Determination of harmane and harmine in human blood using reversed-phased high-performance liquid chromatography and fluorescence detection

Abstract

A number of tremorogenic beta-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 essential 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 beta-carboline derivatives, harmane and harmine. Human blood was extracted with ethyl acetate and methyl-t-butyl ether (2:98) under an alkaline condition. After evaporation of organic solvent, the samples were reconstructed in methanol. The samples were fractionated on a 250 x 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 beta-carboline toxicities.

FIG. 1

Typical HPLC traces of harmane and harmine. (A) Harmane and harmine standards in methanol (2.5 ng/50 μl); (B) harmane and harmine (6 ng) added in 10 ml of human blood; and (C) human blood sample from a patient (ID # 1803-0) without addition of harmane and harmine. Arrows indicate (1) harmane and (2) harmine.

FIG. 2

Emission spectra of harmane and harmine in collected HPLC fractions. Harmane and harmine standards were made in either methanol or blood. The blood standards underwent extraction. followed by HPLC. The methanol standards were directly injected onto HPLC without extraction. The peak fractions at 7–8 and 10–11 min of each injection were collected and scanned for emission spec trum at an excitation wavelength of 300 nm. (A) Fraction of harmane in methanol; (B) fraction of harmane in blood; (C) fraction of harmine in methanol; and (D) fraction of harmine in blood.

FIG. 3

Stability of harmane (A) and harmine (B) (25 ng/ml) in HPLC assay solution. Standard harmane and harmine were added to the assay solution at day 0. The samples were processed for HPLC analyses at the time indicated. Data represent means of three to four separate assays.