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Review

Liquid Chromatography-Mass Spectrometry (LC-MS) Derivatization-Based Methods for the Determination of Fatty Acids in Biological Samples

by
Christiana Mantzourani
and
Maroula G. Kokotou
*
Laboratory of Chemistry, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
*
Author to whom correspondence should be addressed.
Molecules 2022, 27(17), 5717; https://doi.org/10.3390/molecules27175717
Submission received: 1 August 2022 / Revised: 30 August 2022 / Accepted: 1 September 2022 / Published: 5 September 2022
(This article belongs to the Special Issue Lipids–Lipolytic Enzymes–Lipid Analysis: A Themed Issue Dedicated to Prof. Edward A. Dennis for His 80th Birthday)
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Versions Notes

Abstract

:
Fatty acids (FAs) play pleiotropic roles in living organisms, acting as signaling molecules and gene regulators. They are present in plants and foods and may affect human health by food ingestion. As a consequence, analytical methods for their determination in biological fluids, plants and foods have attracted high interest. Undoubtedly, mass spectrometry (MS) has become an indispensable technique for the analysis of FAs. Due to the inherent poor ionization efficiency of FAs, their chemical derivatization prior to analysis is often employed. Usually, the derivatization of the FA carboxyl group aims to charge reversal, allowing detection and quantification in positive ion mode, thus, resulting in an increase in sensitivity in determination. Another approach is the derivatization of the double bond of unsaturated FAs, which aims to identify the double bond location. The present review summarizes the various classes of reagents developed for FA derivatization and discusses their applications in the liquid chromatography-MS (LC-MS) analysis of FAs in various matrices, including plasma and feces. In addition, applications for the determination of eicosanoids and fatty acid esters of hydroxy fatty acids (FAHFAs) are discussed.

1. Introduction

Fatty acids (FAs) constitute one of the most important classes of lipids, and they are ubiquitous in every living organism. They are natural carboxylic acids with aliphatic chains, mostly found in esterified form as triglycerides (triacylglycerols, TAGs) or phospholipids, but also in their free form as carboxylic acids. In humans, they are present in biological fluids (for example, human plasma) and in various tissues. However, they are also present in almost every natural source, including plants and foods. They can be classified in various classes, depending on their chemical structural characteristics. According to their chain length, they are classified into short-chain FAs (SCFAs, 2–6 carbon atoms); medium-chain FAs (MCFAs, 7–12 carbon atoms); long-chain FAs (LCFAs, 13–22 carbon atoms); and very-long-chain FAs (VLFAs, >22 carbon atoms). According to their saturation degree, they are classified into saturated FAs (SFAs); monounsaturated FAs (MUFAs, a single double bond, usually cis); and polyunsaturated FAs (PUFAs, several double bonds, usually cis). PUFAs are further divided into ω-6 (or n-6) and ω-3 (or n-3) FAs, based on the distance of the first double bond from the terminal carbon atom.
In addition to their roles as structural elements in cell membranes as phospholipids or as the energy reservoir in adipose tissue in the form of TAGs, FAs play diverse roles in human health, acting as signaling molecules and gene regulators [1,2]. Intracellular free FAs (FFAs) together with their metabolites are able to affect gene expression upon interaction with the nuclear peroxisome proliferator-activated receptors (PPARs) [3], while several seven-transmembrane domain receptors have also been identified for the direct interaction of various classes of FFAs with them [4]. Long-chain FFAs are ligands for the FFA1 receptor (also known as GPR40) and FFA4 receptor (also known as GPR120), which indirectly affect energy homeostasis via hormonal signaling, thus, linking FAs and diet with metabolic disorders and type 2 diabetes (T2D) [5,6]. Short-chain FFAs bind to the FFA2 receptor (also known as GPR41) and FFA3 receptor (also known as GPR43), which are related to the gut-microbiome-mediated effects on health [7].
Apart from the common FAs, a great number of FAs bearing additional functional groups are present in many natural sources and living organisms. Such uncommon (rare) FAs are usually found at low concentrations; however they exert interesting bioactivities. These uncommon FAs include odd-chain, branched and cyclic FAs [8] and FAs bearing functional groups such as hydroxy [9,10,11,12], oxo [13,14], epoxy [15,16] and nitro [17,18].
Another very important class of lipidic carboxylic acids, which has attracted high medicinal interest, is the class of eicosanoids. These bioactive metabolites are generated by the oxidation of arachidonic acid, following its release from membrane glycerophospholipids by the enzymatic action of phospholipase A2 (PLA2) [19]. As shown in Figure 1, a variety of lipid messengers are produced by the activity of various enzymes on arachidonic acid, leading to compounds known as prostaglandins, leukotrienes, etc. [20]. All these carboxylic acids are involved in inflammatory diseases, including atherosclerosis, diabetes and cancer [21].
Recently, a new class of endogenous bioactive lipids has been identified, which is known as fatty acid esters of hydroxy fatty acids (FAHFAs) [22]. Each of the various classes of FAHFAs consist of multiple regio-isomers. As shown in the general structure of FAHFAs depicted in Figure 2, the hydroxy group connecting the hydroxy FA with the other FA chain by an ester bond may be at different positions (e.g., 5- or 9-). The bioactivities of FAHFAs and the methods for their analysis are summarized in recent review articles [23,24].
Due to the great importance of FAs and related lipid compounds such as eicosanoids and FAHFAs as bioactive agents and food ingredients, their determination in various matrices, including biological fluids, plants and foods, is of high interest. The classical method for the determination of FAs in various samples employs the use of gas chromatography combined with either flame ionization detection (GC-FID) or mass spectrometry detection (GC-MS), which requires the conversion of FAs into the corresponding methyl esters (FAMEs) [25,26]. However, the conversion of FAs to FAMEs has also been used for the analysis of monounsaturated branched chain FAs by electron ionization and covalent adduct chemical ionization tandem MS [27]. Dennis and coworkers presented a highly sensitive quantitative lipidomics analysis of FAs in biological samples by GC-MS, involving the conversion of FAs into corresponding esters by treatment with pentafluorobenzyl bromide [28]. Adopting various analytical methods, they demonstrated that lipidomics reveals a remarkable diversity of lipids in human plasma [29].
Liquid chromatography-mass spectrometry (LC/MS) methods for the determination of FAs in various samples have also been developed, involving or avoiding a derivatization step. Koletzko et al. developed a straightforward quantitative LC–tandem mass spectrometry (LC–MS/MS) method for the determination of non-esterified FAs (NEFAs) in plasma [30]. Masoodi et al. described a high-throughput quantitative lipidomics analysis of NEFAs in plasma by LC coupled to high-resolution mass spectrometry (LC-HRMS) [31]. Positional isomers such as polyunsaturated and branched-chain species were sufficiently separated and the possibility to perform untargeted screening was also demonstrated. Recently, we presented LC-HRMS methods for the rapid and direct determination of FFAs in milk [32] and human plasma from healthy and diabetic subjects [33]. Thus, the direct determination of common FFAs is possible by avoiding derivatization. However, when FFAs are present in samples at very low concentrations, for example in the case of eicosanoids or FAHFAs, methods offering high sensitivity are required.
Although FAs can be analyzed as [M–H]− in negative ion mode using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), as presented above in the previous representative examples, in some cases, insufficient ionization in negative ion mode has been observed, possibly due to the moderate gas-phase acidity of the carboxy group. This may result in limited analytical sensitivity. In addition, the characteristic and sufficient fragmentation of both saturated and unsaturated FAs is not usually observed via collision-induced dissociation [34]. To overcome these drawbacks, a strategy has been developed, which employs the charge reversal of the analytes by derivatization of the carboxyl group and conversion to a derivative, which can easily accept positive charge (Figure 3). Thus, MS may operate in positive ion mode, monitoring [M+H]+ [34,35].
Another derivatization approach is the functionalization of the double bond of unsaturated FAs, which is discussed in Section 3. Such a derivatization allows the identification of the position of the double bond in a lipidic chain.
The aim of the present review article is to summarize the reagents which have been used for the derivatization of FAs and, in particular, those used in order to reverse their charge and make them suitable for MS detection and quantification in positive ESI mode, instead of negative ESI mode. The derivatization reagents are categorized on the basis of their chemical structure and their functional groups. Various applications of these derivatization approaches for the determination of FAs in biological samples are discussed. The most recent advances in this growing field are summarized in the present review article, including the very recently reported approaches for the determination of the double bond location in FAs.

