A method for resolving norfloxacin enantiomers

Abstract

The present application relates to the technical field of pharmaceutical analysis, in particular to a method for resolving and detecting norfloxacin enantiomers. The method adopts high performance liquid chromatography to resolve sample solutions containing norfloxacin enantiomers and detects with a diode array detector; wherein the high performance liquid chromatography adopts an Agilent ZORBAX Eclipse XDB-C8 chromatographic column and uses L-isoleucine copper acetate as a chiral mobile phase additive. The above method realizes the resolution of norfloxacin enantiomers, and the analysis time is only 22 min, which is shortened by nearly half of the analysis time, the resolution is much greater than 1.5, and the resolution and determination requirements are met. The method is simple, economical and practical, has good linear relationship, precision and sensitivity, and can be used for the resolution and quality control of norfloxacin enantiomers.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and specifically to a method for the separation and detection of enantiomers of naflufloxacin. Background Technology

[0002] Nadifloxacin is a third-generation quinolone antibacterial drug developed by Otsuka Pharmaceutical Co., Ltd. of Japan in the early 1980s. Structurally, nadifloxacin has a unique benzoquinazine structure with a chiral carbon bonded to the parent ring via a C-C bond. This unique structure significantly distinguishes its overall molecular configuration, rigidity, and hydrophobicity from those of the previous three generations of quinolone drugs. The two enantiomers of nadifloxacin exhibit significant differences in antibacterial activity, pharmacokinetics, and toxicity both in vivo and in vitro. High-performance liquid chromatography (HPLC) is an effective method for separating and detecting the enantiomers of nadifloxacin, determining the purity of individual enantiomers. The applicant previously used C... 18 A chromatographic column was used, with hydroxypropyl-β-cyclodextrin as the chiral mobile phase additive and potassium dihydrogen phosphate / ethanol added to prepare the mobile phase for resolving naflufloxacin enantiomers. However, this method requires 40 minutes for a single injection to separate naflufloxacin enantiomers, resulting in a long separation time. Furthermore, potassium dihydrogen phosphate in the mobile phase is prone to crystallization, easily causing instrument clogging and damage, leading to high system maintenance costs. Therefore, there is an urgent need to find different resolution and detection methods suitable for naflufloxacin enantiomers. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for the separation and detection of enantiomers of naflufloxacin. This method can achieve complete separation of the two enantiomers of naflufloxacin, and significantly shortens the time required for a single separation, thereby enabling the separation and quality control of naflufloxacin enantiomers.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: This application provides a method for the separation and detection of enantiomers of naflufloxacin, which uses high performance liquid chromatography (HPLC) to separate sample solutions containing naflufloxacin enantiomers and a diode array detector for detection; wherein the HPLC method uses an Agilent ZORBAX Eclipse XDB-C8 column and L-isoleucine copper acetate as a chiral mobile phase additive.

[0005] Furthermore, the method for preparing the mobile phase in the high performance liquid chromatography is as follows: L-isoleucine and copper acetate are dissolved in water to prepare an L-isoleucine-copper acetate solution; the pH value is adjusted to 5.6-7.0 with ammonia water as mobile phase A; and methanol is used as mobile phase B.

[0006] Furthermore, the volume ratio of the mobile phase A to the mobile phase B is 57-60:40-43.

[0007] Preferably, the method for preparing the mobile phase in the high performance liquid chromatography is as follows: L-isoleucine and copper acetate are dissolved in water to prepare an L-isoleucine copper acetate solution; the pH value is adjusted to 7.0 with ammonia water as mobile phase A; and methanol is used as mobile phase B, wherein the volume ratio of mobile phase A to mobile phase B is 60:40.

[0008] Furthermore, the molar concentration of L-isoleucine in the L-isoleucine copper acetate solution is 4–8 mmol / L, the molar concentration of copper acetate is 4–8 mmol / L, and the concentration ratio of L-isoleucine to copper acetate is 1.5–2.5:1.

[0009] Preferably, the L-isoleucine copper acetate solution has a molar concentration of 8 mmol / L for L-isoleucine and a molar concentration of 4 mmol / L for copper acetate, and the concentration ratio of L-isoleucine to copper acetate is 2:1.

