A RP-HPLC-RID method for detecting the (R)-isomer of pregabalin

Abstract

The invention discloses an RP-HPLC-RID method for detecting the (R)-isomer of pregabalin, belonging to the field of drug analysis. The method uses a polysaccharide derivative coated chiral reversed-phase chromatographic column and a differential refractometer to directly and effectively separate pregabalin and the (R)-isomer, and the detection result has good accuracy and high sensitivity. Compared with the existing evaporative light scattering detector method and pre-column derivatization reversed-phase chromatography method, the method is simpler to operate, has lower cost and is more environmentally friendly. The invention provides a better guarantee for the quality control of pregabalin and its preparations.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and in particular relates to a reverse phase high performance liquid chromatography differential refractometry (RP-HPLC-RID) method for detecting pregabalin (R)-isomers. Background Art

[0002] Pregabalin is a γ-aminobutyric acid (GABA) receptor antagonist, which was developed by Pfizer and applied for registration in August 2003. It was officially approved by the FDA in December 2008. Pregabalin is a drug for post-herpetic neuralgia and fibromyalgia, and has shown significant effects in clinical trials at home and abroad, and can effectively relieve pain and improve sleep quality.

[0003] The chemical name of pregabalin is (S)-3-(aminomethyl)-5-methylhexanoic acid, and its molecular formula is C8H 17 NO2. Its (R)-isomer chemical name is (R)-3-(aminomethyl)-5-methylhexanoic acid, and its molecular formula is C8H 17 NO2, the structural formula is shown in Table 1. Since its isomers exhibit different pharmacological activities and toxicities, the (R)-isomer of pregabalin and its preparations is an important indicator in quality control during the production process, which is of great significance for its safe use.

[0004] Table 1 Structural formula of pregabalin and its (R)-isomer

[0005]

[0006] Since pregabalin has weak ultraviolet absorption, derivatization treatment is often used in the prior art: 1. CN1786703A discloses a method for separating and determining chiral isomers of pregabalin, wherein one of Nα-(5-fluoro-2,4-dinitrophenyl)-L-amino acid compounds is selected as a derivatization reagent, and the reaction is carried out at 20 to 60° C. for 20 to 120 minutes. The reactants need to be completely dried in a drying system of phosphorus pentoxide and potassium hydroxide, and dimethyl sulfoxide or N,N-dimethylformamide is added to dissolve the dried product and then filtered, and high performance liquid chromatography or high performance liquid chromatography and mass spectrometry is used. The combined separation and determination method is cumbersome to operate, prone to errors, and takes too long; 2. CN102375037A discloses a method for separating and determining chiral isomers of pregabalin using electrophoresis-mediated microanalysis technology, wherein Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide is selected as a derivatization reagent, and electrophoresis-mediated microanalysis technology is used to treat a derivatization reagent solution and a derivatization reagent solution containing pregabalin with voltage, so that pregabalin and the derivatization reagent are mixed and reacted due to different electrophoretic mobilities, and then non-chiral capillary micellar electrokinetic chromatography is used to separate the derivatives. Although this method saves reaction time, the operation is also very cumbersome, and the popularity of electrophoresis is not high, which is not conducive to promotion; 3. CN104777263A discloses a method for separating and detecting chiral isomers of pregabalin, using fluorenylmethoxycarbonyl chloride as a pre-column derivatization reagent, a chiral chromatographic column, and a mixture of n-hexane and anhydrous ethanol as a mobile phase, and separating and determining the chiral isomers of pregabalin by high performance liquid chromatography. Although this method shortens the reaction time, it uses normal phase chromatography and has high instrument requirements; 4. The quality standards of pregabalin in USP (United States Pharmacopoeia) and EP (European Pharmacopoeia) both use Marfey reagent for derivatization treatment, where USP reacts at 55°C for one hour and EP reacts at 40°C for one hour, and the steps are cumbersome and time-consuming. Although the methods used in the above patents and pharmacopoeias of various countries are different, they all require the use of derivatization reagents for derivatization, with high analysis costs, complex sample processing, and high instrument requirements.