2. Derivatization of Carboxyl Group—Classes of Derivatizing Agents

2.1. Primary Amines

Coupling with primary amines has been a very popular approach in FA derivatization reactions for LC-MS analysis. This is attributed to the nucleophilicity of primary amines, which can result in the quantitative formation of amides under mild conditions. Numerous reagents have been developed for this purpose, bearing additional functional groups for charge reversal, mainly tertiary amines. Typical examples are primary aliphatic amines with simple structures such as DMED (2-dimethylaminoethylamine) (Figure 4) [36,37]. Zhu et al. used DMED and d4-DMED (d4-2-dimethylaminoethylamine) in eicosanoid labeling. The detection sensitivities of DMED labeled eicosanoids showed a 5–138-fold improvement in serum matrix compared to unlabeled analytes, and a good separation of different isomers was accomplished [36]. Later, the same team developed another method for the quantification of FAHFAs (PAHSAs, OAHSAs, SAHSAs and POHSAs) in various biological samples based on a SAX-SPE-CL-UHPLC–MS/MS analysis. Due to anion exchange interactions between the SAX cartridge and the analytes, the FAHFAs were selectively extracted and purified, while the DMED labeling enhanced their detection sensitivities in a UHPLC–MS analysis. The corresponding deuterium-labeled derivatives were used as internal standards and this method was implemented on the quantification of seven endogenous FAHFAs in different organs and tissue of rats and human serum [37]. Additionally, a DMED-FAHFA in silico library of 4290 high-resolution tandem mass spectra from 264 different FAHFA classes was constructed based on the MS/MS fragmentation patterns of DMED-FAHFA authentic standards, which were applied to computer-generated DMED-FAHFAs [38]. Recently, the same team established a derivatization-based in-source fragmentation-information-dependent acquisition (DISF-IDA) strategy for the profiling of submetabolomes by LC-ESI-Q-TOF MS. In particular, 36 carboxylated compounds labeled with DMED were used as model compounds for the profiling of carboxylated submetabolome in mice feces [39]. In 2020, DMED was also utilized for the labeling of short chain fatty acid esters of hydroxy fatty acids (SFAHFAs). With their method, Gowda et al. were able to identify new SFAHFAs in rat colon contents with high mass accuracy using UHPLC/LTQ Orbitrap MS in positive ion ESI mode with collision-induced dissociation (CID) [40]. In 2022, Gowda et al. also presented a sensitive method for the quantification of SCFAs and their hydroxyl derivatives, by LC–MS/MS, once again employing DMED as a derivatization reagent. The method was validated by analyzing spiked intestinal content samples, with limits of detection and quantification of SCFAs at 0.5 and 5 fmol, respectively [41].
Bian et al. developed a method for determining LCFAs in biological samples, including a derivatization step with cholamine (Figure 4), using HATU as the coupling reagent in the presence of HOBt. A 2000-fold increase in sensitivity was observed in comparison to the non-derivatization method, as well as an enhanced ionization and LCFA separation [42]. In 2018, the same group compared different primary amines for the derivatization of multiple carboxyl-containing metabolites. As a result, DIAAA (5-(diisopropylamino)amylamine) (Figure 4) proved to be the best choice, allowing the simultaneous determination of SCFAs and LCFAs among other metabolites, with satisfactory separation resolution using UHPLC-Q-TOF/MS [43]. Recently, Yang et al. developed a method for the quantitative analysis of n-3 PUFAs using cholamine-d0 and cholamine-d9 as labeling agents for biological samples and internal standards, respectively. Their method was applied to mouse serum and brain tissue with improved MS sensitivity and chromatographic separation [44].
Another approach in FA derivatization is the conversion to bis(hydroxymethyl) oxazoline derivatives using THAM (tris(hydroxymethyl)aminomethane) (Figure 4). Williams et al. established a solvent free, microwave assisted reaction for the conversion of FAs to the corresponding 2-oxazoline products in a single step. Using LC-APCI-MS, a 200-fold improvement in the limit of quantitation (LOQ) for palmitic and oleic acid was observed, and a 2-fold improvement for arachidonic acid [45]. Additionally, primary amines containing benzofurazan moieties were synthesized and utilized for a LC-MS/MS analysis of FAs. Specifically, DAABD-AE ({4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole}) (Figure 4) was applied to an FA analysis in rat plasma samples. The derivatization reaction took place at 60 °C for 30 min and the derivatized FAs were separated on a reversed-phase column, with LODs in the femtomole range [46]. In 2020, Zheng et al. developed a fluorous derivatization method for the quantification of LCUFAs in biological samples. LCUFAs were perfluoroalkylated with PFPA (3-(perfluorooctyl)-propylamine) (Figure 4) and retained on a fluorous phase LC column, achieving a limit of detection (LOD) at an atto-molar level and determining eight LCUFAs with high sensitivity [47].