[0010] Furthermore, the chromatographic column is an Agilent ZORBAX Eclipse XDB-C 85μm, 4.6×250 mm.

[0011] Furthermore, the method for preparing the sample solution containing the enantiomer of naflufloxacin is to dissolve the naflufloxacin sample in acetonitrile to prepare a sample solution with a naflufloxacin concentration of 500-1500 μg / ml.

[0012] Furthermore, the column temperature is 40℃, the detection wavelength is 345nm, the injection volume is 20μL, and the flow rate is 1.0ml / min.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses L-isoleucine copper acetate as a chiral mobile phase additive, screens the chromatographic column, and optimizes various experimental parameters, finally achieving the separation of naflufloxacin enantiomers on an Agilent ZORBAX Eclipse XDB-C8 chromatographic column. The resolution between enantiomer 1 and enantiomer 2 is much greater than 1.5, which can achieve complete separation of naflufloxacin enantiomers. At the same time, the analysis time is shortened by nearly half compared with the previous application. The method of this application can meet the need for rapid and complete separation and determination of naflufloxacin enantiomers. This method has excellent practical effect and economic value. Methodological experiments show that the method of this application has good linearity, precision and sensitivity for the separation and detection of naflufloxacin enantiomers, and can be well used for the separation and quality control of naflufloxacin enantiomers. Attached Figure Description

[0014] Figure 1 The HPLC chromatogram of column ① used in this embodiment of the invention; Figure 2 The HPLC chromatogram of column ② used in this embodiment of the invention; Figure 3 The HPLC chromatogram of column ③ used in this embodiment of the invention; Figure 4 The HPLC chromatogram of column ④ used in this embodiment of the invention; Figure 5 This is a spectrum of naflurane from an embodiment of the present invention. Detailed Implementation

[0015] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise specified, all methods described herein are conventional methods, and all raw materials described herein are available from publicly available commercial sources.

[0017] Example 1: Establishment of a method for the resolution and detection of enantiomers of naloxamine 1. Experimental Section 1.1 Instruments and Reagents The system used was an LC-20AD XR high-performance liquid chromatograph with a diode array detector (Shimadzu Corporation, Japan), and a METTLERMS 205DU electronic balance. Naflufloxacin reference standard (source: China National Institutes for Food and Drug Control, batch number: 130462-201001, purity: 97.0%; racemic mixture), purified water, L-isoleucine (99% purity), chromatographic grade methanol, and all other reagents were analytical grade.

[0018] 1.2 Chromatographic conditions Mobile phase: L-isoleucine copper acetate solution (containing 8 mmol / L L-isoleucine and 4 mmol / L copper acetate, with the pH adjusted to 7.0 by ammonia) - methanol (60:40); Flow rate: 1.0 mL / min; Column: Agilent ZORBAX Eclipse XDB-C8 5 μm, 4.6 × 250 mm; Column temperature: 40℃; Injection volume: 20 μL; Detection wavelength: 345 nm.

[0019] 1.3 Solution Preparation Accurately weigh 51.45 mg of naflufloxacin reference standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well to obtain the naflufloxacin reference solution (naflufloxacin concentration is 998.13 μg / mL, and the concentration of each enantiomer of naflufloxacin is half the concentration of naflufloxacin). Accurately measure an appropriate amount of the above solution and dilute it with the mobile phase in “1.2 Chromatographic Conditions” to obtain naflufloxacin concentrations of 499.07, 299.44, 199.63, 99.81, 49.91, 19.96, 9.981, 4.991, 1.996, and 0.599 μg / mL.

[0020] 2 Results and Discussion 2.1 Selection of Chromatographic Column A 199.63 μg / mL naflufloxacin reference solution was used, and the determination was performed on columns ① through ④ according to the method described in "1.2 Chromatographic Conditions". Column ① was an Agilent ZORBAX Eclipse XDB-C 85 μm, 4.6 × 250 mm column (the column described in "1.2 Chromatographic Conditions"); column ② was an Agilent ZORBAX SB-C 85 μm, 4.6 × 250 mm column; and column ③ was a Waters Symmetry C... 18 5μm, 4.6×250mm, column ④ is InertSustain AQ-C 18 5μm, 4.6×250mm, test results are shown in [link to test results]. Figures 1 to 4 , Figure 1 This is the HPLC chromatogram of column ①. Figure 2 This is the HPLC chromatogram of column ②. Figure 3 This is the HPLC chromatogram of column ③. Figure 4 The figure shows the HPLC chromatogram of column ④. In the figure, 1 represents enantiomer 1 of naflufloxacin and 2 represents enantiomer 2 of naflufloxacin.

