全自动二维高效液相色谱测定霉酚酸血药浓度的研究

Measurement of mycophenolic acid in blood by two-dimensional liquid chromatography

目的:采用全自动二维液相色谱(2D-LC-UV)建立测定霉酚酸(MPA)血药浓度的方法,并应用于临床。方法:样品去除蛋白后直接进样,待测物在一维柱AstonSC2T(3.5 mm×50 mm,5μm)上初步分离,通过中间柱Aston SBR(3.0 mm×10 mm,5μm)截取保留,转移到二维柱Aston SBX4(4.6 mm×150 mm,5μm)上进一步分离。一维流动相为MPA-1A移动相,流速0.8 mL·min-1;二维流动相为MPA-2A移动相,流速1.2 mL·min-1;柱温40℃;紫外检测波长304 nm。结果:在所建立的色谱条件下,霉酚酸与各杂质分离良好,在0.32~25.03μg·mL-1范围内与峰面积呈良好的线性关系(r=0.9999),可在13 min内完整出峰,方法回收率高于95%;日内、日间的精密度RSD值均小于5%;稳定性试验RSD小于5%。结论:本方法简便快速、结果准确、稳定性良好、自动化程度高、检测成本低,适用于临床霉酚酸快速检测。

OBJECTIVE To establish a method for rapid determination of mycophenolic acid(MPA)blood concentration by automated two-dimensional liquid chromatography(2 D-LC-UV)and apply it in clinical practice.METHODS The samples were directly injected after protein removal.The analytes were preliminarily separated on a one-dimensional column Aston SC2 T(3.5 mm×50 mm,5μm),intercepted and retained by an intermediate column Aston SBR(3.0 mm×10 mm,5μm),and transferred to a two-dimensional column Aston SBX4(4.6 mm×150 mm,5μm)for further separation.The one-dimensional mobile phase was MPA-1 A mobile phase at a flow rate of 0.8 mL·min-1;the two-dimensional mobile phase was MPA-2 A mobile phase at a flow rate of 1.2 mL·min-1;the column temperature was 40℃;and the UV detection wavelength was 304 nm.RESULTS Under the established chromatographic conditions,mycophenolic acid was well separated from each impurity,showed a good linear relationship with the peak area in the range of 0.32-25.03μg·mL-1(r=0.9999),could completely peak within 13 min,the method recovery was higher than 95%;the intra-day and inter-day precision RSD was less than 5%;the RSD of stability test was less than 5%.CONCLUSION The method is simple,rapid,accurate,stable,automatic and low cost,and suitable for rapid detection of mycophenolic acid in clinic.