Chiral detection method for (r)-4,4,4-trifluoro-3-hydroxy-3-methylbutan-2-one

Abstract

The application provides a chiral detection method of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbutan-2-one, and belongs to the field of drug analysis. The method utilizes the characteristics that the benzylhydrazone derivative obtained by the reaction of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbutan-2-one and its chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone with phenylhydrazine under weak acid conditions has strong ultraviolet absorption, chromatographic separation is carried out by using a chiral chromatographic column, and detection analysis is carried out by using a high performance liquid chromatograph. The method has the advantages of simple operation, short reaction time, mild reaction condition, clean and complete reaction, good specificity and accuracy, high sensitivity and the like.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis, specifically to a chiral detection method for (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one. Background Technology

[0002] (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one is a commonly used small-molecule chiral introducing reagent with advantages such as high activity, good stereospecificity, wide availability, moderate price, and environmental friendliness. Its structural formula is: .

[0003] Its chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone has the following structural formula: .

[0004] Meanwhile, (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one is also a key starting material for the novel non-opioid analgesic suzetril, whose structural formula is: .

[0005] The structure of suzetrol contains a large number of chiral carbon atoms, and the chiral purity of each introduced chiral center directly affects its final chiral purity. Therefore, the chiral purity control of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one, as a key starting material for introducing chiral centers into suzetrol, is particularly important. (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one and its chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone lack ultraviolet chromophores, therefore their contents cannot be directly determined using high-performance liquid chromatography with an ultraviolet detector.

[0006] Currently, common methods for detecting the chirality of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one include specific rotation measurement and gas-phase chiral column analysis. Specific rotation is not sensitive enough and can only be used for preliminary qualitative analysis. Gas-phase chiral columns are expensive and time-consuming, limiting their widespread use. Reaction of the hydroxyl functional group in the structure with benzoyl chloride or benzenesulfonyl chloride is also an option, but due to the insufficient reactivity of the alcohol hydroxyl group, the resulting derivatives are few and unstable, making accurate determination of chiral purity impossible.

[0007] Therefore, there is currently a lack of rapid, stable, and accurate methods for detecting the chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone in (R)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a chiral detection method for (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one. This method utilizes the reaction of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one and its chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone with phenylhydrazine under weakly acidic conditions. The resulting phenylhydrazone derivative exhibits strong ultraviolet absorption. Chromatographic separation is performed using a chiral column, and detection and analysis are performed using high-performance liquid chromatography (HPLC). This method offers advantages such as simple operation, short reaction time, mild reaction conditions, clean and complete reaction, good specificity and accuracy, and high sensitivity.

[0009] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: a chiral detection method for (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one, which utilizes the reaction of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one and its chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone with phenylhydrazine under weakly acidic conditions to obtain a phenylhydrazone derivative with strong ultraviolet absorption. Chromatographic separation is performed using a chiral chromatographic column, and detection and analysis are performed using a high-performance liquid chromatograph.

[0010] Specifically, the steps include the following: (1) Preparation of blank solution Take an appropriate amount of diluent n-hexane:isopropanol (v / v 95:5) as a blank solution; (2) System suitability solution preparation Dissolve the chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone reference standard in ethanol to obtain the chiral isomer stock solution. Take a headspace vial, add phenylhydrazine, (R)-4,4,4-trifluoro-3-hydroxy-3-methylbutanone reference standard and the chiral isomer stock solution, then add ethanol and acetic acid, cap and seal the vial, react in a water bath at 80-90℃, let it return to room temperature, concentrate under reduced pressure, add dichloromethane, wash with water, take the dichloromethane layer, dry it and concentrate it to a yellow solid, take the yellow solid and add diluent to prepare a system-suitable solution. (3) Preparation of the test solution; Take a headspace vial, add phenylhydrazine, (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one, ethanol and acetic acid, cap and seal the vial, react in a water bath at 80-90℃, let it return to room temperature, concentrate under reduced pressure, add dichloromethane, wash with water, take the dichloromethane layer, dry it and concentrate it to a yellow solid, take the yellow solid and add diluent to prepare the test solution; (4) Determination method: Take blank solution, system suitability solution and test solution respectively, inject them into high performance liquid chromatograph for HPLC detection, record the chromatogram, and calculate the content of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one and impurity (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone by area normalization method.

