Analysis method of pregabalin related substances and application of pregabalin related substances

Abstract

The invention provides an analysis method of pregabalin related substances and application of the pregabalin related substances in drug quality control. According to the method, the content of pregabalin and / or related substances is determined by adopting a high performance liquid chromatography, and the method for analyzing the related substances of the pregabalin phenylethylamine process is established by repeatedly exploring chromatographic conditions such as different types of chromatographic columns, pH of a mobile phase and the proportion of the mobile phase. A methodological verification result proves that the analysis method is high in precision, good in repeatability, stable and free of interference, and can be used for quality control of the pregabalin bulk drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to an analysis method for pregabalin-related substances and application thereof in drug quality control. Technical Background

[0002] Pregabalin is a gamma-aminobutyric acid (GABA) receptor agonist that blocks voltage-dependent calcium channels and reduces neurotransmitter release. It is commonly used clinically to treat neuropathic pain conditions such as herpes zoster neuralgia and fibromyalgia syndrome, as well as partial epileptic seizures and generalized anxiety disorder in adult patients. Pregabalin is the world's best-selling analgesic, with FDA approval for the treatment of neuralgia associated with diabetic peripheral neuropathy, postherpetic neuralgia, spinal cord injury-related neuralgia, fibromyalgia-related neuralgia, and partial-onset seizures in certain adult patients with epilepsy.

[0003]

[0004] At present, there are two main industrial production processes for pregabalin: chemical synthesis and enzymatic method. The former is more widely used in industrialization. Among them, the chemical synthesis method is mainly based on the use of a resolving agent to split and then process to obtain S-pregabalin. The process using R-phenylethylamine as a resolving agent (reference WO1996038405A1, hereinafter referred to as the phenylethylamine process) can be used to obtain the isomers obtained by splitting, which can be recovered by racemization to obtain its racemate, thereby being recycled (reference CN109761838A, CN104086439B). Compared with other chemical synthesis processes, it shows obvious cost advantages and is one of the representative processes for producing pregabalin.

[0005] The substances involved in the phenylethylamine process include the active ingredient pregabalin and its related substances. The information is shown in Table 1.

[0006] Table 1 Main substance information of pregabalin API (phenylethylamine process)

[0007]

[0008]

[0009] Currently, the Pharmacopoeia of the People's Republic of China does not include a quality control method for pregabalin API. The quality control methods used in the United States Pharmacopoeia and the European Pharmacopoeia are unable to effectively detect byproducts and degradation impurities produced by the phenylethylamine process. When the inventors used the United States Pharmacopoeia method to conduct quality testing on pregabalin API produced by the phenylethylamine process, they found that the peak time of the impurity PGB-3 was interfered with by the main peak, the process impurity trimer could not elute within the gradient elution time, and the lactam was interfered with by the solvent peak. When testing using the European Pharmacopoeia method, they found that the intermediate PGB-5 could not be separated from the main peak, and the process impurity trimer had a long retention time (over 100 minutes).

[0010] Therefore, it is necessary to develop a detection method for pregabalin-related substances in the phenylethylamine process. Summary of the Invention

[0011] The first aspect of the present invention provides a method for analyzing pregabalin-related substances, which uses high performance liquid chromatography to determine the content of pregabalin-related substances;

[0012] The high performance liquid chromatography method uses mobile phase A and mobile phase B for gradient elution, wherein mobile phase A is a mixed solution of buffer and organic solvent, and mobile phase B is an organic solvent;

[0013] The pregabalin-related substances include:

[0014]

[0015] In some embodiments of the present invention, the buffer is a phosphate buffer, and the phosphate includes ammonium dihydrogen phosphate, diammonium hydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate or sodium dihydrogen phosphate, preferably ammonium dihydrogen phosphate and / or diammonium hydrogen phosphate.

[0016] In some embodiments of the present invention, the pH of the buffer solution is 4-7, preferably 5-7, and more preferably, the pH of the buffer solution is 5.5-6.5.