[0021] from Figures 1 to 4 As can be seen, using column ① to separate and detect the enantiomers of naflufloxacin, the resolution reached 1.857, and the analysis time was only 22 min. Column ② had a resolution of 1.750 and an analysis time of 60 min. Columns ③ and ④ could not separate the two enantiomers of naflufloxacin. The method in this application, using an Agilent ZORBAX EclipseXDB-C 85μm, 4.6×250 mm column, can not only achieve complete separation of the enantiomers of naflufloxacin, but also greatly shorten the analysis time.

[0022] 2.2 Selection of detection wavelength The higher the pH value of the L-isoleucine and copper acetate solution, the darker the solution color, resulting in greater background interference (higher noise) in the mobile phase. Figure 5 The spectrum of naflufloxacin is shown. As can be seen from the figure, the maximum absorption wavelength of naflufloxacin is 298 nm. In this application, a naflufloxacin reference solution with a concentration of 1.996 μg / mL was used, and the results were obtained at 298 nm. Other measurements were performed according to the chromatographic conditions in "1.2". The results showed that the signal-to-noise ratio of naflufloxacin enantiomer 1 and enantiomer 2 was low. To screen for the optimal detection wavelength, a naflufloxacin reference solution with a concentration of 1.996 μg / mL was used, and the results were obtained according to the chromatographic conditions in "1.2", detecting at 345, 350, and 360 nm. The signal-to-noise ratios of naflufloxacin enantiomer 1 and enantiomer 2 were statistically analyzed, and the results are shown in Table 1 below. Table 1. Signal-to-noise ratio data for naflufloxacin

[0023] As can be seen from Table 1, although the spectrum of naflufloxacin shows that its maximum absorption wavelength is 298 nm, the signal-to-noise ratio of the naflufloxacin enantiomer peaks at wavelengths of 298 nm and 360 nm are both below 10, while the signal-to-noise ratio is the highest and the sensitivity is the highest at wavelength of 345 nm. Therefore, this application selected 345 nm as the measurement wavelength for the remaining studies.

[0024] 2.3 Selection of Organic Phase In section 1.2 Chromatographic Conditions, methanol was replaced with acetonitrile while keeping other conditions unchanged. Chromatograms of naflufloxacin reference solutions with a concentration of 199.63 μg / mL were measured and recorded to investigate the effect of different organic phases on separation. The results showed that when acetonitrile was used as the mobile phase, naflufloxacin enantiomers 1 and 2 overlapped into a single chromatographic peak and could not be separated.

[0025] 2.4 Selection of Methanol Ratio Adjust the methanol ratio in “1.2 Chromatographic Conditions” while keeping other conditions unchanged. Measure the concentration of 199.63 μg / mL of naflufloxacin reference solution and record the chromatograms to investigate the effect of methanol ratio changes on the separation effect. The results are shown in Table 2.

[0026] Table 2. Effect of Methanol Ratio in Mobile Phase on Separation Efficiency

[0027] Note: t R1 Both N1 and N2 are enantiomer 1 data, t R2 Both N2 and N2 are enantiomer 2 data. As shown in Table 2, a higher methanol ratio results in a shorter analysis time, but a decrease in resolution and theoretical plate number. Reducing the methanol ratio to 30% only improves the resolution to 2.5, but increases the analysis time by more than four times. Considering all factors, 40% methanol is selected as the optimal determination condition.

[0028] 2.5 Effect of mobile phase additives on separation effect The chiral separation capabilities of different chiral additives vary considerably. The chiral additives in "1.2 Chromatographic Conditions" were adjusted while keeping other conditions constant. Chromatograms of naflufloxacin reference solutions with a concentration of 199.63 μg / mL were measured and recorded to investigate the effects of L-phenylalanine, L-isoleucine, and L-proline as chiral additives on the separation effect. The results showed that using only L-isoleucine from this invention resulted in two chromatographic peaks for the separation of naflufloxacin, achieving the separation of naflufloxacin enantiomers. The chromatographic peaks of the two naflufloxacin isomers using L-phenylalanine and L-proline overlapped.