[0011] The chromatographic conditions used for the HPLC detection are as follows: Chiral chromatographic column: CHIRALCEL OD-H, column length 150-250 mm; Column temperature: 25-35℃; Flow rate: 0.8-1.2 ml / min; Injection volume: 5-10 μl; UV detector: 260-280nm; Mobile phase v / v: Elute with gradients of n-hexane:isopropanol (99:1-95:5) for 20-30 min.

[0012] Preferably, in steps (2) and (3), the equivalence ratio of phenylhydrazine to (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one is 1:1, and the reaction time is 60 minutes.

[0013] Preferably, step (2) specifically involves: taking 25 mg of the chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone reference standard, placing it in a 25 ml volumetric flask, adding ethanol to dissolve and dilute to the mark, as the chiral isomer stock solution; taking another 20 ml headspace vial, adding 140 mg of phenylhydrazine, 200 mg of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbutanone reference standard, 1.0 ml of the above chiral isomer stock solution, then adding 4 ml of ethanol and 1.5 ml of acetic acid, capping and sealing the vial, reacting in an 85°C water bath for 60 minutes, allowing it to return to room temperature, concentrating under reduced pressure, adding 5 ml of dichloromethane, washing three times with 5 ml of water, taking the dichloromethane layer, drying it, and concentrating it to a yellow solid, taking the yellow solid and using a diluent to prepare a 1.0 mg / ml solution as the system suitability solution.

[0014] Preferably, step (3) is as follows: Take a 20ml headspace vial, add 140mg of phenylhydrazine, 200mg of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one test sample, add 5ml of ethanol and 1.5ml of acetic acid, cap and seal the vial, react in an 85℃ water bath for 60 minutes, let it return to room temperature, concentrate under reduced pressure, add 5ml of dichloromethane, wash three times with 5ml of water, take the dichloromethane layer, dry it and concentrate it to a yellow solid, take the yellow solid and dilute it with a diluent to prepare a 1.0mg / ml solution as the test sample solution.

[0015] Preferably, the chromatographic conditions are as follows: column temperature of 28-32℃, flow rate of 0.9-1.1 ml / min, and UV detector wavelength of 268-272 nm.

[0016] Preferably, the chromatographic column used is 4.6mm*250mm, 5μm, with a column temperature of 30℃; the flow rate is 1.0ml / min, the injection volume is 10μl, the UV detector detection wavelength is 270nm, and the mobile phase is n-hexane:isopropanol (v / v 98:2), with isogradient elution for 20min.

[0017] The beneficial effects of this invention are as follows: This invention provides an HPLC detection method for the chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone in (R)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone. The analytical method of this invention was determined by optimizing and screening derivatization methods and chromatographic conditions. This method utilizes the reaction of (R)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone and its chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone with phenylhydrazine under weakly acidic conditions. The resulting phenylhydrazone derivative exhibits strong ultraviolet absorption. Chromatographic separation is then performed using a chiral column, and detection and analysis are performed using high-performance liquid chromatography (HPLC). This method has the advantages of short reaction time, mild reaction conditions, clean and complete reaction, and simple operation. It also has good experimental results in terms of specificity, detection limit, quantitation limit, linear range, accuracy, precision, repeatability, and robustness. It can be used for the detection of chiral isomers of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one or its chiral enantiomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone as key materials for introducing chirality into molecules in industrial production or research and development. Attached Figure Description

[0018] Figure 1 Chiral blank spectrum; Figure 2 Applicability map for chiral systems; Figure 3 The chromatogram of the chiral test sample; Figure 4 The graph shows the chiral linear relationship of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one. Figure 5 The graph shows the chiral linear relationship of (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone. Figure 6 The chromatogram shows the purity of the relevant substances in the test sample. Detailed Implementation

[0019] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0020] Instruments: Agilent 1260 Infinity II with UV detector; Digital display constant temperature water bath: Jiangsu Jinyi Instrument Technology Co., Ltd., model HH-S1; Diluent v / v: n-hexane-isopropanol (95:5).

[0021] Example 1: Preparation of blank solution and system suitability solution Take an appropriate amount of diluent v / v: hexane-isopropanol (95:5) as a blank solution.

