Cell is the basic unit of structure and function of all living bodies. The study of intracellular drug concentration distribution is helpful to understand the drug efficacy of target site. Pemetrexed is a new multitarget folate antagonist with pyrrolidine group as its core structure. An ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for rapid quantification of pemetrexed concentration in human breast cancer cells (MCF-7). Sample pretreatment was completed by protein precipitation using methanol. The optimized chromatographic separation was achieved on a ZORBAX SB-C₁₈ column (2.1 × 150 mm, 3.5 μm). The column was equilibrated with initial mobile phase and eluted under gradient phases containing 0.1% formic acid in water (phase A) and in acetonitrile (phase B). The gradient program started at 5% B, increased to 95% B in 2 min, and then held at 95% B for 1.5 min. The linear range of pemetrexed in cells and nucleus was 2.0–200.0 ng/mL, while in the medium sample it was 50.0–5000.0 ng/ml, and the correlation coefficients were all greater than 0.99. The recovery was 47.50–67.55% (2.0–200.0 ng/mL) and 82.72–97.15% (50.0–5000.0 ng/mL), and the matrix effect was 98.10–100.62% (2.0–200.0 ng/mL) and 89.78–97.65% (50.0–5000.0 ng/mL). Interday precision and intraday precision (RSD%) were less than 15.0% (for LLOQ, less than 20%), and accuracy (RE%) was within ±15%; the deviation of stability was within ±15%, all meeting the requirements of biological sample analysis. The results of intracellular samples showed that the concentration of pemetrexed reached its peak at 3 h after administration. The concentration of pemetrexed in the nucleus continued to increase 2 h after administration and may have reached the maximum concentration at 6 h.

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基于UHPLC-MS / MS的人乳腺癌细胞（McF-7）中培美曲塞浓度和分布的快速测定

细胞是所有生物的结构和功能的基本单位。细胞内药物浓度分布的研究有助于了解靶点的药物疗效。培美曲塞是一种以吡咯烷基为核心结构的新型多靶点叶酸拮抗剂。开发了一种超高效液相色谱串联质谱法（UHPLC-MS / MS），用于快速定量人类乳腺癌细胞（MCF-7）中的培美曲塞浓度。样品的预处理通过使用甲醇进行蛋白沉淀来完成。优化的色谱分离在ZORBAX SB-C达到₁₈柱（2.1×150毫米，3.5  μm）。用初始流动相平衡色谱柱，并在含有0.1％甲酸的水（A相）和乙腈（B相）梯度相中洗脱。梯度程序从5％B开始，在2分钟内增加到95％B，然后在95％B下保持1.5分钟。培美曲塞在细胞和细胞核中的线性范围为2.0–200.0 ng / mL，而在中等样品中为50.0–5000.0 ng / mL，相关系数均大于0.99。回收率为47.50–67.55％（2.0–200.0 ng / mL）和82.72–97.15％（50.0–5000.0 ng / mL），基质效应为98.10–100.62％（2.0–200.0 ng / mL）和89.78–97.65 ％（50.0–5000.0 ng / mL）。日间精度和日内精度（RSD％）小于15.0％（对于LLOQ，小于20％），精度（RE％）在±15％以内；稳定性偏差在±15％以内，全部满足生物样品分析的要求。细胞内样品的结果表明，培美曲塞的浓度在给药后3小时达到峰值。给药后2小时，培美曲塞的核浓度继续增加，并可能在6小时达到最大浓度。