Method for determining aureomycin premix content and related substances through high-performance liquid chromatography method

Abstract

The present invention relates to a method for determining the aureomycin premix content and related substances through a high-performance liquid chromatography method, wherein the method adopts the following chromatography conditions that the chromatographic column is (C18, 5 [mu]m) chromatographic column, a mixed solution of water, phosphoric acid and N,N-dimethylformamide according to a volume ratio of 63-77:1.8-2.2:25-30 is adopted as a mobile phase, the phosphoric acid concentration is 2 mol / L, the flow rate is 0.8-1.2 ml / min, the detection wavelength is 270-400 nm, the column temperature is 30-40 DEG C, the sample injection volume is 10-25 [mu]L, and the detector is an ultraviolet detector. According to the method, an aureomycin standard substance solution, a tetracycline standard substance solution, a chlorquatrimycin standard substance solution and an aureomycin premix sample solution are prepared, high-performance liquid chromatography analysis is performed according to the chromatography conditions, and the content of the related substances in the premix is calculated through the peak area. The detection method of the present invention has characteristics of relatively mild chromatography conditions (the mobile phase does not contain the easily-crystallizing salt and does not use the strong acid), easy equipment maintenance, and high test accuracy.

Description

technical field [0001] The invention belongs to the field of analysis and testing, and relates to a method for detecting the content of a premix and related substances, in particular to a method for measuring the content of aureomycin premix and related substances by high performance liquid chromatography. Background technique [0002] Aureomycin premix is ​​a chlortetracycline calcium salt made from the whole fermentation broth of the chlortetracycline-producing bacteria and an appropriate amount of calcium carbonate. The main drug ingredient is aureomycin, but it also contains some related fermentation by-products. Such as tetracycline, aureomycin and so on. The determination of the aureomycin content generally adopts the microbiological detection method (recorded in the national standard of veterinary medicine). Practice has shown that the use of microbiological methods to determine the content has several disadvantages: 1) the method is cumbersome and takes a long time,...

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Problems solved by technology

Practice has shown that the use of microbiological methods to determine the content has several disadvantages: 1) the method is cumbersome and takes a long time, resulting in low work efficiency; 2) the method has many influencing factors, which have a greater impact on the accuracy of the test. The performance and reproducibility are also relatively poor; 3) the test cost is high; 4) the related substances in it cannot be determined at the same time

[0003] "Chinese Pharmacopoeia" (2005 edition) has recorded aureomycin hydrochloride, and its content and related substance detection adopt HPLC method, can be used for the mensuration of aureomycin premix, but, its chromatographic condition is more complicated, harsh, mobile phase Use a mixed solution of ammonium oxalate, diammonium hydrogen phosphate and dimethylformamide (use ammonia test solution to adjust pH to 8.3)

Although this method is more convenient and faster than the microbial detection method, it has the following disadvantages: 1) mobile phase pH8.3 is an alkaline condition, and aureomycin is extremely unstable under alkaline conditions, and is easily oxidatively degraded; phase, salts are easy to crystallize and suck out, causing wear and tear on the accessories of the instrument system, shortening the life of the instrument, which is not good for the maintenance of the chromatographic system, and is not suitable for long-term use; The pH of the mobile phase of the method is 8.3, and it needs to be equipped with an alkali-resistant special chromatographic column, and the cost is higher than that of ordinary chromatographic columns.

[0004] "Chinese Pharmacopoeia" (2010 edition) records the aureomycin hydrochloride, content and related impurity detection still adopts HPLC method, also can be used for the detection of aureomycin premix, although this method overcomes the shortcoming of 2005 edition, adjustment The chromatographic conditions were changed, and the mobile phase was changed to acidic conditions; however, the mobile phase was still relatively harsh, and a mixed solution of perchloric acid, water and dimethyl sulfoxide was used

Perchloric acid is a strong oxidizing acid. Long-term use will cause corrosion of the instrument system, shorten the life of the chromatographic column and the instrument, and is still unfavorable to the maintenance of the instrument. It is not suitable for long-term use; another disadvantage is that the price of dimethyl sulfoxide The more expensive is about twice the price of dimethylformamide, and the cost is higher for long-term use

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