A method for separating epimeric isomers of pharmaceutical lactose by capillary electrophoresis

Abstract

The present invention belongs to the field of pharmaceutical analysis, and specifically discloses a method for separating anomeric isomers of pharmaceutical lactose by capillary electrophoresis. First, a lactose sample is dissolved in an electrophoresis buffer solution. During the mutarotation process of lactose molecules in an aqueous solution, the two anomeric isomers, α-form and β-form, undergo reversible interconversion and finally reach a dynamic equilibrium. Taking the lactose solution containing both α and β configurations as the sample to be analyzed, the baseline separation of the two configurations can be achieved by using capillary electrophoresis ultraviolet direct detection technology without the need for derivatization and addition of organic reagents. The above technical solution is simple to operate, environmentally friendly and economical, and has high repeatability, thus establishing a new type of rapid and efficient separation method for lactose anomeric isomers.

Description

Technical Field

[0001] The present invention relates to a method for separating anomeric isomers of pharmaceutical lactose by capillary electrophoresis, and belongs to the technical field of pharmaceutical analysis. Background Art

[0002] At present, lactose is one of the most commonly used pharmaceutical excipients, and diverse pharmaceutical lactose products can be further applied to the research and development of various types of preparations. The properties of lactose molecules are mainly reflected in the differences in the specific rotation and solubility of its different anomeric isomers (α-type and β-type), and this property has important practical guiding significance for the pharmaceutical industry. The chromatographic analysis method commonly used to separate the α and β configurations of lactose nowadays is high performance liquid chromatography (HPLC), but this method has cumbersome operations, large consumption of reagents, and expensive chromatographic columns, and has limitations in the detection of trace samples.

[0003] The application of capillary electrophoresis (HPCE) technology in pharmacy, biology, medicine, and environmental science reflects its unique advantages. Compared with traditional gel electrophoresis, HPCE can operate under high voltage conditions without generating heat effects due to the use of a capillary with a small inner diameter. Compared with HPLC, HPCE has many advantages. One of the major features of HPCE is rapid analysis. Under the action of an externally applied high electric field, the analysis time of the analyte generally does not exceed 30 minutes. HPCE can improve separation by adjusting conditions such as the pH, voltage, and temperature of the buffer solution during the operation process. Without the high-pressure pump transport system, large amounts of organic reagents, and expensive chromatographic columns of HPLC, HPCE can use microliter-level reagents to achieve rapid separation of multiple samples, so capillary electrophoresis is more environmentally friendly and economical. In addition, HPCE uses an externally applied voltage as the electroosmotic flow driving force, eliminating the influence of the radial diffusion of the parabolic flow pattern in liquid chromatography driven by pressure on the column efficiency of the finally obtained chromatographic peak, and has a higher separation column efficiency.

[0004] The application of capillary electrophoresis technology in pharmaceutical analysis is mainly reflected in the following aspects: small molecule drugs and their related substances; chiral separation; traditional Chinese medicine and natural products; in vivo drug analysis; macromolecule and biological product analysis. However, there are few studies on the use of HPCE for the analysis of pharmaceutical excipients.

[0005] In view of the above-mentioned defects, the present invention aims to create a method for separating anomeric isomers of pharmaceutical lactose by capillary electrophoresis, making it more valuable for industrial utilization. Summary of the Invention

[0006] To solve the above technical problems, the object of the present invention is to provide a method for capillary electrophoresis separation of anomeric isomers of pharmaceutical lactose. By using a borax electrophoresis buffer, a negatively charged complex is formed with lactose, which can improve the absorbance of lactose at 195 nm in ultraviolet light, enabling direct detection of lactose without going through a complex derivatization process or using other expensive detectors. At the same time, this charged complex can migrate in an electric field, and the complexes formed by α-lactose and β-lactose are separated based on their different electrophoretic mobilities without adding organic reagents, which is environmentally friendly and economical.

[0007] A method for capillary electrophoresis separation of anomeric isomers of pharmaceutical lactose according to the present invention, the specific separation steps are as follows:

[0008] (1) Prepare a borax electrophoresis buffer, and adjust the borax electrophoresis buffer to different pH values using boric acid or saturated sodium hydroxide solution;

[0009] (2) Use the above borax electrophoresis buffer to dissolve the pharmaceutical lactose sample, and prepare a lactose solution at room temperature. The lactose molecules undergo mutarotation in the aqueous solution, and its two anomeric isomers, α-form and β-form, undergo reversible interconversion and finally reach a dynamic equilibrium;

[0010] (3) Use the above-obtained lactose solution containing both α and β configurations as the sample to be analyzed, construct a capillary electrophoresis ultraviolet direct detection method, and achieve baseline separation of the anomeric isomers of pharmaceutical lactose.

