Chromatographic resolution method for four chiral isomers in loxoprofen sodium racemate

Abstract

The invention discloses a chromatographic resolution method for four chiral isomers of loxoprofen sodium, which comprises the following steps: adopting a high performance liquid chromatography method, and adopting a normal-phase chiral chromatographic column as a chromatographic column; the mobile phase is a mixed solution of alkane, tertiary butanol, trifluoroacetic acid and water. According to the technical scheme, the method for splitting the four chiral isomers in the loxoprofen sodium racemate in a normal-phase chiral mode is established for the first time, the separation degree and accuracy of the isomers can be improved, the separation degree is high, the peak pattern symmetry is good, and meanwhile the reproducibility and the stability effect are excellent.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a chromatographic separation method for four chiral isomers in a loxoprofen sodium racemate. Background Art

[0002] Loxoprofen sodium is a propionic acid precursor nonsteroidal anti-inflammatory drug. After being converted into activated metabolites by the liver, it exerts antipyretic, analgesic and anti-inflammatory effects by inhibiting the rate-limiting enzyme redoxase in the production of prostaglandins. The drug was launched in Japan in 1986 and in my country in 1999. It is widely used for anti-inflammatory and analgesic treatment of rheumatoid arthritis after surgery, trauma and tooth extraction, as well as antipyretic and analgesic treatment of acute upper respiratory tract inflammation. The chemical structure of loxoprofen sodium is:

[0003]

[0004] As can be seen from the above structural formula, loxoprofen sodium has two chiral centers and four chiral isomers. Its two chiral centers are located at the α position (1' position) of propionic acid and the α position (2 position) of cyclopentanone, respectively.

[0005] Currently, loxoprofen sodium is marketed in the form of racemates. Existing nonsteroidal anti-inflammatory drugs containing chiral centers, such as ibuprofen and naproxen, have been shown to have different pharmacological effects between their enantiomers. Therefore, it is very necessary to establish a detection method that can strictly control the ratio of the four isomers of loxoprofen sodium.

[0006] The detection method of chiral isomers is generally based on the normal phase mode of chiral columns. Compared with the reverse phase mode, the normal phase mode has better reproducibility and durability, and the stability of the chromatographic column is also better. However, in the prior art, there is no method for detecting and separating loxoprofen sodium in the normal phase chiral mode. Therefore, it is necessary to establish a chiral column normal phase detection and separation method for loxoprofen sodium chiral isomers. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a chromatographic separation method and application of four chiral isomers in the racemate of loxoprofen sodium. The technical solution of the present invention detects and separates the isomers in the racemate of loxoprofen sodium by normal phase chiral mode, and the method can improve the separation degree and accuracy between isomers, and has excellent reproducibility and stability.

[0008] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0009] The first aspect of the present invention provides a chromatographic separation method for four chiral isomers in a loxoprofen sodium racemate. The loxoprofen sodium racemate solution is separated by high performance liquid chromatography, the chromatographic column is a normal phase chiral chromatographic column, and the mobile phase is a mixed solution of alkanes, tert-butyl alcohol, trifluoroacetic acid and water.

[0010] In mobile phase, the selection of tert-butyl alcohol is particularly important, and is not selected at random. If tert-butyl alcohol is replaced by other alcohols, such as isopropanol, methanol, ethanol etc., then all can not reach the effect that 4 chiral isomers can be separated as in the present application. This is because different alcohols can have different selectivities in chiral separation and cause. In mobile phase, water is also one of necessary conditions, and the addition of water can play the effect of stabilizing mobile phase system, and is conducive to improving separation effect. Trifluoroacetic acid, as additive, can improve the separation effect of the isomers in the loxoprofen sodium racemate, and is conducive to improving the symmetry of peak type.

[0011] Preferably, in the mobile phase, the volume of tert-butanol is 5-15% of the total volume of the mobile phase, the volume of water is 0.005-0.5% of the total volume of the mobile phase, the volume of trifluoroacetic acid is 0.02-0.2% of the total volume of the mobile phase, and the remainder is alkane.

