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9, 2011

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Gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry in quantifying fatty acids
Gao-Ling Wei, Eddy Y. Zeng
This critical overview covers current analytical methods and future developments in quantitative determination of fatty acids (FAs), emphasizing sample extraction, derivatization and instrumental analysis with gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS2). We compare the benets and the drawbacks of these two analytical techniques. We consider the well-established GC/MS method with pre-derivatization to be a traditional technique in terms of highly standardized sample-preparation procedures, affordability and readily available library searching for compound identication. However, the complicated derivatization steps required prior to instrumental analysis with GC/MS take a long time, with loss and transformation of FAs, low recovery and poor reproducibility. HPLC/MS2 without derivatization shows the benets of simple, mild sample-processing conditions, satisfactory recovery, short running time and high selectivity and sensitivity, which may allow it to become a viable alternative to GC/MS for the analysis of FAs in the years ahead. 2011 Elsevier Ltd. All rights reserved.
Keywords: Derivatization; Fatty acid; Gas chromatography-mass spectrometry; High-performance liquid chromatography-tandem mass spectrometry; Instrumental analysis; Limit of detection; Pre-derivatization; Sample extraction; Sample preparation; Sensitivity

Gao-Ling Wei State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China Graduate School, Chinese Academy of Sciences, Beijing 100049, China Eddy Y. Zeng* State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China

1. Introduction The occurrence of fatty acids (FAs) in the environment has been extensively investigated for various purposes. As biomarkers of environmental implications [14], FAs are used to identify the sources and the fates of organic matter, to reect microbial activity, and to reveal changes in the sedimentary environment and diagenesis. FAs play an important role in pathology in diagnosing inherited disease, diabetes and cancer, because of the close relationship between trans FAs and the risk of these diseases [5]. In addition to their biological signicance, determination of FAs in food samples is of great interest for nutritional assessment and food-quality control [68].

Corresponding author. Tel.: +86 20 85291421; Fax: +86 20 85290706; E-mail: eddyzeng@gig.ac.cn

In recent years, numerous analytical techniques have been utilized for measurement of FAs {e.g., thin-layer chromatography (TLC), electrophoresis, gas chromatography (GC), and liquid chromatography (LC), coupled to detectors [e.g., ame ionization, spectrophotometric, mass spectrometry (MS) and nuclear magnetic resonance]} [9]. Of these techniques, GC/ MS with derivatization to enhance sensitivity has been the most common and popular for quantifying FAs in environmental [1,3,9], biological [1012] and food [8,13] samples. In addition, LC/ultraviolet (UV) spectrometry and LC/MS with derivatization have also been utilized to enhance the detection sensitivity for FAs [14,15]. However, the complicated derivatization procedures can cause a variety of 1429