Other primary amines that have been employed for the derivatization of FAs bear pyridine or quinoline moieties that can be easily protonated. For example, AMPP (N-(4-aminomethylphenyl)-pyridinium) (Figure 4) is a common derivatization reagent containing a pyridine moiety. Bollinger et al. first described the synthesis of AMPP in 2010, reporting that the derivatization of eicosanoids with AMPP improved the sensitivity of detection by 10- to 20-fold compared to the negative mode ESI detection of the underivatized analytes. This protocol was then used for the detection of eicosanoids in complex biological samples with LOQs in the 200–900 fg range [48]. The same team later demonstrated that coupling FAs with AMPP using EDCI as a coupling reagent resulted in a 60000-fold increase in sensitivity in comparison to the non-derivatization method. Moreover, analytical specificity was improved due to the significant fragmentation of the precursor ions, and their method was successfully applied to mouse serum [49]. Liu et al. used AMPP in their charge-switch derivatization protocol in an effort to label linoleic acid, arachidonic acid and docosahexaenoic acid metabolites. Their method yielded a 10- to 30-fold increase in ionization efficiency, with LOQs ranging between 0.05 and 6.0 pg [50]. Furthermore, Narreddula et al. developed dual-function derivatization reagents based on AMPP, in order to improve FA detection. The 4-I-AMPP (1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium) (Figure 4) derivatives include a selectively photoactivated aryl-iodide moiety, in order to generate structurally diagnostic ions via radical-directed dissociation. The derivatization of diverse FA structures yielded photodissociation mass spectra with characteristic radical-driven fragmentation patterns, enabling the distinction of isomers [51]. On the other hand, one derivatization reagent that contains a quinoline moiety is AMQ (4-aminomethylquinoline) (Figure 4). AMQ has been used in SCFA analysis in human fecal samples by UPLC/MS/MS. Fu et al. published a new method with a short reaction and analysis time, which enabled the quantitation of SCFAs with improved sensitivity [52]. Finally, APBQ (1-(3-aminopropyl)-3-bromoquinolinium bromide) (Figure 4) is a reagent that possesses a permanent positive charge as well as a bromine atom in its structure. Mochizuki et al. achieved a qualitative and quantitative determination of a series of FAs in human plasma and saliva after derivatization with APBQ, taking advantage of the bromine isotope pattern and the improved sensitivity caused by the permanent positive charge [53].
Picolylamines are primary amines attached to a pyridine ring, a characteristic that renders them promising agents for FA tagging. 3-PA (3-picolylamine) (Figure 4) was employed by Li et al. in a derivatization method for FA analysis using orbitrap mass spectrometry in positive ESI mode, which provided enhanced sensitivity and selectivity. They reported that this method had an LOD in the low femtomole range, and 14 saturated and unsaturated FAs were separated in a 15 min run [54]. In addition, Nagatomo et al. used 2-PA (Figure 4) in their method for the determination of 10 SCFAs in the fecal samples of obese type II diabetes mice [55]. Lastly, Wu et al. also used 2-PA for the derivatization of FAs and other metabolites in seminal plasma samples. With their developed method, they achieved a 44 to 1500-fold enhancement of FA signals in positive ESI mode [56].
Interestingly, (R)-(+)-1-phenylethylamine (R-PEA) (Figure 4) has been also tested as a derivatization reagent for FFA detection in soil samples. Soil sample extracts were first treated with ethyl chloroformate and triethylamine to afford anhydride intermediates that were then reacted with R-PEA. This protocol resulted in chiral FA derivatives that could be separated with the use of chiral chromatographic columns [57]. In 2022, Zhu et al. developed a two-step derivatization strategy for the identification of 2/3-OHUFAs and 2/3-OHFAs. The first step was the derivatization with ADMI (4-amino-1,1-dimethylpiperidin-1-ium iodide hydrochloride) (Figure 4), followed by mCPBA-oxidation. This protocol enabled the fast and accurate determination of 2/3-OHUFAs and 2/3OHFAs in complex matrices without the use of standards and was applied in a mouse melanoma model analysis [58]. Another interesting approach involves the use of derivatization reagents that contain a bromine atom. In particular, APBP (1-(3-aminopropyl)-3-bromopyridinium bromide hydrochloride) (Figure 4) has been used as a labeling agent, and a subsequent cluster analysis of the derivatized mixture of compounds can be employed in order to resolve the individual derivatized species [59]. Recently, an isotope-free method with dual derivatization using LC-MS was reported for the quantification of SCFAs. According to this method, DMED or Dns-HZ (N,N-dimethyldansulfonyl hydrazide) were used to label the samples, and their structural analogues DEEA (N,N-diethyl ethylene diamine) (Figure 4) or Dens-HZ (N,N-diethyldansulfonyl hydrazide) tagged standard mixtures. Specifically, DMED/DEEA was employed for the dual derivatization and quantification of fecal SCFAs from hepatocellular carcinoma patients and healthy individuals [60].