[0007] Although there are also literatures later (Li Jing et al., Determination of the content of R-enantiomer of pregabalin in pregabalin raw materials by reversed-phase high performance liquid chromatography-evaporative light scattering detector, Physical and Chemical Testing-Chemical Volume, 2017, 53(6): 659-663.), directly using a chiral chromatographic column in series with an evaporative light scattering detector, the mobile phase is methanol-acetonitrile-water-formic acid-diethylamine (450:450:100:2.0:2.5), the drift tube temperature is 70°C, and the carrier gas flow rate is 2.0L / min for detection, although this method avoids derivatization treatment, it has high requirements for instruments and equipment, and the adjustable range of the mobile phase is narrow. Therefore, in order to facilitate enterprises to control product quality, it is necessary to invent a convenient, feasible, and widely applicable method that meets the daily detection of pregabalin isomers. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for efficiently analyzing the (R)-isomer of pregabalin after repeated experiments and explorations. The method adopts RP-HPLC-RID, is simple and easy to operate, is suitable for popularization and use, can achieve high sensitivity, and can better control the product quality of pregabalin.

[0009] To achieve the above object, the present invention uses the following technical solutions:

[0010] A RP-HPLC-RID method for detecting pregabalin (R)-isomers comprises: diluting a sample to be tested and injecting it into a high performance liquid chromatograph for detection; the high performance liquid chromatograph comprises a differential refractive index detector, the setting temperature of the differential refractive index detector is 25-40° C., the chromatographic column of the high performance liquid chromatograph is a chiral reverse phase chromatographic column coated with a polysaccharide derivative, the mobile phase is a potassium hexafluorophosphate solution and acetonitrile, the pH of the potassium hexafluorophosphate solution is 4.5-5.5, accounting for 70-80% of the volume of the mobile phase, isocratic elution, and the flow rate is 0.1-1 ml / min.

[0011] Preferably, the filler of the polysaccharide derivative coated chiral reverse phase chromatography column is silica gel coated with cellulose tris(4-methylbenzoate).

[0012] Further preferably, the model of the polysaccharide derivative coated chiral reverse phase chromatography column is CHIRALCEL OJ-RH, or a model with similar performance.

[0013] Preferably, the setting temperature of the differential refractive index detector is 30°C.

[0014] Preferably, the pH of the potassium hexafluorophosphate solution is 5.

[0015] Preferably, the potassium hexafluorophosphate solution contains triethylamine, and the preparation method is to add 0.1% triethylamine to 100 mM potassium hexafluorophosphate solution and adjust the pH to 5 with phosphoric acid.

[0016] Preferably, the potassium hexafluorophosphate solution accounts for 75% of the volume of the mobile phase.

[0017] Preferably, the flow rate is 0.6 ml / min.

[0018] All percentages involved in the present invention are volume percentages.

[0019] Compared with the prior art, the present invention has the following advantages and positive effects:

[0020] 1. Compared with the use of derivatization reagents, the present invention has the advantages of simple and quick operation, reduced error generation, and no introduction of excessive impurities.

[0021] 2. Compared with the normal phase system, the reverse phase chromatography system has the advantages of stable chromatographic medium performance, economy and environmental protection.

[0022] 3. The present invention realizes the effective separation and detection of the (R)-isomer of pregabalin. The reversed-phase liquid chromatography-differential refractometer determination method used has the advantages of strong separation ability, simple operation process, wide applicability, etc., which can more conveniently and quickly control product quality, improve product quality, and ensure the safety and effectiveness of clinical medication.

[0023] 4. The quantitative limit of the present invention is 0.084 μg / ml, the detection limit is 0.033 μg / ml, and the sensitivity is high, which can meet the daily detection needs of controlling the limit of pregabalin (R)-isomer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a system suitability solution map.

[0025] Figure 2 This is the blank excipient spectrum in the specificity test.

[0026] Figure 3 This is the standard curve of pregabalin (R)-isomer.

[0027] Figure 4 To detect the content of pregabalin (R)-isomer in pregabalin oral solution. DETAILED DESCRIPTION

[0028] To facilitate those skilled in the art to understand the contents of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection of the present invention in any way.

[0029] In the following examples, the instruments and reagents used are as follows:

[0030] High performance liquid chromatography mainframe model: Waters e2695 (including 120-position automatic sampler).

[0031] Detector model: Waters 2414.