[0017] In some embodiments of the present invention, the volume percentage of the buffer in the mobile phase A is greater than 75%, preferably greater than 80%, and more preferably, the volume percentage of the buffer in the mobile phase A is 90%.

[0018] In some embodiments of the present invention, the organic solvent includes methanol, acetonitrile, propanol, isopropanol or tetrahydrofuran, preferably methanol and / or acetonitrile; more preferably, the organic solvent of mobile phase A is a mixed solution of methanol and acetonitrile, and the organic solvent of mobile phase B is acetonitrile. Further preferably, the volume percentage of methanol in the mixed solution of mobile phase A is greater than 70%.

[0019] In some embodiments of the present invention, the analysis method comprises the following steps:

[0020] (1) Accurately measure an appropriate amount of pregabalin API and use mobile phase A as diluent to prepare a test solution;

[0021] (2) Setting up a high performance liquid chromatograph, using a chromatographic column filled with octadecylsilane bonded silica gel; the mobile phase flow rate is 0.5-1.5 mL / min, the detection wavelength is 200-280 nm, the column oven temperature is 10-40°C, the elution method is gradient elution, the mobile phase system is mobile phase A and mobile phase B, and the injection volume is 30-80 μL;

[0022] (3) Accurately measure the test solution of step (1), inject the sample under the conditions of step (2), and obtain the detection results of pregabalin and / or related substances.

[0023] In some embodiments of the present invention, the flow rate of the mobile phase is 0.5-1.0 mL / min, preferably 0.8 mL / min.

[0024] In some embodiments of the present invention, the detection wavelength is 200-250 nm, preferably 210 nm.

[0025] In some embodiments of the present invention, the column temperature of the column oven is 20-35°C, preferably 30°C.

[0026] In some embodiments of the present invention, the injection volume is 40-60 μL, preferably 50 μL.

[0027] In some embodiments of the present invention, the gradient elution procedure includes: 0-9 min, the volume fraction of the mobile phase A is 95-100%, 9-27 min, the volume fraction of the mobile phase A is uniformly reduced from 95-100% to 10-30%, 27-27.1 min, the volume fraction of the mobile phase A is uniformly reduced from 10-30% to 95-100%, and 27.1-37 min, the volume fraction of the mobile phase A is 95-100%.

[0028] In some embodiments of the present invention, the chromatographic column is a reverse phase chromatographic column, preferably an ODS column, more preferably an Inertsil ODS-3 150×4.0 mm, 3 μm chromatographic column.

[0029] Another aspect of the present invention provides a use of the analytical method according to the first aspect of the present invention in drug quality control, characterized in that the drug is pregabalin.

[0030] This study established a method for analyzing process-related substances in pregabalin phenylethylamine by repeatedly exploring chromatographic conditions such as different chromatographic column models, mobile phase pH, and mobile phase ratio. Methodological validation results demonstrated that this analytical method is highly precise, reproducible, stable, and interference-free, and can be used for quality control of pregabalin API. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Attachment Figure 1 :Relationship between degradation time and degradation amount of pregabalin lactam

[0032] Attachment Figure 2 :Pregabalin lactam degradation equilibrium equation diagram under alkaline degradation conditions

[0033] Attachment Figure 3 : Chromatograms of blank solution, selective solution 1, and selective solution 2 (A. Blank solution B. Selective solution 1 C. Selective solution 2 1. 4-ene pregabalin 2. 5-ene pregabalin 3. Pregabalin 4. R-phenylethylamine 5. PGB-3 6. PGB-5 7. Lactam 8. Trimer (two peaks) 9. PGB-5D 10. PGB-5B 11. PGB-5C)

[0034] Definition and Description

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0036] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0037] In the present invention, "optional" or "optionally" means that the subsequently described event or situation may or may not occur, and the description includes cases where the event or situation occurs and cases where it does not occur.

