高效液相色谱法测定生物样品中3PO的方法建立与验证

目的: 建立并验证用于大鼠血清、脑脊液、大脑和脊髓中3-(3-吡啶)-1-(4-吡啶)-2-丙烯-1-酮(3PO)含量测定的HPLC-UV法。方法: 样品处理使用蛋白沉淀的方法；色谱条件：色谱柱为Chromolith RP-18e(100 mm×4.6 mm)，流动相A为乙腈-水(2.5∶97.5，v/v)添加0.1%TFA，流动相B为乙腈添加0.1%TFA，梯度洗脱[0~2.3 min，0%B→100%B(线性梯度)；2.3~2.6 min，100%B(等度)；2.6~2.7 min，100%B→0%B(线性梯度)；2.7~5 min，0%B(等度)]，流速2 mL·min^-1，检测波长304 nm，进样量10 μL。结果: 大鼠的血清、脑脊液、大脑和脊髓中内源性物质不干扰3PO的测定，在上述4种基质中，该方法的检测限分别为0.2、0.1、0.1、0.1 μg·mL^-1；定量限分别为0.8、0.2、0.5、0.5 μg·mL^-1；线性范围分别为3~150、0.3~150、0.9~150、0.6~150 μg·mL^-1，相关系数均大于0.999；高、低浓度的回收率均在79.9%~100.3%的范围内；方法重复性(n=5)的RSD均大于0.9%。结论: 建立的方法简单，线性范围宽，且选择性、耐用性均佳。

Objective: To develop and validate a high performance liquid chromatographic method with UV detection for the determination of 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one(3PO)in rat's serum,cerebrospinal fluid(CSF),brain and spinal cord.Methods: Protein precipitation was used as sample preparation method.The separation was performed on a Chromolith performance RP-18e column(100 mm×4.6 mm)by gradient elution where mobile phase A consisted of 0.1% trifluoroacetic acid in acetonitrile-water(2.5∶97.5,v/v)and B consisted of 0.1% trifluoroacetic acid in acetonitrile.The gradient program was set as follows:0-2.3 min,0%→100% of B(linear gradient);2.3-2.6 min,100% of B(isocratic);2.6-2.7 min,100%→0% of B(linear gradient);2.7-5 min,0% of B(isocratic).Flow rate was set at 2 mL·min^-1.UV detection was performed at 304 nm.The injection volume was 10 μL.Results: There was no endogenous interference at the retention time of 3PO in blank brain,spinal cord and CSF.LOD and LOQ of the method in serum,CSF,brain and spinal cord were 0.2,0.1,0.1,0.1 μg·mL^-1 and 0.8,0.2,0.5,0.5 μg·mL^-1,respectively.The method was linear in the range from 0.3-150 μg·mL^-1 in CSF,0.9-150 μg·mL^-1 in brain,0.6-150 μg·mL^-1 in spinal cord and 3-150 μg·mL^-1 in serum with coefficient of determination values greater than 0.999.The recovery of high concentration and low concentration were between 79.9%-100.3%.Conclusion: The method is proved to be simple,robust,selective and linear.

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