[0022] Weigh approximately 25 mg of the chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone reference standard accurately, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with ethanol to prepare the chiral isomer stock solution. Take another 20ml headspace vial, add about 140mg of phenylhydrazine, about 200mg of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one reference standard, 1.0ml of the above chiral isomer stock solution, then add 4ml of ethanol and 1.5ml of acetic acid. Cap and seal the vial, react in an 85℃ water bath for 60 minutes, let it return to room temperature, concentrate under reduced pressure, add 5ml of dichloromethane, wash three times with 5ml of water, take the dichloromethane layer, add a small amount of anhydrous magnesium sulfate to dry, filter, concentrate to a yellow solid, take an appropriate amount of the yellow solid, and prepare a solution of about 1.0mg / ml with n-hexane:isopropanol (v / v 95:5) as the system suitability solution.

[0023] Example 2: Preparation of the test solution Take a 20ml headspace vial, add approximately 140mg of phenylhydrazine and approximately 200mg of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one test sample, add 5ml of ethanol and 1.5ml of acetic acid, cap and seal the vial, react in an 85℃ water bath for 60 minutes, let it return to room temperature, concentrate under reduced pressure, add 5ml of dichloromethane, wash three times with 5ml of water, take the dichloromethane layer, add a small amount of anhydrous magnesium sulfate to dry, filter, concentrate to a yellow solid, take an appropriate amount of the yellow solid, and prepare a solution of approximately 1.0mg / ml with n-hexane:isopropanol (v / v 95:5) as the test sample solution.

[0024] Example 3: Chromatographic determination Take the blank solution, system suitability solution, and test solution mentioned above, and inject them into the high-performance liquid chromatograph (HPLC) respectively. Record the chromatograms. If the test sample chromatogram contains a peak with the same retention time as the chiral isomer derivative, calculate the peak area using the peak area normalization method and analyze it under the following chromatographic conditions: Chromatographic column: CHIRALCEL OD-H, 4.6mm*250mm, 5μm; Column temperature: 30℃; Flow rate: 1.0 ml / min; Injection volume: 10 μl; UV detector: 270nm; Elute with a gradient of mobile phase v / v: hexane: isopropanol (98:2) for 20 min.

[0025] The retention times of each substance under these chromatographic conditions are shown in Table 1 and... Figure 2 The chiral blank spectrum, chiral system suitability spectrum, and chiral test sample spectrum are shown below. Figure 1 , Figure 2 and Figure 3 As shown.

[0026] Table 1: Retention times of compounds

[0027] Example 4: Investigation of Sample Preparation Parameters A 1.0 mg / ml solution was prepared as the normal test solution according to Example 2. The test solutions were prepared according to Table 3: (1) The purity of the derivative related substances was detected according to the following methods for detecting related substances of derivatives. The amount of phenylhydrazine remaining was compared by normalizing the peak area and the reaction process was monitored. The results are shown in Table 4-1 (Result 1 of Sample Preparation Parameter Investigation); (2) The effect of preparation parameters on the detection of chiral isomers was investigated according to the chromatographic determination method of Example 3. The results are shown in Table 4-2 (Result 2 of Sample Preparation Parameter Investigation). The results show that when the reaction time is 60 minutes and the equivalent ratio of phenylhydrazine to (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one is 1:1, the reaction time is the shortest, most complete and cleanest, and the purity of the derivative related substances obtained can reach 99.80% (see Figure 6 Apart from the main peak of the derivative, there are basically no other impurity peaks, which can eliminate interference from other reagents; and all changes in preparation parameters have no effect on the detection results of chiral isomers.

[0028] Chromatographic conditions for the detection of related substances in derivatives: Column: Sunniest RP-AQUA C28, 250 mm * 4.6 mm, 5 μm; Diluent v / v: Acetonitrile-water (50-50); Column temperature: 35℃; flow rate: 1.0 ml / min; injection volume: 10 μl; UV detector: 210nm; Mobile phase A: 0.02M diammonium hydrogen phosphate (2.64g diammonium hydrogen phosphate was added per 1000ml of water, and the pH was adjusted to 3.2 with phosphoric acid); Mobile phase B: methanol; Mobile phase A and mobile phase B were used for gradient elution at different volume ratios.

[0029] The gradient elution procedure for the detection method of derivative-related substances is shown in Table 2.