[0011] Further, the pH value of the borax electrophoresis buffer in step (1) is 7 - 10. The borax electrophoresis buffer forms a negatively charged complex with lactose under the condition of pH > 7 and can migrate in an electric field.

[0012] Further, the pH value of the borax electrophoresis buffer in step (2) is 10. The alkaline condition can accelerate the mutarotation process, and finally the α-form and β-form reach a dynamic equilibrium.

[0013] Further, the concentration of the lactose sample in step (2) is 20 mg / mL.

[0014] Further, in step (3), the capillary electrophoresis separation voltage for constructing the capillary electrophoresis ultraviolet direct detection method is 5 - 20 kV; the concentration of the borax electrophoresis buffer is 55 - 95 mM; the injection volume condition is 5 kPa, 3 - 12 s; the pH value of the borax electrophoresis buffer is 7 - 9.

[0015] Further, the capillary electrophoresis separation voltage for constructing the capillary electrophoresis ultraviolet direct detection method in step (3) is 10 kV. As the voltage increases, the retention time of the anomers of lactose gradually decreases. This is because as the voltage increases, the electroosmotic flow in the column increases, and the retention of lactose decreases, so its peak emergence time gradually shortens. As the voltage increases, the column efficiency of the peak shows a gradually increasing trend. As the voltage increases, the resolution shows an increasing trend. When the voltage is 5 kV, the electroosmotic flow is small, the retention time is long, and the separation selectivity is strong, but the column efficiency is low. When the voltage is 20 kV, the column efficiency increases, but the resolution decreases. Finally, 10 kV is determined as the optimal voltage value.

[0016] Further, the concentration of the borax electrophoresis buffer for constructing the capillary electrophoresis ultraviolet direct detection method in step (3) is 75 mM. As the concentration of the buffer increases, the retention time of lactose molecules gradually increases. This is because the increase in the buffer concentration causes a decrease in the electroosmotic flow, and the retention time of the anomers of lactose shows an increasing trend. When the buffer concentration is 55 mmol L -1 , the column efficiency is low and the resolution is low. Continuing to increase the ionic strength of the solution, the column efficiency gradually increases. When the buffer concentration is 75 mmol L -1 , the resolution is the largest because when the buffer concentration is too small or too large, the change in the ionic strength of the solution will affect the electroosmotic flow in the column. When the buffer with the optimal concentration is used, the separation ability of the capillary column is optimal.

[0017] Further, the injection volume condition for constructing the capillary electrophoresis ultraviolet direct detection method in step (3) is 5 kPa, 3 s. As the injection volume increases, it will cause peak tailing and a decrease in column efficiency. The theoretical plate number of the electrophoretogram of lactose molecules decreases with the increase in the injection volume. As the injection volume increases, the resolution shows a downward trend. This is because a higher sample volume will cause peak tailing and a decrease in column efficiency. Therefore, the resolution of the anomers of lactose decreases with the increase in the sample volume. Finally, the preferred value of the injection volume is determined as 5 kPa, 3 s.

[0018] Further, the pH value of the borax electrophoresis buffer for constructing the capillary electrophoresis ultraviolet direct detection method in step (3) is 8.5. As the pH value increases, the retention time of the anomers of lactose shows an increasing trend. This is because as the pH value increases, it is more conducive to the complexation of borate with lactose molecules to form a negatively charged complex, and its apparent mobility decreases with the increase in the pH value, so the retention time increases. As the pH value increases, the column efficiency of the electrophoretogram gradually decreases because the enhanced retention of the complex in the column causes peak tailing effect. When the pH value is 8.5, this system has the best separation performance.

[0019] By means of the above solution, the present invention has at least the following advantages:

[0020] The method for separating epimers of pharmaceutical lactose provided by the present invention is simple in operation and high in repeatability, further expanding the application of capillary electrophoresis technology in the field of separation of pharmaceutical excipients and providing new ideas for the establishment of industry standards for pharmaceutical lactose.

[0021] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and be able to implement it according to the content of the specification, the following further details the preferred embodiments of the present invention in conjunction with the accompanying drawings. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 : Intra-day repeatability of separation of lactose epimers by capillary electrophoresis.

[0024] Figure 2 : Inter-day repeatability of separation of lactose epimers by capillary electrophoresis.

[0025] Figure 3 : Influence of pH value of electrophoresis buffer on the separation of pharmaceutical lactose epimers. Among them, the pH value of the buffer is a, 7; b, 7.5; c, 8; d, 8.5; e, 9.

[0026] Figure 4 : Influence of separation voltage on the separation of pharmaceutical lactose epimers. Among them, the separation voltage is a, 5 kV; b, 10 kV; c, 15 kV; d, 20 kV.