[0012] For example, the volume of tert-butyl alcohol can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0013] The volume of water is more preferably 0.01-0.5%, such as 0.01-0.08%, 0.01-0.12%, 0.01-0.15%, 0.01-0.2%, 0.01-0.5%; most preferably 0.01-0.05%.

[0014] The volume of the trifluoroacetic acid is more preferably 0.02-0.2%, such as 0.02-0.08%, 0.08-0.12%, 0.12-0.15%, 0.15-0.2%.

[0015] The content of tert-butyl alcohol and water is not arbitrary, but is a very important parameter. If the volume of tert-butyl alcohol in the mobile phase is less than 5%, the four isomers cannot be separated. If it is higher than 15%, the retention time of each isomer is too short and the complete separation cannot be achieved.

[0016] In addition, if the mobile phase does not contain water, the separation effect will be poor and the four chiral isomers cannot be completely separated. If too much water is added, such as more than 0.5%, the mobile phase will be stratified and cannot be a uniform phase, which will affect subsequent separation experiments.

[0017] Preferably, the alkane is selected from one or more of n-hexane, n-heptane and isooctane. More preferably, the alkane is n-hexane.

[0018] Preferably, the matrix of the chromatographic column is silica gel, and the surface of the silica gel is coated with cellulose-tris(4-methylbenzoate).

[0019] Preferably, the mobile phase further comprises other alcohols, the other alcohols are selected from one or more of isopropanol, methanol and ethanol, and the volume of the other alcohols is 1-10% of the total volume of the mobile phase, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0020] Preferably, the concentration of the loxoprofen sodium racemate solution is 0.5-5 mg / ml, wherein the solvent is a mixture of n-hexane and ethanol. For example, the concentration of the sample to be tested can be 0.5-2 mg / ml, 0.5-3 mg / ml, or 0.5-4 mg / ml.

[0021] Preferably, the volume ratio of n-hexane to ethanol is 1:0.5-2. For example, it may be 1:0.5-1.8, 1:0.5-1.5, 1:0.8-1.2.

[0022] By selecting a mixture of n-hexane and ethanol as the solvent, not only can the racemic form of loxoprofen sodium be diluted and dissolved to complete the injection, but the influence of the solvent effect on the final separation effect can also be avoided.

[0023] Preferably, the detection wavelength in the high performance liquid chromatography is 200-280 nm, such as 200-230 nm, 230-280 nm.

[0024] Preferably, the column temperature in the HPLC method is 20-35° C. For example, it can be 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C.

[0025] Preferably, the elution mode in the high performance liquid chromatography method includes isocratic elution.

[0026] Preferably, the mobile phase flow rate in the high performance liquid chromatography is 0.6 to 2.0 ml / min. For example, it can be 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, 1.3 ml / min, 1.4 ml / min, 1.5 ml / min, 1.6 ml / min, 1.7 ml / min, 1.8 ml / min, 1.9 ml / min, 2.0 ml / min. Preferably, the mobile phase flow rate in the high performance liquid chromatography is 0.8 to 1.5 ml / min.

[0027] Preferably, the injection volume in the high performance liquid chromatography method is 2 to 20 μl, such as 5 to 12 μl, 8 to 15 μl, or 5 to 20 μl.

[0028] The second aspect of the present invention provides a method for chromatographically resolving the four chiral isomers in the loxoprofen sodium racemate as described above for chiral resolution of the loxoprofen sodium racemate.

[0029] The technical solution of the present invention provides a chromatographic separation method and use of four chiral isomers in the racemic form of loxoprofen sodium. The technical solution of the present application establishes a chiral column normal phase detection separation method for chiral isomers of loxoprofen sodium for the first time. The separation method is simple, has good separation effect, and has excellent reproducibility and stability, and has extremely high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown is the separation spectrum of Example 1 of the present invention.