[0030] Table 2: Gradient elution procedure for the detection of related substances in derivatives

[0031] Table 3: Sample preparation parameters

[0032] Table 4-1: Results of Sample Preparation Parameter Investigation

[0033] Table 4-2: Results of Sample Preparation Parameter Investigation

[0034] Example 5: Specificity Investigation The diluent was used as a blank solution, and the determination was performed under the chromatographic conditions of Example 3. The chromatogram is shown below. Figure 1 It does not interfere with the detection of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one and (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone. Example 6: Examination of Linear Relationships Take a 20 ml headspace vial, add about 140 mg of phenylhydrazine and about 200 mg of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one reference standard, add 5 ml of ethanol and 1.5 ml of acetic acid, cap and seal the vial, react in an 85 °C water bath for 60 minutes, let it return to room temperature, concentrate under reduced pressure, add 5 ml of dichloromethane, wash three times with 5 ml of water, take the dichloromethane layer, add a small amount of anhydrous magnesium sulfate to dry, filter, concentrate to a yellow solid, and obtain the (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one derivative reference standard.

[0035] Take another 20ml headspace vial, add about 140mg of phenylhydrazine and about 200mg of the chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone, add 5ml of ethanol and 1.5ml of acetic acid, cap and seal the vial, react in an 85℃ water bath for 60 minutes, let it return to room temperature, concentrate under reduced pressure, add 5ml of dichloromethane, wash three times with 5ml of water, take the dichloromethane layer, add a small amount of anhydrous magnesium sulfate to dry, filter, concentrate to a yellow solid, and obtain the (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone derivative reference standard.

[0036] Weigh 50 mg each of the above-mentioned (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one derivative reference standards and (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone derivative reference standards accurately, place them in the same 50 ml volumetric flask, add diluent solvent and dilute to the mark, shake well, and use as linear stock solution 1. Then accurately measure 1.0 ml of the above linear stock solution 1 and place it in a 100 ml volumetric flask, add diluent solvent and dilute to the mark, shake well, and use as linear stock solution 2. Then dilute to each concentration according to Table 5 (linear dilution table), and determine according to the chromatographic conditions of Example 3. Plot a regression curve with concentration versus corresponding peak area, and calculate the regression curve and correlation coefficient. The linearity results are shown in Table 6. (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one showed good linearity in the range of 0.1004–5.0203 μg / ml, and (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone showed good linearity in the range of 0.0996–4.9778 μg / ml. (See Table 6 for more details.) Figure 4 and Figure 5 .

[0037] Table 5: Linear Dilution Table

[0038] Table 6: Linearity Results

[0039] Example 7: Investigation of Limit of Detection and Limit of Quantification The linear stock solutions of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one derivative reference standards and (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone derivative reference standards were serially diluted and determined according to the chromatographic conditions of Example 3. The signal-to-noise ratio (S / N) was measured. The concentration with an S / N of approximately 10 was the limit of quantitation (LOQ) concentration; the concentration with an S / N of approximately 3 was the limit of detection (LOD) concentration. The LOD and LOD results are shown in Table 7. This invention can detect 0.002% of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one (abbreviated as R in Table 7) at the level of (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone (abbreviated as S in Table 7), with high sensitivity.

[0040] Table 7: Results of Limit of Detection and Limit of Quantitation

[0041] Example 8: Repeatability Test According to Example 6, approximately 25 mg of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one derivative reference standard was accurately weighed and placed in a 25 ml volumetric flask. The solution was dissolved and diluted to the mark with diluent, and then shaken well to obtain six parallel test solutions. Then, 1.0 ml of each test solution was accurately measured and placed in a 100 ml volumetric flask, diluted to the mark with diluent, and shaken well to obtain six 1% self-control test solutions.

[0042] 25 mg of (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone derivative reference standard was prepared according to Example 6. The standard was accurately weighed and placed in a 25 ml volumetric flask. The diluent was dissolved and diluted to the mark, and the solution was shaken well. Then, 1.0 ml of the solution was accurately measured and placed in a 100 ml volumetric flask. The diluent was diluted to the mark, and the solution was mixed well. This solution was used as the chiral isomer reference standard solution for external standard method.

[0043] The chromatographic conditions of Example 3 were followed for determination. Repeatability of related substances was calculated by peak area using the external standard method, the self-comparison method with correction factors, and the area normalization method. The repeatability results are shown in Table 8 below. The variations in chiral enantiomer content of the six test samples calculated by the external standard method, the self-comparison method, and the area normalization method were within ±0.1% of the average value. There were no significant differences in the results calculated by the external standard method, the self-comparison method, and the area normalization method. This method exhibits good repeatability.

