柱前衍生化-高效液相色谱法测定白念珠菌中多胺的含量

Determination of polyamines in Candida albicans by Pre-column derivation HPLC

目的建立同时测定白念珠菌中4种多胺（腐胺、尸胺、亚精胺和精胺）的HPLC方法。方法样品经苯甲酰氯衍生化，资生堂C18色谱柱（100mm×3．0mm，3．0μm）分离，以甲醇（A）-0．1％甲酸水（B）为流动相进行梯度洗脱，0～5min，30％-48％B；5—12min，48％B；12～15min，48％～70％B；15～25min，70％b，流速0．6ml／min，检测波长254nm。结果腐胺、尸胺、亚精胺和精胺浓度分别在0．775—77．50μg／ml（r=0．9999）、1．160～116．0μg／ml（r=0、9999）、5．800～580．0μg／ml（r=0．9998）、2．533～192．7μg/ml（r=0．9999）的范围内线性关系良好。4种多胺的加样回收率为94．27％～109．3％，精密度RSD〈4％。结论该含量测定方法灵敏、重现性好，能同时测定白念珠菌中4种多胺的含量。

Objective To develop a HPLC method for simultaneous determination of four polyamines （putrescine, cadaverine, spermidine, spermine） in Candida albicans. Methods HPLC analysis was performed on a Shiseido C18 column（ 100 mm × 3.0 mm, 3.0 μm） after benzoyl chloride derivatization. The mobile phase consist of methanol （A） and water containing 0.1% methane acid （B）. The gradient elution program was as follow. 0 -5 min,30% -48% 70% B, the flow rate was 0.6 ml/min, the detection wavelength B; 5 -12 min,48% B;12 -15 min,48% -70% B;15 -25 min, was 254 nm. Results The linearity was obtained over 0. 775 77.50 μg/ml （ r = 0. 999 9 ） for putrescine, 1. 160 - 116.0 μg/ml （ r = 0. 999 9） for cadaverine ,5. 800 - 580.0 μg/ml （ r = 0. 999 8 ） for spermidine and 2. 533 - 192.7 μg/ml （ r = 0. 999 9） for spermine. The average recoveries were between 94.27% - 109.3% . The RSDs of precision were less than 4%. Conclusion The present study provided a sensitive and reproducible method for the simultaneously determination of four polyamines levels in Candida albicans.