[0031] Figure 2 Shown is the separation spectrum of Example 2 of the present invention.

[0032] Figure 3 Shown is the separation spectrum of Example 3 of the present invention.

[0033] Figure 4 Shown is the separation spectrum of Example 4 of the present invention.

[0034] Figure 5 Shown is the separation spectrum of Example 5 of the present invention.

[0035] Figure 6 It shows the separation spectrum of Comparative Example 1 of the present invention.

[0036] Figure 7 It shows the separation spectrum of Comparative Example 2 of the present invention.

[0037] Figure 8 It shows the separation spectrum of Comparative Example 3 of the present invention.

[0038] Fig. 9 It shows the separation spectrum of Comparative Example 4 of the present invention.

[0039] Fig.10 It shows the separation spectrum of Comparative Example 5 of the present invention. DETAILED DESCRIPTION

[0040] The following is an explanation of the embodiments of the present invention by specific examples to verify the practical feasibility of the method of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0041] It should be noted that the process equipment or devices not specifically specified in the following examples are all conventional equipment or devices in the art. In addition, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below, and one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified. It should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, not to limit the scope of protection of the present invention. The test methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under the conditions recommended by the manufacturers.

[0042] In the following examples, a specific chromatographic separation method for the four chiral isomers in the loxoprofen sodium racemate is adopted, wherein the high performance liquid chromatographs used are: Welch PUP2000 high performance liquid chromatography system and workstation; the chromatographic columns used are: Ultimate Cellu-J silica gel surface coated normal phase chromatographic columns, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the particle size of the silica gel filler in the chromatographic column is 5 μm.

[0043] The tert-butyl alcohol used in the following examples and comparative examples of the present application was all freshly opened and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0044] Example 1

[0045] In this embodiment, a chromatographic separation method for four chiral isomers in the loxoprofen sodium racemate is provided, comprising the following steps:

[0046] 1) Test conditions:

[0047] Mobile phase: n-hexane, tert-butyl alcohol, trifluoroacetic acid and water in a volume ratio of 92:8:0.1:0.05;

[0048] Detection conditions: isocratic elution, detection wavelength of 220 nm, mobile phase flow rate of 1.0 ml / min, column temperature of 25 °C.

[0049] 2) Detection steps:

[0050] Preparation of test solution: Dissolve the racemic form of loxoprofen sodium in a mixture of n-hexane and ethanol to obtain a test solution with a concentration of 1 mg / ml, wherein the volume ratio of n-hexane to ethanol is 1:1;

[0051] Detection: Take 10 μl of the above test solution, inject it into the high performance liquid chromatograph, perform isocratic elution at the above flow rate, and record the chromatogram. The specific chromatogram is shown in the attached Figure 1 The details are shown in Table 1.

[0052] Depend on Figure 1 It can be seen that the method of Example 1 can completely separate the four chiral isomers in the loxoprofen sodium racemate. At the same time, it can be seen from Table 1 that the chromatogram has good peak symmetry and high separation, indicating that the separation effect is good.

[0053] Table 1

[0054]

[0055]

[0056] Example 2

[0057] In this embodiment, a chromatographic separation method for four chiral isomers in the loxoprofen sodium racemate is provided, comprising the following steps:

[0058] 1) Test conditions:

[0059] Mobile phase: n-hexane, tert-butyl alcohol, trifluoroacetic acid and water in a volume ratio of 90:10:0.1:0.05;

[0060] Detection conditions: isocratic elution, detection wavelength of 220 nm, mobile phase flow rate of 1.5 ml / min, column temperature of 25 °C.

[0061] 2) Detection steps:

[0062] Preparation of test solution: Same as Example 1, except that the final concentration of the test solution was 0.5 mg / ml;

[0063] Detection: Same as Example 1, see attached chromatogram for details Figure 2 The details are shown in Table 2.

