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[摘要]
目的 建立HPLC-MS/MS法测定NB4细胞培养液中全反式维甲酸的浓度。方法 采用ACQUITY UPLCTM BEH C18(50 mm×2.1 mm,1.7 μm)色谱柱,以布洛芬为内标,流动相为乙腈-水(含0.1%乙酸),体积流量为0.2 mL/min,梯度洗脱方式分离全反式维甲酸,同时采用ESI源负离子检测方式,定量分析时的离子反应分别为m/z 299.2→m/z 255.2(全反式维甲酸)和m/z 205.4→m/z 161.3(内标布洛芬)。结果 全反式维甲酸在2.55~255 ng/mL线性关系良好,定量限为2.55 ng/mL,日内精密度RSD ≤ 10.3%,日间精密度RSD ≤ 12.9%。结论 建立的HPLC-MS/MS法可用于测定NB4细胞含维甲酸的培养液中全反式维甲酸的浓度,以及在加入维甲酸代谢阻断剂时全反式维甲酸浓度的变化。
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[Abstract]
Objective To develop an HPLC-MS/MS method for determination of all-trans retinoic acid (ATRA) in NB4 cell medium. Methods Following liquid-liquid extraction with anhydrous ether, the analyte (ATRA) and internal standard (ibuprofen) were separated from medium using an gradient elution with acetonitrile and aqueous phase, containing 0.1% acetic acid, on an ACQUITY UPLCTM BEH C18 (50 mm×2.1 mm, 1.7 μm) column at the flow rate of 0.2 mL/min. The detection was carried out by means of electrospray ionization mass spectrometry in negative ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 299.2→m/z 255.2 and m/z 205.4→m/z 161.3 was used to quantify ATRA and internal standard, respectively. Results The calibration curves were linear in the range of 2.55-255 ng/mL. The limit of quantification was 2.55 ng/mL. The intra-and inter-day precisions (RSD) were less than 10.3% and 12.9%. Conclusion It can be used to determinate all-trans retinoic acid (ATRA)in NB4 cell medium, and the variety of ATRA with metabolic inhibitor of ATRA.
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