Objective To establish a method for the determination of related substances in thymalfasin for injection by reversed phase high performance liquid chromatography(RP-HPLC). Methods The chromatographic analysis was adopted after the sample was dissolved by phosphate buffer solution(PBS),and the Agilent ZORBAX SB-C18 chromatographic column(250.0 mm× 4.6 mm,5μm) was applied;the gradient elution was adopted with the mobile phase A[acetonitrile-water-orthophosphoric acid (140∶860∶1)] and mobile phase B[acetonitrile-water-orthophosphoric acid(250∶750∶1)] with a flow rate of 1.0 mL/min;the column temperature was 40 ℃;the detected wavelength was 210 nm,and the injection volume was 20 μL. Results This RP-HPLC method was strong specificity,with good reproducibility and stability. The related substances showed a good linear relationship between peak area and concentration within 0.709 8~23.615 0μg/mL(r=0.999 3). Conclusion The RP-HPLC method is simple, accurate and reliable,which can be applied in determination the related substances of thymalfasin for injection.%目的：建立以反相高效液相色谱法（RP-HPLC）测定注射用胸腺法新有关物质的方法研究。方法将样品用磷酸盐缓冲液（PBS）溶解后进行色谱分析，采用Agilent ZORBAX SB-C18（250.0 mm×4.6 mm，5μm）色谱柱，以流动相A[乙腈-水-磷酸（140∶860∶1）]和流动相B[乙腈-水-磷酸（250∶750∶1）]进行梯度洗脱，流速为1.0 mL/min，柱温40℃，检测波长210 nm，进样量20μL。结果 RP-HPLC法专属性强，重现性好，稳定性佳；有关物质在0.7098～23.6150μg/mL范围内与峰面积有良好的线性关系（r=0.9993）。结论 RP-HPLC法简单、准确、可靠，可用于测定注射用胸腺法新中有关物质的含量。
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