Abstract Methylation of carboxylic acids upon syringe injection of a mixture of the acid sample and phenyl- trimethylammonium hydroxide (PTMAH) into the GC in- jection port is a convenient but under-utilized derivatiza- tion procedure. To minimize potential instrumental prob- lems due to the sample matrix, it was shown that solid- phase microextraction (SPME) is effective for the absorp- tion of both the carboxylic acid (RCOOH) and PTMAH permitting on-line methylation from the fiber. A compari- son of three fibers, polydimethylsiloxane (PDMS), poly- acrylate (PA), and carboxene/PDMS for decanoic and stearic acids showed the carboxene/PDMS fiber was about five times more effective for the extraction of the RCOOH-PTMAH mixture dissolved in methanol. The optimum fiber absorption time was about 20 min and the optimum desorption time in the injection port held at 280 °C was about 5–10 min. The optimum PTMAH/RCOOH ratio was about 125:1. Linearity for C18:0 at 3.3×10 –6 – 3.3×10 –4 M was demonstrated by GC-MS with a detection limit of 1 μM. This SPME method is also effective for the methylation of C18:1, C18:2, and C18:3 fatty acids. Transesterification of olive oil using PTMAH and then on-line methylation either by the syringe method or by SPME gave comparable fatty acid methyl ester profiles. Keywords Solid-phase microextraction · Pyrolytic methylation Introduction Carboxylic acids play an important role in biological and environmental processes. For example, the fatty acid syn- thesis in plants is important to understanding the regula- tion of biosynthesis of trigylcerides, the primary compo- nent of vegetable oils [1]. For long-chain aliphatic and aromatic carboxylic acids, esterification is generally re- quired to make volatile and non-adsorptive derivatives suitable for determination by gas chromatography (GC). Many of these esterification reactions such as silylation using hexamethyldisilazane/trimethylchlorosilane involve non-aqueous reaction conditions; prior extraction of the carboxylic acids from an aqueous solution is often re- quired [2]. Most common methylation methods using methanol in hydrochloric acid or in sulfuric acid require extraction with pentane after derivatization to prevent acid from entering the GC [2]. A classical method using diazomethane requires the in situ generation of this de- rivatizing reagent from a nitroso-substituted organic com- pound and KOH as well as a post organic solvent extrac- tion [2]. One method not requiring an extraction step is pyro- lytic methylation. This reaction occurs readily between a carboxylic acid and a quaternary ammonium compound (QUAT) such as the tetramethylammonium cation in the GC injection port [3]. The tertiary amine demethylation product is easily separated from the more non-polar methyl esters. Improved reproducibility of the procedure has been demonstrated by first drying the solution to a salt and insertion of the residue using a probe injection device [4]. Use of trimethylanilium hydroxide permitted the in- jection port temperature to be decreased by 100 °C to 265 °C eliminating possible degradation reactions [5]. Determi- nation of fatty acids derived from edible fats and oils using methanolic (m-fluoromethylphenyl)-trimethylam- monium hydroxide was convenient since the derivatizing reagent could do the transesterification reaction in one step [6]. Pyrolytic methylation GC has been reviewed for a wide variety of organic compounds with NH, OH, or COOH groups [7]. Because the QUAT is in excess and a significant volume is injected, contamination of the injec- tion port liner and column can be a problem upon QUAT decomposition in the hot injection port. Solid-phase microextraction (SPME) utilizes a short (about 1 cm), fused-silica fiber coated with a thin (about 50–100 μm) polymeric coating to extract the analyte from Yaqian Liu · Seong R. Cho · Neil D. Danielson Solid-phase microextraction and on-line methylation gas chromatography for aliphatic carboxylic acids Anal Bioanal Chem (2002) 373 : 64–69 DOI 10.1007/s00216-002-1271-5 Received: 14 November 2001 / Revised: 18 February 2002 / Accepted: 18 February 2002 / Published online: 13 April 2002 SPECIAL ISSUE PAPER Y. Liu · S.R. Cho · N.D. Danielson (✉) Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA e-mail: danielnd@muohio.edu © Springer-Verlag 2002