2.2. Secondary Amines

To increase the hydrophobicity and retention times of analytes in biological samples, various secondary amines have been designed and tested as derivatization reagents for FA analysis. As an example, DMPP (2,4-dimethoxy-6-piperazin-1-yl pyrimidine) (Figure 5) was introduced by Leng et al. in a protocol with EDCI as the coupling reagent [61], and in a subsequent publication, with oxalyl chloride, which was added prior [62]. This protocol, followed by LC-ESI-MS/MS, was applied in human thyroid samples, where 17 FFAs exhibited differences in quantity in thyroid carcinoma samples and para-carcinoma samples [62]. Other reagents that contain a piperazine moiety are Dns-PP (5-dimethylamino-naphthalene-1-sulfonyl piperazine) and Dens-PP (5-diethylamino-naphthalene-1-sulfonyl piperazine) (Figure 5). These structural analogs have been used in twins derivatization protocols for the quantification of FFAs, where Dens-PP is used for labeling the internal standards. Jiang et al. compared this protocol to a non-derivatization protocol and indicated that the detection sensitivities of the analytes were 50 to 1500-fold increased. With their method, they managed to quantify 38 FFAs in rat serum [63]. A similar protocol was used by ** in the differentiation of FA isomers and the identification of new FAs.

Author Contributions

Conceptualization, M.G.K.; investigation, C.M. and M.G.K.; writing—original draft preparation, C.M. and M.G.K.; writing—review and editing, C.M. and M.G.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Molecules 27 05717 g001 550
Figure 1. Generation of eicosanoid mediators from arachidonic acid.
Figure 1. Generation of eicosanoid mediators from arachidonic acid.
Molecules 27 05717 g001
Molecules 27 05717 g002 550
Figure 2. Structure of fatty acid esters of hydroxy fatty acids (FAHFAs).
Figure 2. Structure of fatty acid esters of hydroxy fatty acids (FAHFAs).
Molecules 27 05717 g002
Molecules 27 05717 g003 550
Figure 3. Basic principle of FA derivatization for its charge reversal.
Figure 3. Basic principle of FA derivatization for its charge reversal.
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Molecules 27 05717 g004 550
Figure 4. Primary amines as derivatization reagents for LC-HRMS analysis of FFAs.
Figure 4. Primary amines as derivatization reagents for LC-HRMS analysis of FFAs.
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Molecules 27 05717 g005 550
Figure 5. Secondary amines as derivatization reagents for LC-HRMS analysis of FFAs.
Figure 5. Secondary amines as derivatization reagents for LC-HRMS analysis of FFAs.
Molecules 27 05717 g005
Molecules 27 05717 g006 550
Figure 6. Aromatic amines, hydrazines and hydrazides as derivatization reagents for LC-HRMS analysis of FFAs.
Figure 6. Aromatic amines, hydrazines and hydrazides as derivatization reagents for LC-HRMS analysis of FFAs.
Molecules 27 05717 g006
Molecules 27 05717 g007 550
Figure 7. Bromides, hydroxylamines and other derivatization reagents for LC-HRMS analysis of FFAs.
Figure 7. Bromides, hydroxylamines and other derivatization reagents for LC-HRMS analysis of FFAs.
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Molecules 27 05717 g008 550
Figure 8. Principle of the combination of the Paternò–Büchi reaction with tandem mass spectrometry (MS/MS) for the determination of the double bond location in UFAs.
Figure 8. Principle of the combination of the Paternò–Büchi reaction with tandem mass spectrometry (MS/MS) for the determination of the double bond location in UFAs.
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Molecules 27 05717 g009 550
Figure 9. Ketones and aldehydes used for the derivatization of UFAs and determination of their double bond location.
Figure 9. Ketones and aldehydes used for the derivatization of UFAs and determination of their double bond location.
Molecules 27 05717 g009
Table
Table 1. Summary of FA carboxyl group derivatization reagents and their applications using LC-HRMS.
Table 1. Summary of FA carboxyl group derivatization reagents and their applications using LC-HRMS.
Derivatization ReagentAnalytical TechniqueInstrumental AnalysisColumn/Mobile PhaseSample Preparation—
