ABSTRACT: The determination of FAME by GC is among the most commonplace analyses in lipid research. Quantification of FAME by GC with FID has been effectively performed for some time, whereas detection with MS has been used chiefly for quali- tative analysis of FAME. Nonetheless, the sensitivity and selectiv- ity of MS methods advocate a quantitative role for GC���MS in FAME analysis���an approach that would be particularly advanta- geous for FAME determination in complex biological samples, where spectrometric confirmation of analytes is advisable. To as- sess the utility of GC���MS methods for FAME quantification, a comparative study of GC���FID and GC���MS methods has been conducted. FAME in prepared solutions as well as a biological standard reference material were analyzed by GC���FID and GC���MS methods using both ion trap and quadrupole MS systems. Quantification by MS, based on total ion counts and processing of selected ions, was investigated for FAME ionized by electron impact. Instrument precision, detection limits, calibration behav- ior, and response factors were investigated for each approach, and quantitative results obtained by each technique were com- pared. Although there were a number of characteristic differences between the MS methods and FID with respect to FAME analysis, the quantitative performance of GC���MS compared satisfactorily with that of GC���FID. The capacity to combine spectrometric ex- amination and quantitative determination advances GC���MS as a powerful alternative to GC���FID for FAME analysis. Paper no. L9654 in Lipids 40, 419���428 (April 2005) The first of many important advances in the early development of GLC for analytical purposes was the separation and deter- mination of FA reported by James and Martin in 1952 (1). Soon after, the analytical separation of FAME by vapor-phase chro- matography was described by Cropper and Heywood (2). Since then, the characterization of FA composition by esterification to FAME and subsequent determination by GC has become one of the most widely performed analyses in lipid research labora- tories and has found broad application to biochemical, biomed- ical, microbiological, agricultural, and ecological research. Presently, a large number of lipid analysts use the FID to measure FAME separated by GC. Although FID has proven to be a robust tool for FAME determination, the lack of any se- lectivity can limit the usefulness of this detector when applied to complicated samples, since only instrument response and re- tention time information may be gathered. Owing in large part to this limitation, misidentification of FAME in the presence of contaminants, artifacts, or coeluting compounds is still of con- cern when using FID (3���5). Thus, much work has been done to maximize the usefulness of retention time for FAME identifi- cation through methods such as retention time locking, reten- tion time prediction, and the dependence of retention time on FAME equivalent chain lengths (6���8) however, FAME identi- ties assigned by such methods are generally considered tenta- tive (9). Hence, FID analysis of complex biological extracts may prove inadequate in some situations, particularly for FAME of relatively low abundance (10). With the coupling of MS methods to GC, much has been ac- complished in the area of qualitative characterization of FAME mixtures. Since GC���MS provides spectrometric information on separated compounds, it provides a means of analyte selec- tivity thus, detection with MS also represents a potentially powerful tool for quantitative analysis of FAME, especially in the presence of a convoluted biochemical background. Despite the prospective benefits of GC���MS methodologies for quanti- tative FAME analysis, the more familiar FID is still favored in many laboratories, particularly among lipid specialists. GC���MS offers a host of tools for qualitative characteriza- tion of FA. For example, standard 70 eV EI ionization of pico- linyl, dimethyloxazoline, pyrrolidide, pentafluorodimethylsilyl, and trimethylsilyl derivatives of FA has been extensively studied for the purpose of structural determination (11���17). EI ioniza- tion of these less common FA derivatives yields fragments of di- agnostic value in locating the positions of branching and in some cases the positions of unsaturation in FA (although the geomet- ric configuration of the double bond is not forthcoming). The simplest method of FAME quantification by EI���MS may be carried out by monitoring a range of mass-to-charge (m/z) values that will encompass the fragments expected of the analytes, then determining amount based on integration of Copyright �� 2005 by AOCS Press 419 Lipids, Vol. 40, no. 4 (2005) Present address of first two authors: Department of Chemistry, University of California Davis, One Shields Ave., Davis, CA 95616. *To whom correspondence should be addressed at University of Alaska An- chorage, Department of Chemistry, 3211 Providence Dr., Anchorage, AK 99508. E-mail address: kennish@uaa.alaska.edu Abbreviations: AR, area ratio EI, electron impact IT, ion trap LOD, limit of detection QP, quadrupole RF, response factor RSD, relative SD SIE, selected ion extraction SIM, selective ion monitoring SRM, Standard Ref- erence Material TIC, total ion counts. Gas Chromatographic Quantification of Fatty Acid Methyl Esters: Flame Ionization Detection vs. Electron Impact Mass Spectrometry Eric D. Doddsa, Mark R. McCoya,d, Lorrie D. Reac,d, and John M. Kennisha,b,* aApplied Science, Engineering, and Technology Laboratory and bDepartment of Chemistry, University of Alaska Anchorage, Anchorage, Alaska 99508, cEnvironment and Natural Resources Institute, University of Alaska Anchorage, Anchorage, Alaska 99501, and dAlaska Department of Fish and Game, Physiological Ecology Laboratory, Anchorage, Alaska 99518

