Davis T P, Gehrke C W, Gehrke C W, Cunningham T D, Kuo K C, Gerhardt K O, Johnson H D, Williams C H
J Chromatogr. 1979 Mar 1;162(3):293-310. doi: 10.1016/s0378-4347(00)81516-9.
A remarkably sensitive, simple and selective reversed-phase high-performance liquid chromatographic (HPLC) method has been developed, allowing, for the first time, the direct measurement of histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin and tyramine in a single sample of plasma (2 ml), tissue (0.2 g), or urine. The biogenic amines were modified by pre-column derivatization with o-phthalaldehyde which stabilizes the molecules, aids in extraction, and improves HPLC detection at the nanogram level. To minimize losses during the sampling procedure a careful collection procedure was designed. We developed a simple sample cleanup in which the samples were thawed, neutralized with KOH, immediately derivatized, extracted into ethyl acetate (EtOAc) and then chromatographed by HPLC. The derivatives were stable in EtOAc for more then 24 h. Interfering amino acids were removed from the EtOAc by partitioning twice with Na2HPO4 buffer (pH 10.0). Complete separation was achieved in ca. 60--90 min on a muBondapak phenyl column using a stepwise gradient of acetonitrile and/or methanol-phosphate buffer (pH 5.1). A variable wavelength fluorometer with a 5-microliter flow-cell was used (excitation 340 nm; emission 480 nm). Linearity ranged from 200 pg to 50 ng onto the column. Precision (R.S.D.) for retention times was 1% and for derivatization and injection 2.5%. Recoveries of the seven biogenic amines from plasma spiked with 25 ng/ml averaged 70%, with a relative standard deviation of 6%. Separation studies were also done using a muBondapak C18 column. The effects of various eluents are presented. Gas-liquid chromatography was also investigated but lacked the sensitivity achieved by HPLC. The HPLC method is used routinely for the determination of biogenic amines in plasma from pigs with malignant hyperthemia and thermally stressed bovine. Significant differences in levels of biogenic amines were noted between stressed and non-stressed animals. Data on rat brain tissue samples were compared with the trihydroxyindole method and canine heart tissue was analyzed for ventricular norepinephrine and dopamine. Application of the method to urine from normal persons and a patient with a brain tumor has been demonstrated.
已开发出一种灵敏度极高、简单且具选择性的反相高效液相色谱（HPLC）方法，首次实现了在2毫升血浆、0.2克组织或尿液的单个样本中直接测定组胺、去甲肾上腺素、章鱼胺、去甲变肾上腺素、多巴胺、5-羟色胺和酪胺。通过邻苯二甲醛进行柱前衍生化对生物胺进行修饰，这可使分子稳定、有助于萃取并改善纳克级别的HPLC检测。为尽量减少采样过程中的损失，设计了仔细的采集程序。我们开发了一种简单的样品净化方法，即先将样品解冻，用氢氧化钾中和，立即进行衍生化，萃取到乙酸乙酯（EtOAc）中，然后通过HPLC进行色谱分析。衍生物在EtOAc中可稳定超过24小时。通过用磷酸氢二钠缓冲液（pH 10.0）进行两次分配，可从EtOAc中去除干扰性氨基酸。在μBondapak苯基柱上使用乙腈和/或甲醇 - 磷酸盐缓冲液（pH 5.1）的梯度洗脱，约60 - 90分钟内可实现完全分离。使用配有5微升流通池的可变波长荧光计（激发波长340纳米；发射波长480纳米）。柱上线性范围为200皮克至50纳克。保留时间的精密度（相对标准偏差）为1%，衍生化和进样的精密度为2.5%。在添加了25纳克/毫升的血浆中，七种生物胺的平均回收率为70%，相对标准偏差为6%。还使用μBondapak C18柱进行了分离研究。介绍了各种洗脱剂的效果。也对气相色谱进行了研究，但缺乏HPLC所达到的灵敏度。该HPLC方法常规用于测定患有恶性高热的猪和热应激牛的血浆中的生物胺。在应激和非应激动物之间，生物胺水平存在显著差异。将大鼠脑组织样本的数据与三羟基吲哚法进行了比较，并对犬心脏组织中的心室去甲肾上腺素和多巴胺进行了分析。已证明该方法可应用于正常人尿液和一名脑肿瘤患者的尿液。
