Pfizer, Inc., Groton, CT 06340, USA.
J Mass Spectrom. 2011 Jul;46(7):705-11. doi: 10.1002/jms.1941.
Quantitation of urinary tetranor PGDM or tetranor PGEM (tPGDM and tPGEM) in the past was performed separately using off-line SPE LC-MS/MS methods. The manual SPE procedure is generally time-consuming and cost-ineffective. In addition, simultaneous quantitation of tPGDM and tPGEM is favorable yet very challenging because of the similar chemical structures and identical MRM transitions. This work describes the development and validation of a high-throughput online SPE-LC-MS/MS method, allowing simultaneous and high-throughput measurement of tPGDM and tPGEM in human urine. The reportable range of the assay was 0.2-40 ng/ml for tPGDM and 0.5-100 ng/ml for tPGEM. Intra- and inter-assay precision and accuracy determined using quality control samples were all within acceptable ranges (% CV and % Bias < 15%). Tetranor PGDM was stable under all tested conditions while tPGEM was stable at 4 °C and after three F/T cycles but not stable at room temperature for 24 h (recovery below 80%). The assay was applied to measure urinary tPGDM and tPGEM among healthy volunteers, smokers and COPD patients. Significantly higher urinary levels of both tPGDM and tPGEM were observed in COPD patients than those of non-smoking healthy volunteers. These results demonstrated that the high-throughput online SPE-LC-MS/MS assay provides sensitive, reproducible and accurate measurement of urinary tPGDM and tPGEM as biomarkers for assessing inflammatory diseases such as COPD.
过去，尿中四氢去甲 PGDM 或四氢去甲 PGEM（tPGDM 和 tPGEM）的定量分析分别采用离线 SPE-LC-MS/MS 方法进行。手动 SPE 程序通常耗时且成本效益低。此外，由于化学结构相似且 MRM 跃迁相同，同时定量分析 tPGDM 和 tPGEM 是有利的，但极具挑战性。本工作描述了一种高通量在线 SPE-LC-MS/MS 方法的开发和验证，该方法允许同时高通量测量人尿中的 tPGDM 和 tPGEM。该测定法的报告范围为 tPGDM 的 0.2-40ng/ml 和 tPGEM 的 0.5-100ng/ml。使用质控样品测定的日内和日间精密度和准确度均在可接受范围内（%CV 和 %Bias<15%）。四氢去甲 PGDM 在所有测试条件下均稳定，而 tPGEM 在 4°C 和经过三个 F/T 循环后稳定，但在室温下 24 小时不稳定（回收率低于 80%）。该测定法用于测量健康志愿者、吸烟者和 COPD 患者的尿中 tPGDM 和 tPGEM。与非吸烟的健康志愿者相比，COPD 患者的尿中 tPGDM 和 tPGEM 水平均显著升高。这些结果表明，高通量在线 SPE-LC-MS/MS 测定法可作为评估 COPD 等炎症性疾病的生物标志物，灵敏、重现性和准确地测量尿中 tPGDM 和 tPGEM。
