目的：采用星点设计-效应面法优选哈蟆油中腺嘌呤的提取工艺。方法采用HPLC测定腺嘌呤含量,以Inertsil ODS-SP Extend C18(250 mm ×4．6 mm,5．0μm)为色谱柱,流动相甲醇-水,梯度洗脱,流速1．0 mL/min,检测波长260 nm,柱温25℃,进样量10μL。以腺嘌呤为考察指标,通过星点设计-效应面法考察甲醇浓度、提取时间、提取温度对哈蟆油腺嘌呤提取工艺的影响。结果腺嘌呤对照品进样量在17．73~110．33μg/mL呈良好线形关系,回归方程为Y=0．1045 X+0．1001,R2=0．9996,平均回收率为97．84%,RSD为1．76%(n=6)。最佳提取工艺为甲醇浓度73%,提取时间68 min,提取温度61℃,哈蟆油得率为465．30μg/g。结论优选的哈蟆油腺嘌呤提取工艺简便、稳定。%Objective To optimize the process of extracting adenine from oviductus ranae by central compos-ite design/response surface methodology. Methods The analysis was achieved with an Inertsil ODS-SP Extend C18 column (250 mm × 4. 6 mm,5. 0 μm) by gradient elution of methanol and water at 25 ℃. The flow rate was 1. 0 mL/min,injection volume was 10 μL,and detection wave length was set at 260 nm. The effects of influence factors such as methanol concentration,extraction time and extraction temperature on extraction rate of adenine was investigated using box-behnken design and response surface method. Results The results indicate that the calibration curves of adenine have good linearity over the range from 17. 73 to 110. 33 μg/mL with a regression equation of Y=0. 104 5 X+0. 100 1,R2 =0. 999 6. The mean recovery was 97. 84% (RSD=1. 76%,n=6). Optimum processing conditions were: the concentration of methanol 73%,the extraction time was 68 min,the extraction temperature was 61 ℃ as the rate of ade-nine was 465. 30 μg/g. Conclusion Optimized processing technology is simple and stable.
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