Method for detecting glucose residues in amphotericin b bulk drug

Abstract

The application discloses a method for detecting glucose residues in amphotericin B bulk drug, and a specific implementation manner is as follows: liquid chromatography-mass spectrometry technology is adopted, an amide group bonded phase chromatographic column is used for hydrophilic interaction chromatography separation; acetonitrile and 0.1% ammonia solution are used as mobile phases, and alkaline conditions are used for promoting glucose ionization; in a negative ion mode, a multiple reaction monitoring mode is adopted to detect specific ion pairs of glucose. The method can realize effective separation of glucose and amphotericin B, the detection limit of glucose can reach 5 ng / mL, the method has strong specificity and high sensitivity, and can be used for accurately monitoring the glucose residue in amphotericin B bulk drug, so as to guarantee the purity and quality safety of the drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting glucose residues in amphotericin B raw material. Background Technology

[0002] Amphotericin B is an antibiotic composed of 37 carbon atoms and is a macrocyclic lactose-heptaene. It is the only effective drug for treating systemic deep fungal infections. The amphotericin B raw material is produced by microbial fermentation. Under certain conditions (including culture medium, temperature, pH, aeration, and stirring), the amphotericin B-producing bacteria are allowed to grow and multiply, and produce amphotericin B during metabolism. The amphotericin B is then extracted from the fermentation broth by solvent crystallization, purified and dried to obtain the amphotericin B raw material.

[0003] Currently, pharmacopoeias of various countries, such as the 2025 edition of the Chinese Pharmacopoeia, the current edition of the United States Pharmacopeia (USP), the European Pharmacopoeia EP2024, and the European Pharmacopoeia EP11.0, all include quality standards for amphotericin B and control related substances generated during the production process. These controlled related substances are mainly impurities with similar chemical structures to amphotericin B, such as impurity A (amphotericin A), impurity B (1R-oxy-methyl-amphotericin B), impurity C (1-oxy-ethyl-amphotericin B), and impurity D (1S-oxy-methyl-amphotericin B). However, relevant literature, patents, and pharmacopoeia standards do not control for glucose residues that may exist during the fermentation production of this drug, and no methods for detecting glucose residues in amphotericin B raw materials have been reported.

[0004] When preparing drugs using biocatalysis, glycogen substrates, such as glucose and sucrose, are frequently used, followed by microbial fermentation or direct enzymatic catalysis to obtain the target product. Besides serving as substrates, sucrose and glucose are often used in fermentation-based drug preparation to provide carbon and glycogen for microbial growth. In the preparation of amphotericin B, glucose is added to meet basal metabolic needs, participating in glycolysis and the tricarboxylic acid cycle to generate energy, supporting cell growth and primary metabolism. If subsequent purification processes are not thorough, the final product is highly likely to contain such sugars and their derivatives. Research on pharmaceutical manufacturing processes and quality control methods for sugar-based drugs has revealed that common residual sugars, such as fructose, can be dehydrated under acidic and high-temperature conditions to yield 5-hydroxymethylfurfural (5-HMF), which has been shown to cause intestinal oxidative stress, disrupt glucose and lipid metabolism, and induce intestinal damage.

[0005] Currently, regulatory agencies in some countries have recognized the importance of fermentation residues in fermentation-produced drugs. For example, the relevant general rules of the European Pharmacopoeia (EP12.0) clearly stipulate that, in addition to routine impurity studies, drugs produced by biocatalysis also require research and control of residues such as sugar and carbon sources (e.g., glucose), proteins, and nucleic acids generated during the fermentation catalysis process. Therefore, it is necessary to propose relevant technical solutions to fill this gap, further improve the quality standards of amphotericin B drugs, and ensure their quality.