0165-9936/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2011.05.005

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problems [11], limiting their applications. High-performance liquid chromatography (HPLC)/MS [1619] and HPLC/tandem mass spectrometry (MS2) [6,20] have also been reported to quantify FAs directly in shellsh, phospholipid formulation, process water, blood samples, and chocolate and fermentation-broth samples with sensitivity similar to GC/MS with a derivatization step. Such simple, rapid analytical techniques are expected to become increasingly popular in the years ahead. This critical overview covers currently available methods for determination of FAs, including extraction and derivatization procedures, as well as the benets and the drawbacks of GC/MS and HPLC/MS or HPLC/MS2. We aim to discuss the technical trends in FA-method development and to demonstrate that HPLC/MS2 with a simplied sample-treatment procedure can be a viable alternative to GC/MS with derivatization steps. 2. Sample extraction A typical sample-processing protocol commonly comprises an extraction step and a derivatization process required prior to instrumental analysis (Fig. 1). 2.1. Extraction of fatty acids Numerous techniques have been developed for the extraction of FAs from different matrices [12]. These techniques include Soxhlet extraction, accelerated solvent extraction (ASE), supercritical uid extraction (SFE), ultrasound-assisted extraction (UAE), microwaveassisted solvent extraction (MASE), liquid-liquid extraction (LLE), solid-phase extraction (SPE) and solid-phase microextraction (SPME). Organic solvents [12] suitable for these extraction processes include chloroformmethanol, dichloromethane-methanol, dichloromethane, diethylether, hexane and isopropanol-hexane. It is therefore necessary to assess the method performance of a specic protocol in terms of extraction efcacy, cost effectiveness and solvent consumption before it can be selected. Table 1 compares the performance of the abovementioned extraction techniques in processing various types of samples, and it sets out features useful in selection of an appropriate extraction method. As a classic extraction method, Soxhlet extraction has been successfully applied to extract FAs from environmental samples {e.g., sediment [1,2,4,21] and water-column particulate matter [22]}. The low cost of apparatus and highly standardized extraction procedures of Soxhlet extraction have made it popular in routine laboratory analyses. However, Soxhlet extraction is rather time consuming and consumes large amounts of organic solvents. Typically, up to 72 h of extraction time and 200500 mL of organic solvent may be needed to achieve satisfactory extraction efciency [4,21]. Extraction of FAs from lacustrine and marine sediments [23], cheese and sh [13] has also been conducted 1430

with ASE, which requires short extraction time (usually <1 h), moderate solvent consumption (<200 mL) and simple treatment steps. Lee et al. [24] and McDaniel et al. [25] utilized SFE to extract FAs from sediment and sand samples. Compared with conventional methods, SFE shows distinct benets of short extraction time (<15 min), low solvent consumption (<10 mL), and high recovery of volatile analytes. However, the applications of ASE and SFE in routine analysis have been limited because of the high costs of equipment and maintenance. Both UAE and MASE are also prevalent and effective techniques for extracting FAs from solid samples [3,9,2628], due to their short extraction cycles, low solvent consumption (<60 mL), easy sample handling, and low costs of equipment and maintenance. However, the application of MASE may be somewhat restricted due to transesterication and oxidation of unsaturated FAs under microwave irradiation [29] and limited choice of solvents suitable for microwave absorbance. In recent years, SPE and SPME have gradually been applied to enrichment and purication of FAs [16,30]. These two techniques are robust and are quite economic in terms of time, labor and solvent consumption, compared to more conventional methods, and they can be automated [31]. LLE has also been used in extracting FAs from saponated solution after acidication [13,19]. In addition, direct derivatization has been performed on biological samples without an extraction step [8]. In conclusion, ASE, UAE and SPE seem to be simpler in operation and more cost effective than other extraction techniques. 2.2. Derivatization of fatty acids Derivatization of FAs is a common step to increase the volatility of FAs, to enhance detection sensitivity, to improve chromatographic resolution, and to reduce peak tailing for long-chain FAs [32]. Eder [11] and Brondz [12] extensively reviewed various approaches for derivatization of FAs. Here, we focus on the most widely used derivatization route [i.e. transesterication of FAs into methyl esters (FAMEs)]. The main methods of methylation involve acid-catalyzed and base-catalyzed reactions. Anhydrous methanol (MeOH) with an acid catalyst [e.g., hydrogen chloride (MeOH-HCl), concentrated sulfuric acid (MeOH-H2SO4), boron triuoride (MeOH-BF3) or boron trichloride (MeOH-BCl3)] is a common reagent used in acidcatalyzed esterication. MeOH-HCl (5%, w/v) is used most commonly and is the mildest reagent [12]. As an alternative to MeOH-HCl, MeOH-H2SO4 (12%, v/v), which can be used in the same manner as MeOH-HCl, can also achieve the same rate of FA esterication [11]. Furthermore, both MeOH-BF3 and MeOH-BCl3 (1214%,

