A method for simultaneous determination of (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD

Abstract

The present application discloses a method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on the RP-HPLC-NQAD method, and specifically relates to the field of drug detection technology. The method comprises the following steps: preparing a standard curve of ethambutol hydrochloride and (+)-2-aminobutanol; taking ethambutol hydrochloride tablets, grinding them into powder, and obtaining ethambutol hydrochloride tablet powder; adding pure water to the ethambutol hydrochloride tablet powder, ultrasonically dissolving, and diluting with water to 2 mg / ml and 0.25 mg / ml, respectively, to obtain two machine analysis liquids; after the machine analysis liquid is detected by a reversed-phase high-performance liquid chromatography-tandem hydrogel particle laser technology detector, the concentrations of ethambutol hydrochloride and (+)-2-aminobutanol are calculated. The method has the advantages of being able to simultaneously detect the contents of ethambutol hydrochloride and (+)-2-aminobutanol compounds, and realizing the results of non-interference in the detection process of the above two compounds, no derivatization is required for pre-treatment, high accuracy, high sensitivity, and good durability.

Description

Technical Field

[0001] The present application relates to the technical field of detection of ethambutol hydrochloride and (+)-2-aminobutanol in pharmaceutical products, and in particular to a method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method. Background Art

[0002] Ethambutol hydrochloride is the hydrochloride form of ethambutol, an ethylenediamine derivative with antibacterial activity and special effects on mycobacteria. Ethambutol hydrochloride inhibits the binding of mycolic acid to the bacterial cell wall, thereby hindering the growth of bacterial cells. In addition, it can inhibit other cellular metabolic processes, ultimately preventing cell proliferation and inducing cell death. Today, the World Health Organization recommends that ethambutol hydrochloride be widely used in the treatment of tuberculosis. The content and impurities of drugs are key quality attributes that affect the quality, safety and efficacy of drugs. For ethambutol hydrochloride drugs, ethambutol hydrochloride and (+) 2-aminobutanol are common detection parameters. However, since ethambutol hydrochloride and (+) 2-aminobutanol are both polar medicinal compounds and lack ultraviolet chromophores, it undoubtedly increases the difficulty of analysis.

[0003] In the pharmacopoeias of various countries, thin layer chromatography or fluorescence spectroscopy is usually used to determine (+) 2-aminobutanol. Under specific pH conditions, ethambutol hydrochloride can react with Cu 2 + coordination occurs to form a complex, so that the complex can be measured by a UV detector. For example, the Chinese Pharmacopoeia uses RP-HPLC to detect the content of ethambutol hydrochloride tablets, with acetate buffer solution (containing ammonium acetate and copper acetate) with a pH value of 5.0 and methanol as the mobile phase, and ethambutol is measured at a wavelength of 270 nm. The defects of this method are: copper ions in the mobile phase will cause tailing of the chromatographic peak and shorten the service life of the chromatographic column.

[0004] For example, it is reported in the literature that by using the derivatization reagent (R)-(+)-α-methylbenzyl isocyanate for pre-column derivatization, RP-HPLC coupled with a UV detector was used to determine the content and solubility of ethambutol hydrochloride in anti-tuberculosis drug compound preparations. The defects of this method are: the pre-treatment process of the derivatization analysis is complicated, and the derivatization reagent is highly toxic.

[0005] Therefore, how to achieve a method that is suitable for detecting both ethambutol hydrochloride and (+)-2-aminobutanol compounds, achieves high detection sensitivity, and ensures that the two compounds do not interfere with the detection results is a technical problem that needs to be solved urgently. Summary of the invention

[0006] The present invention provides a method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method. The method has the advantages of being able to simultaneously detect the contents of ethambutol hydrochloride and (+)-2-aminobutanol compounds, achieving the results of the two compounds not interfering with each other during the detection process, having no derivatization step, high accuracy, high sensitivity and good durability.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method, the method comprising the following steps:

[0009] S01: Prepare standard curves of ethambutol hydrochloride and (+)-2-aminobutanol;

[0010] S02: Grind ethambutol hydrochloride tablets into powder to obtain ethambutol hydrochloride tablet powder;

[0011] S03: adding purified water to the ethambutol hydrochloride tablet powder, dissolving by ultrasonication, diluting with phase water to 2 mg / ml and 0.25 mg / ml respectively, and filtering the two ethambutol hydrochloride tablet solutions obtained above through a 0.22 μm water membrane to obtain two analytical solutions for the machine respectively;