[0038] The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0039] The term "HPLC" includes high performance liquid chromatography, as well as a device or method for analyzing a sample by using a liquid as the mobile phase and a high-pressure infusion system to pump a single solvent of different polarities or a mixed solvent of different proportions, a buffer, or other mobile phase into a chromatographic column containing a stationary phase. After the components are separated in the column, they enter a detector for detection.

[0040] The term "detection limit" refers to the lowest concentration or amount of the analyte that can be detected by an analytical method under specified experimental conditions.

[0041] The term "limit of quantitation" refers to the lowest concentration or amount of an analyte in a sample that can be quantitatively determined by an analytical method. The lower the limit of quantitation, the higher the sensitivity.

[0042] The term "external standard method" refers to a method in which a pure sample of the analyte is used as a reference substance, and the response signals of the analyte in the sample are compared to those of the reference substance for quantitative determination. For details on the external standard method, please refer to General Chapter 0512 of Part IV of the Chinese Pharmacopoeia (2020 edition).

[0043] The term "ODS" refers to octadecylsilyl silica gel.

[0044] The parameters of the Inertsil ODS-3 chromatographic column used in the present invention may be as follows, or a chromatographic column having equivalent effects:

[0045] Silica gel: high purity spherical silica gel

[0046] Particle size: 2μm, 3μm, 4μm, 5μm

[0047] Surface area: 450m 2 / g

[0048] Purity: 99.999%

[0049] Aperture: 100A

[0050] Pore volume: 1.05mL / g

[0051] Chemical bonding group: octadecyl

[0052] End capping: Yes

[0053] Carbon content: 15%

[0054] USP code: L1. DETAILED DESCRIPTION

[0055] In order to better understand the technical solution of the present invention, the following is further described in conjunction with specific embodiments of the present invention, but it is not limited to the present invention.

[0056] Example 1: Optimization of chromatographic conditions

[0057] 1. Investigation of mobile phase ratio

[0058] To elute and effectively separate pregabalin and its ten impurities in a short period of time, a gradient elution method was required. Because the ten impurities have a wide range of polarity, an isocratic elution method using buffer plus methanol and acetonitrile was used in the initial elution phase to retain and separate the highly polar impurities. This was followed by a gradient elution with acetonitrile to retain and separate the less polar impurities. Through trial and error, mobile phase A was ultimately determined to be phosphate buffer: acetonitrile: methanol = 900:20:80, with a gradient elution with acetonitrile.

[0059] 2. Investigation of mobile phase buffer pH

[0060] According to the analytical methods for related substances in the United States Pharmacopoeia, during the impurity spike recovery process, it was found that the elution time of the PGB-3 impurity differed significantly when it was spiked alone and when it was spiked with high concentrations of pregabalin. Experimenting with the buffer pH revealed that under slightly acidic buffer conditions, the elution of PGB-3 was no longer affected by high concentrations of pregabalin. However, under these conditions, R-phenylethylamine was prone to solvent effects, resulting in bifurcation. Ultimately, the buffer pH was set at approximately 6.0, which effectively separated the impurities without peak drift or solvent effects.

[0061] Example 2: Detection conditions and solution preparation

[0062] 1. Instruments and Reagents

[0063] Agilent 1260 high-performance liquid chromatograph (Agilent); XP205 1 / 100,000 electronic balance (Mettler Toledo); XP26 1 / 100,000 electronic balance (Mettler Toledo); S400 pH meter (Mettler Toledo); Inertsil ODS-3 150×4.0 mm, 3 μm chromatographic column; ammonium dihydrogen phosphate (ACS, J&K Technology Co., Ltd.); diammonium hydrogen phosphate (chromatographic grade, ACROS); acetonitrile (chromatographic grade, MERCK); methanol (chromatographic grade, MERCK); ultrapure water (prepared by MiLLi-Q water purifier).