[0032] Column oven model: Waters Alliance.

[0033] Reagents: Potassium hexafluorophosphate, phosphoric acid and triethylamine were all analytical grade, and acetonitrile was of chromatography grade.

[0034] Pregabalin reference substance and pregabalin (R)-isomer reference substance were purchased from QCS by the applicant.

[0035] Example 1 Screening of stationary phase

[0036] First, the system suitability solution was used to compare the normal phase and reverse phase chiral chromatographic columns. The results showed that normal phase chromatography could not effectively separate pregabalin and its isomers, and the separation degree between the two was less than 1.5, while the separation degree of reverse phase chromatography was higher. Then, the sensitivity solution was used to compare the reverse phase chiral chromatographic columns with three different fillers, and the stationary phase with higher sensitivity was selected. The specific method is as follows:

[0037] Detector and temperature: RI-30℃

[0038] Flow rate: 0.6ml / min

[0039] Injection volume: 10 μl

[0040] Column temperature: 30°C

[0041] Diluent: mobile phase.

[0042] Preparation of sensitivity solution: Accurately weigh the pregabalin (R)-isomer reference substance, prepare a solution with a concentration of 0.10 μg / ml with diluent, and shake well.

[0043] Preparation of system suitability solution: Take 2 mg of (R)-isomer reference substance, place it in a 10 ml volumetric flask, add 1 ml of pregabalin oral solution, dissolve and dilute to the scale with diluent, and shake well.

[0044] (1) Screening of positive phase and reverse phase

[0045] Reversed Phase Chromatography:

[0046] Chromatographic column: CHIRALCEL OJ-RH (150*4.6mm, 5μm)

[0047] Mobile phase: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine, adjust pH to 5.0 with phosphoric acid)-acetonitrile (75:25, v / v)

[0048] Normal Phase Chromatography:

[0049] Chromatographic column: CHIRALCEL OJ-H (150*4.6mm, 5μm)

[0050] Mobile phase: n-hexane-isopropanol (98:2, v / v)

[0051] (2) Filler selection

[0052] Mobile phase: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine, adjust pH to 5.0 with phosphoric acid)-acetonitrile (75:25, v / v)

[0053] Chromatographic column 1: Silica gel coated with cellulose-tri[4-methylbenzoate] CHIRALCEL OJ-RH (150*4.6mm, 5μm)

[0054] Chromatographic column 2: Silica gel coated with cellulose-tris (3,5-diphenylcarbamate) CHIRALCEL OD-RH (150*4.6mm, 5μm)

[0055] Chromatographic column 3: Silica gel surface coated with amylose-tri (3,5-xylylcarbamate) CHIRALPAKAD-RH (150*4.6mm, 5μm)

[0056] The results are shown in Table 2.

[0057] Table 2 Stationary phase selection

[0058]

[0059] Conclusion: Under normal phase chromatography conditions, the resolution between the main component and the (R)-isomer was less than 1.5, and effective separation was not achieved. Under reverse phase chromatography conditions, the resolution of the three chromatographic columns was high, but when the CHIRALCEL OJ-RH column was used, the signal-to-noise ratio of the main peak in the sensitive solution was the largest (S / N>10), and the detection effect was better.

[0060] Example 2 Screening of mobile phase

[0061] Three mobile phase ratios and three mobile phase pH values ​​were compared to select the mobile phase with the best detection effect. The specific method is as follows:

[0062] Chromatographic column: CHIRALCEL OJ-RH (150*4.6mm, 5μm)

[0063] Detector and temperature: RI-30℃

[0064] Flow rate: 0.6ml / min

[0065] Injection volume: 10 μl

[0066] Column temperature: 30°C

[0067] Mobile phase: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine, adjust pH to 4.5-5.5 with phosphoric acid)-acetonitrile (70-80:30-20, v / v)

[0068] Diluent: mobile phase.

[0069] Preparation of sensitivity solution: Accurately weigh the pregabalin (R)-isomer reference substance, prepare a solution with a concentration of 0.10 μg / ml with diluent, and shake well.

[0070] Preparation of system suitability solution: Take 2 mg of (R)-isomer reference substance, place it in a 10 ml volumetric flask, add 1 ml of pregabalin oral solution, dissolve and dilute to the scale with diluent, and shake well.

