Pretreatment and one-shot separating analysis of whole catecholamine metabolites in plasma by using LC/MS

Catecholamines are biogenic amines that play an important role in the nervous system. Some catecholamines have been used as tumor makers of phenochromocytoma, paraganglioma and neuroblastoma. The analysis of total catecholamine metabolites should be useful for one-shot screening of multiple aspects of diseases; however, it is difficult to do this, because the catecholamine metabolites are divided into three groups: five amines, one amino acid and three carbonic acids. Catecholamines and small molecules were separated from plasma proteins by an internal-surface reversed-phase column (protein-coated octadeyclsilica column) and were analyzed by liquid chromatography (LC)/mass spectrometry (MS) using electrospray ionization time-of-flight MS. Using a reversed-phase column and hydrophilic mobile phases, we succeeded in the separation of nine catecholamines, all of which had similar structures. These nine substances were eluted in the following order: norepinephrine, epinephrine, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylalanine, vanillomandelic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. The reproducibility of this method was acceptable. The highest coefficient of variation was 7.4%. In addition, various types of compounds were separated from and detected in plasma proteins by applying LC/MS. The plasma direct injection method, which uses an internal-surface reversed-phase column and an ion-pair reagent, allowed us to separate small molecules from plasma proteins. MS detected some compounds that high-performance LC could not succeed in separating and detecting with UV detection. We think that the method can be applied to find new markers in neuroblastoma, by comparing the plasma of patients with that of normal infants. The method can be also used to help in making a diagnosis of other diseases and finding their new makers.