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Figure 1. Available methods of sample extraction, derivatization and instrumental analysis for quantifying fatty acids. ASE, Accelerated solvent extraction; SFE, Supercritical uid extraction; UAE, Ultrasound-assisted extraction; MASE, Microwave-assisted solvent extraction; SPE, Solid-phase extraction; SPME, Solid-phase microextraction; LLE, Liquid-liquid extraction; TLC, Thinlayer chromatography; HPTLC, High-performance TLC; CE, Capillary electrophoresis; GC, Gas chromatography; LC, Liquid chromatography; UV, Ultraviolet; LIF, Laser-induced uorescence; FID, Flame-ionization detection; ECD, Electrochemical detection; DAD, Diode-array detector; ELSD, Evaporative light-scattering detection; NMR, Nuclear magnetic resonance; MS, Mass spectrometry; and MS2, Tandem MS.

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Table 1. Procedures of extraction, derivatization, and instrumental analysis typically employed in the measurement of fatty acids in various matrices and related detection sensitivity Matrix Sediment Sediment Sediment Sediment Particulate matter Sediment Cheese and sh Sediment Sand Sediment Sludge Peat Water Sputum specimen Herbage Meat Shellsh Formulation Process water Blood sample Chocolate Fermentation broth Meconium Cemetery soil Shewanella pealeana Atherosclerotic plaque Sediment and soil Extraction Soxhlet Soxhlet Soxhlet Soxhlet Soxhlet ASE ASE SFE SFE UAE UAE UAE SPME DI-SPME DTE DTE Shake LLE MSPD CHCl3/MeOH 20 Time 2436 h 72 h 72 h 48 h 24 h 30 min 30 min 15 min 10 min 1h 6h 1.2 h NR NR NR NR 30 min CHCl3/MeOH MeOH/CHCl3 Solvent DCM/MeOH DCM/MeOH DCM/MeOH CHCl3/MeOH DCM DCM/MeOH EAC/CYCH DCM/MeOH DCM/MeOH MeOH/CHCl3 DCM DCM/MeOH 180 10 90 100 Usage 200 200 200 200 200 Derivatization BCl3-MeOH,110C, 8 min BF3-MeOH BF3-MeOH BSTFA Diazomethane HCl-MeOH, 70C, 12 h BF3-MeOH PFE HCl-MeOH BF3-MeOH BF3-MeOH BSTFA,70C, 1.5 h PDAM H2SO4-MeOH, 80C, 1 h HCl-MeOH, 70C, 2 h HCl-MeOH, 70C, 1.5 h Column HP-5 SE-54 HP-5 DB-5 DB-5 CP-Sil 5 CP-Sil 19 DB-17 DB-5 TM 32 HP-1 HP-5 SPB-5 HP-1 CP-Sil 88 HP-88 C8 C8 C18 RP-18e C18 C18 C8 RP-18 Instrument GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC-ECD GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS HPLC/MS HPLC/MS HPLC/MS HPLC/MS HPLC/MS2 HPLC/MS2 HPLC/MS2 HPLC/MS HPLC/MS2 HPLC/MS2 HPLC/MS2 60560 lg/g 2.2911.48 ng/L 0.53.1 lg/L Low-pg range 21153 ng/g 5137 lg/L 29.351.2 ng/g 5 pg 4.040.0 pg 250 pg LOQ Ref. [1] [4] [21] [2] [22] [23] [13] [24] [25] [3] [9] [26] [43] [28] [44] [8] [16] [17] [18] [19] [6] [20] [39] [42] [10] [45] [36]