[0071] (1) Screening of mobile phase ratio

[0072] Ratio 1: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine, adjust pH to 5.0 with phosphoric acid) - acetonitrile (80:20, v / v)

[0073] Ratio 2: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine, adjust pH to 5.0 with phosphoric acid) - acetonitrile (75:25, v / v)

[0074] Ratio 3: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine, adjust pH to 5.0 with phosphoric acid) - acetonitrile (70:30, v / v)

[0075] (2) Screening of pH of buffered salt solution

[0076] pH 1: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine and adjust pH to 4.5 with phosphoric acid) - acetonitrile (75:25, v / v)

[0077] pH 2: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine and adjust pH to 5.0 with phosphoric acid) - acetonitrile (75:25, v / v)

[0078] pH 3: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine and adjust pH to 5.5 with phosphoric acid) - acetonitrile (75:25, v / v)

[0079] The results are shown in Table 3.

[0080] Table 3 Mobile phase selection

[0081]

[0082] Conclusion: Various ratios of mobile phase and pH can achieve effective separation of system suitability solutions, and the separation of main components and isomers meets the detection requirements. The mobile phase ratio and pH have a great influence on the signal-to-noise ratio of the sensitive solution. When the potassium hexafluorophosphate solution-acetonitrile ratio is 75:25 and the pH of the potassium hexafluorophosphate solution is 5.0, the S / N is greater than 10, which can meet the detection requirements of the sensitive solution.

[0083] Example 3 Screening of differential detector temperature

[0084] The temperature of the differential detector has a great influence on the stability of the baseline. At the same time, the refractive index of the optical medium changes with temperature, which ultimately leads to different detection sensitivities. This experiment compares a variety of different detection temperatures and selects the best one. The specific method is as follows:

[0085] Chromatographic column: CHIRALCEL OJ-RH (150*4.6mm, 5μm)

[0086] Detector and temperature: RI-25-40℃

[0087] Flow rate: 0.6ml / min

[0088] Injection volume: 10 μl

[0089] Mobile phase: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine, adjust pH to 5.0 with phosphoric acid)-acetonitrile (75:25, v / v)

[0090] Diluent: mobile phase.

[0091] Preparation of sensitivity solution: Accurately weigh the pregabalin (R)-isomer reference substance, prepare a solution with a concentration of 0.10 μg / ml with diluent, and shake well.

[0092] Preparation of system suitability solution: Take 2 mg of (R)-isomer reference substance, place it in a 10 ml volumetric flask, add 1 ml of pregabalin oral solution, dissolve and dilute to the scale with diluent, and shake well.

[0093] Temperature 1: 25℃

[0094] Temperature 2: 30℃

[0095] Temperature 3: 40℃

[0096] The results are shown in Table 4.

[0097] Table 4 Temperature selection

[0098]

[0099] Conclusion: When the detector temperature is set to 30℃, the S / N is greater than 10, which can meet the detection requirements of the sensitive solution, and the corresponding chromatographic column temperature should also be set to 30℃.

[0100] Example 4 Methodological Investigation

[0101] 1. Test methods

[0102] 1.1 Chromatographic conditions

[0103] Chromatographic column: CHIRALCEL OJ-RH (150*4.6mm, 5μm)

[0104] Detector and temperature: RI-30℃

[0105] Flow rate: 0.6ml / min

[0106] Injection volume: 10 μl

[0107] Column temperature: 30°C

[0108] Mobile phase: 100 mM potassium hexafluorophosphate solution (add 0.1% triethylamine, adjust pH to 5.0 with phosphoric acid)-acetonitrile (75:25, v / v)

[0109] Diluent: mobile phase.

[0110] 1.2 Detection

[0111] According to the above chromatographic conditions, the sensitivity solution, system suitability solution, test solution, control solution, and blank auxiliary material solution were sampled and tested and the chromatograms were recorded. The preparation methods of each solution are as follows:

[0112] Preparation of sensitivity solution: Accurately weigh the pregabalin (R)-isomer reference substance, prepare a solution with a concentration of 0.10 μg / ml with diluent, and shake well.