Solvent Extraction/
Cartridge-Column		SampleAnalyteRef.
DMEDUPLC–ESI–MS/MS
(+) ESI mode		ABI/SCIEX
4500 Triple QuadTM coupled to
Shimadzu LC-30AD UPLC.		Acquity UPLC BEH phenyl column (2.1 mm × 50 mm, 1.7 μm, Waters) / (Solvent A) HCOOH in H2O (0.1%, v/v) and (Solvent B) ACN/MeOH (7/3, v/v); flow rate 0.4 mL/min; temperature 40 °C.Extraction with EtOAc containing 10 μL of BHT (0.10 mM) and 10 μL of 0.5% HCOOHSerumCytochrome P450 metabolites of arachidonic acid[36]
DMEDUHPLC-ESI–MS/MS
(+) ESI mode		Shimadzu MS-8040 mass spectrometer (Tokyo, Japan) with an electrospray ionization source (Turbo Ionspray) coupled to a Shimadzu LC-30AD UPLC system (Tokyo, Japan)Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm, Waters). The mobile phase consisted of (A) HCOOH in ACN/H2O (0.1%, 6/4, v/v) and (B) HCOOH in IPA/ACN (0.1%, 9/1, v/v); flow rate 0.4 mL/min; temperature 40 °CExtraction with cold saline solution and ACN containing 0.1% NH3/ SAX SPE-cartridge (1 mL, 50 mg) Weltech Co. (Wuhan, China)Rat tissue Human serumFAHFAs[37]
DMEDUHPLC/LTQ-Orbitrap MS
(+) ESI mode		LTQ-Orbitrap-MS coupled to Shimadzu UHPLC.Atlantic T3 C18 reverse-phase column (2.1 mm × 150 mm, 3 µm, Waters, Milford, MA, USA) / (Solvent A) 10 mM of aqueous CH3COONH4 with 0.1% CH3COOH, (Solvent B) IPA, (Solvent C) MeOH; flow rate 0.2 mL/min; temperature 40 °C.Folch methodColon contentsShort chain FAHFAs[40]
DMEDLC-MS/MS
(+) ESI mode		Prominence UFLC (Shimadzu, Kyoto, Japan) coupled to TSQ Quantum Mass Spectrometer System (Thermo Fisher Scientific, San Jose, CA, USA)Hypersil gold C8 column (50 mm × 2.1 mm, 5 μm; Thermo Fisher Scientific) / Mobile phase A: 20 mM CH3COONH4, B: MeOH and ACN (1:1); flow rate 0.4 mL/min; temperature 40 °C.Extraction with ACNIntestinal contentsSCFAs and hydroxy SCFAs[41]
CholamineUHPLC-Q-TOF/MS
(+) ESI mode		Agilent 6550 UHD accurate-mass Q-TOF/MS system coupled to Agilent 1290 Infinity LC system (UHPLC, Santa Clara, CA, USA).Agilent Eclipse XDB-C18 column (2.1 mm × 100 mm, 1.8 μm) / Mobile phase A and B were 0.1% HCOOH-containing H2O and 0.1% HCOOH-containing ACN; temperature 40 °C.Extraction with EtOAc
SerumLong chain FFAs[42]
CholamineLC-MS/MS
(+) ESI mode		Xevo TQD triple-quadrupole tandem mass spectrometry coupled to an ACQUITY UPLC system (UPLC-QQQ-MS/MS, Waters Corp., Manchester, UK).ACQUITY BEH C18 column (150 mm × 2.1 mm i.d., 1.7 μm) / ACN containing 0.1% HCOOH (A, v/v) and 0.1% aqueous HCOOH solution (B, v/v); flow rate 0.3 mL/min; temperature 45 °C.Extraction with cold EtOAc containing 0.5% HCOOHSerumn-3 PUFAs and their metabolites[44]
DIAAAUHPLC-Q-TOF/MS
(+) ESI mode		Agilent 6550 UHD accurate-mass Q-TOF/MS coupled to an Agilent 1290 Infinity LC system (UHPLC, Santa Clara, CA, USA)Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) / Mobile phase A and B were 0.1% HCOOH containing H2O and 0.1% HCOOH containing ACN; flow rate 0.3 mL/min; temperature 40 °C.Extraction with cold MeOHSerumSCFAs and LCFAs[43]
PFPAUPLC-MS/MS
(+) ESI mode		5500 QTRAP mass spectrometer (AB Sciex, Foster City, CA, USA) coupled to a Shimadzu LC-30AD UHPLC system (Tokyo, Japan).Thermo Scientific Accucore pentafluorophenyl (PFP) column (2.1 mm × 15 mm, 2.6 μm) / 0.1% HCOOH in H2O/MeOH (3:7, v/v) as mobile phase A and 0.1% HCOOH in MeOH/IPA (6:4, v/v) as mobile phase B; flow rate 0.3 mL/min; temperature 40 °C.Extraction with cold CH2Cl2/MeOH (2:1, v/v)Serum, lung tissueLCUFAs[46]
DAABD-AELC–ESI-MS
(+) ESI mode		HP 1090 series II system (Hewlett-Packard GmbH) coupled to an ESI ion trap spectrometer (Esquire 3000+, Brucker Daltonics, Billeria, MA, USA).Capcellpak C18 (35 mm × 2.0 mm, i.d., 5 µm; Shiseido, Tokyo, Japan) / Mobile phase A: ACN–H2O (10:90, v/v) containing 0.1% HCOOH, and mobile phase B: ACN–H2O (90:10, v/v); flow rate 0.2 mL/min.Bligh and Dyer methodRat plasmaFAs[47]
AMPPLC/ESI-MS/MS
(+) ESI mode		Waters Xevo TQ triple quadrupole mass spectrometer coupled to an Acquity UPLC.Waters Acquity UPLC BEH Shield RP18 (2.1 mm × 100 mm, 1.7 μm) / Solvent A: 100% H2O/0.1% HCOOH, solvent B: ACN/0.1% HCOOH; flow rate 0.4 mL/min; temperature 45 °C.Extraction with MeOH/1N HCl and isooctaneSerumFAs[48]
AMPPLC/ESI-MS/MS