[0006] Because amphotericin B has extremely poor solubility in common solvents, such as water, anhydrous ethanol, or ether, and the residual glucose content in the sample requires high control (limit of 0.1%), it is particularly important to improve the sensitivity of the detection method to ensure that trace amounts of glucose in the sample can be effectively detected. Summary of the Invention

[0007] To address the above problems, the present invention aims to provide a method for detecting glucose residues in amphotericin B raw material.

[0008] This invention optimizes the chromatographic separation mode by using a HILIC separation column instead of conventional C18, C8, or amino columns. Furthermore, it optimizes the mobile phase composition and ratio (acetonitrile is preferred over methanol due to its lower background noise in negative ion mode and better compatibility with HILIC mode; 0.1% ammonia solution is used, as its alkaline mobile phase conditions favor the formation of negative glucose ions) and detector parameters (such as acquisition mode and ion source parameters). The final proposed technical solution achieves a glucose detection limit of 5 ng / mL.

[0009] The specific technical solution is as follows:

[0010] A method for detecting glucose residues in amphotericin B raw material, the method employing liquid chromatography-mass spectrometry (LC-MS) and comprising the following steps:

[0011] (1) Preparation of test solution: Amphotericin B raw material is dissolved in solvent and then processed to obtain test solution;

[0012] (2) Preparation of reference solution: Dissolve glucose reference standard in solvent to obtain reference solution;

[0013] (3) Liquid chromatography separation: An amide-bonded phase column was used for chromatographic separation with a mobile phase consisting of acetonitrile and 0.1% ammonia solution;

[0014] (4) Mass spectrometry detection: Multiple reaction monitoring mode was used to detect glucose ion pairs in negative ion mode, where the quantitative ion pair was m / z 179.1→89.1 and the qualitative ion pair was m / z 179.1→119.0.

[0015] Furthermore, the amide-bonded phase column in step (3) is a Waters Xbridge BEH Amide column.

[0016] Furthermore, in step (3), the volume ratio of acetonitrile to 0.1% ammonia solution in the mobile phase is 68-70:30-32.

[0017] Furthermore, in step (3), the column temperature of the liquid chromatography is 30-40℃, the flow rate is 0.6-1.0mL / min, and the injection volume is 15-25μL.

[0018] Furthermore, in step (4), the mass spectrometry detection uses an electrospray ionization source with an atomizing gas pressure of 33-37 psi, a drying gas temperature of 280-320℃, a drying gas flow rate of 4.5-5.5 L / min, and a capillary voltage of 3300-3700 V.

[0019] Further, the preparation of the test solution in step (1) includes: weighing amphotericin B raw material, adding methanol, sonicating in a 50°C water bath, cooling and then adjusting the volume and centrifuging, and taking the supernatant as the test solution.

[0020] Further, the preparation of the reference solution in step (2) includes: weighing glucose reference standard, dissolving it in water, diluting it to the mark with methanol to obtain glucose stock solution, and then further diluting the stock solution with methanol to obtain reference solution.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention establishes a complete method for detecting glucose residues, a specific impurity, in amphotericin B raw material. This method exhibits good separation between glucose and amphotericin B, strong specificity and sensitivity, and can effectively monitor the glucose content in the crude raw material. This ensures the removal of glucose impurities during the purification and refining process, and guarantees that the residual glucose content in the purified product is below the limit (0.05%), thereby improving the quality control of amphotericin B. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0024] Example 1:

[0025] A method for detecting glucose residues in amphotericin B raw material includes the following steps:

[0026] Sample solution: Accurately weigh about 10 mg of amphotericin B raw material, place it in a 100 ml volumetric flask, add an appropriate amount of methanol, sonicate in a 50 °C water bath for 30 min to dissolve, cool to room temperature, dilute to volume with methanol, centrifuge, and take the supernatant as the test solution.

[0027] Reference solution: Accurately weigh approximately 10 mg of glucose reference standard and place it in a 100 ml volumetric flask. Add approximately 1 ml of water to dissolve the glucose, then dilute to the mark with methanol and mix well. This is the glucose reference standard stock solution (100.0 μg / ml). Accurately measure 1.0 ml of the above reference standard stock solution into a 1000 ml volumetric flask, dilute to the mark with methanol, and mix well. This is the reference solution (glucose approximately 100 ng / ml).