[0113] Preparation of system suitability solution: Take 2 mg of (R)-isomer reference substance, place it in a 10 ml volumetric flask, add 1 ml of pregabalin oral solution, dissolve and dilute to the scale with diluent, and shake well.

[0114] Preparation of test solution: Accurately measure 1 ml of pregabalin oral solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0115] Preparation of control solution: Accurately measure 0.1 ml of the test solution, place it in a 50 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0116] Blank excipient solution: Take 1 ml of blank excipient, place it in a 10 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0117] 2. Test results

[0118] (1) System applicability

[0119] In the system suitability solution chromatogram, the peak elution order was pregabalin and (R)-isomer, with retention times of 17.526 min and 21.580 min, respectively. The resolution between the pregabalin peak and the (R)-isomer peak was 5.89, meeting the resolution requirements of USP not less than 3.0 and EP not less than 4.4.

[0120] (2) Sensitivity

[0121] In the sensitivity solution chromatogram, the signal-to-noise ratio of the pregabalin peak was 13.1, which was greater than 10, and met the requirements.

[0122] (3) Exclusivity

[0123] from Figure 2 The results showed that the blank excipient had no interference with the detection of (R)-isomer.

[0124] (4) Linearity and range

[0125] Standard curve: external standard method.

[0126] Solution preparation: Accurately measure 0.25, 0.50, 1.00, 1.50, 2.00, and 3.00 ml of 50 μg / ml pregabalin (R)-isomer reference solution and place them in 10 ml volumetric bottles, add diluent to the scale, shake well, and make a series of standard solutions with concentrations of approximately 1.25, 2.50, 5.00, 7.50, 10.00, and 15.00 μg / ml. Analyze and record the peak area according to the above chromatographic conditions, and make a linear regression curve based on the peak area versus the concentration. The results are shown in Table 5, and the regression curve is shown in Figure 3 The regression equation is y = 8523.2x-1558, R 2 =0.9996, with good linearity in the concentration range of 0.084 to 14.865 μg / ml.

[0127] Table 5 Pregabalin (R)-isomer standard curve

[0128]

[0129] (5) Limit of quantification and limit of detection

[0130] The pregabalin (R)-isomer reference solution was diluted stepwise to a certain concentration. The concentration when the signal-to-noise ratio was about 3 was the detection limit, and the concentration when the signal-to-noise ratio was about 10 was the quantification limit.

[0131] Table 6 Quantitation limit results

[0132]

[0133] Table 7 Detection limit results

[0134]

[0135] It can be seen from Tables 6 and 7 that the quantitative limit concentration of the present invention is 0.084 μg / ml, the ratio of which to the concentration of the test solution is 0.004%, and the detection limit concentration is 0.033 μg / ml, which has high sensitivity and can meet the daily detection requirements for controlling the limit of pregabalin (R)-isomer.

[0136] (6) Accuracy (sample recovery rate)

[0137] Solution preparation: Accurately measure 0.5, 1.0, and 1.5 ml of 40 μg / ml pregabalin (R)-isomer reference solution and place them in 10 ml volumetric bottles, then add 1 ml of pregabalin oral solution, add diluent to the scale, shake well, and prepare the recovery test solution. Analyze according to the above chromatographic conditions and calculate the detection amount according to the main component self-control method, calibrate with the test solution, and calculate the recovery. The recovery test results are shown in Table 8.

[0138] Table 8 (R)-isomer recovery test results

[0139]

[0140] At the three concentrations of 50%, 100% and 150% of the limit concentration, the average recovery rate of the (R)-isomer was 99.30%. In the range of 80% to 120%, RSD=0.45%<10%, indicating that the method has high accuracy.

[0141] (7) System precision

[0142] The pregabalin (R)-isomer reference solution was diluted step by step to the quantification limit concentration, and the quantification limit solution was injected 6 times continuously to calculate the relative standard deviation of the (R)-isomer peak area and retention time. The results are shown in Table 9.

[0143] Table 9 (R)-Isomer system precision test results

[0144]

[0145]

[0146] The quantitative limit solution was injected six times continuously, and the RSDs of the peak area and retention time of the (R)-isomer were both less than 10%, indicating that the method had good system precision.

