Separation and detection method for enantiomer and diastereoisomer of dextroborneol

Abstract

The invention relates to a method for separating and detecting enantiomers and diastereoisomers of dextroborneol. The method comprises the following steps: S1, preparing a test solution; the method comprises the following steps: dissolving and diluting a dextroborneol sample with an organic solvent to obtain a test solution; s2, preparing a reference substance solution; the method comprises the following steps: respectively putting a levoisoborneol reference substance, a dextroisoborneol reference substance, a levocamphol reference substance and a dexcamphol reference substance into the same measuring flask, and dissolving and diluting by using an organic solvent to obtain a reference substance solution; and S3, respectively detecting the contents of isomers in the reference substance solution and the test solution by adopting a capillary gas chromatographic column gas chromatography, and recording chromatograms. The method can be used for simultaneously detecting the impurity contents of other three isomers of dextroborneol, has good linear relation, precision, accuracy and sensitivity, and is suitable for scenes requiring high-precision chiral purity analysis and impurity control.

Description

Technical Field

[0001] The invention relates to the field of separation and detection of isomers, and in particular to a method for separation and detection of dextroborneol enantiomers and diastereomers. Background Art

[0002] Dextroborneol, also known as dextral borneol, has important application value in the fields of medicine and fine chemicals. It is the main active ingredient of edaravone dextroborneol concentrated solution for injection (an injection solution that improves neurological symptoms, daily living activities and functional disorders caused by acute ischemic stroke). There are two chiral centers in the molecular structure of dextroborneol, which constitute four isomers, namely dextroborneol (dextral borneol), levoborneol (levoborneol), levorotatory isoborneol (levorotatory isoborneol), and dextrorotatory isoborneol (dextral isoborneol) (see the formula below). Different chiral configurations may have completely different physiological activities, pharmacological effects and chemical reaction characteristics. Often one of the chiral isomers may have the expected therapeutic effect, while the other may be ineffective or even produce toxic side effects, such as levorotatory isoborneol and dextrorotatory isoborneol have liver damage effects.

[0003]

[0004] One existing detection method is to use CYDEX-B chiral capillary column (25m×0.22mm, 0.25μm) as the analytical column and use gas chromatography to determine the content of dextranol and its isomers. Another detection method is to use CYCLOSIL-B chiral capillary column (30m×0.25mm×0.25μm) as the analytical column and use gas chromatography to determine the content of dextranol and levotranol in Xingnaojing injection.

[0005] The first detection method is for the determination of the content of dextrokanol and levokanol. The peak shape of dextrokanol and levokanol in the method is poor, and the sensitivity is insufficient; and the separation degree of levorotatory isoborneol and dextrorotatory isoborneol is not ideal. Separation degree: dextrokanol and levokanol can be completely separated, but levorotatory isoborneol and dextrorotatory isoborneol can only be partially separated, and the separation degree is not ideal. Peak shape: Because the CYDEX-B stationary phase is pre-methylated cyclodextrin, and dextrokanol and its isomers contain hydroxyl groups, these hydroxyl groups hydrogen bond with the oxygen atoms in cyclodextrin to produce peak tailing, resulting in poor peak shape. Sensitivity: The low ionization efficiency of dextrokanol and its isomers leads to broadening of the chromatographic peak, which makes the sensitivity low in GC detection and requires a larger injection volume; however, the increase in injection volume will make the chromatographic separation effect worse. Therefore, the injection volume was set at 0.5 μL and the sample concentration was set at 2-5 mg / mL in this method.

[0006] In the second detection method, only the contents of dextroborneol and levokanol are measured, and the contents of levokanol and dextroborneol cannot be detected simultaneously. The linear ranges of dextroborneol and levokanol measured in the method are 0.048-0.960 mg / mL and 0.050-1.008 mg / mL, respectively. The minimum detection concentration is about 0.05 mg / mL, and the sensitivity is insufficient.

[0007] In summary, there is currently a lack of a method that can efficiently, accurately, sensitively, quickly and stably separate and detect dextran and its three isomers. Accurate determination of impurity content is particularly important, especially in industrial production and research and development and preparation of high-purity dextran, which require strict control of product quality. Summary of the invention

[0008] In order to overcome the shortcomings of the prior art, the present invention provides a method for separating and detecting dextroborneol enantiomers and diastereomers, which can simultaneously detect the impurity content of the other three isomers of dextroborneol, and the method is efficient, accurate, sensitive, rapid and stable.