[0044] Table 8: Repeatability Results

[0045] Example 9: Recovery Rate Study 25 mg of (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone derivative reference standard was prepared according to Example 6. The standard was accurately weighed and placed in a 25 ml volumetric flask. The diluent was dissolved and diluted to the mark, and the solution was shaken well. Then, 1.0 ml of the solution was accurately measured and placed in a 100 ml volumetric flask. The diluent was diluted to the mark, and the solution was mixed well. This solution was used as the chiral isomer reference standard solution for external standard method.

[0046] Weigh approximately 50 mg of (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone accurately, place it in a 100 ml volumetric flask, add ethanol to dissolve and dilute to the mark, and use it as a stock solution for the chiral isomer.

[0047] Nine portions of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one reference standard, each approximately 200 mg, were accurately weighed and placed in separate 20 ml headspace vials. Three portions of each chiral isomer stock solution (1.0 ml, 2.0 ml, and 3.0 ml) were added to each vial. Approximately 140 mg of phenylhydrazine, 4 ml of ethanol, and 1.5 ml of acetic acid were added to each vial. The vials were capped and sealed, and the reaction was carried out in an 85°C water bath for 60 minutes. After being allowed to return to room temperature, the solution was concentrated under reduced pressure, and 5 ml of dichloromethane was added. The solution was washed three times with 5 ml of water. The dichloromethane layer was collected, dried with a small amount of anhydrous magnesium sulfate, filtered, and concentrated to a yellow solid. Weigh 50 mg of the yellow solid accurately and place it in a 50 ml volumetric flask. Dissolve and dilute to the mark with diluent, and shake well to obtain chiral enantiomer test solutions of low, medium and high concentrations. Then accurately measure 1.0 ml of each chiral enantiomer test solution and place it in a 100 ml volumetric flask. Dilute to the mark with diluent and shake well to obtain 9 self-control solutions (i.e., self-control test solutions with a recovery rate of 1%).

[0048] Accurately measure the chiral enantiomer test solutions, 1% self-control test solutions, and chiral isomer reference solutions at the above-mentioned concentration ratios, and inject them for analysis according to the chromatographic conditions of Example 3. Based on the repeatability test results, the recoveries of each substance at low, medium, and high concentrations were calculated using the 1% self-control method, the external standard method for impurity references, and the area normalization method, respectively, using peak area. The recovery results are shown in Tables 9-1, 9-2, and 9-3 below. The recoveries of this method are all between 92.0% and 105%, and the RSDs are all less than 3.0%, indicating good recovery of this method.

[0049] Table 9-1: Recovery rate of external standard method

[0050] Table 9-2: Recovery rate of self-control method

[0051] Table 9-3: Recovery rates of samples added using the area normalization method

[0052] Example 10: Solution Stability Study The system suitability solution was analyzed at room temperature at 0, 2.5, 5, 7, and 10 chromatographic conditions as in Example 3. Peak areas and retention times were recorded to assess solution stability. The results are shown in Table 10 below. Under room temperature storage conditions, the RSD of the main component peak area was no greater than 2.0% within 10 hours, and the change in total impurity content was within ±0.1% of the average value. The system suitability solution was stable at room temperature for 10 hours.

[0053] Table 10: Solution stability results

[0054] Example 11: Durability Test Using normal conditions, different mobile phase ratios, different wavelengths, flow rates, and column temperatures, 0.5% linear mixed solutions were tested after the instrument system stabilized (see Example 6). Specific parameter changes are shown in Table 11, with only one parameter changed at a time. The system suitability index under each parameter change condition was statistically analyzed, and the results are shown in Table 12. The results indicate that the chromatographic conditions are robust under minor changes.

[0055] Table 11: Durability Parameters

[0056] Table 12: Durability Test Results

[0057] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be used to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A chiral detection method for (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one, characterized in that, The phenylhydrazone derivative obtained by reacting (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one and its chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone with phenylhydrazine under weakly acidic conditions has strong ultraviolet absorption. Chromatographic separation was performed using a chiral column, and HPLC analysis was performed using a high-performance liquid chromatograph.

