Determination of 4-Hydroxy-3-methoxyphenylethylene Glycol 4-Sulfate in Human Urine Using Liquid Chromatography−Tandem Mass Spectrometry

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Abstract
A major metabolite of norepinephrine (NE) in brain is 4-hydroxy-3-methoxyphenylethylene glycol (MHPG). In many species, a large fraction of MHPG formed in brain is converted to the sulfate conjugate. Consequently, MHPG sulfate has been proposed as a biomarker for NE metabolism in the central nervous system. As part of the clinical trials of the monoamine oxidase inhibitor selegiline for treating cocaine addiction, we required a method for measuring urine concentrations of MHPG sulfate. Using a deuterium-labeled analogue as an internal standard, we developed a liquid chromatography−electrospray ionization tandem mass spectrometry (LC−MS/MS) method for determination of MHPG sulfate in human urine. Sample preparation involves simply diluting 50 μL of urine with 1 mL of ammonium formate buffer and adding the internal standard. The sample is centrifuged, the supernate is transferred to an autosampler vial, and 10 μL is injected into the LC−MS/MS system. Standard curves from 50 to 10 000 ng/mL are generated. Only one sample of 277 clinical samples analyzed had a concentration outside of this range. Precision (coefficient of variation) ranged from 1.9 to 9.7%, and accuracy ranged from 97 to 103% of expected values for controls prepared by spiking sulfatase-treated urine with MHPG sulfate.