Ueyama Jun, Kitaichi Kiyoyuki, Iwase Mitsunori, Takagi Kenji, Takagi Kenzo, Hasegawa Takaaki
Department of Medical Technology, Nagoya University School of Health Sciences, 1-1-20 Daikominami, Higashi-ku, Nagoya 461-8673, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Dec 5;798(1):35-41. doi: 10.1016/j.jchromb.2003.08.045.
In order to develop a reliable, simple and routine method using small sample volume to determine norepinephrine (NE) and epinephrine (E) concentrations in plasma of humans and rodents, we utilize the ultrafiltration (UF) method by Ultrafree-MC filter device and a high-performance liquid chromatography equipped with electrochemical detector (HPLC-ECD) to detect NE and E. Optimum UF and HPLC conditions were as follows: the filter nominal molecular weight limit size is 30,000, the pH of added phosphate buffer to each plasma sample for UF is 3.0, and the mobile phase is 0.1M phosphate buffer (pH 3)/acetonitrile (98:2) containing 0.05% sodium disulfite and 0.001% EDTA 2Na. The plasma samples and 1.0M phosphate buffer (pH 3) containing 3,4-dihydroxybenzylamine (DHBA), as an internal standard, was mixed and poured into the UF units. After the centrifugation for 60 min at 13,000 x g at 4 degrees C, the filtrate was directly injected into HPLC. The calibration curve of NE and E was linear for the concentrations studied (20-400 pg) with a correlation coefficient of >0.999. Intra-assay coefficients of variation for NE and E using this method were less than 3%. The method also correlated well with the well-established alumina method (r=0.954). The present findings suggest that a newly-developed UF method with HPLC-ECD would apply successfully to measure plasma NE and E concentrations in humans and rodents.
为了开发一种可靠、简单且常规的方法，使用小样本量来测定人和啮齿动物血浆中去甲肾上腺素（NE）和肾上腺素（E）的浓度，我们利用Ultrafree-MC过滤装置的超滤（UF）方法以及配备电化学检测器的高效液相色谱法（HPLC-ECD）来检测NE和E。最佳超滤和高效液相色谱条件如下：过滤器标称分子量截留尺寸为30,000，用于超滤的每个血浆样本中添加的磷酸盐缓冲液的pH值为3.0，流动相为含有0.05%亚硫酸钠和0.001%乙二胺四乙酸二钠的0.1M磷酸盐缓冲液（pH 3）/乙腈（98:2）。将血浆样本与含有内标3,4-二羟基苄胺（DHBA）的1.0M磷酸盐缓冲液（pH 3）混合，然后倒入超滤装置中。在4℃下以13,000×g离心60分钟后，将滤液直接注入高效液相色谱仪。在所研究的浓度范围（20 - 400 pg）内，NE和E的校准曲线呈线性，相关系数>0.999。使用该方法测定NE和E的批内变异系数小于3%。该方法与成熟的氧化铝方法也具有良好的相关性（r = 0.954）。目前的研究结果表明，新开发的结合HPLC-ECD的超滤方法将成功应用于测量人和啮齿动物血浆中NE和E的浓度。