2. The chirality detection method as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of blank solution Take an appropriate amount of diluent as a blank solution; the diluent is n-hexane:isopropanol in a volume ratio of 95:

5. (2) System suitability solution preparation Dissolve the chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone reference standard in ethanol to obtain the chiral isomer stock solution. Take a headspace vial, add phenylhydrazine, (R)-4,4,4-trifluoro-3-hydroxy-3-methylbutanone reference standard and the chiral isomer stock solution, then add ethanol and acetic acid, cap and seal the vial, react in a water bath at 80-90℃, let it return to room temperature, concentrate under reduced pressure, add dichloromethane, wash with water, take the dichloromethane layer, dry it and concentrate it to a yellow solid, take the yellow solid and add diluent to prepare a system-suitable solution. (3) Preparation of the test solution; Take a headspace vial, add phenylhydrazine, (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one, ethanol and acetic acid, cap and seal the vial, react in a water bath at 80-90℃, let it return to room temperature, concentrate under reduced pressure, add dichloromethane, wash with water, take the dichloromethane layer, dry it and concentrate it to a yellow solid, take the yellow solid and add diluent to prepare the test solution; (4) Determination method: Take blank solution, system suitability solution and test solution respectively, inject them into high performance liquid chromatograph with chiral column for HPLC detection, record the chromatogram, and calculate the content of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one and impurity (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone.

3. The chirality detection method as described in claim 1 or 2, characterized in that, The chiral chromatographic column is a CHIRALCEL OD-H column.

4. The chirality detection method as described in claim 3, characterized in that, The chromatographic conditions used for HPLC detection are as follows: Chiral chromatographic column: CHIRALCEL OD-H, column length 150-250 mm; Column temperature: 25-35℃; Flow rate: 0.8-1.2 ml / min; Injection volume: 5-10 μl; UV detector: 260-280nm; Mobile phase v / v: Use a gradient elution of n-hexane:isopropanol = 99:1-95:5 for 20-30 min.

5. The chirality detection method as described in claim 4, characterized in that, The chromatographic conditions were as follows: column temperature 28-32℃, flow rate 0.9-1.1 ml / min, and UV detector wavelength 268-272 nm.

6. The chirality detection method as described in claim 5, characterized in that, The chiral column used was 4.6mm*250mm, 5μm, with a column temperature of 30℃; the flow rate was 1.0ml / min, the injection volume was 10μl, the UV detector wavelength was 270nm, and the mobile phase was hexane:isopropanol = 98:2 by volume, with isogradient elution for 20min.

7. The chirality detection method as described in claim 2, characterized in that, In steps (2) and (3), the equivalence ratio of phenylhydrazine to (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one is 1:1, and the reaction time is 60 minutes.

8. The chirality detection method as described in claim 7, characterized in that, Step (2) is as follows: Take 25 mg of chiral isomer (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone reference standard, place it in a 25 ml volumetric flask, add ethanol to dissolve and dilute to the mark, as the chiral isomer stock solution; take another 20 ml headspace vial, add 140 mg of phenylhydrazine, 200 mg of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbutanone reference standard, 1.0 ml of the above chiral isomer stock solution, then add 4 ml of ethanol and 1.5 ml of acetic acid, cap and seal the vial, react in an 85°C water bath for 60 minutes, let it return to room temperature, concentrate under reduced pressure, add 5 ml of dichloromethane, wash three times with 5 ml of water, take the dichloromethane layer, dry it, concentrate it to a yellow solid, take the yellow solid and dilute it with diluent to prepare a 1.0 mg / ml solution as the system suitability solution.

9. The chirality detection method as described in claim 7, characterized in that, The specific steps (3) are as follows: Take a 20ml headspace vial, add 140mg of phenylhydrazine, 200mg of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one test sample, add 5ml of ethanol and 1.5ml of acetic acid, cap and seal the vial, react in an 85℃ water bath for 60 minutes, let it return to room temperature, concentrate under reduced pressure, add 5ml of dichloromethane, wash three times with 5ml of water, take the dichloromethane layer, dry it and concentrate it to a yellow solid, take the yellow solid and dilute it with a diluent to prepare a 1.0mg / ml solution as the test sample solution.

10. The chiral detection method according to any one of claims 4-9, characterized in that, The contents of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one and (3S)-4,4,4-trifluoro-3-hydroxy-3-methyl-2-butanone were calculated using the area normalization method.

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