(+) ESI mode		LTQ Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to a Surveyor HPLC system (Thermo Scientific).C18 reverse phase column (Ascentis Express, 2.7 μm particles, 150 mm × 2.1 mm) / Solvent A: 0.1% glacial CH3COOH in H2O) and solvent B: 0.1% glacial CH3COOH in ACN; flow rate 0.2 mL/min; temperature 23 °C.Bligh and Dyer procedure/ Strata-X SPE cartridge (30 mg/mL) Phenomenex (Torrance, CA, USA).Hepatic tissueLinoleic acid, arachidonic acid, docosahexaenoic acid metabolites[49]
AMQUPLC/MS/MS
(+) ESI mode		Triple quadrupole 5500 mass spectrometer (AB SCIEX, Redwood City, CA, USA) coupled to a Waters ACQUITY UPLC I-Class system (Waters, Milford, MA, USA).Waters BEH C18 (2.1 mm × 100 mm I.D., 1.7 μm) UPLC column / H2O:HCOOH (1000:1, v/v; solvent A) and MeOH:HCOOH (1000:1, v/v; solvent B); flow rate 0.4 mL/min; temperature 50 °C.Extraction with ethanolFecesSCFAs[51]
APBQHPLC-ESI-MS/MS
(+) ESI mode		API 3000 triple quadrupole-mass spectrometer (Applied Biosystems, Foster City, CA, USA) coupled to an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA).XbridgeTM C18 column (3.5 μm, 150 mm × 2.1 mm id; Waters, Milford, MA, USA) / H2O / ACN containing 0.1% v/v HCOOH; flow rate 0.2 mL/min; temperature 40 °C.EtOH/SPE cartridge (SOLA-C18, 10 mg/mL, Thermo Scientific, Bellefonte, PA, USA).Plasma, salivaFAs[52]
2-PAUPLC-ESI/MS/MS
(+) ESI mode		Waters Xevo TQD triple quadrupole mass spectrometer coupled to a Waters Acquity H Class UPLC system (Waters Co., Milford, MA, USA).Acquity BEH C18 column (1.7 μm, 2.1 mm × 100 mm) / Mobile phase A and B were 0.1% HCOOH in H2O and 0.1% HCOOH in MeOH; flow rate 0.3 mL/min; temperature 40 °C.Extraction with MeOHFecesSCFAs[56]
(R)-PEALC-DAD-MS
(+) API mode		Finnigan LCQ DECA XP MAX (Finnigan, San Jose, CA, USA) quadrupole IT equipped with an API source coupled to a Finnigan Surveyor series liquid chromatograph.Chromolith RP-18 column (125 4.6 mm) from Merck KGaA / MeOH / H2O (90:10); flow rate 0.5 mL/min; temperature 25 °C.Soxhlet extraction with 1:1 CH2Cl2/ether solutionSoil samplesFFAs[57]
ADMILC–ESI–MS/MS
(+) ESI mode		(UPLC-Q/TOF) MS System (H-class UPLC and Synapt G2-Si MS, Waters, Milford, MA, USA)ACQUITY UPLC BEH C8 column (2.1 mm × 100 mm, 1.8 μm) / Mobile phase A and B were 0.1% HCOOH containing H2O and 0.1% HCOOH containing ACN; flow rate 0.4 mL/min; temperature 45 °C.Addition of H2O and extraction with EtOAcMouse melanoma samples2/3 OHUFAS
2/3 OHFAS		[58]
DMPPLC–ESI–MS/MS
(+) ESI mode		Triple-quadrupole time-of flight (Q-TOF) mass spectrometer (G6500, Agilent) coupled to an HPLC system (1260 Series LC, Agilent) and an ESI source.Agilent Zorbax SB-C8, 2.1 mm × 100 mm, 1.8 μm, Santa Clara, CA, USA) / deionized H2O (solvent A) and ACN (solvent B); flow rate 0.3 mL/min.Oasis HLB cartridge (1 cc, Waters Corporation)UrineFFAs[61]
DMPPLC–ESI–MS/MS
(+) ESI mode		Triple-quadrupole mass spectrometer (G6410A, Agilent, Santa Clara, CA, USA).Agilent Zorbax SB-C8, 2.1 mm × 100 mm, 1.8 μm, Santa Clara, CA, USA) / deionized H2O (solvent A) and ACN (solvent B); flow rate 0.3 mL/min.Extraction with CHCl3–MeOH (4:1, v/v)Human thyroidFFAs[62]
Dns-PP/Dens-PPLC-MS/MS
(+) ESI mode		MS-8040 triple-quadrupole mass spectrometer (Shimadzu Co., Tokyo, Japan) equipped with an ESI source coupled to a Shimadzu Nexera UPLC system.Agilent Zorbax Eclipse XDB-C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent Technologies, Santa Clara, CA, USA)/Mobile phase A: 0.1% HCOOH in H2O, mobile phase B: MeOH; flow rate 0.4 mL/min; temperature 50 °C.Extraction with 0.5% HCOOH in H2O and EtOAcSerumFFAs[63]
Dns-PP/Dens-PPLC-MS/MS
(+) ESI mode		ACQUITY™ ultra performance liquid chromatography (UPLC) coupled to Xevo TQD triple-quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS, Waters, Manchester, UK).Hypersil GOLD™ C18 column (150 mm × 2.1 mm i.d., 1.9 μm) / Mobile phase A (0.1% aqueous HCOOH solution) and mobile phase B (ACN/0.1% HCOOH); flow rate 0.4 mL/min; temperature 40 °C.Oasis HLB cartridge (30 mg, 1 cc, Waters, Manchester, UK)PlasmaEicosanoids[64]
DHPPLC-MS/MS