[0064] Reference substances: pregabalin (EDQM, batch number 1.0, content 99.8%), lactam (TRC, batch number 1-NMA-39-2, content 99.92%), 4-enepregabalin (3-aminomethyl-5-methyl-4-enoic acid), 5-enepregabalin (3-aminomethyl-5-methyl-5-enoic acid), trimer, PGB-3 (3-isobutylglutaric acid), PGB-5 (3-isobutylglutaric acid monoamide) , R-phenylethylamine, PGB-5B (4-isobutyl-2,6-piperidinedione), PGB-5C (monoamide phenylethylamine), PGB-5D (diacid phenylethylamine) (the above nine impurity standards are all from Zhejiang Huahai Pharmaceutical Co., Ltd., with contents of 99.4%, 96.2%, 96.8%, 98.8%, 99.4%, 99.4%, 96.2%, 96.8%, 98.8%, 99.4%), respectively).

[0065] Pregabalin API (Zhejiang Huahai Pharmaceutical Co., Ltd., batch numbers D5248-16-053, D5248-17-086, 087, 088).

[0066] 2. Chromatographic conditions

[0067] An Inertsil ODS-3 column (150×4.0 mm, 3 μm) was used with buffer (7.05 g of ammonium dihydrogen phosphate and 1.45 g of diammonium hydrogen phosphate dissolved in 1000 mL of water): methanol: acetonitrile = 900:80:20 as mobile phase A and acetonitrile as mobile phase B. Gradient elution was performed according to the conditions in Table 2; flow rate was 0.8 mL / min; detection wavelength was 210 nm; column temperature was 30°C; and injection volume was 50 μL.

[0068] Table 2 Gradient elution program

[0069] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0→9 100 0 9→27 100→20 0→80 27→27.1 20→100 80→0 27.1→37 100 0

[0070] 3. Solution Preparation

[0071] Impurity stock solutions: Accurately weigh appropriate amounts of each of the lactam, 4-enepregabalin, 5-enepregabalin, trimer, PGB-3, PGB-5, R-phenylethylamine, PGB-5B, PGB-5C, and PGB-5D reference substances, dissolve in acetonitrile, and dilute to produce a stock solution containing 1 mg of each impurity reference substance per mL (lactam concentration is 1.5 mg per mL).

[0072] Pregabalin reference substance stock solution: Accurately weigh an appropriate amount of pregabalin reference substance and dilute it with mobile phase A to prepare a solution containing 10 mg of pregabalin per 1 mL.

[0073] Reference solution: Accurately measure an appropriate amount of pregabalin reference stock solution and dilute it with mobile phase A to prepare a solution containing 10 μg of pregabalin per 1 mL.

[0074] Test solution: Accurately weigh an appropriate amount of pregabalin bulk drug substance and use mobile phase A as diluent to prepare a solution containing 10 mg of pregabalin per 1 mL.

[0075] Lactam LOQ stock solution: Accurately weigh an appropriate amount of each lactam reference substance, dissolve it in acetonitrile and dilute it to make a lactam LOQ stock solution containing 1 mg per 1 mL.

[0076] Example 3: Method Validation - Specificity

[0077] 1. Impurity research

[0078] Through the study of acid degradation, alkaline degradation, oxidative degradation, high temperature and high humidity, high temperature and low humidity, light degradation, and metal degradation, it is known that the most easily degraded impurity of pregabalin is lactam, and the degree of lactam degradation is the greatest under alkaline decomposition conditions. By changing the alkaline degradation time, the degradation curve of lactam can be obtained, see the attached Figure 1 The equilibrium equation diagram of pregabalin lactam degradation under alkaline degradation conditions is attached. Figure 2 Experimental data confirmed that after the lactam is degraded to a certain amount, it will reach an upper limit and be in equilibrium with the main component pregabalin, as shown in Table 3.

[0079] Table 3 Comparison of degradation time and degradation amount of pregabalin lactam

[0080]

[0081] Process analysis reveals that the possible byproducts, resolving agents, and intermediates produced in the phenylethylamine process include: lactam, 4-ene-pregabalin, 5-ene-pregabalin, trimer, PGB-3, PGB-5, R-phenylethylamine, PGB-5B, PGB-5C, and PGB-5D. The location of these impurities is currently under investigation.

