Dissolved residue detection method of intermediate for synthesizing Arbekacin

Abstract

According to the present invention, the efficient, sensitive and accurate solvent residue determination method is established, the key intermediate product DKB05 in the dibekacin DKB synthesis process is subjected to the solvent residue detection, and the main solvents for the detection are glacial acetic acid and N, N-dimethylformamide; through the establishment of the detection method, the DKB05 solvent residue can be accurately detected, the solvent residue is controlled in the step, a high-quality intermediate is provided for the subsequent synthesis step, the synthesis reaction of the subsequent step is ensured to be smooth, and the quality of the final product Arbekacin sulfate bulk drug is ensured to be controllable.

Description

Technical Field

[0001] The invention relates to arbekacin sulfate, and in particular to a method for detecting solvent residues of key intermediates in the synthesis process of arbekacin sulfate. Background Art

[0002] Residual solvents refer to organic solvents used in the production of raw materials or excipients and in the preparation of preparations, but which are not completely removed during the process. For other solvents, corresponding limits should be established according to the characteristics of the production process to ensure that they comply with product specifications, Good Manufacturing Practice (GMP) or other basic quality requirements.

[0003] The method used to determine residual solvents in drugs in the early stage was the drying loss method. That is, the residual solvent content was determined by the loss of mass of the sample during heating. The biggest disadvantage of this method is that it is non-specific. It can only analyze the total amount but not the qualitative identification, and moisture will also interfere with the determination of residual solvents.

[0004] Spectrophotometry also usually uses the reaction between specific solvents and specific chemical reagents to determine residual solvents in drugs. Although the specificity is acceptable, the sensitivity is low.

[0005] At present, the residual solvent method has been replaced by gas chromatography. Gas chromatography not only has good separation ability and high sensitivity, but also is particularly suitable for the analysis of complex samples such as residual solvents in pharmaceuticals. It is recommended to use a capillary column-headspace injection system, but of course, ordinary packed columns and direct solution injection methods can also be used.

[0006] Common gas chromatography methods include: Direct injection method: Use a packed column or a capillary column with appropriate polarity.

[0007] Isothermal method with capillary column headspace injection: This method is suitable for situations where the number of organic solvents to be examined is small and the polarity difference is small.

[0008] Capillary column headspace injection programmed temperature method: This method is suitable for situations where the number of organic solvents to be examined is large and the polarity differences are large.

[0009] It can basically be divided into three categories: direct injection gas chromatography, headspace gas chromatography and solid phase microextraction gas chromatography.

[0010] Among them, static headspace gas chromatography is the most commonly used method for determining residual solvents.

[0011] Arbekacin sulfate (ABK) is an aminoglycoside antibiotic discovered by Umezepav et al. of the Japan Institute of Microbial Chemistry and developed by Meiji Seika Pharmaceutical Co., Ltd. It is a new generation of aminoglycoside antibiotics and is announced by the World Health Organization as the most promising antibiotic in the 21st century. It has been designated as a wartime antibiotic by Japan, and its status and value in the field of antibiotics are unprecedentedly important.

[0012] The antibacterial mechanism of arbekacin sulfate is to inhibit the synthesis of bacterial proteins, and it belongs to the aminoglycoside antibiotics. Arbekacin is similar to dibekacin, is stable to various inactivating enzymes, and can form a three-dimensional barrier to inactivating enzymes in structure, and has a high antagonism to aminoglycoside inactivating enzymes. The structure of aminoglycoside antibiotics contains multiple hydroxyl groups and amino groups, which are the targets of many inactivating enzymes. Therefore, deleting the hydroxyl groups that are not related to the antibacterial activity or protecting individual amino groups can avoid the attack of these inactivating enzymes, thereby enhancing the antibacterial activity against drug-resistant bacteria. Based on this, Umezawa et al. in Japan carried out structural modification of kanamycin B, deleted the hydroxyl groups at the 3' and 4' positions in kanamycin, and obtained dibekacin (DKB). Using dibekacin as a raw material, all amino groups and hydroxyl groups were protected by trimethylsilyl, and then the 1-amino group was acylated with the synthesized active ester, and the protecting groups were removed with hydrochloric acid and hydrazine hydrate in turn, and finally catalytic hydrogenation was performed to obtain arbekacin.