(+) ESI mode		UHPLC coupled to high-resolution orbitrap fusion mass spectrometer (Ultimate 3000 RSLC-Orbitrap Fusion, Thermofisher scientific, Waltham, MA, USA).Phenomenex polar C18 column (1.6 μm, 2.1 mm × 150 mm) / Mobile phase A: H2O/ 0.1% HCOOH and mobile phase B: ACN; flow rate 0.3 mL/min; temperature 30 °C.Extraction with precooled MeOHFeces, serum, liver tissueSCFAs and OH-SCFAs[65]
AnilineLC-MS/MS
(+) ESI mode		AB Sciex QTRAP 5500 hybrid linear ion-trap quadrupole mass spectrometer equipped with a TurboIonSpray source (Applied Biosystems, Foster City, CA) coupled to an Agilent 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA).Acquity UPLC HSS T3 (1.8 μm, 2.1 mm × 100 mm) / H2O and HPLC-grade IPA, both acidified with 0.1% HCOOH; flow rate 0.35 mL/min; temperature 50 °C.Extraction with 1:1 v/v ACN/H2OHuman stoolSCFAs[66]
3-NPHUPLC-MS/MS
(-) ESI mode		4000 QTRAP triple-quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada) with an ESI source coupled to Ultimate 3000 RSLC system (Dionex Inc., Amsterdam, The Netherlands).Waters BEH C18 (2.1 mm × 100 mm, 1.7 μm) / H2O:HCOOH (100:0.01, v/v; solvent A) and ACN:HCOOH (100:0.01, v/v; solvent B); flow rate 0.35 mL/min; temperature 40 °C.Extraction with 50% aqueous ACNFecesSCFAs[67]
3-NPHLC-MS/MS
(-) ESI mode		AB Sciex 3200 QTRAP (Sciex, Milan, Italy) coupled to an HPLC Dionex 3000 UltiMate system (Thermo Fisher Scientific, MA, USA).Restek Raptor C18 (2.7 μm, 2.1 mm × 100 mm, Bellefonte, PA, USA) / Mobile phase A: H2O +0.1% HCOOH and mobile phase B: ACN; flow rate 0.4 mL/min; temperature 35 °C.Extraction with MeOH/0.05% HCOOH, diluted with deionized H2OSerumSCFAs and MCFAs[68]
2-NPHLC-MS/MS
(-) ESI mode		Thermo Finnigan Surveyor HPLC-TSQ Quantum Quadrupole mass spectrometer system (Thermo Fisher Scientific Inc., Waltham, MA, USA).Ascentis® Express Phenyl-Hexyl column (5 cm × 2.1 mm I.D., 2.7 μm, Supelco, Inc., Bellefonte, PA) / 5 mM aqueous CH3COONH4 (A), IPA (B), and MeOH(C); flow rate 0.2 mL/min; temperature 40 °C.Saponification with 0.3 M KOH-EtOHPlasmaSCFAs, MCFAs, LCFAs, VLCFAs[70]
T3UPLC-MS/MS
(+) ESI mode		Agilent 1290 series UPLC system coupled to an Agilent 6490
triple-quadrupole mass spectrometer (Agilent Technologies, Inc.
Santa Clara, CA, USA) with an AJS electrospray ionization (AJS-ESI) device.		UPLC BEH C18 column (1.7 μm, 100 mm × 2.1 mm i.d.) / Solvent A: H2O (0.2% HCOOH) and solvent B: ACN (0.2% HCOOH); flow rate 0.5 mL/min; temperature 45 °C.Extraction with MeOH (containing 0.001 M of BHT)/ HLB (30 mg, 1 cc) SPE cartridgePlasma, heart tissueEicosanoids[71]
GTLC-MS/MS
(+) ESI mode		Agilent 6420 triple quadrupole LC/MS (Santa Clara, CA, USA) coupled to an Agilent 1260 Infinity Binary LC (Santa Clara, CA, USA).Agilent Zorbax HILIC plus column (4.6 mm × 100 mm, 3.5 μm) / Solvent A: H2O containing 20 mM of CH3COONH4 and 20 mM of CH3COOH, solvent B: 100% ACN; flow rate 0.5 mL/min.Dilution with distilled H2OGut bacteria E. rectale culture mediumSCFAs[72]
PABrLC-MS/MS
(+) ESI mode		LC-MS/MS 8050, Shimadzu Corporation, (Kyoto, Japan).xBridge C18 column (100 mm × 2.1 mm × 3.5 μm, Waters, Milford, MA, USA) / HCOOH:H2O (0.1:100, v/v) (phase A) and MeOH (phase B); flow rate 0.4 mL/min; temperature 30 °C.Dilution in H2O and saturated sodium carbonate solutionPlasma, fecesSCFAs[76]
DmPABrUPLC-MS/MS
(+) ESI mode		AB Sciex QTrap 6500 mass spectrometer (Framingham, MA, USA) coupled to a Waters Acquity UPLC Class II (Milford, MA, USA)AccQ-tag C18 column (2.1 mm × 100 mm, 1.4 μm,
Milford, MA, USA) / Mobile phase A: H2O containing 10 mM of NH4COOH and 0.1% HCOOH, mobile phase B: 100% ACN; flow rate 0.7 mL/min; temperature 60 °C.		Extraction with H2O/MeOH
(1:4 v/v)		HepG2 cellsFFAs[77]
O-BHALC-MS/MS
(+) ESI mode		5500 triple-quad mass spectrometer (Sciex, Concord, ON, Canada) equipped with Turbospray ESI source coupled to a Shimadzu Nexera X2 UHPLC system (Shimadzu, Kyoto, Japan).Kinetex C18 (100 mm × 2.1 mm 2.6 μm, Phenomenex, Torrance, CA, USA) / 0.1% HCOOH in H2O with 10 mM of NH4COOH (solvent A) and 0.1% HCOOH in MeOH: IPA (9:1 v/v) (solvent B); flow rate 0.4 mL/min; temperature 45 °C.Extraction with MeOHPlasmaSCFAs[78]
AABD-SHLC-MS/MS
(+) ESI mode		QTRAP 5500 (ABSciex, Framingham, MA, USA) coupled to a 1290 HPLC instrument (Agilent Technologies, Glostrup, Denmark).Pursuit 5 C18 (150 × 2.0 mm; Agilent Technologies, Santa Clara, CA, USA) / Mobile phase A (0.1 % HCOOH in H2O) and mobile phase B (0.1% HCOOH in ACN); flow rate 0.5 µL/min; temperature 40 °C.Dilution with H2OFeces, plasmaSCFAs[80]