[0064] Depend on Figure 2It can be seen that the method of Example 2 can completely separate the four chiral isomers in the loxoprofen sodium racemate. At the same time, it can be seen from Table 2 that the chromatogram has good peak symmetry and high separation, indicating that the separation effect is good.

[0065] Table 2

[0066] Peak number Retention time Separation Tailing Factor 1 13.417 1.039 2 15.173 1.4 1.239 3 17.772 1.8 1.110 4 24.893 3.6 1.172

[0067] Example 3

[0068] In this embodiment, a chromatographic separation method for four chiral isomers in the loxoprofen sodium racemate is provided, comprising the following steps:

[0069] 1) Test conditions:

[0070] Mobile phase: n-hexane, tert-butyl alcohol, isopropanol, trifluoroacetic acid and water in a volume ratio of 85:10:5:0.1:0.05;

[0071] Detection conditions: isocratic elution, detection wavelength of 220 nm, mobile phase flow rate of 0.8 ml / min, column temperature of 25 °C.

[0072] 2) Detection steps:

[0073] Preparation of test solution: Same as Example 1, except that the final concentration of the test solution was 1 mg / ml;

[0074] Detection: Same as Example 1, see attached chromatogram for details Figure 3 The details are shown in Table 3.

[0075] Depend on Figure 3 It can be seen that the method of Example 3 can completely separate the four chiral isomers in the loxoprofen sodium racemate. At the same time, it can be seen from Table 3 that the chromatogram has good peak symmetry and high separation, indicating that the separation effect is good.

[0076] Table 3

[0077] Peak number Retention time Separation Tailing Factor 1 15.522 1.078 2 17.025 1.4 1.250 3 18,905 1.6 1.154 4 24.622 3.7 1.278

[0078] Example 4

[0079] In this embodiment, a chromatographic separation method for four chiral isomers in the loxoprofen sodium racemate is provided, comprising the following steps:

[0080] 1) Test conditions:

[0081] Mobile phase: n-hexane, tert-butyl alcohol, trifluoroacetic acid and water in a volume ratio of 85:15:0.1:0.05;

[0082] Detection conditions: isocratic elution, detection wavelength of 220 nm, mobile phase flow rate of 1.0 ml / min, column temperature of 25 °C.

[0083] 2) Detection steps:

[0084] Preparation of test solution: Same as Example 1, except that the final concentration of the test solution was 1 mg / ml;

[0085] Detection: Same as Example 1, see attached chromatogram for details Figure 4 The details are shown in Table 4.

[0086] Depend on Figure 4 It can be seen that the method of Example 4 can completely separate the four chiral isomers in the loxoprofen sodium racemate. At the same time, it can be seen from Table 4 that the chromatogram has good peak symmetry and high separation, indicating that the separation effect is good.

[0087] Table 4

[0088] Peak number Retention time Separation Tailing Factor 1 12.332 1.016 2 13.573 1.3 1.306 3 15.770 1.9 1.132 4 20.860 3.1 1.246

[0089] Example 5

[0090] In this embodiment, a chromatographic separation method for four chiral isomers in the loxoprofen sodium racemate is provided, comprising the following steps:

[0091] 1) Test conditions:

[0092] Mobile phase: n-hexane, tert-butyl alcohol, trifluoroacetic acid, and water in a volume ratio of 95:5:0.1:0.01;

[0093] Detection conditions: isocratic elution, detection wavelength of 220 nm, mobile phase flow rate of 0.8 ml / min, column temperature of 25 °C.

[0094] 2) Detection steps:

[0095] Preparation of test solution: Same as Example 1, except that the final concentration of the test solution was 1 mg / ml;

[0096] Detection: Same as Example 1, see attached chromatogram for details Figure 5 The details are shown in Table 5.

[0097] Depend on Figure 5 It can be seen that the method of Example 5 can completely separate the four chiral isomers in the loxoprofen sodium racemate. At the same time, it can be seen from Table 5 that the chromatogram has good peak symmetry and high separation, indicating that the separation effect is good.