[0012] Among them, the ethambutol hydrochloride tablet powder solution with a concentration of 2 mg / ml is used as the on-machine analysis liquid for detecting the content of (+)-2-aminobutanol; the ethambutol hydrochloride tablet powder solution with a concentration of 0.25 mg / ml is used as the on-machine analysis liquid for detecting the content of ethambutol hydrochloride;

[0013] S04: After the analytical liquid is detected by reverse phase high performance liquid chromatography coupled with hydrogel particle laser technology detector, the contents of ethambutol hydrochloride and (+)-2-aminobutanol are calculated.

[0014] Preferably, step S01 comprises: accurately weighing 20 mg of (+)-2-aminobutanol and ethambutol hydrochloride reference substances, dissolving them in 20 ml and 10 ml of water, respectively, to prepare (+)-2-aminobutanol stock solution and ethambutol hydrochloride stock solution respectively; diluting the (+)-2-aminobutanol stock solution and ethambutol hydrochloride stock solution with water to prepare a series of linear solutions, with concentration ranges corresponding to 4 μg / ml to 60 μg / ml and 5 to 500 μg / mL, respectively.

[0015] Preferably, in step S04, the chromatographic separation conditions are:

[0016] The stationary phase was a Capcell pak C18 column 4.6×250 mm, 5 μm, and the mobile phase was a mixture of 0.05-0.2% trifluoroacetic acid solution and methanol, with isocratic elution and a flow rate of 1.0 mL / min;

[0017] The column temperature was maintained at 30 °C, and the injection volume was 10 μL;

[0018] NQAD uses nitrogen with a flow rate of 4.7 L / min as the atomizing gas, the atomizing temperature is 15℃-30℃, the vaporization temperature is 20℃-90℃, and the filtration time is 5 seconds.

[0019] Preferably, the volume ratio of the trifluoroacetic acid solution to methanol is 94:6.

[0020] Preferably, the volume percentage of the trifluoroacetic acid solution is 0.1%.

[0021] Preferably, the atomization temperature is 30°C.

[0022] Preferably, the vaporization temperature is 70°C.

[0023] Preferably, in step S03, the ultrasonic power is 360 W and the ultrasonic time is 5 min.

[0024] Compared with the prior art, the advantages or beneficial effects of the technical solution of the present application include:

[0025] 1. This method is suitable for the simultaneous determination of (+) 2-aminobutanol and ethambutol hydrochloride that lack ultraviolet chromophores. There is no need to use different experimental steps for the detection of (+) 2-aminobutanol and ethambutol hydrochloride, which greatly improves the experimental efficiency.

[0026] 2. In the detection steps of this method, no derivatization operation is required during sample pretreatment, which simplifies the detection steps, shortens the experimental time, improves the analysis efficiency, and avoids the experimenters from contacting toxic derivatization reagents.

[0027] 3. When this method is used to detect (+) 2-aminobutanol and ethambutol hydrochloride, (+) 2-aminobutanol and ethambutol hydrochloride will not interfere with each other and affect the test results.

[0028] 4. The detection results of this method are highly accurate, sensitive and durable. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the chromatogram of ethambutol hydrochloride and (+)-2-aminobutanol in ion exchange mode;

[0030] Figure 2This is the chromatogram of ethambutol hydrochloride and (+)-2-aminobutanol in HILIC separation mode;

[0031] Figure 3 The chromatograms of ethambutol hydrochloride and (+)-2-aminobutanol in RP-HPLC mode;

[0032] Figure 4 The retention time and resolution of ethambutol hydrochloride and (+)-2-aminobutanol vary with the concentration of trifluoroacetic acid solution;

[0033] Figure 5 The retention time and resolution of ethambutol hydrochloride and (+)-2-aminobutanol vary with acetonitrile concentration;

[0034] Figure 6 This is a statistical diagram of the effect of changes in atomization temperature on sensitivity;

[0035] Figure 7 It is a statistical diagram of the response sensitivity of ethambutol hydrochloride and (+)-2-aminobutanol to the increase of evaporator temperature;

[0036] Figure 8 HPLC-NQAD detection chromatograms of ethambutol hydrochloride and (+)-2-aminobutanol under different conditions;

[0037] Fig. 9 This is a statistical table of the analysis results of ethambutol hydrochloride and (+)-2-aminobutanol;

[0038] Fig.10 is the correlation coefficient and linear equation table. DETAILED DESCRIPTION

[0039] The following will describe the implementation methods of the present application in detail with reference to the accompanying drawings and embodiments, so that the implementation process of how the present application applies technical means to solve technical problems and achieve corresponding technical effects can be fully understood and implemented accordingly. The embodiments of the present application and the various features in the embodiments can be combined with each other without conflict, and the technical solutions formed are all within the protection scope of the present application.