[0009] The technical solution adopted by the present invention to solve the technical problem is: to provide a method for separating and detecting the enantiomers and diastereomers of dextroborneol, comprising the following steps: S1. Preparation of a test solution: Take a dextroborneol sample, dissolve and dilute it with an organic solvent to obtain a test solution; S2, preparation of reference solution: respectively take left-handed isoborneol reference substance, right-handed isoborneol reference substance, left-handed alcohol reference substance, right-handed alcohol reference substance and place them in the same volumetric flask, dissolve and dilute with an organic solvent to obtain a reference solution; S3, the prepared test solution and reference solution are sequentially injected according to the following chromatographic mass spectrometry conditions, the chromatogram is recorded, and the impurity content of the other three isomers of dextroborneol is detected; The chromatographic conditions are: The chromatographic column adopts a capillary gas chromatography column; Injection volume: 0.2~5μl; Injection port temperature: 200~240℃; Split ratio: (10~50):1; Use H2 as combustion gas, Air as combustion-supporting gas, and N2 as carrier gas and protective gas; Use programmed temperature rise; The detector is a hydrogen flame ionization detector FID; the detector temperature is 200~240℃.

[0010] Furthermore, the organic solvent is methanol or acetonitrile.

[0011] Furthermore, the chromatographic column model is CYCLOSIL-B, the column length is 30 m, the inner diameter is 0.25 mm, and the film thickness is 0.25 μm.

[0012] Furthermore, the concentration of dextroborneol in the reference solution is 4-6 mg / ml, and the concentrations of levoborneol, levorotatory isoborneol, and dextrorotatory isoborneol are all 7-8 μg / ml.

[0013] Furthermore, column flow rate: 0.8~1.2ml / min.

[0014] Furthermore, the heating procedure is as follows: the initial temperature is 70-90°C, the temperature is increased to 130-150°C at a rate of 1-2°C per minute, and then the temperature is increased to 200-220°C at a rate of 15-25°C per minute, and maintained for 1-5 minutes.

[0015] Furthermore, the temperature was raised as follows: the initial temperature was 80°C, the temperature was raised to 140°C at a rate of 2°C per minute, and then the temperature was raised to 210°C at a rate of 20°C per minute, and maintained for 3 minutes.

[0016] Furthermore, the detector temperature was 220°C.

[0017] Furthermore, the flow rate of H2 is 28-32 ml / min, the flow rate of Air is 380-420 ml / min, and the flow rate of N2 is 28-32 ml / min.

[0018] Furthermore, injection volume: 1 μl; injection port temperature: 220° C.; split ratio: 20:1.

[0019] The beneficial effects of the present invention are: (1) By optimizing the chromatographic conditions, the present invention achieves baseline separation of all four isomers, especially for the difficult-to-separate levorotatory isocarboxylic acid and dextrorotatory isocarboxylic acid, the separation degrees reach 2.4 and 2.6 respectively, far exceeding the partial separation that can only be achieved in the prior art.

[0020] (2) The method of the present invention has extremely high sensitivity and can accurately determine the presence of trace isomers. The limits of quantification of 1-isocarboxylic acid, 2-isocarboxylic acid and 1-isocarboxylic acid are 3.02 ng, 2.97 ng and 3.44 ng, respectively, which are much lower than the minimum detection concentration in the prior art, making this method suitable for scenarios requiring highly accurate chiral purity analysis and impurity control. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a gas chromatogram of the separation solution in an embodiment of the present invention; Figure 2 It is the linear regression diagram of the L-isoborneol of the separation solution in the embodiment of the present invention; Figure 3 It is the linear regression diagram of dextrorotatory isoborneol of the separation solution in the embodiment of the present invention; Figure 4It is the linear regression diagram of levoborneol of the separation solution in the embodiment of the present invention; Figure 5 It is the linear regression diagram of dextroborneol of the separation solution in the embodiment of the present invention; Figure 6 It is the gas chromatogram of the separation solution in the comparative example of the present invention; Figure 7 The present invention is attached Figure 6 A partial enlarged gas chromatogram. DETAILED DESCRIPTION

[0022] The following will be combined with the accompanying drawings in the embodiments of the invention to clearly and completely describe the technical solutions in the embodiments of the invention. It is understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. It should also be noted that, for the convenience of description, only the parts related to the present invention are shown in the accompanying drawings, rather than all structures. Example

[0023] 1. Test substances: Dextroborneol samples, i.e., the test products to be tested, are substances whose specific contents of L-isoborneol, D-isoborneol, L-borneol and D-borneol are unknown; The reference substances include L-isoborneol reference substance, D-isoborneol reference substance, L-canned alcohol reference substance and D-canned alcohol reference substance. The reference substances are calibrated and have clear content.

[0024] 2. Preparation of reference solution: Take appropriate amounts of L-isoborneol, D-isoborneol, L-isoborneol and D-isoborneol reference substances respectively, place them in the same volumetric flask, dissolve and dilute with methanol to make reference substance solutions containing 5 mg / ml of D-isoborneol and 7.5 μg / ml each of L-isoborneol, L-isoborneol and D-isoborneol in each ml.

