广地龙饮片的HPLC特征图谱及5个核苷类成分的测定

目的：建立广地龙饮片及其2种常见伪品的HPLC特征图谱并测定其中尿嘧啶、次黄嘌呤、黄嘌呤、腺苷、肌苷的含量，为广地龙饮片的真伪鉴别和质量控制提供参考。方法：采用Thermo TSK-GEL C₁₈色谱柱（4.6 mm×250 mm，5 μm），以0.1%甲酸水溶液-甲醇（99：1）为流动相，等度洗脱40 min，流速为1.0 mL·min^-1，检测波长260 nm，柱温30℃，进样量10 μL。采用"中药色谱指纹图谱相似度评价系统"建立指纹图谱并进行相似度评价，并对尿嘧啶，次黄嘌呤，黄嘌呤，腺苷和肌苷的含量测定进行方法学考察。结果：建立了广地龙饮片及其伪品的特征图谱，正品（参环毛蚓）中标定了7个共有峰，伪品大腔蚓和暗孔远盲蚓中均标定了8个共有峰。对不同来源的广地龙饮片及其伪品中5个核苷成分进行含量测定。尿嘧啶、次黄嘌呤、黄嘌呤、腺苷、肌苷线性范围分别为3.40~170、4.40~220、5.00~250、3.40~170、4.20~210 μg·mL^-1，线性关系良好（r ≥ 0.999 9）；重复性和稳定性的RSD均<3.0%，平均加样回收率在95.5%~107.0%之间。18批广地龙饮片中均未检测到腺苷，尿嘧啶、次黄嘌呤、黄嘌呤、肌苷含量分别为0.021~0.408、0.309~6.133、0.825~4.714和1.413~23.382 mg·g^-1。结论：所建立特征图谱专属性强，结合5个主要成分含量测定，能够为广地龙饮片的真伪鉴别和质量控制提供科学依据。

Objective: To establish the HPLC characteristic chromatogram and to determine the contents of uracil, hypoxanthine, xanthine, adenosine and inosine in Guang Dilong decoction pieces simultaneously, and provide the reference for authentication and quality control of Guang Dilong decoction pieces.Methods: HPLC analysis was performed on Thermo TSK-GEL C₁₈ column (4.6 mm×250 mm, 5 μm).The mobile phase consisted of 0.1% formic acid solution-methanol (99:1) with isocratic elution for 40 minutes.The flow rate was 1.0 mL·min^-1, the detection wavelength was 260 nm, the column temperature was 30℃ and the injection volume was 10 μL.Similarity evaluation was performed by Similarity Evaluation System for Fingerprint of Traditional Chinese Medicine.Assay method for uracil, hypoxanthine, xanthine, adenosine and inosine was validated.Results: HPLC characteristic chromatogram of Guang Dilong decoction pieces and its adulterants from different sources were established and five characteristic peaks were marked.Seven characteristic peaks were simultaneously determined in Pheretima aspergillum, and 8 characteristic peaks were simultaneously determined in Metaphire magna and Amynthas obscuritoporus.The linear relationships for uracil, hypoxanthine, xanthine, adenosine and inosine were observed in the ranges of 3.40-170 μg·mL^-1, 4.40-220 μg·mL^-1, 5.00-250 μg·mL^-1, 3.40-170 μg·mL^-1 and 4.20-210 μg·mL^-1, respectively, with r ≥ 0.999 9.The RSDs of repeatability and stability tests were<3.0% and the average recoveries varied from 95.5%-107.0%.Adenosine in 18 batches of Guang Dilong decoction pieces was not detected.The contents of uracil, hypoxanthine, xanthine, adenosine and inosine were 0.021-0.408 mg·g^-1, 0.309-6.133 mg·g^-1, 0.825-4.714 mg·g^-1 and 1.413-23.382 mg·g^-1, respectively.Conclusion: The characteristic chromatogram and the assay method of five nucleoside components in Guang Dilong decoction pieces and its adulterants can provide scientific basis for the authentication and quality control of Guang Dilong decoction pieces and its adulterants.

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