[0040] It should be clear that the embodiments described below are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by technicians in the relevant field without making creative work are within the scope of protection of the present application. Example 1

[0041] The method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method is characterized by comprising the following steps:

[0042] S01: Prepare standard curves of ethambutol hydrochloride and (+)-2-aminobutanol.

[0043] Specifically, 20 mg of (+)-2-aminobutanol and ethambutol hydrochloride reference substances were accurately weighed and dissolved in 20 ml and 10 ml of water, respectively, to prepare (+)-2-aminobutanol stock solution and ethambutol hydrochloride stock solution. The (+)-2-aminobutanol stock solution and ethambutol hydrochloride stock solution were diluted with water to prepare a series of linear solutions with concentration ranges of 4 μg / ml to 60 μg / ml and 5 to 500 μg / mL, respectively.

[0044] S02: Take ethambutol hydrochloride tablets, grind them into powder, and obtain ethambutol hydrochloride tablet powder.

[0045] S03: Add pure water to the ethambutol hydrochloride tablet powder, dissolve it by ultrasonication, dilute it with phase water to 2 mg / ml and 0.25 mg / ml respectively, and filter the two ethambutol hydrochloride tablet solutions obtained above through a 0.22 μm water membrane to obtain two analytical liquids, respectively. The ultrasonic power is 360 W and the ultrasonic time is 5 min.

[0046] Among them, the ethambutol hydrochloride tablet powder solution with a concentration of 2 mg / ml is used as the on-machine analysis liquid for detecting the content of (+)-2-aminobutanol; the ethambutol hydrochloride tablet powder solution with a concentration of 0.25 mg / ml is used as the on-machine analysis liquid for detecting the content of ethambutol hydrochloride.

[0047] S04: After the analytical fluid is detected by reverse phase high performance liquid chromatography coupled with a hydrogel particle laser technology detector, the concentrations of ethambutol hydrochloride and (+)-2-aminobutanol are calculated.

[0048] The chromatographic analysis conditions were as follows: the stationary phase was a Capcell pak C18 column 4.6×250 mm, 5 μm, the mobile phase was a mixture of 0.1% trifluoroacetic acid solution and methanol, isocratic elution, and a flow rate of 1.0 mL / min; the volume ratio of trifluoroacetic acid solution to methanol was 94:6.

[0049] The column temperature was maintained at 30 °C, and the injection volume was 10 μL;

[0050] (NQAD) Nitrogen with a flow rate of 4.7 L / min was used as the nebulizing gas, the nebulizing temperature was 30°C, the vaporization temperature was 70°C, and the filtration time was 5 seconds.

[0051] Example 2 (Establishment and Optimization of Analytical Methods)

[0052] 1. Establishment of high performance liquid chromatography

[0053] Under acidic conditions (pH 2-5), most of the nitrogen of ethambutol hydrochloride carries two positive charges, and its logarithmic distribution coefficient is less than -5. It is highly polar and has weak retention under general reversed phase conditions. NQAD is an aerosol-based detector and should be used with a volatile mobile phase. In order to achieve the best separation and analysis performance, three separation modes compatible with NQAD were studied and evaluated:

[0054] 1) Ion exchange chromatography (IEC);

[0055] 2) Hydrophilic interaction liquid chromatography (HILIC);

[0056] 3) Reverse phase chromatography in which an ionizing agent is added to the mobile phase.

[0057] Ion exchange mode: Ethambutol hydrochloride can carry a positive charge. If a strong cation exchange column is used, it may not be eluted from the chromatographic column. Therefore, a strong cation exchange and reversed phase chromatography mixed filler is selected as the chromatographic column. CapcellpakCR1:20 chromatographic column is used for separation with ammonium formate buffer solution and acetonitrile as the mobile phase. Introducing a small amount of ion exchange groups in the reversed phase chromatographic column can increase the retention time of ethambutol hydrochloride, but the peak shape is very wide and the tailing is severe (with Figure 1 as shown).

