Objective To establised a method for the determination of 4‐hydroxyisoleucine in the seeds of Trigonella foenum‐grae‐cum L .from different habitats .Method Precolumn derivatization HPLC was adopted using phenylisothiocyanate as the derivative reagent .Zorbax Eclipse XDB‐C18 column was used .The mobile phase was A [acetonitrile]∶B [1 mL · L -1 phosphoric acid in wa‐ter] (0‐20 min ,20∶80 ;20 min ,60∶40) ,with a flow rate of 1 .5 mL · min-1 at 30 ℃ .Result A good linearity was obtained in the range of 4 .2‐20 .9μg · mL-1 (r=0 .999 3) for 4‐hydroxyisoleucine .The average recovery of 4‐hydroxyisoleucine was 96 .8% ,RSD=1 .7% .Conclu‐sion Using phenylisothiocyanate as the derivative reagent ,the precolumn derivatization HPLC method is simple ,reproducible and accurate .%目的：柱前衍生化法测定4‐羟基异亮氨酸含量。方法采用异硫氰酸苯酯作为衍生化试剂，色谱柱（Zorbax Eclipse XDB‐C18，150 mm ×4．6 mm ，5μm ，Agile）；流动相A ：乙腈，流动相B：1 mL · L －1磷酸水，梯度洗脱（A相∶B相）：（0～20 min ，20∶80；20 min，60∶40）；流速：1．5mL·min－1；进样体积：20μL；检测波长：254nm；柱温：30℃。结果4‐羟基异亮氨酸在质量浓度4．2～20．9μg · mL －1范围内线性关系良好（r＝0．9993），平均回收率为96．8％，RSD为1．7％。结论该方法简便、稳定，结果准确，可作为4‐羟基异亮氨酸的含量测定方法。
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