35 lg/g

1.68150.4 lg/L

Vortex Soxhlet DTE Shake USE

15 min 3h

Hexane Ether/DCM Hexane/CHCl3 CHCl3/MeOH DCM/MeOH

10 100

Amides FAPE TMAE

15 min 30 min

5 60

Diphenyl C18 and C8

LOQ, Limit of quantication; ASE, Accelerated solvent extraction; SFE, Supercritical uid extraction; UAE, Ultrasound-assisted extraction; SPME, Solid-phase microextraction; DI-SPME, Direct solid-phase microextraction; DTE, Direct transesterication; LLE, Liquid-liquid extraction; MSPD, Matrix solid-phase dispersion; EAC, Ethyl acetate; CYCH, Cyclohexane; BSTFA, N,O-Bis trimethylsilyltriuoroacetamide; PFE, Pentauorobenzyl ester; PDAM, 1-pyrenyldiazomethane; FAPE, Fatty acid phenacyl ester; TMAE, Trimethylaminoethyl ester.

w/v) have been successfully applied to methylation of FAs [1,3,4]. For base-catalyzed transestericaiton, the most popular reagent is sodium methoxide in anhydrous methanol, which can yield satisfactory recoveries of short-chain FAs [11]. Despite the vast benets of using derivatization in analyses of FAs, there are also drawbacks [11]: (1) tedious and time-consuming; (2) difcult to achieve complete transesterication of FAs and extraction of FAMEs; (3) difcult to prevent decomposition, isomerization and oxidation of some unsaturated FAs upon heat-

ing and loss of highly volatile short-chained FAs during solvent evaporation; and, (4) easy to result in side products that may interfere with subsequent qualitative and quantitative analyses. In addition, further derivatization into picolinyl esters or 4,4-dimethyloxazoline derivatives is still needed for conrmatory purposes, because FAMEs cannot give information about the position and the conguration of the double bonds. With the above drawbacks of derivatization, it is urgent to develop a simple, rapid method for extracting FAs without this tedious step.

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Figure 2. Total ion chromatograms of (A) fatty acid methyl esters (FAMEs) obtained from GC/MS; (B) and (C) free fatty acids (FAs) obtained from UHPLC/ MS2. FAs are abbreviated in Cm:bxn, where m: the number of carbon, b: the number of double bond, xn: the position of the last double bond, and n: the carbon of the last double bond counted from the methyl terminal. The conguration isomers of unsaturated FAs are represented in cis (c) and trans (t). Isomers of branched FAs are represented in iso (i) and anteiso (a) to demonstrate the methyl group at the second last and the third last carbon, respectively. GC/MS analysis of FAMEs was performed on a Shimadzu Model 2010 gas chromatograph-electron impact-mass spectrometer with a DB-1MS (60 m 0.32 mm i.d. with 0.25 lm lm thickness) capillary column. The column oven temperature was programmed from 70C to 290C (held for 20 min) at a rate of 3C/min (A). FA analysis with HPLC/MS2 was performed on a C18 column (2.1 100 mm and 1.8 lm particle size) and C8 column (2.1 50 mm and 1.8 lm particle size) for (B) and (C), respectively. For (B), H2O (1 mM NH4Ac) and ACN were used as the mobile phase, and the elution gradient was started at 60% ACN and held for 5 min, increased to 85% ACN in 11 min, then stepped to 98% ACN in 1 min and held for 7 min. For (C), H2O (1 mM NH4Ac + 0.1% HCOOH) and ACN were used as mobile phase, programming as follows: initial 90% ACN maintained for 4 min, then increased to 98% ACN in 0.5 min and held for 8.5 min.