[0098] Table 5

[0099]

[0100]

[0101] Comparative Example 1

[0102] 1) Test conditions:

[0103] Mobile phase: n-hexane, isopropanol, and water in a volume ratio of 90:10:0.05;

[0104] Detection conditions: exactly the same as in Example 2.

[0105] 2) Detection steps:

[0106] Preparation of test solution: exactly the same as in Example 2;

[0107] Detection: Exactly the same as in Example 2; and record the chromatogram, see the attached chromatogram for details Figure 6 .Depend on Figure 6 It can be seen that the chromatogram contains only one peak, that is, only one peak can be detected for the four isomers of the loxoprofen sodium racemate, indicating that under the detection conditions of Comparative Example 1, the four isomers in the loxoprofen sodium racemate cannot be separated and there is no separation trend.

[0108] Comparative Example 2

[0109] 1) Test conditions:

[0110] Mobile phase: n-hexane, isopropanol, trifluoroacetic acid, and water in a volume ratio of 90:10:0.1:0.05;

[0111] Detection conditions: exactly the same as in Example 2.

[0112] 2) Detection steps:

[0113] Preparation of test solution: same as in Example 2;

[0114] Detection: Exactly the same as in Example 2; and record the chromatogram, see Figure 7 .Depend on Figure 7 It can be seen that the chromatogram contains two peaks. Compared with Comparative Example 1, the separation method of Comparative Example 2 can detect two peaks of the four isomers of the loxoprofen sodium racemate, which shows that the addition of trifluoroacetic acid is beneficial to the separation of the chiral isomers of loxoprofen sodium. At the same time, it shows that under the detection conditions of Comparative Example 2, the four isomers in the loxoprofen sodium racemate cannot be separated and there is no separation trend.

[0115] Comparative Example 3

[0116] 1) Test conditions:

[0117] Mobile phase: n-hexane, ethanol, trifluoroacetic acid, and water in a volume ratio of 90:10:0.1:0.05;

[0118] Detection conditions: exactly the same as in Example 2.

[0119] 2) Detection steps:

[0120] Preparation of test solution: same as in Example 2;

[0121] Detection: Exactly the same as in Example 2; and record the chromatogram, see Figure 8 .Depend on Figure 8 It can be seen that the chromatogram contains two peaks, that is, the four isomers of the loxoprofen sodium racemate can detect two peaks, indicating that the addition of trifluoroacetic acid is beneficial to the separation of the chiral isomers of loxoprofen sodium. At the same time, it shows that under the detection conditions of Comparative Example 3, the four isomers in the loxoprofen sodium racemate cannot be separated and there is no separation trend.

[0122] Comparative Example 4

[0123] 1) Test conditions:

[0124] Mobile phase: n-hexane, methanol, trifluoroacetic acid, and water in a volume ratio of 90:10:0.1:0.05;

[0125] Detection conditions: exactly the same as in Example 2.

[0126] 2) Detection steps:

[0127] Preparation of test solution: same as in Example 2;

[0128] Detection: Exactly the same as in Example 2; and record the chromatogram, see Fig. 9 .Depend on Fig. 9 It can be seen that the chromatogram contains two peaks, that is, the four isomers of the loxoprofen sodium racemate can detect two peaks, indicating that the addition of trifluoroacetic acid is beneficial to the separation of the chiral isomers of loxoprofen sodium. At the same time, it shows that under the detection conditions of Comparative Example 4, the four isomers in the loxoprofen sodium racemate cannot be separated and there is no separation trend.

[0129] Comparative Example 5

[0130] 1) Test conditions:

[0131] Mobile phase: n-hexane, tert-butyl alcohol, and trifluoroacetic acid in a volume ratio of 95:5:0.1;

[0132] Detection conditions: exactly the same as in Example 5.

