Department of Chemistry, Soochow University, Taipei, Taiwan.
J Chromatogr A. 2011 Apr 15;1218(15):2085-90. doi: 10.1016/j.chroma.2010.10.091. Epub 2010 Oct 30.
Urinary 8-isoprostaglandin F(2α) (8-isoPGF(2α)) has been reported as an important biomarker to indicate the oxidative stress status in vivo. In order to quantitatively determine the low contents of 8-isoPGF(2α) (in sub- to low ng mL(-1) range) in physiological fluids, a sensitive detection method has become an important issue. In this study, we employed a microfluidic chip-based nano liquid chromatography (chip-nanoLC) with on-chip sample enrichment coupled to triple quadrupole mass spectrometer (QqQ-MS) for the quantitative determination of 8-isoPGF(2α) in human urine. This chip-nanoLC unit integrates a microfluidic switch, a chip column design having a pre-column (enrichment column) for sample enrichment prior to an analytical column for separation, as well as a nanospray emitter on a single polyimide chip. The introduction of enrichment column offers the advantages of online sample pre-concentration and reducing matrix influence on MS detection to improve sensitivity. In this study, the chip-nanoLC consisting of Zorbax 300A SB-C18 columns and Agilent QqQ Mass spectrometer were used for determining 8-isoPGF(2α) in human urine. Gradient elution was employed for effective LC separation and multiple reaction monitoring (MRM) was utilized for the quantitative determination of 8-isoPGF(2α) (m/z 353→193). We employed liquid-liquid extraction (LLE)/solid-phase extraction (SPE) for extracting analyte and reducing matrix effect from urine sample prior to chip-nanoLC/QqQ-MS analysis for determining urinary 8-isoPGF(2α). Good recoveries were found to be in the range of 83.0-85.3%. The linear range was 0.01-2 ng mL(-1) for urinary 8-isoPGF(2α). In addition, the proposed method showed good precision and accuracy for 8-isoPGF(2α) spiked synthetic urine samples. Intra-day and inter-day precisions were 1.8-5.0% and 4.3-5.8%, respectively. The method accuracy for intra-day and inter-day assays ranged from 99.3 to 99.9% and 99.4 to 99.7%, respectively. Due to its rapidity, enhanced sensitivity, and high recovery, this chip-nanoLC/QqQ-MS system was successfully utilized to determine the physiological biomarkers such as 8-isoPGF(2α) in human urine for clinical diagnosis.
尿 8-异前列腺素 F(2α)（8-isoPGF(2α)）已被报道为指示体内氧化应激状态的重要生物标志物。为了定量测定生理流体中低含量的 8-isoPGF(2α)（亚至低 ng mL(-1)范围内），一种灵敏的检测方法已成为一个重要问题。在本研究中，我们采用基于微流控芯片的纳流液相色谱（chip-nanoLC）与芯片上样品富集相结合，结合三重四极杆质谱仪（QqQ-MS），用于定量测定人尿中的 8-isoPGF(2α)。该 chip-nanoLC 单元集成了微流控开关、具有预柱（富集柱）的芯片柱设计，用于在分析柱分离之前对样品进行富集，以及在单个聚酰亚胺芯片上的纳米喷雾发射器。富集柱的引入具有在线样品预浓缩和减少基质对 MS 检测影响的优点，以提高灵敏度。在本研究中，使用 Zorbax 300A SB-C18 柱和安捷伦 QqQ 质谱仪的 chip-nanoLC 用于测定人尿中的 8-isoPGF(2α)。采用梯度洗脱进行有效的 LC 分离，并采用多反应监测（MRM）进行定量测定 8-isoPGF(2α)（m/z 353→193）。我们采用液-液萃取（LLE）/固相萃取（SPE）提取分析物，并在 chip-nanoLC/QqQ-MS 分析前从尿液样品中去除基质效应，用于测定尿液中的 8-isoPGF(2α)。发现回收率在 83.0-85.3%范围内。尿 8-isoPGF(2α)的线性范围为 0.01-2 ng mL(-1)。此外，该方法对合成尿液样品中添加的 8-isoPGF(2α)具有良好的精密度和准确度。日内和日间精密度分别为 1.8-5.0%和 4.3-5.8%。日内和日间测定的方法准确度范围分别为 99.3-99.9%和 99.4-99.7%。由于其快速、灵敏度高和回收率高，该 chip-nanoLC/QqQ-MS 系统成功用于测定人尿中的生理生物标志物，如 8-isoPGF(2α)，用于临床诊断。
