Development of a Fully-automated On-line Oxidation Column-switching HPLC System for the Determination of Endogenous Melatonin in Human Clinical Samples

Abstract

A fully-automated on-line oxidation column-switching HPLC system has been developed for the determination of endogenous melatonin in human plasma and saliva. This HPLC system consists of four processes. In the first step, melatonin is fractionated using a reversed-phase C4 column (Proteonavi, 75 mm × 1.0 mm i.d.). In the second step, the obtained melatonin fraction is on-line collected, and oxidized to a highly-fluorescent compound, N- [(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), by mixing with hydrogen peroxide under alkaline conditions. In the third step, the produced 6-MOQMA is concentrated, and the oxidation reagents are removed using an alkaline resistive reversed-phase column, Asahipak ODP (35 mm × 1.0 mm i.d.). In the final fourth step, the 6-MOQMA is determined by a microbore-ODS column packed with ultrafine particles (CAPCELL PAK C18 IF, 250 mm × 1.0 mm i.d.). The limit of detection of melatonin using this system is about 200 amol/injection, and the determination of endogenous melatonin in a small volume of human physiological fluids, such as 100 μL of plasma and 300 μL of saliva, was successfully accomplished.

Rumen-Protected 5-Hydroxytryptophan Improves Sheep Melatonin Synthesis in the Pineal Gland and Intestinal Tract

“…While an injection of 5-HTP can directly make it into the bloodstream, oral administration of RP 5-HTP might have delayed effects, and the 5-HTP plasma might therefore be cleared in the liver and kidneys [20]; thus, the synthetic MLT precursor content in sheep might be reduced. Trp is not only an important indirect precursor of MLT, but also plays an important biological role in vertebrates [21–23]. The present study indicated that RP 5-HTP administration did not affect the plasma Trp content in sheep.…”

Comprehensive two-dimensional ion chromatography (2D-IC) coupled to a post-column photochemical fluorescence detection system for determination of neonicotinoids (imidacloprid and clothianidin) in food samples

“…19,38,39,[41][42][43][44] The matrix interferences and analyte loss problems can be resolved by making sample preparation method online with the 2D-IC system just like SPE methods aer making online proved more efficient, versatile, reproducible, selective, reduce labor, human error, costs, solvent and time. [45][46][47][48] Since last two decades, an online column-switching technique for heart-cutting is extensively utilized for complex samples clean-up and concentration to reduce matrix interferences. [49][50][51][52] However, most of the reported online extraction techniques use the mini hydrophobic column as pre-treatment column which unable to complete elimination of matrix interferences from analytes of interest and matrices co-elute and transfer along the analytes of interest in second dimension column.…”