[0013] The detection method of the present invention is to detect the residual solvent of the key intermediate product DKB05 in the synthesis process of dibekacin (DKB), and the main solvents detected are glacial acetic acid and N, N-dimethylformamide. Since it contains multiple solvents and the polarity difference is not large, the "capillary column headspace injection program temperature rise method" is adopted for detection. Through the establishment of the detection method, the residual solvent of DKB05 can be accurately detected, the residual solvent is controlled in this step, and a high-quality intermediate is provided for the subsequent synthesis step, so as to ensure that the synthesis reaction of the subsequent step is smooth and the quality of the final product arbekacin sulfate raw material is controlled. Summary of the invention

[0014] Purpose of the Invention The present invention aims to create an efficient, sensitive and accurate method for determining residual solvents to detect the residual glacial acetic acid and N,N-dimethylformamide in the key intermediate dibekacin 05 (DKB05) in the synthesis process of arbekacin sulfate, control the residual solvent in this step, provide high-quality intermediates for subsequent synthesis steps, and obtain high-quality raw material arbekacin sulfate.

[0015] Technical content In order to accurately detect the residual solvent amount of arbekacin sulfate intermediate dibekacin 05 (DKB05) after using glacial acetic acid and N,N-dimethylformamide, based on the reference method of solvent residual determination, precise gas chromatography was used. According to the properties of the intermediate, the type of residual solvent, combined with the conditions of detection requirements, referring to the solvent residual requirements and instructions of the "Chinese Pharmacopoeia" and "Japanese Pharmacopoeia", combined with the production requirements of arbekacin sulfate, an efficient, sensitive and accurate residual solvent detection method was created using a hydrogen flame ionization detector (FID) and a capillary column equipped with polyethylene glycol filler, with comprehensive settings of temperature, equilibrium time, flow rate, sample type, concentration and other parameters.

[0016] Technical Solution This method uses gas chromatography to determine the residual glacial acetic acid and N,N-dimethylformamide in dibekacin 05 (DKB05), a key intermediate of arbekacin sulfate, using a gas chromatograph with a hydrogen flame ionization detector (FID) and headspace injection.

[0017] Chromatographic column: Agilent INNOWAX (30m* 0.53mm* 1μm) or equivalent column; The concentration of the sample solution is 50 mg / mL.

[0018] The carrier gas ratio is air: hydrogen: tail gas at a ratio of 300:50:60; Flow rate 1.5ml / min The headspace conditions were set at 120 °C, equilibration time of 15 min, loop temperature of 135 °C, injection time of 0.5 min, transfer line temperature of 145 °C, and cycle time of 35 min.

[0019] The temperature was raised from 50 to 200°C for 5 minutes, and then raised to the required temperature of the detector at a rate of 10°C / min for another 6 minutes.

[0020] The injection port temperature was 190 °C and the detector temperature was 260 °C.

[0021] Beneficial Effects This method can efficiently, sensitively and accurately determine the residual glacial acetic acid and N,N-dimethylformamide in debekacin 05 (DKB05) at 1 part per million (ppm) or even lower concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Residual spectra of glacial acetic acid and N,N-dimethylformamide determined by gas phase FID detector DETAILED DESCRIPTION Example

[0024] 1. Instruments and reagents

[0025]

[0026] 2 Chromatographic conditions

[0027] 3. Solution Preparation 3.1 Test solution (DKB05 concentration is 40 mg / ml) Weigh 0.2 g of DKB05 sample accurately and place it in a headspace bottle, add 3.00 ml of N-methylpyrrolidone to dissolve it, then add 2 ml of water, seal it with a cap, and mark it as SP#1 and SP#2. Prepare two copies in parallel.

[0028] 3.2 Reference solution (concentration 0.005 mg / ml) Accurately weigh 670 mg of glacial acetic acid and N,N-dimethylformamide residue respectively and place them in a 50 mL volumetric flask containing a certain amount of N-methylpyrrolidone, and dilute to the mark with N-methylpyrrolidone. Marked as STD-STOCK#1 Accurately pipette 5.00 ml of the above STD-STOCK#1 solution into a 50 mL volumetric flask, dilute to the mark with N-methylpyrrolidone, mix well, accurately pipette 3.00 ml of the above solution into a headspace bottle, accurately add 2.00 ml of water, seal with a cap, and repeat 5 times, labeling STD#1 to STD#5.

[0029] 3.3 Blank solution N-Methylpyrrolidone 3.00ml + water 2.00ml 4. Sampling and calculation 4.1 Injection sequence Please confirm that the system suitability has been passed before sample analysis. The injection sequence is as follows:

[0030] 4.2 System suitability 4.2.1 Blank solution The blank solution has no interference at the retention time of the glacial acetic acid and N,N-dimethylformamide peaks in the reference and sample solutions. If there is any interference, it should be deducted in the calculation.