[0147] (8) Repeatability

[0148] Accurately measure 1 ml of pregabalin oral solution and place it in a 10 ml volumetric flask. Accurately add 1 ml of 40 μg / ml pregabalin (R)-isomer reference solution, dilute to the scale with diluent, shake well, prepare 6 portions in parallel, and inject them into the liquid chromatograph respectively. Calculate the detection amount of the (R)-isomer according to the main component self-reference method, and calibrate it with the test solution to calculate the recovery rate, and calculate the relative standard deviation of the recovery rate of 6 determinations. The results are shown in Table 10.

[0149] Table 10 (R)-isomer repeatability test results

[0150] Sample No. Measurement results (%) 1 98.88 2 99.03 3 98.87 4 99.29 5 98.91 6 98.46 average value(%) 98.91 RSD(%) 0.28

[0151] As shown in Table 10, the detection method provided by the present invention has good repeatability. The recovery rate of the (R)-isomer was detected 6 times in parallel, and the RSD was 0.28%, which met the requirements.

[0152] (9) Intermediate precision

[0153] Accurately measure 1 ml of pregabalin oral solution and place it in a 10 ml volumetric flask. Accurately add 1 ml of 40 μg / ml pregabalin (R)-isomer reference solution, dilute to the mark with diluent, shake well, prepare 6 portions in parallel, and inject them into the liquid chromatograph respectively. Calculate the detected amount of (R)-isomer according to the principal component self-reference method, and calibrate with the test solution to calculate the recovery rate. Calculate the relative standard deviation of the 6 measurement results and the 12 measurement results by different test personnel. The results are shown in Table 11.

[0154] Table 11 (R)-isomer repeatability and intermediate precision test results

[0155]

[0156] As shown in Table 11, the intermediate precision of the detection method provided by the present invention is good. The recovery rate of the (R)-isomer was detected 12 times by different personnel, and the RSD was 0.34%, which meets the requirements.

[0157] (10) Determination of isomers in pregabalin oral solution

[0158] Take the test solution for testing, and calculate the detection amount of (R)-isomer according to the main component self-control method. Figure 4 It can be seen that the (R)-isomer was not detected in this batch of pregabalin oral solution.

Claims

1. A RP-HPLC-RID method for detecting the (R)-isomer of pregabalin, characterized in that: The sample to be tested is diluted and then injected into a high performance liquid chromatograph for detection. The high performance liquid chromatograph contains a differential refractive index detector, the setting temperature of the differential refractive index detector is 25-40°C, the chromatographic column of the high performance liquid chromatograph is a polysaccharide derivative coated chiral reverse phase chromatographic column, the filler of the polysaccharide derivative coated chiral reverse phase chromatographic column is a silica gel surface coated with cellulose-tri (4-methylbenzoate), the mobile phase is a potassium hexafluorophosphate solution and acetonitrile, the pH of the potassium hexafluorophosphate solution is 4.5-5.5, accounting for 70-80% of the volume of the mobile phase, isocratic elution, and the flow rate is 0.1-1 ml / min.

2. The RP-HPLC-RID method for detecting the (R)-isomer of pregabalin according to claim 1, characterized in that: The model of the polysaccharide derivative coated chiral reverse phase chromatography column is CHIRALCEL OJ-RH, or a model with similar performance.

3. The RP-HPLC-RID method for detecting the (R)-isomer of pregabalin according to claim 1, characterized in that: The setting temperature of the differential refractive index detector was 30°C.

4. The RP-HPLC-RID method for detecting the (R)-isomer of pregabalin according to claim 1, characterized in that: The pH of the potassium hexafluorophosphate solution is 5.

5. The RP-HPLC-RID method for detecting the (R)-isomer of pregabalin according to claim 4, characterized in that: The potassium hexafluorophosphate solution contains triethylamine, and the preparation method is to add 0.1% triethylamine to 100 mM potassium hexafluorophosphate solution and adjust the pH to 5 with phosphoric acid.

6. The RP-HPLC-RID method for detecting the (R)-isomer of pregabalin according to claim 4, characterized in that: The potassium hexafluorophosphate solution accounts for 75% of the volume of the mobile phase.

7. The RP-HPLC-RID method for detecting the (R)-isomer of pregabalin according to claim 4, characterized in that: The flow rate is 0.6 ml / min.

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