[0082] 2. Solution Preparation

[0083] Selective solution 1: Accurately weigh an appropriate amount of pregabalin reference substance and an appropriate amount of impurity stock solution. In the same volumetric flask, add mobile phase A to dissolve and dilute to a solution containing 10 mg of pregabalin, 15 μg of lactam, 10 μg of 4-enepregabalin, 10 μg of 5-enepregabalin, 10 μg of trimer, 10 μg of PGB-3, 10 μg of PGB-5, and 1 μg of R-phenylethylamine per 1 mL.

[0084] Selective solution 2: Accurately weigh an appropriate amount of pregabalin reference substance and an appropriate amount of impurity stock solution. In the same volumetric flask, add mobile phase A to dissolve and dilute to a solution containing 10 mg of pregabalin, 10 μg of R-phenylethylamine, 10 μg of PGB-5B, 10 μg of PGB-5C, and 10 μg of PGB-5D per 1 mL.

[0085] 3. Detection and Analysis

[0086] Under the conditions of Example 2, 50 μL of each of the selective solution 1 and the selective solution 2 were accurately measured and injected into the HPLC instrument to record the spectra. The spectra of the blank solution, the selective solution 1 and the selective solution 2 are shown in the attached Figure 3 .

[0087] The results showed that pregabalin could be completely separated from its impurities and degradation products (resolution greater than 2.0).

[0088] Example 4: Method Validation - Limit of Detection and Limit of Quantitation

[0089] 1. Solution Preparation

[0090] Gradient concentration solution: Accurately measure appropriate amounts of each impurity stock solution, pregabalin reference substance stock solution, and lactam LOQ stock solution, and dilute them step by step to obtain gradient concentration solutions.

[0091] 2. Detection and Analysis

[0092] Under the conditions of Example 2, each gradient concentration solution was precisely measured and injected. The concentration of pregabalin and each impurity at a signal-to-noise ratio of approximately 10 was used as the limit of quantification; the concentration of pregabalin and each impurity at a signal-to-noise ratio of approximately 3 was used as the limit of detection. The measurement results are shown in Table 4.

[0093] Table 4 Results of quantification limits and detection limits of pregabalin and its impurities

[0094] Compound LOQ (%) LOD (%) Pregabalin 0.05 0.02 lactam 0.01 0.003 4-Enpregabalin 0.01 0.003 5-Enpregabalin 0.03 0.01 trimer 0.01 0.003 PGB-3 0.05 0.02 PGB-5 0.03 0.01 R-phenylethylamine 0.01 0.003 PGB-5B 0.01 0.003 PGB-5C 0.01 0.003 PGB-5D 0.01 0.003

[0095] Example 5: Method Validation - Linear Relationship

[0096] 1. Solution Preparation

[0097] Linear concentration series: Accurately measure appropriate amounts of each impurity stock solution, pregabalin reference stock solution, and lactam LOQ stock solution into a volumetric flask. Dilute each to the mark with mobile phase A and shake well to prepare a linear concentration series. The impurity concentration points are: 50%, 80%, 100%, 120%, and 150% of the LOQ and the target, respectively.

[0098] 2. Detection and Analysis

[0099] Under the conditions of Example 2, each series of linear concentration solutions was precisely measured, injected, and a chromatogram was recorded. A standard curve was plotted with pregabalin and each impurity concentration as the abscissa and peak area as the ordinate. The regression equation and linear range are shown in Table 5.

[0100] Table 5 Linearity test results

[0101]

[0102]

[0103] The results showed that the linear correlation coefficients between pregabalin and lactam, 4-ene pregabalin, 5-ene pregabalin, trimer, PGB-3, and PGB-5 were all greater than 0.99, indicating a good linear relationship.