TMAELC-MS/MS
(+) ESI mode		Finnigan TSQ Quantum Ultra AM mass spectrometer (Thermo Electron Corporation, San Jose, CA, USA) coupled to a Hitachi L-2130 pump equipped with Hitachi Autosampler L-2200 (Hitachi, San Jose, CA, USA)Varian Pursuit Diphenyl 3μm column (150 mm × 2 mm i.d., 3μm) / Solvent A: 5 mM of CH3COONH4 in H2O, solvent B: 5mM of CH3COONH4 in ACN; flow rate 0.5 mL/min; temperature 25 °C.Hydrolysis with 40% aqueous KOH and extraction with diethyl ether/hexaneAtherosclerotic plaques from carotid arteriesFAs[81]
Table
Table 2. Summary of UFA derivatization reagents and their applications.
Table 2. Summary of UFA derivatization reagents and their applications.
Derivatization ReagentAnalytical TechniqueInstrumental AnalysisColumn/Mobile PhaseSample Preparation—
Solvent Extraction/
Cartridge-Column		SampleAnalyteRef.
AcetoneMS/MS
(-) ESI mode		4000 QTRAP triple quadrupole/linear ion trap (LIT) hybrid mass spectrometer (Sciex, Toronto, Canada) -Extraction with MeOH/iso-octaneRat brain tissueUFAs[83]
AcetoneMS/MS
(-) ESI mode		4000 QTRAP (Applied Biosystems, Thornhill, Ontario, Canada)-Bligh−Dyer methodRAW 264.7 cellsPUFAs[84]
AcpyMS/MS
(+) ESI mode		Orbitrap Q Exactive HF instrument (Thermo Fisher Scientific GmbH, Germany)-Extraction with MTBEMouse brain tissueMUFAs and PUFAs[85]
TriFAPMS/MS
(+) ESI mode		QTRAP 4500 mass spectrometer (SCIEX, Toronto, Canada)HILIC column Sigma-Aldrich, MO, USA (150 mm × 2.1 mm, 2.7 μm) / 10 mM of aqueous CH3COONH4 (A), ACN (B); flow rate 0.2 mL/min; temperature 30 °C.Extract from Avanti Polar Lipids, Inc. (Alabaster, AL, USA)Lipid extract from bovine liverUFAs[87]
2-AcpyMS/MS
(+) ESI mode		X500R QTOF mass spectrometer (Sciex, Toronto, Canada) coupled to a Shimadzu LC-20AD system (Kyoto, Japan)C18 column Sigma-Aldrich, MO, USA (150 mm × 3.0 mm, 2.7 μm) / H2O:ACN (40:60, v/v with 20 mM ofHCOONH4) (A), IPA:ACN (40:60, v/v with 0.2% FA).(B); flow rate 0.45 mL/min.Folch methodHuman plasmaFAs and UFAs[88]
3-PYAUHPLC–MS
(+) ESI mode		QTRAP 6500 + (AB SCIEX, Framingham, MA, USA) coupled to a Shimadzu LC-30AD system (Shimadzu, Kyoto, Japan)ACQUITY UPLC BEH C 18 column (Waters, 1.7 μm, 2.1 mm × 100 mm) / H2O (A), ACN (B). both mobile phases contained 5 mM of CH3COONH4; flow rate 0.3 mL/min.Extract from Aladdin Trading Co., Ltd. (Shanghai, China)Lipid extract from bovine liverUFAs[90]
BIQD
DF-BIQD		LC–MS/MS
and
MALDI–MS/MS
(+) ESI mode		ESI-Q-TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) and MALDI-TOF/TOF mass spectrometer (rapiflex TM, Bruker Daltonics, Billerica, MA, USA)ACQUITY UPLC HSS T3 column (Waters, 3 mm ×100 mm, 1.8 μm) / H2O, 0.1% FA (A), ACN/IPA, 9:1, v/v, 0.1% FA, (B); flow rate 0.25 mL/min; temperature 35 °C.Extraction with saline solution and ACNRat heart, brain, lung, spleen, thymus, kidney, liver and plasma samplesUnsaturated lipids[91]
Table
Table 3. Advantages of derivatization for the analysis of FAs.
Table 3. Advantages of derivatization for the analysis of FAs.
Type of derivatizationSensitivityFragmentation
Carboxylic group derivatizationIncreaseIncrease structural information
Double bond derivatizationIncreaseIdentification of the double bond location
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Mantzourani, C.; Kokotou, M.G. Liquid Chromatography-Mass Spectrometry (LC-MS) Derivatization-Based Methods for the Determination of Fatty Acids in Biological Samples. Molecules 2022, 27, 5717. https://doi.org/10.3390/molecules27175717

AMA Style

Mantzourani C, Kokotou MG. Liquid Chromatography-Mass Spectrometry (LC-MS) Derivatization-Based Methods for the Determination of Fatty Acids in Biological Samples. Molecules. 2022; 27(17):5717. https://doi.org/10.3390/molecules27175717

Chicago/Turabian Style

Mantzourani, Christiana, and Maroula G. Kokotou. 2022. "Liquid Chromatography-Mass Spectrometry (LC-MS) Derivatization-Based Methods for the Determination of Fatty Acids in Biological Samples" Molecules 27, no. 17: 5717. https://doi.org/10.3390/molecules27175717

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