[0058] HILIC separation mode: Usually, in the HILIC mode, a combination of polar stationary phase and highly organic mobile phase is used. This experiment uses a highly polar silica stationary phase and a solution composed of ammonium formate buffer solution and acetonitrile as the mobile phase to determine ethambutol hydrochloride and (+)-2-aminobutanol. The high proportion of organic phase in the mobile phase helps the aerosol spray to vaporize, so the peak height of ethambutol is not bad (with a peak height of 1.37 mm / s). Figure 2 ). However, in HILLIC mode, the baseline noise is high, which may affect the quantitative detection of (+)-2-aminobutanol at trace concentration levels. The signal-to-noise ratio of ethambutol also shows that the method is not sensitive enough to detect (+)-2-aminobutanol. In HILLIC mode, in order to achieve the best chromatographic performance, the sample needs to be dissolved in a high proportion of organic solvent. Ethambutol hydrochloride is a highly water-soluble substance. When the sample is dissolved in a high organic phase solvent, it is easy to cause the sample to precipitate, so this is not suitable for the analysis of this sample.

[0059] Adding ion pair reagents to the RP-HPLC system: Trifluoroacetic acid is a volatile acid and is compatible with NQAD. Therefore, trifluoroacetic acid is usually added to the mobile phase as a volatile ion pair reagent to extend the retention time of the analyte. A preliminary study of the reverse phase system was conducted, using 0.1% TFA solution and acetonitrile as the mobile phase and separation on a C18 column. The results showed that ethambutol hydrochloride was fully retained during the elution process and the chromatographic peak shape was good. (With Figure 3 Therefore, RPHPLC mode was used for analysis in this study.

[0060] Example 3 (Effect of buffer concentration)

[0061] Trifluoroacetic acid is a volatile acid that can be used as a mobile phase additive to improve the retention of ethambutol hydrochloride. We investigated three concentrations of 0.05%, 0.1%, and 0.2% (volume ratio) TFA in the aqueous mobile phase. The chromatographic behavior of ethambutol hydrochloride and (+)-2-aminobutanol showed that the retention time and resolution of both analytes increased with increasing TFA concentration (measured in Figure 4 As shown in Figure 2 ). In a 0.05% TFA solution, (+)-2-aminobutanol could not be separated from ethambutol hydrochloride, and the peak shape was poor. However, as the TFA concentration increased to 0.1% or 0.2%, the separation and peak shape improved. As the concentration of trifluoroacetic acid in the mobile phase increased, the pH value of the mobile phase also decreased, which put higher requirements on the performance of the chromatographic column. Since the pH value range of traditional chromatographic columns is 2-9, a 0.1% trifluoroacetic acid concentration was selected as the aqueous phase of the mobile phase.

[0062] Example 4 (Effect of organic solvents)

[0063] In order to study the effect of organic solvents on the retention and separation of ethambutol hydrochloride and (+)-2-aminobutanol, different organic solvent compositions and ratios were investigated. As the content of organic solvent in the mobile phase increased from 0% to 10%, the retention and separation of ethambutol hydrochloride and (+)-2-aminobutanol decreased. When the concentration of methanol (MeOH) was between 0% and 6%, the separation of impurity 1 and ethambutol was very good, with the corresponding separation degrees ranging from 2.5 to 6.8 (in terms of Figure 5 The results also show that the sensitivity of (+)-2-aminobutanol increases significantly with the increase of methanol content. In RPHPLC, under low concentration organic phase conditions, the elution capacity of acetonitrile is comparable to that of methanol. Figure 5 Considering the sensitivity and resolution factors, 0.1% TFA aqueous solution-methanol (96:4) was selected as the mobile phase.

