Pittertschatscher Klaus, Hochreiter Romana, Thalhamer Josef, Hammerl Peter
Institute of Chemistry and Biochemistry, University of Salzburg, Austria.
Anal Biochem. 2002 Sep 15;308(2):300-6. doi: 10.1016/s0003-2697(02)00260-9.
A novel one-step ethylchloroformate (ECF) derivatization of histamine in biological liquid matrices that allows the sensitive quantification by gas chromatography and mass spectroscopic detection (GC-MS) from small volumes of blood plasma or cell culture supernatants within 15 min is described. After addition of ECF/chloroform directly to the crude sample, histamine has been found to be quantitatively derivatized within seconds. Following centrifugation, the organic phase is transferred to a fresh vial, dried by addition of anhydrous sodium sulfate, and subjected to GC-MS analysis. The reliability of the results is verified by use of two different ion pairs for detection. The method is validated according to DIN 38402. Linearity is given from 0.0054 to 13 microg/ml and the limit of detection is 2 ng/ml (10 pg absolute, at a signal to noise ratio of 3:1). The limit of quantification, as calculated at a confidence level of 95%, is 15.6 ng/ml. Practical application is exemplified by the determination of the histamine content in blood plasma of birch pollen-sensitized mice and in the culture supernatant of rat basophil leukemia cells after Ca(2+) ionophore-mediated degranulation.
本文描述了一种新型的一步法用氯甲酸乙酯（ECF）对生物液体基质中的组胺进行衍生化的方法，该方法能够在15分钟内从小体积血浆或细胞培养上清液中通过气相色谱和质谱检测（GC-MS）进行灵敏定量分析。将ECF/氯仿直接加入粗样品后，发现组胺在数秒内即可定量衍生化。离心后，将有机相转移至新的小瓶中，加入无水硫酸钠干燥，然后进行GC-MS分析。通过使用两种不同的离子对进行检测来验证结果的可靠性。该方法按照德国工业标准38402进行了验证。线性范围为0.0054至13微克/毫升，检测限为2纳克/毫升（绝对量为10皮克，信噪比为3:1）。在95%置信水平下计算得出的定量限为15.6纳克/毫升。通过测定桦树花粉致敏小鼠血浆中的组胺含量以及钙离子载体介导脱颗粒后大鼠嗜碱性粒细胞白血病细胞培养上清液中的组胺含量，举例说明了该方法的实际应用。