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3. Instrumental analysis Because Carrasco-Pancorbo et al. [33] comprehensively compared various detection techniques for FAs analysis, emphasizing HPLC, GC and other related techniques, this review selectively discusses the applicability of GC/MS and HPLC/MS or HPLC/MS2 in measurement of FAs, and summarizes the benets and drawbacks of these techniques (Table 1). The development of analytical techniques for quantifying FAs is shown in Fig. 1. 3.1. GC/MS analysis It seems difcult to analyze free FAs by GC/MS without derivatization, because of their high polarity and low volatility [12]. Derivatizing FAs into more volatile derivatives is compulsory to enhance detection sensitivity and to improve separation, especially for samples containing trace levels of FAs. The main derivatives involved in GC/MS analysis are quite diverse, including methyl esters, pyrrolidide, picolinyl esters, 4,4-dimethyloxazoline and TMS [33]. With derivatization, GC/MS has been considered a conventional, powerful analytical technique and broadly utilized in determination of FAs in different sample matrices (Table 1). With acid-catalyzed derivatization into FAMEs, measurements of FAs in sediment [1,3,4,21,23] and sludge [9] were often performed with GC/MS, achieving limits of detection (LODs) of 35 lg/g for FAs (C8C22) in sludge. In addition, GC/MS was regarded as a reliable technique for processing biological samples [30,34] and food samples [8,13,27]. Beyond doubt, the main drawback of GC/MS analysis is the complexity of sample extraction involving derivatization, and we discussed the issues related to derivatization above. Eder [11] reviewed the methods for determination of FAs with derivatization into methyl esters and suggested that sample introduction to GC should be carefully progressed to minimize discrimination between high-boiling and low-boiling FAMEs. Eder [11] also commented that polar stationary phases (e.g., CP Sil-88, SP-2340 and SP-2330) had low thermal stability and long retention time for FAMEs despite excellent separation of isomers, while non-polar phases (e.g., CP-Sil 5CB, DB-5 and DB-1) had high thermal stability but inferior selectivity. Consequently, a column with median polarity (OV-225, DB-1701 and DB-225) can be chosen to balance between thermal stability and good separation. Van der Steege et al. [35] obtained a retention time of 47 min for C24:0-FAME on a CP-Sil-5 cross-linked methylsilicone-coated column (50 m 0.20 mm i.d.). Fig. 2A shows separation of FAMEs (C6:0C30:0) on a DB-1 column (60 m 0.32 mm i.d. with 0.25 lm lm thickness) with the oven temperature programmed from 70C to 290C at a rate of 3C/min, and retention times of 67 min for C24:0 and up to 83 min for C30:0 were 1434

obtained [36]. In addition to extended retention times for C26:0C30:0, detection sensitivity was another issue because these components were not detectable at 0.5 lg/mL [36]. 3.2. HPLC/MS and HPLC/MS2 analysis Thanks to the low volatility and high polarity of FAs, use of HPLC enables FAs to be analyzed as underivatized components. HPLC coupled with MS has also been applied to FA measurement without derivatization (Fig. 1). Novakova et al. [37] summarized the HPLC methods with diverse detectors for simultaneous determination of ascorbic and dehydroascorbic acids. They revealed that HPLC/MS was a preferred analytical technique with high selectivity and sensitivity without a derivatization step. Sajiki and Yonekubo [38] presented an overview of the methods for measuring free polyunsaturated FAs in biological materials using HPLC and HPLC/MS. Analyses of FAs by reversed-phase LC/MS were conducted with both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in the selective ion monitoring (SIM), single reaction monitoring (SRM) and pseudo-multiple reaction monitoring (PMRM) modes to obtain sufcient detection sensitivity. The limit of quantication (LOQ) was 60560 lg/g wet weight for determination of free FAs in shellsh with a reverse C8 column [16]. Concentrations of FAs in phospholipid formulations and raw materials were directly measured without prederivatization with LOQs of 2.2911.48 lg/mL [17]. In addition, Rigol et al. [18] developed an HPLC-APCIMS method for direct analysis of FAs in process waters from paper industries, achieving good recoveries of 75 86% and low method LODs in the range 0.53.1 lg/L. Furthermore, Nagy et al. [19] reported a sensitive method for analyzing long-chain FAs (C16C26) in blood samples, with LOQs at the low-pg level. Detection of FAs in chocolate was done by HPLC-ESIMS2 with LOQs in the range of 21153 ng/g. Analyses of spiked chocolate samples at about 50150% of the background levels of FAs yielded recoveries of 79103% [6]. With PMRM mode to reduce the noise base, FAs in fermentation broths were detected with LOQs of 0.005 0.137 mg/L [20]. HPLC/MS2 methods with prior derivatization have also been established to enhance ionization. Recoveries of FA ethyl esters spiked in meconium by HPLC/MS2 were in the range 5487% with LOQs of 29.351.2 ng/g [39]. With pre-derivatization of FAs into trimethylaminoethyl ester, the composition of FAs in atherosclerotic plaque tissue samples was determined by HPLC-ESI(+)MS2, with LOQs of 4.040.0 pg. This technique was considered superior to GC/MS [40].