[0028] High-performance liquid chromatography (HPLC): Inject the test solution and the control solution separately under the following detection conditions:

[0029] Chromatographic column: Waters Xbridge BEH Amide column (4.6 mm × 250 mm, 5 μm);

[0030] Mobile phase: Acetonitrile-0.1% ammonia aqueous solution (volume ratio 70:30);

[0031] Column temperature: 35℃;

[0032] Flow rate: 0.8 mL / min;

[0033] Injection volume: 20 μL.

[0034] Mass spectrometer detector: Acquisition mode: MRM, polarity: negative ion mode, ion source: electrospray ionization source (ESI), mass spectrometry acquisition time: 8.2 min; nebulizer gas pressure: 35 psi, drying gas temperature: 300 ℃, drying gas flow rate: 5.0 L / min, capillary voltage: 3500 V.

[0035] The test results showed that glucose, a specific impurity, could be effectively separated from amphotericin B under the chromatographic conditions. This method can effectively detect glucose residue in the sample as low as 0.007%.

[0036] Example 2: A method for detecting glucose residues in amphotericin B raw material

[0037] The sample solution and the control solution were the same as in Example 1.

[0038] High-performance liquid chromatography (HPLC): Inject the test solution and the control solution separately under the following detection conditions:

[0039] Chromatographic column: Waters Xbridge BEH Amide column (4.6 mm × 250 mm, 5 μm);

[0040] Mobile phase: Acetonitrile-0.1% ammonia aqueous solution (volume ratio 68:32);

[0041] Column temperature: 40℃;

[0042] Flow rate: 0.8 mL / min;

[0043] Injection volume: 20 μL.

[0044] Mass spectrometer detector: Acquisition mode: MRM, polarity: negative ion mode, ion source: electrospray ionization source (ESI), mass spectrometry acquisition time: 7.5 min; nebulizer gas pressure: 35 psi, drying gas temperature: 300 ℃, drying gas flow rate: 5.0 L / min, capillary voltage: 3500 V.

[0045] The test results showed that glucose, a specific impurity, could be effectively separated from amphotericin B under the chromatographic conditions. This method can effectively detect glucose residue in the sample as low as 0.008% (n=6, RSD=3.1%).

[0046] Example 3: Limit of Detection and Limit of Quantification

[0047] Limit of Quantitation Solution: Transfer 5.0 ml of the above reference solution to a 50 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain the sensitivity solution (glucose concentration approximately 10 ng / ml).

[0048] Detection limit solution: Transfer 5.0 ml of the above reference solution to a 100 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain the sensitivity solution (glucose concentration approximately 5 ng / ml).

[0049] The sample was injected and analyzed under the preferred chromatographic conditions given in Example 1, and the chromatogram was recorded. The limit of quantitation (LOQ) of glucose was 10 ng / ml, equivalent to 0.01% of the amphotericin B sample; the limit of detection (LOD) was 5 ng / ml, equivalent to 0.005% of the amphotericin B sample. The results indicate that the detection sensitivity of the method in this application meets the requirements, enabling accurate quantification of glucose, a specific fermentation residue, in amphotericin B raw material, thus satisfying the relevant requirements for drug quality control.

[0050] Comparative Example

[0051] A method for detecting glucose residues in amphotericin B raw material includes the following steps:

[0052] Sample solution: Accurately weigh about 10 mg of amphotericin B raw material, place it in a 100 ml volumetric flask, add an appropriate amount of methanol, sonicate in a 50 °C water bath for 30 min to dissolve, cool to room temperature, dilute to volume with methanol, centrifuge, and take the supernatant as the test solution.

