柱前衍生高效液相色谱法测定人血浆中尿囊素浓度

Pre-column derivatized HPLC determination of allantoin in plasma

目的:建立血浆中尿囊素的浓度测定方法。方法:采用柱前衍生反相高效液相色谱法,尿囊素与2,4-二硝基苯肼(DNPH)反应生成2,4-二硝基苯腙,色谱柱为Phenomenex Luna C18色谱柱(150mm×4.6mm,5μm),柱温为室温。流动相A为0.2%冰醋酸(用三乙胺调pH至5.0),流动相B为乙腈。梯度洗脱程序:A与B的体积比变化为0～3min 85:15～65:35(曲线系数为7),3-5min 65:35～15:85(曲线系数为7),5～9min 15:85～15:85(曲线系数为6),9～10min 15:85～85:15(曲线系数为6),10～15min 85:15(曲线系数为6)。PAD检测波长:340nm,流速1.0mL·min-1。内标为加替沙星。结果:在此色谱条件下,尿囊素衍生物与血浆中其他成分的分离完全,线性检测范围为2.5-40mg·L-1,最低检测浓度为2.5mg·L-1,相对回收率在99.35%～103.14%之间,日内及日间精密度RSD小于2.1%。结论:柱前衍生反相高效液相色谱法稳定、灵敏、可靠,可用于人体血浆中尿囊素浓度的测定。

Objective:To establish a method for determination of allantoin in plasma. Methods: Allantoin sample was derivatized by reaction with 2,4-dinitrophenyhydrazine and then separated in a Phenomenex(?) Luna C18 column of room temperature by programmed gradient elution procedure using mobile phase A (0.2% acetic acid pH 5.0) and mobile phase B (acetonitrile) with a flow rate of 1.0 mL·min-1 and detected with a UV detector at 340nm. Gatifloxacin was adopted as the internal standard . Results: Under this chromatographic conditions,allantoin could be completely separated from oth-er components in the sample with a linear range of 2.5-40mg·L-1,a detection limit of 2.5mg·L-1, a relative recovery range of 99.35 %-103.14 % , and an intra- and inter-day RSD of < 2.1% . Conclu-sion:A simple,accurate,and reproducible method is established for determination of allantoin in plasma.