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A new analytical method for measurement of 3,4-dihydroxyphenylalanine (DOPA) in human urine was developed. DOPA from an aqueous solution was converted into an ethoxycarbonyl (EOC) derivative. A tertbutyldimethylsilyl (TBDMS) reaction under anhydrous conditions was then attempted for analysis by gas chromatography-mass spectrometry in selected ion monitoring mode. A new mass spectral data on DOPA as a tri-EOC/mono-TBDMS derivative was built. This method showed good linearity (r ≥ 0.999), precision (% relative standard deviation = 3.1-9.2), and accuracy (% relative error = −7.2-8.8), with a detection limit of 0.05 ng/mL. This selective and accurate method of DOPA analysis will be useful for biochemical monitoring of various neurological disorders including Parkinson's disease in biological fluids.Key Words : 3,4-Dihydroxyphenylalanine (DOPA), Ethoxycarbonyl/tert-butyldimethylsilyl derivatives, Gas chromatography-mass spectrometry, Urine sample IntroductionAs a metabolite of tyrosine, 3,4-dihydroxyphenylalanine (DOPA) is consecutively metabolized to catecholamines, such as dopamine, norepinephrine, and epinephrine, [1][2][3] which have been suggested as neurotransmitter candidates in the central nervous system.1 Thus, accurate measurement of DOPA level in biological fluids of various neurological disorders, including Parkinson's disease, 4-7 has been useful for biochemical monitoring of disease states.Analytical methods for detection of DOPA in platelets, plasma, and adrenal glands have been mostly reported by high performance liquid chromatography (HPLC), 8-13 liquid chromatography-mass spectrometry (LC-MS) or LC-MS/ MS, 14-16 only a few studies have been performed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). 17-19In GC and GC-MS analysis, derivatization is generally required to block all active protons of hydroxyl, carboxyl, and amine groups present in DOPA, for improvement of its thermal stability and volatility, as well as GC and GC-MS properties.A variety of derivatization methods based on acylation, [18][19][20] or methylation 17 have been investigated for analysis of DOPA. Among them, trimethylsilyl (TMS) derivatives are unstable under moist conditions; acylation and methylation usually require a clean-up step for removal of excess reagent and side products. 17Most of previous reports for DOPA analysis by GC and GC-MS were performed under anhydrous conditions, even though it contains a catechol group and can easily oxidize and decompose in sample preparations. Thus, in this work, we first attempted DOPA analysis in human urine, as in our previous reports.21-23 It includes direct conversion as a stable solvent-extractable form by a two-phase ethoxycarbonyl (EOC) derivative with ethyl chloroformate (ECF) in the dichloromethane phase for catechol and amine groups of DOPA in aqueous solution, followed by a tert-butyldimethylsilyl (TBDMS) derivative for carboxylic acid function in anhydrous conditions with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), which i...