[0027] Figure 5 : Influence of sample injection volume on the separation of pharmaceutical lactose epimers. Among them, the sample injection volume is a, 5 kPa, 3 s; b, 5 kPa, 6 s; c, 5 kPa, 9 s; d, 5 kPa, 12 s.

[0028] Figure 6 : Influence of buffer concentration on the separation of pharmaceutical lactose epimers. Among them, the buffer concentration is a, 55 mM; b, 65 mM; c, 75 mM; d, 85 mM; e, 95 mM. Detailed Embodiments

[0029] The specific embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0030] A method for capillary electrophoresis separation of anomeric isomers of pharmaceutical lactose, the steps of the method are as follows:

[0031] (1) Prepare a borax electrophoresis buffer solution, and adjust it to a pH value of 7-10 with boric acid or saturated sodium hydroxide solution; the borax electrophoresis buffer solution forms a negatively charged complex with lactose under the condition of pH>7, so as to migrate in an electric field. At the same time, borax forms a negatively charged complex with lactose, which can increase the absorbance of lactose at 195 nm in ultraviolet light, realizing the direct detection of lactose without derivatization.

[0032] (2) Use the borax electrophoresis buffer solution described in step (1) to dissolve the pharmaceutical lactose sample, and prepare a lactose solution with a concentration of 20 mg / mL at room temperature. Lactose molecules exhibit mutarotation in aqueous solution, and its two anomeric isomers, α-form and β-form, undergo reversible interconversion. Alkaline conditions can accelerate the mutarotation process, and finally the α-form and β-form reach a dynamic equilibrium;

[0033] (3) Use the lactose solution containing α and β configurations obtained in step (2) as the sample to be analyzed, construct a capillary electrophoresis ultraviolet direct detection method, and achieve baseline separation of the two configurations, where the capillary electrophoresis separation voltage is 5-20 kV; the concentration of the borax electrophoresis buffer solution is 55-95 mM; the injection volume is 5 kPa, 3-12 s; the pH value of the borax electrophoresis buffer solution is 7-9.

[0034] Example 1

[0035] 1. Preparation of the pharmaceutical lactose sample solution:

[0036] Prepare a borax electrophoresis buffer solution, weigh 3.0183 g of anhydrous sodium tetraborate and dissolve it in ultrapure water, adjust it to pH 10 with saturated sodium hydroxide solution, and prepare 200 mL of borax electrophoresis buffer solution with a concentration of 75 mM. Dissolve the purchased anhydrous lactose (Maclean (Shanghai, China)) (Ph Eur, USP, NF, JP) in the electrophoresis buffer solution (pH 10), and prepare a lactose solution with a concentration of 20 mg / mL at room temperature. Lactose molecules exhibit mutarotation in aqueous solution, and alkaline conditions can increase its mutarotation rate, promote the reversible interconversion of the α-form and β-form, and finally reach a dynamic equilibrium. This lactose solution containing α and β configurations is further used as the sample to be analyzed, and capillary electrophoresis ultraviolet direct separation and detection are adopted.

[0037] 2. Capillary electrophoresis analysis:

[0038] An uncoated quartz capillary with an effective length of 31.5 cm (total length 40 cm, inner diameter 50 μm) was used. The running buffer was a borate electrophoresis buffer solution, which could increase the absorbance of lactose at 195 nm in ultraviolet light, enabling direct ultraviolet detection of lactose. The borate electrophoresis buffer forms a negatively charged complex with lactose under the condition of pH > 7, which can migrate in an electric field, and baseline separation of the α and β configurations is achieved based on different electrophoretic mobilities. By optimizing the pH value (pH 7 - 9) of the borate electrophoresis buffer, the electroosmotic mobility and electrophoretic mobility are adjusted to improve the separation efficiency. At the same time, by optimizing the electrophoresis separation conditions including voltage (5 - 20 kV), concentration of borate electrophoresis buffer (55 - 95 mM), and injection volume (5 kPa, 3 - 12 s), the optimal electrophoresis conditions are obtained for the separation of lactose anomers. The analysis of lactose anomers by this electrophoresis system shows high separation repeatability. As Figure 1 and Figure 2 shown, this separation system has high intra-day and inter-day stability.

[0039] Example 2

[0040] According to the method of Example 1, the preferred value of the pH value of the borate electrophoresis buffer was determined. From Figure 3 the preferred results, it can be seen that as the pH value increases, the retention time of lactose anomers shows an increasing trend. This is because as the pH value increases, it is more conducive to the complexation of borate with lactose molecules to form a negatively charged complex, and its apparent mobility decreases as the pH value increases, so the retention time increases. As the pH value increases, the column efficiency of the electrophoretogram gradually decreases because the enhanced retention of the complex in the column causes peak tailing effect. When the pH value is 8.5, this system has the best separation performance.

