Abstract: Shenmai injection is a derivative formula of the ancient prescription "Shengmaisan". It is an intravenous injection developed by two scientific materials, red ginseng and Ophiopogon japonicus, which is used in the treatment of shock, coronary heart disease Viral myocarditis, chronic pulmonary heart disease, neutropenia, etc.
Objective To establish a method for the determination of 5-hydroxymethylfurfural in Shenmai injection. Methods The HPLC method was used. The chromatographic column was AlltimaC18 (4.6mm × 150mm, 5μm), the mobile phase was methanol-water solution (8:92), the detection wavelength was 284nm, the flow rate was 1ml · min-1, and the column temperature was 30 ℃. Results 5-hydroxymethylfurfural showed a good linear relationship (R2 = 0.999992) within the injection range of 0.0306903 ～ 0.6138054μg. The average recovery rate of this method was 103.19% and RSD 0.7%. Conclusion The method is simple, rapid, accurate and reliable, and can be used for the determination of 5-hydroxymethylfurfural in Shenmai injection.
1 Instruments and reagents
Watersalliance 2695 HPLC instrument; Waters 2489 dual-wavelength ultraviolet detector; Sartorius MC21S electronic analytical balance. The 5-hydroxymethylfurfural reference substance (081216) was purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd. and its content was 99.45% by normalization method. Methanol is chromatographically pure (Merck) and water is distilled water. Shenmai injection (Zhengda Qingnianbao Pharmaceutical Co., Ltd., batch number 0612141, 0707241, 0809252, 0901201, 0903281).
2 Methods and results
2.1 HPLC conditions The chromatographic column was AlltimaC18 (4.6mm × 150mm, 5μm); the mobile phase was methanol-water solution (8:92); detection wavelength was 284nm; flow rate: 1ml · min-1; column temperature was 30 ℃. The number of theoretical plates is not less than 9000 in terms of 5-hydroxymethylfurfural peak.
2.2 Preparation of reference substance solution Weigh accurately 3.086mg of 5-hydroxymethylfurfural reference substance, place it in a 100ml volumetric flask, add distilled water to dissolve and bring to volume, and shake to obtain.
2.3 Preparation of test solution The sample of Shenmai injection should be shaken and sampled directly through the microporous filter membrane (0.45μm).
2.4 Specific test Take 5-hydroxymethylfurfural reference solution for blank and control determination. Under the same conditions, there is no interference peak in the absorption peak position of 5-hydroxymethylfurfural in HPLC of Shenmai injection.
2.5 Investigation of the linear relationship Precision pipette reference solution 1, 2, 4, 8, 12, 16 and 20 μl were injected into the liquid chromatograph for measurement, with the peak area of ​​5-hydroxymethylfurfural as the ordinate (Y) and the injection volume X (μg) is the abscissa, and the working curve is drawn. The regression equation is: Y = 7.19 × 106X-5.88 × 104, R2 = 0.999992, indicating that 5-hydroxymethylfurfural is within the range of 0.0306903 ～ 0.6138054μg injection volume and peak area A good linear relationship.
2.6 Precision experiment Accurately draw 12μl of the same reference solution and repeat the injection analysis 6 times under the above chromatographic conditions. The results show that the method has good precision and the relative standard deviation of 5-hydroxymethylfurfural content (μg · ml-1) ( RSD) is 0.1%.
2.7 Repeatability experiment Accurately absorb 20 μl of the same test solution and repeat the injection analysis 5 times under the above chromatographic conditions. The results show that the repeatability is good, and the relative standard deviation of 5-hydroxymethylfurfural content (μg · ml-1) ( RSD) is 0.2%.
2.8 Recovery rate experiment Accurately measure the same batch of 2ml of the test Shenmai injection solution with known content into 5 10ml volumetric flasks, and then add 5ml of the reference solution accurately, add distilled water to the mark, shake well, precise Draw 20μl and measure under the above chromatographic conditions, the average recovery is 103.19%, RSD = 0.7%.
2.9 Sample measurement results Directly take 5 batches of Shenmai injection, after passing through the microporous filter membrane (0.45μm), accurately take 20μl of each injection into the HPLC instrument, measure under the above chromatographic conditions, record the peak area of ​​the chromatogram, calculate content.
3 Discussion
In this paper, a method for determining the content of 5-hydroxymethylfurfural in Shenmai injection is established by HPLC. This method requires no pretreatment, no interference, is fast, simple, accurate and reliable, and provides a basis for the quality control of Shenmai injection.
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