile phase, and the following chromatographic separation conditions were used for separation: The mobile phase flow rate was 1 mL / min; The column temperature was 40°C; The injection volume was 10 μL; The detection wavelength is 220nm; The mobile phase was 0.1% phosphoric acid in water and acetonitrile; The gradient elution program was: 。 2. The method for separating guanfacine hydrochloride intermediate 2,6-dichlorophenylacetic acid and impurities by high performance liquid chromatography according to claim 1, characterized in that: The high performance liquid chromatograph adopts Agilent 1260 high performance liquid chromatograph.

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