[0104] Example 6: Method Validation - Repeatability and Intermediate Precision

[0105] 1. Solution Preparation

[0106] Precision solution: Accurately weigh an appropriate amount of pregabalin API test sample and an appropriate amount of each impurity stock solution. In the same volumetric flask, add mobile phase A to dissolve and dilute to make a precision solution containing 10 mg of pregabalin API test sample per 1 mL and the target concentrations of other impurities. Prepare 6 portions in the same manner.

[0107] 2. Detection and Analysis

[0108] Under the conditions of Example 2, each precision solution was accurately measured and injected once.

[0109] The results showed that the RSDs of the peak areas of lactam, 4-enepregabalin, 5-enepregabalin, trimer, PGB-3, and PGB-5 were 0.34%, 0.28%, 0.34%, 0.70%, 0.45%, and 0.47%, respectively.

[0110] The personnel, chromatographic column, instrument and experimental date were changed. Six portions of intermediate precision solution were prepared in the same manner and injected once each under the conditions of Example 2.

[0111] The results showed that the RSDs of the peak areas of lactam, 4-enepregabalin, 5-enepregabalin, trimer, PGB-3, and PGB-5 were 0.46%, 0.57%, 0.40%, 1.80%, 0.54%, and 0.40%, respectively, indicating that the method had good precision.

[0112] Example 7: Method Validation - Solution Stability

[0113] Take the reference solution and the test solution and inject them at room temperature at 0, 24, and 48 hours under the conditions of Example 2.

[0114] The results showed that the peak area of pregabalin in the reference solution at 48 h was 1.03 higher than that at 0 h, and there was no significant change in the contents of pregabalin, lactam, 4-enepregabalin, 5-enepregabalin, trimer, PGB-3 and PGB-5 in the test sample, indicating that the reference solution and the test solution were stable within 48 h.

[0115] Example 8: Method Validation - Accuracy

[0116] 1. Solution Preparation

[0117] Accuracy solution: Accurately weigh an appropriate amount of pregabalin test sample and an appropriate amount of impurity stock solution. In the same volumetric flask, add mobile phase A to dissolve and dilute to make 10 mg of pregabalin per 1 mL. The concentrations of other impurities are 80%, 100%, and 120% of the target, respectively. Prepare 3 portions of accuracy solution for each concentration point in the same way, for a total of 9 portions.

[0118] 2. Detection and Analysis

[0119] Under the conditions of Example 2, precisely measure each accuracy solution, inject it into the sample, measure it, record the chromatogram, and calculate the experimental results.

[0120] The results showed that the average recoveries of lactam, 4-enepregabalin, 5-enepregabalin, trimer, PGB-3, and PGB-5 were 100.6% (RSD = 0.56%, n = 9), 100.2% (RSD = 0.38%, n = 9), 100.5% (RSD = 0.46%, n = 9), 101.1% (RSD = 1.13%, n = 9), 100.0% (RSD = 0.63%, n = 9), and 100.0% (RSD = 0.54%, n = 9), respectively, indicating that the method has high accuracy.

[0121] Example 9: Method Validation - Robustness

[0122] Selective solution 1 was detected under the conditions of Example 2 by replacing chromatographic columns of different batches and high performance liquid chromatographs of different numbers, adjusting the column temperature to ±2°C and the flow rate to ±0.1 mL / min.

[0123] The results showed that there was no significant difference in the separation and peak areas of pregabalin, lactam, 4-enepregabalin, 5-enepregabalin, trimer, PGB-3, and PGB-5, indicating that the method was robust.

[0124] Example 10: Stability Study of API Samples

[0125] 1. Solution Preparation

[0126] For three different batches of pregabalin drug substance, accelerated stability samples (storage conditions: T = 40 ° C ± 2 ° C, RH = 75% ± 5%; storage time: 0 day, 1 month, 2 months, 3 months, 6 months) and long-term stability samples (storage conditions: T = 25 ° C ± 2 ° C, RH = 60% ± 5%; storage time: 0 day, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 36 months, 48 months, 60 months) were prepared according to the method for preparing the test solution in Example 2.