[0041] Example 3

[0042] According to the method of Example 1, the preferred value of the separation voltage was confirmed. Since an increase in the running voltage will cause an increase in electroosmotic flow, which thus plays an inhibitory role in the retention of lactose molecules in the capillary column, the column efficiency of the separation changes, further affecting the separation degree. Therefore, for this experiment, the influence of voltage change on its separation performance was further investigated. From Figure 4 the preferred results, it can be seen that as the voltage increases, the retention time of lactose anomers gradually decreases because as the voltage increases, the electroosmotic flow in the column increases and the retention of lactose decreases. Therefore, its peak emergence time gradually shortens. As the voltage increases, the column efficiency of the peak shows a gradually increasing trend. As the voltage increases, the separation degree shows an increasing trend. When the voltage is 5 kV, the electroosmotic flow is small, the retention time is long, and the separation selectivity is strong, but the column efficiency is low. When the voltage is 20 kV, the column efficiency increases, but the separation degree decreases. Finally, 10 kV was determined as the optimal voltage value.

[0043] Example 4

[0044] According to the method of Example 1, the preferred value of the sample injection volume was confirmed. In this example, the influence of the sample injection volume on the separation of lactose anomers was investigated. As can be seen from Figure 5 the preferred test results, as the sample injection volume increases, it will cause chromatographic peak tailing and a decrease in column efficiency. The theoretical plate number of the electrophoretogram of lactose molecules shows a decreasing trend with the increase of the sample injection volume. As Figure 5 shown, as the sample injection volume increases, the resolution shows a downward trend. This is because a higher sample volume will cause peak tailing and a decrease in column efficiency. Therefore, the resolution of lactose anomers decreases with the increase of the sample volume. Finally, the preferred value of the sample injection volume was determined to be 5 kPa, 3 s.

[0045] Example 5

[0046] According to the method of Example 1, the preferred value of the borax electrophoresis buffer concentration was confirmed. As Figure 6 shown, the influence of the borax electrophoresis buffer concentration on the separation of lactose anomers was analyzed. As can be seen from Figure 6 the preferred value results, as the concentration of the borax electrophoresis buffer increases, the retention time of lactose molecules gradually increases. This is because the increase in the concentration of the borax electrophoresis buffer will cause a decrease in electroosmotic flow, and the retention time of lactose anomers shows an increasing trend. As Figure 6 shown, when the concentration of the borax electrophoresis buffer is 55 mmol / L -1 , the column efficiency is low and the resolution is low. Continuing to increase the ionic strength of the solution, the column efficiency gradually increases. When the buffer concentration is 75 mmol / L -1 , the resolution is the largest. This is because when the concentration of the borax electrophoresis buffer is too small or too large, the change in the ionic strength of the solution will affect the electroosmotic flow in the column. When the borax electrophoresis buffer with the optimal concentration is used, the separation ability of the capillary column is optimal.

[0047] It can be seen from the above examples that capillary electrophoresis technology provides a rapid and efficient analysis platform for the separation of lactose anomers in pharmaceuticals.

[0048] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for separating the anomers of pharmaceutical lactose by capillary electrophoresis, characterized in that The specific separation steps are as follows: (1) Prepare a borax electrophoresis buffer solution, and adjust the borax electrophoresis buffer solution to a pH value of 7-10 using boric acid or saturated sodium hydroxide solution; (2) Use the above-mentioned borax electrophoresis buffer solution with a pH value of 10 to dissolve the pharmaceutical lactose sample, and prepare a lactose solution with a concentration of 20 mg / mL at room temperature. The lactose molecules undergo mutarotation in the aqueous solution, and the two anomeric isomers, α-form and β-form, undergo reversible interconversion until a dynamic equilibrium is finally reached; (3) Use the above-obtained lactose solution containing both α and β configurations as the sample to be analyzed, construct a capillary electrophoresis ultraviolet direct detection method, and maintain and achieve baseline separation of the anomeric isomers of pharmaceutical lactose; In step (3), the capillary electrophoresis separation voltage for constructing the capillary electrophoresis ultraviolet direct detection method is 10 kV; In step (3), the concentration of the borax electrophoresis buffer solution for constructing the capillary electrophoresis ultraviolet direct detection method is 75 mM; In step (3), the injection volume condition for constructing the capillary electrophoresis ultraviolet direct detection method is 5 kPa, 3 s; In step (3), the pH value of the borax electrophoresis buffer solution for constructing the capillary electrophoresis ultraviolet direct detection method is 8.5.

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