Nano-LC-MS/MS for the quantitation of prostanoids in immune cells
Nano-LC-MS/MS for the quantitation of prostanoids in immune cells
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DOI：
10.1007/s00216-014-8134-8
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发表时间：
2014-09
影响因子：
4.3
通讯作者：
D. Thomas;J. Suo;T. Ulshöfer;H. Jordan;N. Bruin;K. Scholich;G. Geisslinger;N. Ferreirós
中科院分区：
文献类型：
--
作者：
D. Thomas;J. Suo;T. Ulshöfer;H. Jordan;N. Bruin;K. Scholich;G. Geisslinger;N. Ferreirós
前列腺素是花生四烯酸的衍生物，参与炎症和免疫反应。为了了解不同免疫细胞产生的前列腺素的作用，开发了一种采用纳米液相色谱-串联质谱法对前列腺素 E2（PGE2）、前列腺素 D2（PGD2）、6-酮前列腺素 F1α（6-酮 PGF1α）、前列腺素 F2α（PGF2α）和血栓素 B2（TXB2）进行高灵敏定量的方法。它根据美国食品和药物管理局 (FDA) 的指南在线性、精密度、准确度、回收率、稳定性和定量下限 (LLOQ) 方面进行了验证。 PGE2 和 PGD2 注射溶液中的 LLOQ 为 25 pg/mL（柱上 75 fg（o.c.）），6-酮 PGF1α、PGF2α 和 TXB2 的 LLOQ 分别为 37.5 pg/mL（柱上 112.5 fg）。它已成功应用于注射酵母聚糖后从爪子中分离出的鼠类肥大细胞，以及在迟发型超敏反应模型中来自致敏与非致敏小鼠血液中的 CD4+ 和 CD8+T 淋巴细胞。大约 5,000 个（T 细胞）到 40,000 个（肥大细胞）细胞足以进行定量。在肥大细胞中，PGE2 的产生量明显高于其他前列腺素的合成量。无论是从致敏小鼠还是非致敏小鼠中获得，T 淋巴细胞在前列腺素生成方面均未表现出任何差异。
Prostanoids, derivatives of arachidonic acid, are involved in inflammation and immune reactions. To understand the role of prostanoids produced by diverse immune cells, a highly sensitive quantitation method for prostaglandin E2(PGE2), prostaglandin D2(PGD2), 6-keto prostaglandin F1α(6-keto PGF1α), prostaglandin F2α(PGF2α), and thromboxane B2(TXB2) by means of nano-liquid chromatography-tandem mass spectrometry has been developed. It was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of linearity, precision, accuracy, recovery, stability, and lower limit of quantitation (LLOQ). The LLOQ were 25 pg/mL in the injected solution (75 fg on column (o.c.)) for PGE2and PGD2and 37.5 pg/mL (112.5 fg on column) for 6-keto PGF1α, PGF2α, and TXB2, respectively. It was successfully applied to murine mast cells isolated from paws after zymosan injection and to CD4+and CD8+T lymphocytes from blood of sensitized versus non-sensitized mice in context of a delayed type hypersensitivity model. About 5,000 (T cells) to 40,000 (mast cells) cells were sufficient for quantitation. In the mast cells, the production of PGE2increased at a significantly higher extent than the synthesis of the other prostanoids. The T lymphocytes did not show any difference in prostanoid production, no matter whether they were obtained from sensitized mice or non-sensitized mice.