[0064] Example 5 (Optimizing NQAD parameters)

[0065] The key parameters that affect the response of NQAD include the nebulizer gas flow rate, nebulizer temperature, and evaporator temperature. The nebulizer gas flow rate of NQAD is not adjustable, so we changed the nebulizer temperature and evaporator temperature to explore their effects on the response of NQAD. In terms of nebulizer temperature, the nebulizer temperature was increased from 15℃ to 45℃ (with a step size of 5℃). The results showed that within the range of 15-30℃, the change of nebulizer temperature had little effect on the sensitivity (with a step size of 15℃). Figure 6 As the atomization temperature increases, the response (peak area) increases slightly, while the sensitivity decreases sharply at 40°C (as shown in Figure 2). Figure 6 In order to achieve the best response of NQAD, the evaporation temperature was between 20℃ and 90℃ (in steps of 10℃). The results showed that increasing the evaporator temperature slightly reduced the response of ethambutol hydrochloride and (+)-2-aminobutanol (in Figure 7 The sensitivity remains stable until the evaporation tube temperature reaches 40°C, then reaches the highest point at 70-80°C, and then decreases at 90°C (as shown in Figure 2). Figure 7 As shown in Figure 2). This may be because at a certain temperature, the increase in evaporation temperature promotes the evaporation of the mobile phase, making the baseline smoother and thus improving the sensitivity. When the evaporation temperature reaches 70°C, the background decrease of the mobile phase has tended to equilibrium. As the evaporation temperature increases further, the background interference remains stable, while the peak area continues to decrease, resulting in a decrease in sensitivity. Based on the above results, the atomization temperature and evaporation temperature were set to 30°C and 70°C, respectively. Example 6

[0066] The performance of the established RP-HPLC-NQAD method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines.

[0067] 1. Exclusivity

[0068] Forced degradation studies were performed on ethambutol hydrochloride to demonstrate the selectivity of the analytical method for ethambutol hydrochloride and its impurities. The following experiments were performed under acidic, alkaline, thermal, photo, and oxidative conditions: the samples under acidic and alkaline conditions were neutralized and then injected. The results showed that ethambutol hydrochloride and (+)-2-aminobutanol were well separated and free of interference (with Figure 8 ). Figure 8 In the figure, the chromatographic elution curves represent the experimental results under the following conditions from top to bottom: acid-base neutralization blank, acid, base, light, oxidation, high temperature, undestroyed, and system.

[0069] 2. Accuracy

[0070] A certain amount of ethambutol hydrochloride and (+)-2-aminobutanol standard solution were added to the ethambutol hydrochloride fine powder. Sample solutions were prepared at 50%, 100% and 150% of the standard limit concentration of (+)-2-aminobutanol (1.0%), and each concentration was prepared in triplicate. The ethambutol hydrochloride detection solution was prepared in triplicate, each at 80%, 100% and 120% of the labeled concentration, and each concentration was prepared in triplicate for recovery determination. The average recoveries of ethambutol hydrochloride and (+)-2-aminobutanol were 98.62% and 99.66%, respectively. The RSD of ethambutol hydrochloride was 1.350%, and the RSD of (+)-2-aminobutanol was 10.97%. The results are shown in Fig. 9 .

[0071] 3. Injection precision

[0072] The mixed standard solutions of (+)-2-aminobutanol and ethambutol hydrochloride at 20 μg / mL and 0.25 mg / mL were injected 6 times. The results showed that the RSDs of (+)-2-aminobutanol and ethambutol hydrochloride were 1.83% and 1.30%, respectively.

[0073] 4. Linear

[0074] A series of standard solutions with different concentration levels were analyzed to determine the linear relationship. The concentration range of ethambutol hydrochloride standard solution was 5μg / mL-500μg / mL, and the concentration range of (+)-2-aminobutanol was 4μg / mL-60μg / mL. The peak areas at different concentrations were plotted into a calibration curve. Both ethambutol hydrochloride and (+)-2-aminobutanol showed good linear relationships, with a correlation coefficient (R 2 ) are greater than 0.999. Correlation coefficient and linear equation are shown in Fig.10 .

[0075] 5. Sensitivity (LOD and LOQ)

[0076] The limit of detection (LOD), limit of quantification (LOQ) and LOQ values ​​were calculated under the conditions of signal-to-noise ratio of 3 and 10. The limit of detection and limit of quantification of ethambutol hydrochloride were 5.6 ng and 18.8 ng, respectively, and the limit of detection and limit of quantification of IMP1 were 9.8 ng and 32.5 ng, respectively.

[0077] 6. Durability

[0078] Minor changes in chromatographic conditions, such as the use of different C18 columns and changes in column oven temperature (30, 35, and 40 °C), did not result in significant changes in retention time, resolution, or peak symmetry. The results indicate that the analytical method is robust.