peaks in the total ion count (TIC) chromatogram. If the identity and retention time of a given analyte have been established, the sensitivity and specificity of EI���MS can be extended through the use of selective ion monitoring (SIM), which involves the exclusive acquisition of a specified ion or group of ions during a given time frame. When SIM is used in concert with EI, an analyte peak area can be measured reliably regardless of high background or coeluting peaks, provided that ions unique to the analyte are monitored. Ideally, a fragment or fragments of relatively high abundance and characteristic m/z would be monitored. When entire mass spectra are acquired, benefits similar to those offered by SIM may be gained through quan- tification based on only a subset of the acquired ions extracted from the TIC. This selected ion extraction (SIE) allows com- plete spectra to be recorded and permits the exclusion of unde- sirable masses for purposes of quantification. Both approaches generally involve the use of relatively few ions for quantifica- tion thus, significant numbers of analyte ions are disregarded and a corresponding loss of signal is experienced. Nonetheless, the net result is an enhancement of the signal-to-noise ratio by elimination of most nonanalyte response from the signal. An investigation of the performance of FID vs. MS in quan- tifying FAME separated by GC has not been conducted since the work of Koza et al. in 1989 (18). These authors compared FID response factors (RF) of FAME with those obtained using EI in quadrupole (QP) as well as sector-type mass spectrome- ters. Unfortunately, only seven FAME were examined, none of which was polyunsaturated. In addition, the study provided no information to users of ion trap (IT) MS systems (since this form of MS was still a relatively recent development at that time). Limits of detection (LOD), calibration behavior, and re- producibility of the instrumental methods were not addressed. The present study provides a comparison of GC���FID and GC���MS techniques for quantitative FAME analysis. Using both IT and QP MS systems, quantification methods based on the TIC and selected ion techniques were applied to FAME ion- ized by EI. LOD, calibration characteristics, RF, and method precision for determination of a broad range of FAME were as- sessed by analysis of standard mixtures with each detection method. To determine the applicability of each approach to the analysis of a biological sample, FAME were prepared from a standard reference fish tissue homogenate with certified values for a group of individual FA [NIST Standard Reference Mater- ial (SRM) 1946] (19). EXPERIMENTAL PROCEDURES Calibration standards. A series of standard mixtures, includ- ing all FAME listed in Table 1, was prepared in residue analy- sis-grade hexane (EM Science, Gibbstown, NJ). These FAME were selected for calibration because of their prevalence in the tissues of fish and marine animals. The FAME were obtained from Sigma-Aldrich (St. Louis, MO), Supelco (Bellefonte, PA), Matreya (State College, PA), and Nu-Chek-Prep (Elysian, MN), and were of the highest purity available. For each level of calibration, all FAME were present at equal concentrations, 420 E.D. DODDS ET AL. Lipids, Vol. 40, no. 4 (2005) TABLE 1 Analyte FAME Present in the Calibration Standards and Their Order of Elutiona Elution FAME order carbon numer Systematic name Common name 1 14:0 Tetradecanoic acid, methyl ester Methyl myristate 2 16:0 Hexadecanoic acid, methyl ester Methyl palmitate 3 16:1n-7 cis-9-Hexadecenoic acid, methyl ester Methyl palmitoleate 4 17:0 Heptadecanoic acid, methyl ester Methyl margarate 5 18:0 Octadecanoic acid, methyl ester Methyl stearate 6 18:1n-9 cis-9-Octadecenoic acid, methyl ester Methyl oleate 7 18:1n-7 cis-11-Octadecenoic acid, methyl ester Methyl cis-vaccenate 8 18:2n-6 cis,cis-9,12-Octadecadienoic acid, methyl ester Methyl linoleate 10 18:3n-3 cis,cis,cis-9,12,15-Octadecatrienoic acid, methyl ester Methyl linolenate 9 19:0 Nonadecanoic acid, methyl ester None 11 20:0 Eicosanoic acid, methyl ester Methyl arachidate 12 20:1n-9 cis-11-Eicosenoic acid, methyl ester Methyl gondoate 13 20:2n-6 cis,cis-11,14-Eicosadienoic acid, methyl ester None 15 20:4n-6 cis,cis,cis,cis-5,8,11,14-Eicosatetraenoic acid, methyl ester Methyl arachadonate 17 20:5n-3 cis,cis,cis,cis,cis-5,8,11,14,17-Eicosapentaenoic acid, methyl ester EPA, methyl ester 14 21:0 Heneicosanoic acid, methyl ester None 16 22:0 Docosanoic acid, methyl ester Methyl behenate 18 22:1n-9 cis-13-Docosenoic acid, methyl ester Methyl erucate 19 22:4n-6 cis,cis,cis,cis-7,10,13,16-Docosatetraenoic acid, methyl ester Methyl adrenate 20 22:5n-3 cis,cis,cis,cis,cis-7,10,13,16,19-Docosapentenoic acid, methyl ester DPA, methyl ester 21 22:6n-3 cis,cis,cis,cis,cis,cis-4,7,10,13,16,19-Docosahexaenoic acid, methyl ester DHA, methyl ester aThe FAME of 21:0 was added pre-analysis as an internal standard.