Liquid Chromatographic–Tandem Mass Spectrometric Method for the Determination of 5-Hydroxyindole-3-acetic Acid in Urine

Abstract

5-Hydroxyindole-3-acetic acid (5-HIAA), the primary metabolite of the neurotransmitter serotonin, is one of the biochemical markers measured in urine by different methods [colorimetric, fluorometric, HPLC, gas chromatography, and immunoassay (1)(2)] in the identification and surveillance of intestinal carcinoid syndrome (3). Usually, 1–3% of dietary tryptophan is metabolized to serotonin. However, as much as 60% of tryptophan is converted to serotonin in patients with the intestinal carcinoid syndrome. Modestly increased urinary 5-HIAA has been seen in Whipple disease, celiac disease, tropical sprue, and some noncarcinoid pancreatic islet tumors. Diet is also a factor affecting urinary excretion of 5-HIAA (4)(5). Our laboratory is actively involved in the conversion to tandem mass spectrometry (MS/MS) of assays currently based on other chromatographic technologies to improve quality and to reduce turnaround time, supply costs, and personnel needs in a high-volume testing environment (6)(7)(8)(9)(10). We directed our efforts toward developing a liquid chromatography (LC)-MS/MS method to replace an interference-prone HPLC procedure currently used for the routine determination of urinary 5-HIAA (2). We purchased 5-HIAA from Sigma. HIAA-d6 was custom-synthesized by Cambridge Isotope Laboratories (Andover, MA). Working solutions of 5-HIAA (0.5–150 mg/L) were prepared in acetic acid (10 mL/L) from an aqueous stock solution (1 g/L). A working solution of HIAA-d6 (10 mg/L) was prepared in acetic acid (10 mL/L) from a methanol stock solution (1 g/L). Bond-Elut® C18 solid-phase extraction (SPE) cartridges were obtained from Varian Inc. All other chemicals and solvents were of the highest purity available from commercial sources and were used without further purification. We mixed 0.5 mL of urine …