Determination of Harmane and Harmine in Human Blood Using Reversed-Phased High-Performance Liquid Chromatography and Fluorescence Detection

Abstract

A number of tremorogenic β-carboline alkaloids have been found in common plant-derived foodstuffs, beverages, and inhaled substances. Because of their natural presence in the food chain, there is a growing concern regarding the potential risks of certain essen tial tremors associated with the long-term low-level dietary exposure to these alkaloids. The purpose of this study was to develop an effective analytical method to determine blood levels of two major β-car boline derivatives, harmane and harmine. Human blood was extracted with ethyl acetate and methyl-tbutyl ether (2:98) under an alkaline condition. After evaporation of organic solvent, the samples were re-constructed in methanol. The samples were fraction ated on a 250 × 4.6-mm C18 reversed-phase column with an isocratic mobile system consisting of 17.5 mM potassium phosphate buffer (ph 6.5) and methanol (30: 70), followed by an on-line fluorescence detection. The method had the detection limit to determine 206 and 81 pg/ml of harmane and harmine, respectively, in 10 ml of human blood. The intraday precision (C.V.) at 25 ng/ml was less than 6.7 and 3.4% for harmane and harmine, respectively. The interday precision was 7.3% for harmane and 5.4% for harmine. The method has proven sensitive, reproducible, and thus useful for both laboratory and clinical studies of β-carboline toxicities.A number of tremorogenic β-carboline alkaloids such as harmane, harmine, harmaline, and ibogaine have been found in common plant-derived foodstuffs (wheat, rice, corn, barley, soybeans, rye, grapes, mushrooms, vinegar). plant -derived beverages (wine. beer. whisky. brandy, sake). and plant-derived inhaled substances (tobacco) (1, 6). These substances are also endogenous to animal tissue (6, 9) and have been isolated in beef and sardines (1). Laboratory animals exposed to these chemicals result in an acute action tremor (5). Because of their natural presence in the food chain, it is conceivable that the route of exposure in humans would be from dietary sources, smoking. and consumption of alcoholic beverages. In fact, the occurrence of β-carbo-lines in human blood under normal physiological Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Rommelspacher et al. employed thin-layer gel-plate separation and mul tiple extraction followed by high-performance liquid chromatography (HPLC) to detect 6-0H-tetrahydronorharmane (6-0H-THN) in rat and human sam ples (9). The substantial precolumn preparation in that study limited its application to clinical monitoring of this and other β-carbolines. Moncrieff also reported an HPLC method for tissue harmane, harmine, and harmaline (4). However, the quantitation of three compounds required three different mobile phases and three separate HPLC runs. A better HPLC method was developed by Adachi et al.(1) for determination norharmane and harmane in foodstuffs. Nevertheless, the adaptability of this method to biological remains uncertain. Moreover, to the best of our knowledge, there has been no report ...

Elevated blood harmane (1-methyl-9H-pyrido[3,4-b]indole) concentrations in essential tremor

“…The intraday precision, measured as a coefficient of variation at 25 ng/mL, was 6.7% for harmane. The interday precision was 7.3% for harmane (Zheng et al, 2000).…”

“…Blood concentrations of harmane were measured blinded to any clinical information, including age, gender, and diagnosis. Harmane concentrations in blood were quantified by a well-established high performance liquid chromatography (HPLC) method in this group and used in our previous studies (Zheng et al, 2000). In short, one volume (9 -12 mL) of whole blood was mixed with half-a-volume (5 -6 mL) of 1 M NaOH.…”

“…A Dell Window based computer equipped with Waters data analysis package was used to collect and analyze the data. The identity of harmane on HPLC chromatographs previously has been clarified (Guan et al, 2001;Zheng et al, 2000). The intraday precision, measured as a coefficient of variation at 25 ng/mL, was 6.7% for harmane.…”

Kinetic tremor: Differences between smokers and non-smokers

“…As documented previously, blood lead concentration was analyzed using graphite furnace atomic absorption spectrophotometry (Fernandez and Hilligoss 1982;Louis et al 2003). The high performance liquid chromatography method to quantify blood harmane concentration was described previously (Louis et al, 2002;Zheng et al, 2000). Serum pesticide concentrations were determined by gas chromatography with electron capture detection following an organochlorine-specific cleanup of the lipid fraction of serum, as detailed elsewhere (Wolff et al 1991;Wolff et al 1982;Wolff et al 2005;Louis et al 2006).…”

Heavy atom enhanced room-temperature phosphorimetry for ultratrace determination of harmane

“…SSRTP may enable selective determination of harmane in the presence of other concominat substances (for instance, zidovudine, thalidomide, reserpine and yohimbine) without the need for previous separations. This feature may impose great advantages in terms of labor and operational cost when compared to HPLC based methodologies 20,21 . Because of its potential clinical applications and the dose-dependent response, the development of a simple and sensitive analytical method for the quality control of harmane based drugs is desirable.…”