Isolation of brain monoamines by high-performance reversed phase liquid chromatography

The development of high-performance liquid chromatography (HPLC), together with the introduction of electrochemical detectors (ED), has made the procedure of determination of monoamines and their precursors and metabolites in brain samples weighing only a few milligrams universally available. Early studies [8, 9] were based on cation-exchange chromatography with glass or metal columns 300-1000 mm long. The effectiveness of these columns for catecholamines was shown to be relatively low. Columns packed w[th a reversed phase, in combination with a mobile phase containing an ion-pair agent, were found to be much more suitable [2, 10]. To analyze monoamines the writers have used a m[crocolumn version of HPLC which has several advantages, especially when tissue samples of small size are used [1]. The internal diameter of the stainless steel columns was 2 mm and their length 60 or 120 ram. The eluant was supplied in some experiments by a "Du Pont 8800" pump (USA), in others by means of a pump of syringe type with electromeehanica l drive from an MSFP-3 microspectrophot ometer (made by Novos[birsk Institute of Organic Chemistry, Siberian Branch, Academy of Sciences of the USSR). The sample was introduced either through a "Rheodyne 7124" injector (USA) or through the sample introducing unit of the "Milikhrom" chromatograph. Concentrations of monoamines in the peaks were measured by means of an ED with glass-carbon working surface of the cell. The cell was designed and made on the basis of drafts and from materials described in the literature [4, 5, 10]. The electronic circuit of the instrument is similar to that given by Keller et aI.[,~], with minor modifications. The glass-carbon plate was cut out of chemical crucible glass of Soviet manufacture. In this paper we examine the questions of packing the columns with sorbent, choice of stationary and mobile phases, preparation of specimens and standards, and reduction of the noise level during chromatography of monoamines in brain samples weighing a few milligrams. Stationary Phase