Method for measuring calcium levofolinate-related substances by using high performance liquid chromatography gradient method

The invention discloses a high performance liquid chromatography of calcium levofolinate. The high performance liquid chromatography comprises the following specific steps of: with octadecylsilane chemically bonded silica (250*4.0mm, 5mum) as a filling agent, with a phosphate buffer as a moving phase A and phosphate buffer-methanol (40: 60) as a moving phase B, carrying out gradient elution; taking calcium levofolinate or calcium levofolinate preparation, adding water to prepare a solution containing 1mg of calcium levofolinate in each 1 ml as a sample solution, and diluting the sample solution with water to prepare a solution containing 0.01mg of calcium levofolinate in each 1ml as a contrast solution; and then respectively injecting the samples, wherein the sum of peak areas of various impurities in a chromatogram of the sample solution is not more than two times of the main peak area of the contrast solution. The high performance liquid chromatography disclosed by the invention overcomes the defects of low number of theoretical plates of main peak, poor separation degree between the main peak and adjacent miscellaneous peaks, low sensitivity and non-durable chromatographic columns of the traditional method, can be used for rapidly detecting impurities and degradation products of calcium levofolinate rapidly and accurately and is simple to operate, good in reproducibility, high in sensitivity and capable of better controlling the quality of the calcium levofolinate product.