[0025] Chromatographic conditions: The chromatographic column used was Agilent CYCLOSIL-B, 30 m × 0.25 mm, 0.25 μm; Injection volume: 1 μl; Inlet temperature: 220°C; Split ratio: 20:1; Column flow rate: 1 ml / min (nitrogen as carrier gas); Detector: FID; Detector temperature: 220°C; H2: 30ml / min; Air: 400ml / min; N2: 30ml / min; Column temperature: programmed temperature, initial temperature 80°C, increase to 140°C at a rate of 2°C per minute, then increase to 210°C at a rate of 20°C per minute, and maintain for 3 minutes.

[0026] Experimental results: as attached Figure 1 As shown, left-rotating isoborneol, right-rotating isoborneol, left-canol, and right-canol eluted in sequence, with separations of 2.4, 2.6, and 1.6, respectively. The results showed that the separation was good.

[0027] 3. Determination of Quantitation Limit and Detection Limit Adjust the sensitivity of the instrument and prepare solutions of left-borneol, left-isoborneol and right-isoborneol with methanol. After gradual dilution, inject the samples according to the chromatographic conditions under the method-specific test item. The detection limit is when the main peak height is 3 times the baseline noise, and the quantification limit is when the main peak height is 10 times the baseline noise.

[0028] Experimental results: The detection limits of L-isoborneol, D-isoborneol and L-isoborneol were measured to be 3.02ng, 2.97ng and 3.44ng respectively, and the detection limits were 1.21ng, 1.19ng and 1.38ng respectively; the results showed that the method had good sensitivity.

[0029] 4. Linearity and range Take L-isoborneol, D-isoborneol, L-borneol, and D-borneol reference substances respectively, weigh them accurately, prepare solutions of different concentrations (3-15 μg / mL), inject them according to the chromatographic conditions under the method-specific test item, use the peak area as the ordinate and the injection volume (ng) as the abscissa, perform linear regression, and calculate the correlation coefficient r.

[0030] Experimental results: Figure 2 As shown, the peak area of ​​levorotatory isoborneol is well linear in the range of 3.07ng~15.11ng, and the linear equation is y=1.0277x-0.4653, r=0.999; Figure 3 As shown, the linearity of right-rotating isoborneol with the peak area is good in the range of 2.97ng~14.86ng, and the linear equation is y=1.042x-0.5677, r=0.999; Figure 4 As shown, levoborneol has good linearity with the peak area in the range of 3.44ng~17.21ng, and the linear equation is y=0.7022x+0.2559, r=0.999; Figure 5 As shown, the dextroborneol has good linearity with the peak area in the range of 2.18 ng to 17.45 ng, and the linear equation is y=0.7951x+0.6469, r=0.999.

[0031] 5. Repeatability test Preparation of test solution: Take 50 mg of dextroborneol sample, weigh accurately, place in a 10 ml volumetric flask, add methanol to dissolve and dilute to scale, shake well to obtain the test solution, prepare 6 test solutions in total; Take 15 mg of dextroborneol reference substance, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with methanol and dilute it to the mark, shake it well, accurately measure 5 ml and place it in a 100 ml volumetric flask, dissolve it with methanol and dilute it to the mark, shake it well, and prepare a solution with a concentration of 7.5 μg / ml as dextroborneol reference substance solution. Accurately measure 1 μl of dextroborneol reference substance solution and test solution respectively, inject them according to the chromatographic conditions under the method specificity test item, record the chromatogram, and evaluate the results.

[0032] Experimental results: For 6 repeated measurements, the average values ​​of L-isoborneol, D-isoborneol and L-isoborneol were 0.04%, 0.03% and 0.05%, respectively, and the RSD% were 3.0%, 3.4% and 2.6%, respectively; the results showed that the method had good repeatability.

[0033] 6. Durability test By making slight changes to the column temperature and carrier gas flow rate of the chromatographic conditions, see Table 1, the tolerance level within which the measurement results are not affected.

[0034] Experimental results: Under different flow rates (0.8 ml / min and 1.2 ml / min) and different initial temperatures (78°C and 82°C), the separation degrees between L-isoborneol, D-isoborneol, L-kanol and D-borneol were all greater than 1.5, and there was no difference in the contents of L-isoborneol, D-isoborneol and L-kanol, as shown in Table 1; the results showed that the method had good durability.

[0035] Table 1 Durability test results of dextroborneol isomers

[0036] Comparative example: The test was conducted with reference to the detection conditions and methods of comparative document 1 "Separation of borneol enantiomers in Xingnaojing injection by gas chromatography and determination of content".