[0133] 2) Detection steps:

[0134] Preparation of test solution: same as in Example 5;

[0135] Detection: Exactly the same as in Example 5; and record the chromatogram, see Fig.10 Compared with Example 5, Fig.10 It can be seen that the chromatogram contains four peaks, but the four peaks cannot be completely separated, indicating that under the detection conditions of Comparative Example 5, when the mobile phase does not contain water, the four isomers in the loxoprofen sodium racemate cannot be completely separated.

[0136] It can be seen from Examples 1 to 5 that the chromatographic separation method provided by the present application can completely separate the four chiral isomers in the loxoprofen sodium racemate under the conditions of the normal phase chiral mode, and the peak shape is good, the separation degree is high, and the effect is excellent; it can be seen from Comparative Examples 1 and 2 that the addition of trifluoroacetic acid in the mobile phase is beneficial to the separation of isomers in the loxoprofen sodium racemate; it can be seen from Example 2 and Comparative Examples 2 to 4 that only when the mobile phase contains tert-butanol can the four chiral isomers in the loxoprofen sodium racemate be completely separated, and replacing tert-butanol with isopropanol, ethanol and methanol cannot achieve the effect of completely separating the four chiral isomers; it can be seen from Example 5 and Comparative Example 5 that it is also necessary to add a certain amount of water to the mobile phase, and only after adding a certain amount of water can the four chiral isomers in the loxoprofen sodium racemate be completely separated.

[0137] In summary, the technical solution provided by the present invention establishes for the first time a chiral column normal phase detection chromatographic separation method for the four chiral isomers of loxoprofen sodium. The chromatographic separation method is simple and effective, and has excellent reproducibility and stability, and has extremely high application value.

[0138] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A chromatographic separation method for four chiral isomers in a loxoprofen sodium racemate, characterized in that: The loxoprofen sodium racemic solution is separated by high performance liquid chromatography, the chromatographic column is a normal phase chiral chromatographic column, and the mobile phase is a mixed solution of alkane, tert-butyl alcohol, trifluoroacetic acid and water.

2. The chromatographic separation method according to claim 1, characterized in that: In the mobile phase, the volume of tert-butyl alcohol is 5-15% of the total volume of the mobile phase, the volume of water is 0.005-0.5% of the total volume of the mobile phase, the volume of trifluoroacetic acid is 0.02-0.2% of the total volume of the mobile phase, and the remainder is alkane.

3. The chromatographic separation method according to claim 1, characterized in that: The alkane is selected from one or more of n-hexane, n-heptane and isooctane.

4. The chromatographic separation method according to claim 1, characterized in that: The matrix of the chromatographic column comprises silica gel, and the surface of the silica gel is coated with cellulose-tris(4-methylbenzoate).

5. The chromatographic separation method according to claim 1, characterized in that: The mobile phase also includes other alcohols, which are selected from one or more of isopropanol, methanol and ethanol. The volume of the other alcohols is 1-10% of the total volume of the mobile phase.

6. The chromatographic separation method according to claim 1, characterized in that: The concentration of the loxoprofen sodium racemate solution is 0.5-5 mg / ml, wherein the solvent is a mixture of n-hexane and ethanol.

7. The chromatographic separation method according to claim 6, characterized in that: The volume ratio of n-hexane to ethanol is 1:0.5-2.

8. The chromatographic separation method according to claim 1, characterized in that: The detection wavelength in the high performance liquid chromatography is 200 to 280 nm; And / or, the column temperature in the high performance liquid chromatography method is 20 to 35°C; And / or, the elution mode in the high performance liquid chromatography includes isocratic elution.

9. The chromatographic separation method according to claim 1, characterized in that: The mobile phase flow rate in the high performance liquid chromatography method is 0.6 to 2.0 ml / min; And / or, the injection volume in the high performance liquid chromatography is 2 to 20 μl.

10. Use of the chromatographic separation method of four chiral isomers in the loxoprofen sodium racemate according to any one of claims 1 to 9 for chiral separation of the loxoprofen sodium racemate.

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