Determining cyclooxygenase-2 activity in three different test systems utilizing online-solid phase extraction-liquid chromatography-mass spectrometry for parallel quantification of prostaglandin E(2), D(2) and thromboxane B(2).
Determining cyclooxygenase-2 activity in three different test systems utilizing online-solid phase extraction-liquid chromatography-mass spectrometry for parallel quantification of prostaglandin E(2), D(2) and thromboxane B(2).
复制标题
DOI：
10.1016/j.chroma.2015.02.059
复制
发表时间：
2015-04
期刊：
影响因子：
--
通讯作者：
Ina Willenberg;Anna K. Meschede;N. Schebb
中科院分区：
文献类型：
--
作者：
Ina Willenberg;Anna K. Meschede;N. Schebb
环氧合酶-2(COX-2)催化花生四烯酸合成PGH2。PGH2进一步转化为不同的前列腺素(PG)，如PGE2、PGD2和TxB2。本研究建立了一种用于COX-2酶分析的在线固相萃取-液质联用同时测定前列腺素E_2、前列腺素D_2和T_xB_2的方法。所有分析物的基线分离仅需7.1分钟，包括在线固相萃取样品制备。该方法具有较高的灵敏度(柱上检出限为0.65~1.25)和精密度(89~113%)。由于在线固相萃取，除了添加IS溶液外，不需要人工制备样品，从而允许将该方法用作COX-2活性测定的快速读出方法。这一点在三种体外试验系统中得到了证明：无细胞酶试验、用组成性表达COX-2的Hca-7细胞和原代人单核细胞进行的试验。在这些分析中，三种流行药物塞来昔布、吲哚美辛和地塞米松的效力被成功地用新的在线LC-MS方法进行了表征。结果表明，PG生成的抑制效果强烈依赖于测试系统。因此，我们建议，至少应该在两个检测系统中表征测试化合物对COX-2活性的调节。随着在线固相萃取-LC-MS的描述在这里，我们提出了一个通用的工具作为读出这些类型的分析。
Cyclooxygenase-2 (COX-2) catalyzes the formation of PGH2from arachidonic acid. PGH2is further converted to different prostaglandins (PG), such as PGE2, PGD2and TxB2. In this study a rapid online-SPE-LC-MS method for the simultaneous quantification of PGE2, PGD2and TxB2streamlined for COX-2 enzyme assays is presented. Baseline separation of all analytes was achieved in only 7.1 min per sample, including sample preparation by online SPE. The method showed high sensitivity (LODs of 0.65–1.25 fmol on column) and accuracy (89–113%) in protein containing media. Because of online-SPE, no manual sample preparation was required, except for addition of IS solution, allowing to use the approach as rapid read-out in COX-2 activity assays. This was demonstrated by applying the method on threein vitrotest systems: a cell-free enzyme assay, an assay using HCA-7 cells constitutively expressing COX-2 and primary human monocytes. In these assays, the potency of three popular drugs celecoxib, indomethacin and dexamethasone was successfully characterized with the new online-LC-MS method. The comparison of the results showed that the inhibitory effects of PG formation strongly depend on the test system. Thus we suggest that the modulation of COX-2 activity of a test compound should be at least characterized in two assay systems. With the online-SPE-LC-MS described in here we present a versatile tool as read-out for these types of assays.