[0029] Adjusting the copper acetate in "1.2 Chromatographic Conditions" to copper sulfate and adjusting the pH to between 4.6 and 7.0 showed that adding copper sulfate to the mobile phase would cause solid precipitation, making it unusable.

[0030] Effect of pH value of 2.6L-Isoleucine copper acetate solution on the separation effect Adjust the pH value of the L-isoleucine copper acetate solution in “1.2 Chromatographic Conditions”, keep other conditions unchanged, and measure and record the chromatograms of the naflufloxacin reference solution with a concentration of 199.63 μg / mL to investigate the effect of pH value change of L-isoleucine copper acetate solution on the separation effect. The results are shown in Table 3.

[0031] Table 3. Effect of pH of L-Isoleucine Copper Acetate Solution on Separation Efficiency

[0032] The results showed that the higher the pH value, the greater the resolution, theoretical plate number, and retention time. Considering all factors, a pH value of 7.0 was selected as the optimal measurement condition.

[0033] The effect of 2,7-L-isoleucine copper acetate concentration on the separation effect The concentration of L-isoleucine copper acetate solution in “1.2 Chromatographic Conditions” was adjusted, while keeping the L-isoleucine copper acetate concentration ratio (2:1) and other conditions unchanged. Chromatograms of naflufloxacin reference solution with a concentration of 199.63 μg / mL were measured and recorded to investigate the effect of L-isoleucine copper acetate solution concentration on separation effect. The results are shown in Table 4.

[0034] Table 4. Effect of L-Isoleucine Copper Acetate Concentration on Separation Efficiency

[0035] The results showed that the higher the molar concentration of L-isoleucine and copper acetate, the greater the theoretical plate number and resolution. At a concentration of 2 mmol / L, the resolution was less than 1.5, which was not suitable. At a concentration of 8 mmol / L, the resolution was only 0.2 higher than that at 4 mmol / L. Considering cost savings and instrument protection, the optimal molar concentrations were 4 mmol / L for L-isoleucine and 2 mmol / L for copper acetate.

[0036] Effect of 2,8-L-Isoleucine Copper Acetate Concentration Ratio on Resolution Adjust the concentration ratio of L-isoleucine in copper acetate solution in “1.2 Chromatographic Conditions”, keeping other conditions constant, and measure and record the chromatograms of naflufloxacin reference solution with a concentration of 199.63 μg / mL. Keep the L-isoleucine concentration constant at 8 mmol / L, and investigate the effect of different copper acetate solution concentrations on the separation effect. The results are shown in Table 5.

[0037] Table 5. Effects of L-Isoleucine-Copper Acetate Concentration Ratio on Retention Time and Resolution

[0038] The results showed that when the concentration of copper acetate solution was 8 mmol / L, the resolution was less than 1.5 and baseline separation could not be achieved. As the concentration of copper acetate solution decreased, the retention time of the chromatographic peak increased and the resolution showed an upward trend. However, when the concentration was reduced to 2.67 mmol / L, the resolution decreased instead. Therefore, the optimal ratio of L-isoleucine to copper acetate concentration was 2:1, at which point the resolution was optimal.

[0039] 2.9 Effect of column temperature on separation effect Only the column temperature in "1.2 Chromatographic Conditions" was adjusted. Naflufloxacin reference solution with a concentration of 199.63 μg / mL was injected and chromatograms were recorded to investigate the effect of changing the column temperature on enantiomer separation. When the flow rate and column temperature were 25℃, the enantiomer elution times were 27.728 min and 30.208 min, respectively, with theoretical plate numbers of 6834 and 7712, and a resolution of 1.818. The resolution was not significantly different from that at a column temperature of 40℃, but the retention time was prolonged and the theoretical plate number was reduced. Therefore, a column temperature of 40℃ was ultimately selected as the optimal setting.

