目的: 建立液相色谱-串联质谱法测定与利那洛肽共孵育后人结肠腺癌肺转移细胞（T84细胞）裂解液中环磷酸鸟苷（cGMP）的浓度。方法: 采用Kromasil 100-5-C18（150 mm×2.1 mm,5 μm）色谱柱,以0.1%甲酸水溶液和0.1%甲酸甲醇溶液为流动相,梯度洗脱,柱温50 ℃;采用电喷雾离子源（ESI-）和多反应监测（MRM）模式检测,cGMP和内标8-Br-cGMP检测离子通道分别为m/z 344.20 → 150.00和m/z 423.90 → 230.00。结果: cGMP线性范围为1~500 ng · mL-1（r ≥ 0.999）,方法精密度、准确度、基质效应、提取回收率均符合分析要求,使用该方法准确检测了2种利那洛肽胶囊制剂溶液给药后细胞中cGMP水平。利那洛肽与T84细胞孵育30 min后,cGMP水平发生显著的浓度依赖性变化,参比制剂及受试制剂的EC50值分别为167.6、112.1 nmol · L-1。结论: 本研究所建立的方法具有优异的选择性和准确性,能够有效定量人结肠腺癌肺转移细胞裂解液中的cGMP水平,为相关药理学研究提供了一种可靠的分析工具。
Objective: To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining the concentration of cyclic guanosine monophosphate (cGMP) in lysates of human colon adenocarcinoma lung metastasis cells (T84 cells) after co-incubation with linaclotide. Methods: A Kromasil 100-5-C18 column (150 mm×2.1 mm, 5 μm) was used with a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in methanol, employing a gradient elution at a column temperature of 50 ℃. The detection was performed using an electrospray ionization (ESI-) source and multiple reaction monitoring (MRM) mode, with the monitored ion transitions for cGMP and the internal standard 8-Br-cGMP being m/z 344.20 → 150.00 and m/z 423.90 → 230.00, respectively. Results: The linear ranges for cGMP were 1 to 500 ng · mL-1 (r≥ 0.999). The method demonstrated precision, accuracy, matrix effects, and extraction recovery rates that met analytical requirements. The method was successfully applied to accurately detect cGMP levels in cells after administration of two types of linaclotide capsule formulations. A significant concentration-dependent change in cGMP levels was observed after co-incubation of linaclotide with T84 cells for 30 min, with EC50 values of 167.6 and 112.1 nmol · L-1 for the reference and test formulations, respectively. Conclusion: The method established in this study demonstrates excellent selectivity and accuracy, effectively quantifying cGMP levels in lysates of human colon adenocarcinoma lung metastasis cells, providing a reliable analytical tool for related pharmacological research.
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