[0031] 4.2.2 Control solution The RSD% of the peak areas of glacial acetic acid and N,N-dimethylformamide in the initial 5 reference solutions should be ≤10.0%.

[0032] 4.3 Calculation formula

[0033] 5 Methodological Validation 5.1 System suitability and injection precision investigation 5.1.1 Blank solution The blank solution has no interference at the retention time of glacial acetic acid and N,N-dimethylformamide in the reference solution and the sample solution. 5.1.2 Control solution The RSD of glacial acetic acid in the initial 6-pair reference solution was 1.8%, and the RSD of the N,N-dimethylformamide peak area was 3.3%.

[0034] The resolution between the two solvents in the reference solution was greater than 1.5.

[0035] 5.2 Exclusivity Examination 5.2.1 The blank solution should not interfere with the retention times of glacial acetic acid and N,N-dimethylformamide in the reference solution and sample solution.

[0036] 5.2.2 Resolution between solvents in the control solution 19.2 5.2.3 The retention time ratio of glacial acetic acid in the single labeled solution and the standard solution is 1.00, and the retention time ratio of N,N-dimethylformamide in the single labeled solution and the standard solution is 1.00.

[0037] 5.3 Detection limit investigation 5.3.1 The signal-to-noise ratio of 3 consecutive injections of glacial acetic acid is greater than 3 5.3.2 N,N-dimethylformamide 3 consecutive injections with a signal-to-noise ratio greater than 3 5.4 Limit of Quantitation 5.4.1 The signal-to-noise ratio of 6 consecutive injections of glacial acetic acid was greater than 10, and the peak area RSD was 10.5% 5.4.2 N,N-dimethylformamide The signal-to-noise ratio of 6 consecutive injections was greater than 10, and the peak area RSD was 4.1% 5.5 Linearity and range investigation 5.5.1 Linear equation for glacial acetic acid (y=236.71x+5.5375, r=0.9977) 5.5.2 N,N-dimethylformamide linear equation (y=371.33x+4.6703, r=0.9980) 5.6 Accuracy test 5.6.1 Glacial acetic acid

[0038] 5.6.2 N,N-Dimethylformamide

[0039] 5.7 Repeatability Study 5.7.1 RSD of 6 injections of glacial acetic acid is 3.0% 5.7.2 N,N-dimethylformamide 6 injections RSD is 5.0% 5.8 Durability test 5.8.1 Change the injection port temperature to 185℃ and 195℃, and the initial column temperature to 40℃ and 60℃. The peak area of ​​the reference sample is less than 10% for 6 consecutive times.

[0040] 5.8.2 The RAD of the samples is less than 20% compared with the results of the original method.

[0041] 6 Conclusion The present invention is directed to the detection of solvent residues in the key intermediate product DKB05 in the synthesis process of dibekacin (DKB), which is detected by using a "capillary column headspace injection program temperature rise method". From the technical content and methodology research of the method, it can be seen that the method uses simple instruments and reagents, which are easy to obtain, and the conditions are easy to achieve, and efficient, sensitive and accurate detection can be completed. Through the establishment of the detection method, the residual amount in the step is accurately grasped and controlled, a high-quality intermediate is provided for the subsequent synthesis step, the synthesis reaction of the subsequent step is guaranteed to be smooth, and the quality of the final product arbekacin sulfate raw material is guaranteed to be controllable.

Claims

1. The method is characterized in that a capillary chromatographic column with polyethylene glycol (PEG) as the stationary phase is used in the detection process of gas chromatograph hydrogen flame ionization detector (FID) and headspace sampling method.

2. The method is characterized in that the air flow composition ratio is as follows: air: hydrogen: tail blow ratio is 300:50:60; flow rate is 1.5 ml / min.

3. This method is characterized in that the headspace conditions are set to 120-125°C, the equilibration time is 13-18 minutes, the quantitative loop temperature is 130-135°C, the injection time is 0.5 minutes, the transfer line temperature is 140-145°C, and the cycle time is 35-40 minutes.

4. This method is characterized in that the temperature is set to rise for 5 to 8 minutes, 50→200°C, and then for another 6 to 9 minutes at a heating rate of 8 to 10°C / min to reach the temperature required by the detector.

5. This method is characterized in that the injection port temperature is set at 185-195°C and the detector temperature is set at 260-265°C.

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