Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis

More efficient and faster separation, conditions for qualitative as well as quantitative analysis of neuropeptides in human plasma using capillary zon e electrophoresis (CZE) have been developed. The analysis method for neurop eptides has been improved specifically to study thyroid hormone related neu ropetides for the regulation of thyroid disease. In this study, we investig ated the pretreatment methods, composition of the running buffer and rinsin g procedures between runs in order to obtain more sensitive and faster sepa ration of trace neuropeptides in plasma by CZE. The tested neuropeptides we re somatostatin (SOMA), vasopressin (VP), neurotensin (NT), and thyrotropin -releasing hormone (TRH). Plasma samples were pretreated by deproteinizatio n and solid-phase extraction method. The fraction of neuropeptides was reco nstituted in 40% acetonitrile followed by ultrafiltration, and then analyze d by CZE. Resolution and sensitivity was improved using the separation buff er composition with 100 mM Tris-phosphate buffer (pH 2.0) while the sensiti vity was further improved via a stacking method using the sample buffer of 40% acetonitrile. These sample pretreatment methods and buffer condition pe rmit quantitative analysis on tested neuropeptides at the 20 ng/mL level. T he rinsing procedures between runs using 90% ethanol dramatically shortened the rinsing time to 30 min.