[0053] Reference solution: Accurately weigh approximately 10 mg of glucose reference standard and place it in a 100 ml volumetric flask. Add approximately 1 ml of water to dissolve the glucose, then dilute to the mark with methanol and mix well. This is the glucose reference standard stock solution (100.0 μg / ml). Accurately measure 1.0 ml of the above reference standard stock solution into a 1000 ml volumetric flask, dilute to the mark with methanol, and mix well. This is the reference solution (glucose approximately 100 ng / ml).

[0054] High-performance liquid chromatography (HPLC): Inject the test solution and the control solution separately under the following detection conditions:

[0055] Chromatographic column: Ultimate XB amino column (4.6 mm × 250 mm, 5 μm);

[0056] Mobile phase: methanol-water (volume ratio 70:30);

[0057] Column temperature: 35℃;

[0058] Flow rate: 0.8 mL / min;

[0059] Injection volume: 20 μL.

[0060] Mass spectrometer detector: Acquisition mode: MRM, polarity: negative ion mode, ion source: electrospray ionization source (ESI), mass spectrometry acquisition time: 8.0 min; nebulizer gas pressure: 35 psi, drying gas temperature: 300 ℃, drying gas flow rate: 5.0 L / min, capillary voltage: 3500 V.

[0061] The test results showed that glucose, a specific impurity, could be effectively separated from amphotericin B under the chromatographic conditions. The detection limit of this method was 100 ng / mL for glucose concentration. The minimum amount of glucose residue that this method could detect in the sample was 0.1%, and glucose was not detected in the sample.

Claims

1. A method for detecting glucose residues in amphotericin B raw material, characterized in that, The detection method employs liquid chromatography-mass spectrometry (LC-MS) and includes the following steps: (1) Preparation of test solution: Amphotericin B raw material is dissolved in solvent and then processed to obtain test solution; (2) Preparation of reference solution: Dissolve glucose reference standard in solvent to obtain reference solution; (3) Liquid chromatography separation: An amide-bonded phase column was used for chromatographic separation with a mobile phase consisting of acetonitrile and 0.1% ammonia solution; (4) Mass spectrometry detection: Multiple reaction monitoring mode was used to detect glucose ion pairs in negative ion mode, where the quantitative ion pair was m / z 179.1→89.1 and the qualitative ion pair was m / z 179.1→119.

0.

2. The method for detecting glucose residues in amphotericin B raw material as described in claim 1, characterized in that, The amide-bonded phase column in step (3) is a Waters Xbridge BEH Amide column.

3. The method for detecting glucose residues in amphotericin B raw material as described in claim 1, characterized in that, In step (3), the volume ratio of acetonitrile to 0.1% ammonia solution in the mobile phase is 68-70:30-32.

4. The method for detecting glucose residues in amphotericin B raw material as described in claim 1, characterized in that, In step (3), the column temperature of the liquid chromatography is 30-40℃, the flow rate is 0.6-1.0mL / min, and the injection volume is 15-25μL.

5. The method for detecting glucose residues in amphotericin B raw material as described in claim 1, characterized in that, In step (4), the mass spectrometry detection uses an electrospray ionization source with an atomizing gas pressure of 33-37 psi, a drying gas temperature of 280-320℃, a drying gas flow rate of 4.5-5.5 L / min, and a capillary voltage of 3300-3700 V.

6. The method for detecting glucose residues in amphotericin B raw material as described in claim 1, characterized in that, The preparation of the test solution in step (1) includes: weighing amphotericin B raw material, adding methanol, sonicating in a 50°C water bath, cooling and then adjusting the volume and centrifuging, and taking the supernatant as the test solution.

7. The method for detecting glucose residues in amphotericin B raw material as described in claim 1, characterized in that, The preparation of the reference solution in step (2) includes: weighing glucose reference standard, dissolving it in water, diluting it to the mark with methanol to obtain glucose stock solution, and then further diluting the stock solution with methanol to obtain reference solution.