[0127] 2. Detection and Analysis

[0128] Under the conditions of Example 2, each batch of test sample stability solution was accurately measured, injected, and measured.

[0129] The results showed that there was no significant increase in the impurity results of the stability samples, and the impurity results of the three batches of pregabalin APIs for 6 months accelerated and 60 months long-term stability were in compliance with the quality standards.

[0130] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for analyzing pregabalin-related substances, characterized in that: The content of pregabalin-related substances was determined by HPLC; The high performance liquid chromatography method uses mobile phase A and mobile phase B for gradient elution, wherein mobile phase A is a mixed solution of buffer and organic solvent, and mobile phase B is an organic solvent; The pregabalin-related substances include:

2. The analysis method according to claim 1, characterized in that The buffer is a phosphate buffer, and the phosphate includes ammonium dihydrogen phosphate, diammonium hydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate or sodium dihydrogen phosphate, preferably ammonium dihydrogen phosphate and / or diammonium hydrogen phosphate.

3. The analysis method according to claim 1 or 2, characterized in that The pH of the buffer solution is 4-7, preferably 5-7, and more preferably, the pH of the buffer solution is 5.5-6.

5.

4. The analysis method according to any one of claims 1 to 3, characterized in that The volume percentage of the buffer in the mobile phase A is greater than 75%, preferably greater than 80%, and more preferably, the volume percentage of the buffer in the mobile phase A is 90%.

5. The analysis method according to any one of claims 1 to 4, characterized in that The organic solvent includes methanol, acetonitrile, propanol, isopropanol or tetrahydrofuran, preferably methanol and / or acetonitrile; more preferably, the organic solvent of mobile phase A is a mixed solution of methanol and acetonitrile, and the organic solvent of mobile phase B is acetonitrile; further preferably, the volume percentage of methanol in the mixed solution of mobile phase A is greater than 70%.

6. The analysis method according to any one of claims 1 to 5, characterized in that The analysis method comprises the following steps: (1) Accurately measure an appropriate amount of pregabalin API and use mobile phase A as diluent to prepare a test solution; (2) Setting up a high performance liquid chromatograph, using a chromatographic column filled with octadecylsilane bonded silica gel; the mobile phase flow rate is 0.5-1.5 mL / min, the detection wavelength is 200-280 nm, the column oven temperature is 10-40°C, the elution method is gradient elution, the mobile phase system is mobile phase A and mobile phase B, and the injection volume is 30-80 μL; (3) Accurately measure the test solution of step (1), inject the sample under the conditions of step (2), and obtain the detection results of pregabalin and / or related substances.

7. The analysis method according to any one of claims 1 to 6, characterized in that The mobile phase flow rate is 0.5-1.0 mL / min, preferably 0.8 mL / min; or The detection wavelength is 200-250 nm, preferably 210 nm; or The column temperature of the column oven is 20-35°C, preferably 30°C; or The injection volume is 40-60 μL, preferably 50 μL.

8. The analysis method according to any one of claims 1 to 7, characterized in that The gradient elution procedure includes: 0-9 minutes, the volume fraction of the mobile phase A is 95-100%, 9-27 minutes, the volume fraction of the mobile phase A is uniformly reduced from 95-100% to 10-30%, 27-27.1 minutes, the volume fraction of the mobile phase A is uniformly reduced from 10-30% to 95-100%, and 27.1-37 minutes, the volume fraction of the mobile phase A is 95-100%.

9. The analysis method according to any one of claims 1 to 8, characterized in that The chromatographic column is a reverse phase chromatographic column, preferably an ODS column, more preferably an Inertsil ODS-3 150×4.0 mm, 3 μm chromatographic column.

10. Use of the analytical method according to any one of claims 1 to 9 in drug quality control, characterized in that: The drug is pregabalin.

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