[0040] Example 2: Methodological Investigation 1. Examination of linear relationships Solutions of nalofop-p-ethyl with concentrations of 499.07, 299.44, 199.63, 99.81, 49.91, 19.96, 9.981, 4.991, and 1.996 μg / mL were prepared and chromatograms were recorded according to the chromatographic conditions in section 1.2. The peak areas of individual enantiomers of nalofop-p-ethyl are plotted on the ordinate.y ), at the concentration of a single enantiomer of nalofacin ( x Linear regression was performed with π as the abscissa. The results showed that the concentration of a single enantiomer of naflufloxacin in the range of 1–250 μg / mL had a good linear relationship with the corresponding peak area. The regression equation for enantiomer 1 is: y = 31111 x + 9123, R² = 0.9996; the enantiomer 2 regression equation is: y = 31376 x + 8656, R² = 0.9997.

[0041] 2. Precision A 199.63 μg / mL naflufloxacin reference solution was injected six times consecutively under the chromatographic conditions described in "1.2". The chromatograms were recorded. The RSDs of the peak areas of each enantiomer of naflufloxacin were calculated and found to be 0.21% and 0.48%, respectively, indicating that the method has good precision.

[0042] 3. Limit of Quantitation and Limit of Detection Naflufloxacin reference solutions with concentrations of 1.996 μg / mL and 0.599 μg / mL were analyzed according to the chromatographic conditions in section 1.2. The results showed that when the naflufloxacin concentration was 1.996 μg / mL, the signal-to-noise ratio (SNR) of both enantiomer peaks was greater than 10; when the naflufloxacin concentration was 0.599 μg / mL, the SNR of both enantiomer peaks was greater than 3. The limits of detection and quantitation (LOD) for a single enantiomer of naflufloxacin were determined to be 1 μg / mL and 0.3 μg / mL, respectively. 4. Conclusion This study established a simple and rapid method for the resolution and detection of naflufloxacin enantiomers using L-isoleucine and copper acetate as chiral mobile phases and an Agilent C8 column (Agilent ZORBAX Eclipse XDB-C8 5μm, 4.6×250 mm) as the analytical column. This method requires no additional derivatization steps or special chiral columns. The analytical time for resolving naflufloxacin enantiomers can be controlled within 22 min, while maintaining good resolution. This method is simple to operate, economical, practical, and also exhibits good linearity, precision, and sensitivity.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features, all of which should be covered within the scope of the technical solutions claimed in this application.

Claims

1. A method for resolving and detecting enantiomers of naflufloxacin, characterized in that, The sample solution containing naflufloxacin enantiomers was separated by high performance liquid chromatography (HPLC) and detected by a diode array detector. The HPLC method used an Agilent ZORBAX Eclipse XDB-C8 column with L-isoleucine copper acetate as a chiral mobile phase additive. The sample of the enantiomer of naflufloxacin was naflufloxacin reference standard, which was sourced from the National Institutes for Food and Drug Control, batch number 130462-201001. The method for preparing the mobile phase in the high performance liquid chromatography is as follows: L-isoleucine and copper acetate are dissolved in water to prepare L-isoleucine copper acetate solution, and the pH value is adjusted to 5.6-7.0 with ammonia water as mobile phase A, and methanol is used as mobile phase B. The volume ratio of mobile phase A to mobile phase B is 57-60:43-40, and the elution method is isocratic elution. The column temperature was 40℃, the detection wavelength was 345nm, the injection volume was 20μL, and the flow rate was 1.0ml / min.

2. The method for resolving and detecting enantiomers of naflufloxacin according to claim 1, characterized in that, The L-isoleucine copper acetate solution has a molar concentration of 4–8 mmol / L for L-isoleucine and 4–8 mmol / L for copper acetate, and the concentration ratio of L-isoleucine to copper acetate is 1.5–2.5:

1.

3. The method for resolving and detecting enantiomers of naflufloxacin according to claim 1, characterized in that, The chromatographic column was an Agilent ZORBAX Eclipse XDB-C8 5μm, 4.6×250 mm.

4. The method for resolving and detecting enantiomers of naflufloxacin according to claim 1, characterized in that, The method for preparing the sample solution containing the enantiomer of naflufloxacin is as follows: dissolve the naflufloxacin sample in acetonitrile to prepare a sample solution with a naflufloxacin concentration of 500-1500 μg / ml.

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