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In addition, phenacyl esters [41] and admies [42] were additional derivatives of FAs analyzed by HPLC/MS2 with LOQ in the pg range. Despite the above-discussed efforts to employ HPLC/ MS2 for determination of FAs without derivatization, applications of this approach to environmental samples (e.g., sediment, sludge and soil) have not been reported. We recently developed a direct HPLC/MS2 method for measurement of FAs with carbon numbers C10C30 (a total of 41 individual FAs) in sediments and soils, completely eliminating the use of derivatization [36]. The analysis was performed in the ESI negative mode with PMRM. The separation of isomers of unsaturated and moderate-sized FAs (C10C20) was achieved on a reverse C18 column (2.1 100 mm, 1.8 lm particle size) within 23 min. Meanwhile, the elution of long-chained FAs (C21C30) was achieved with a shorter and less polar C8 column (2.1 50 mm, 1.8 lm particle size) in 13 min. Typical total ion chromatograms from these two analytical procedures are depicted in Figs. 2B and C, obtained from the separations of FAs on C18 and C8, respectively, along with a typical total ion chromatogram of FAMEs obtained from GC/MS analysis. The LOQ was 250 pg, indicating that HPLC/MS2 is sensitive for quantifying FAs without derivatization. Moreover, recovery tests with spiked blank samples demonstrated satisfactory recoveries in the range 65119% for all target FAs with relative standard deviations all within 20%. A comparison of the HPLC/MS2 and GC/MS methods suggests that HPLC/MS2 seems to be a favorable choice, because it involves simpler sample extraction, shorter analysis time and better recoveries than GC/MS. Nevertheless, the HPLC/MS2 technique for routine analysis of FAs has not been as popular as GC/MS, probably because of the costs of instruments and maintenance are higher than those of GC/MS. With the increasing affordability of HPLC/MS2 instruments and the availability of diverse LC columns, solvents, and modifying additives, HPLC/MS2 without derivatization is expected to become a viable alternative to GC/MS requiring pre-derivatization with the benets of being simple to operate and time efcient. More efforts should be directed toward exploring wider applications of the HPLC/MS2 technique in analysis of FAs.

free FAs, and incomplete derivatization and extraction). In addition, GC/MS is often inadequate in analyzing high-boiling compounds, and is characterized by serious peak tailing, extremely long retention times and high LODs. The utility of HPLC/MS and HPLC/MS2 may simplify sample extraction without prior derivatization. Our preliminary study demonstrated that HPLC/MS and HPLC/MS2 can be sensitive and economic techniques for determination of FAs in various sample matrices. Acknowledgments This research was financially supported by the Earmarked Fund of the State Key Laboratory of Organic Geochemistry (SKLOG2009A04), the National Natural Science Foundation of China (No. 40821003) and the Chinese Academy of Sciences (KZCX2-YW-Q02-06-01). This is contribution No. IS-1362 from GIGCAS. References
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4. Conclusions This review has summarized the current extraction and instrumentation methods for analysis of FAs to illustrate possible problems and future trends. GC/MS has been the most conventional method for FA analysis with high detection sensitivity, but it requires a complicated, tedious derivatization procedure, resulting in a number of drawbacks (e.g., losses and unexpected oxidation of some FAs, less stability of derivatives than

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