柱前衍生-HPLC法测定大鼠血浆中游离棉酚旋光异构体的含量

Determination of gossypol enantiomers in rat plasma by precolumn derivatization-HPLC

目的建立测定大鼠血浆中棉酚异构体的柱前衍生-高效液相色谱法(HPLC)。方法大鼠血浆加入乙腈,离心,除去蛋白,氮气吹干,加入(R)-(-)-2-胺基-1-丙醇,80℃水浴进行衍生化。采用HPLC进行检测,液相条件:色谱柱Agilent ZORBAX Eclipse XDB-C_(18)(250mm×4.6mm,5μm),流动相甲醇-1%磷酸(85∶15),检测波长247nm,柱温30℃,流速1.0 mL/min,进样量20μL。结果左旋棉酚和右旋棉酚在0.1~2.0μg/mL浓度范围内线性良好,精密度、稳定性、重复性的RSD均<5%。高、中、低回收率分别为(92.8±4.9)%、(94.7±5.3)%和(93.7±3.8)%,大鼠血浆中左旋棉酚的含量为0.23~1.21μg/mL,右旋棉酚的含量为0.67~2.66μg/mL。结论该方法专属性强,灵敏度高,准确度好,可用于生物样品中棉酚异构体含量测定。

Objective To establish pre-column derivatization-HPLC method for determination of gossypol enantionmers in rats plasma.Methods Acetonitrile was added to rat plasma,then centrifugated,removed protein,dried by nitrogen.（R）-（-）-2-amino-1-propanol was added in 80℃ water bath for derivatization.The gossypol enantionmers were determined by HPLC.The column is Agilent ZORBAX Eclipse XDB-C（18）,（250mm×4.6mm,5μm）.The mobile phase is methanol-1%phosphoric acid（85∶15）,and the detection wavelength of 247nm;the column temperature is 30℃;the flow rate is 1.0mL/min,sample volume 20μL.Results The calibration curve of（＋）-gossypol and（-）-gossypol were linear in the range of 0.1μg/mL to 2.0μg/mL.Precision,stability,repeatability and recovery experiments meet the requirements for the determination and RSD≤5%.The average recoveries of（＋）-gossypol and（-）-gossypol（high,medium and low concentration）were（92.8±4.9）%,（94.7±5.3）%and（93.7±3.8）%,respectively.The content of（-）-gossypol in rat plasma is 0.23-1.21g/mL,the content of（＋）-gossypol in rat plasma is 0.67-2.66g/mL.Conclusion This method has strong specificity,high sensitivity and good accuracy,and it can be used for the determination of gossypol enantiomers in biological samples.