[0079] Conclusion: In this study, a non-derivatized RP-HPLC-NQAD method was established and validated for the determination of (+)-2-aminobutanol and ethambutol hydrochloride, which lack UV chromophores. This method can directly determine (+)-2-aminobutanol and ethambutol hydrochloride without derivatization, and the method has high sensitivity and accuracy.

[0080] Unlike other aerosol detectors, the response of NQAD is linear, and no logarithmic transformation is required, which is convenient for calculation. The response of the detector is affected by many factors, such as the composition of the mobile phase, the nebulization temperature and the vaporization temperature. The detector response is optimized to achieve the best performance.

[0081] The method is highly sensitive, with LODs of (+)-2-aminobutanol and ethambutol hydrochloride being 5.6 ng and 9.8 ng, respectively, and LOQs being 18.8 ng and 32.5 ng, respectively.

[0082] The LOQ of ethambutol hydrochloride was determined by NQAD, which showed that its sensitivity was more than 20 times higher than that of the evaporative light scattering detector reported in the literature. (+)-2-aminobutanol and ethambutol hydrochloride obtained good linear relationships in the range of 4μg / mL-60μg / mL and 5μg / mL-500μg / mL, respectively (R 2 =0.999). The average recoveries of (+)-2-aminobutanol and ethambutol hydrochloride were 99.7% and 98.6%, respectively, and the RSDs were 1.4% and 1.0%, respectively, with high accuracy. The validation results showed that NQAD performed well in the analysis of (+)-2-aminobutanol and ethambutol hydrochloride in RPHPLC mode and could be used to evaluate the drug quality of ethambutol hydrochloride tablets.

Claims

1. A method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method, characterized in that: The steps include: S01: Prepare standard curves of ethambutol hydrochloride and (+)-2-aminobutanol; S02: Grind ethambutol hydrochloride tablets into powder to obtain ethambutol hydrochloride tablet powder; S03: adding purified water to the ethambutol hydrochloride tablet powder, dissolving by ultrasonication, diluting with water to 2 mg / ml and 0.25 mg / ml respectively, and filtering the two ethambutol hydrochloride tablet solutions obtained above through a 0.22 μm water membrane to obtain two analytical solutions for the machine respectively; Among them, the ethambutol hydrochloride tablet powder solution with a concentration of 2 mg / ml is used as the on-machine analysis liquid for detecting the content of (+)-2-aminobutanol; the ethambutol hydrochloride tablet powder solution with a concentration of 0.25 mg / ml is used as the on-machine analysis liquid for detecting the content of ethambutol hydrochloride; S04: After the analytical liquid is detected by reverse phase high performance liquid chromatography coupled with hydrogel particle laser technology detector, the contents of ethambutol hydrochloride and (+)-2-aminobutanol are calculated; In step S04, the chromatographic separation conditions are: The stationary phase was a Capcell pak C18 column 4.6×250 mm, 5 μm, and the mobile phase was a mixture of 0.1% trifluoroacetic acid solution and methanol, with isocratic elution and a flow rate of 1.0 mL / min; The column temperature was maintained at 30 °C, and the injection volume was 10 μL; NQAD uses nitrogen with a flow rate of 4.7 L / min as the atomizing gas, the atomizing temperature is 15℃-30℃, the vaporization temperature is 20℃-90℃, and the filtration time is 5 seconds; The volume ratio of the trifluoroacetic acid solution to methanol was 94:

6.

2. The method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method according to claim 1, characterized in that: Step S01 comprises: accurately weighing 20 mg of (+)-2-aminobutanol and ethambutol hydrochloride reference substances, dissolving them in 20 ml and 10 ml of water, respectively, to prepare (+)-2-aminobutanol stock solution and ethambutol hydrochloride stock solution respectively; The (+)-2-aminobutanol stock solution and the ethambutol hydrochloride stock solution were diluted with water to prepare a series of linear solutions with concentration ranges corresponding to 4 μg / ml to 60 μg / ml and 5 to 500 μg / mL, respectively.

3. The method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method according to claim 1, characterized in that: The atomization temperature is 30°C.

4. The method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method according to claim 1, characterized in that: The vaporization temperature is 70 °C.

5. The method for simultaneously determining (+)-2-aminobutanol and ethambutol hydrochloride based on RP-HPLC-NQAD method according to claim 1, characterized in that: In step S03, the ultrasonic power is 360W and the ultrasonic time is 5 minutes.

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