[0037] Preparation of separation solution: Take appropriate amounts of dextroborneol reference substance, levoborneol reference substance, levorotatory isoborneol reference substance, and dextrorotatory isoborneol reference substance, place them in the same volumetric flask, dissolve and dilute them with methanol to make a mixed solution containing approximately 5 mg / ml of dextroborneol and 7.5 ug / ml of levoborneol, levorotatory isoborneol, and dextrorotatory isoborneol per 1 ml.

[0038] Chromatographic conditions: Chromatographic column: CYCLOSIL-B, 30mx0.25mm, 0.25μm; Injection volume: 1 μl; Inlet temperature: 220°C; Split ratio: 1:1; Column flow rate: 1 ml / min (nitrogen as carrier gas); Detector: FID; Detector temperature: 260°C; H2: 30ml / min; Air: 400ml / min; N2: 30ml / min; Column temperature: programmed temperature rise, initial temperature 50℃, hold for 2min, increase to 150℃ at a rate of 5℃ per minute, hold for 5min, increase to 220℃ at a rate of 10℃ per minute, hold for 2min.

[0039] As attached Figure 6 and attached Figure 7 As shown in the figure, under the detection conditions and method of Reference 1, the separation degree of left-handed isoborneol and right-handed isoborneol is 1.2, which does not reach the baseline separation. Left-handed isoborneol is wrapped in right-handed isoborneol and cannot be integrated and quantified. Therefore, this method cannot achieve the simultaneous separation and detection of the impurity content of the other three isomers of right-handed isoborneol.

[0040] The above embodiments are only preferred implementation modes of the present invention, but are not limitations on the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and equivalent substitutions can be made without departing from the principles of the present invention. These technical solutions after improvements and equivalent substitutions to the claims of the present invention all fall within the protection scope of the present invention.

Claims

1. A method for separating and detecting dextroborneol enantiomers and diastereomers, characterized in that: The following steps are involved: S1. Preparation of a test solution: Take a dextroborneol sample, dissolve and dilute it with an organic solvent to obtain a test solution; S2, preparation of reference solution: respectively take left-handed isoborneol reference substance, right-handed isoborneol reference substance, left-handed alcohol reference substance, right-handed alcohol reference substance and place them in the same volumetric flask, dissolve and dilute with an organic solvent to obtain a reference solution; S3. Inject the prepared test solution and reference solution in sequence according to the following chromatographic mass spectrometry conditions and record the chromatogram; The chromatographic conditions are: The chromatographic column adopts a capillary gas chromatography column; Injection volume: 0.2~5μl; Injection port temperature: 200~240℃; Split ratio: (10~50):1; Use H2 as combustion gas, Air as combustion-supporting gas, and N2 as carrier gas and protective gas; Use programmed temperature rise; The detector is a hydrogen flame ionization detector FID; the detector temperature is 200~240℃.

2. The method for separating and detecting dexamethasone enantiomers and diastereomers according to claim 1, characterized in that: The organic solvent is methanol or acetonitrile.

3. The method for separating and detecting dexamethasone enantiomers and diastereomers according to claim 1, characterized in that: The chromatographic column model is CYCLOSIL-B, with a column length of 30m, an inner diameter of 0.25mm, and a film thickness of 0.25μm.

4. The method for separation and detection of dexamethasone enantiomers and diastereomers according to claim 1, characterized in that: The concentration of dextroborneol in the reference solution is 4-6 mg / ml, and the concentrations of levoborneol, levorotatory isoborneol, and dextrorotatory isoborneol are all 7-8 μg / ml.

5. The method for separating and detecting dexamethasone enantiomers and diastereomers according to claim 1, characterized in that: Column flow rate: 0.8~1.2ml / min.

6. The method for separation and detection of dextroborneol enantiomers and diastereomers according to claim 1, characterized in that: Heating procedure: initial temperature 70~90℃, increase the temperature to 130~150℃ at a rate of 1~2℃ per minute, then increase the temperature to 200~220℃ at a rate of 15~25℃ per minute, and maintain for 1~5 minutes.

7. The method for separating and detecting dextroborneol enantiomers and diastereomers according to claim 5, characterized in that: Heating program: initial temperature 80°C, increase the temperature to 140°C at a rate of 2°C per minute, then increase the temperature to 210°C at a rate of 20°C per minute, and maintain for 3 minutes.

8. The method for separation and detection of dexamethasone enantiomers and diastereomers according to claim 1, characterized in that: The detector temperature was 220°C.

9. The method for separation and detection of dextroborneol enantiomers and diastereomers according to claim 1, characterized in that: The flow rate of H2 is 28-32 ml / min, the flow rate of Air is 380-420 ml / min, and the flow rate of N2 is 28-32 ml / min.

10. The method for separation and detection of dextroborneol enantiomers and diastereomers according to claim 1, characterized in that: Injection volume: 1 μl; Injection port temperature: 220°C; Split ratio: 20:1.

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