Chromatographic analysis of lipoxygenase products

Analytica Chimica Acta 465 (2002) 319–335

Review

Chromatographic analysis of lipoxygenase products Manuela Pérez Gilabert, Francisco Garc´ıa Carmona∗ Departamento de Bioqu´ımica y Biolog´ıa Molecular-A, Facultad de Biolog´ıa, Universidad de Murcia, E-30001 Murcia, Spain Received 31 October 2001; received in revised form 22 February 2002; accepted 28 February 2002

Abstract Lipoxygenases (LOXs) are non-heme iron-containing enzymes, widely distributed both in the plant and animal kingdom. They catalyse the regio- and stereospecific dioxygenation of polyunsaturated fatty acids (PUFAs) which contain a (1Z,4Z)-pentadiene system. Although, most LOXs catalyze the formation of one particular regioisomer, it has become apparent that several LOXs exhibit a dual or even multiple positional specificity. Hydroperoxides, the primary products of LOX, are short-lived and are transformed into various families of metabolites. In plants, hydroperoxides are metabolized via several secondary pathways to form bioactive compounds such as jasmonates. In animals, the oxidation of arachidonic acid (AA) by LOX is the source of highly active bioregulators such as leukotrienes and lipoxins. The ability of LOX products to initiate the synthesis of different signaling molecules is determined by the positional and stereospecific nature of the hydroperoxides produced. Thus, the complete characterization of LOX products is essential for establishing the physiological role of this enzyme. Different methods to determine the positional specificity of LOX products have been proposed. In this review, we will describe the different chromatographic methods (RP-, SP- and CP-HPLC, LC/MS and GC/MS) reported to date for analyzing the regio- and stereospecificity of the primary reaction products of LOX. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Lipoxygenase; Lipoxygenase products; Hydroperoxides; Regiospecificity; Stereospecificity; RP-HPLC; SP-HPLC; CP-HPLC; Chirality; GC/MS; LC/MS; Electrospray

Abbreviations: AA, arachidonic acid; AcH, acetic acid; ACN, acetonitrile; ADAM, anthryl diazomethane; APCI, atmospheric pressure chemical ionization; BSTFA, bis(trimethylsilyl)-trifluoroacetamide; CID, collision-induced dissociation; CP-HPLC, chiral-phase HPLC; DL-PC, dilinoleoyl phosphatidylcholine; DNBPG, dinitrobenzoyl phenylglycine; DPPP, diphenyl-1-pyrenylphosphine; diHETE, dihydroxy eicosatetraenoic acid; diHPETE, dihydroperoxy eicosatetraenoic acid; EC, electrochemical; ELSD, evaporative light-scattering detector; EOT, epoxy-octadecatrienoic acid; ES, electrospray; FAB, fast atom bombardment; GC/MS, gas chromatography mass spectrometry; HEPE, hydroxy eicosapentaenoic acid; HETE, hydroxy eicosatetraenoic acid; HODE, hydroxy octadecadienoic acid; HOTE, hydroxy octadecatrienoic acid; HPETE, hydroperoxy eicosatetraenoic acid; HPLC, high performance liquid chromatography; HPOD, hydroperoxy octadecadienoic acid; HPOT, hydroperoxy octadecatrienoic acid; KETE, ketoeicosatetraenoic acid; KODE, ketooctadecadienoic acid; LA, linoleic acid; LC/MS, liquid chromatography mass spectrometry; LOX, lipoxygenase; LT, leukotrienes; LTB4 , leukotriene B4 ; MS/MS, mass spectrometry/mass spectrometry (tandem mass spectrometry); NICI, negative-ion chemical ionization; ODS, octadecylsilyl; PGB2 , prostaglandin B2 ; PUFA, polyunsaturated fatty acids; RP-HPLC, reversed-phase HPLC; SP-HPLC, straight-phase HPLC; TFA, trifluoroacetic acid; THETE, trihydroxy eicosatetraenoic acid; TMS, trimethyl silyl derivative; TSP, thermospray; UV, ultraviolet ∗ Corresponding author. Tel.: +34-968-36-47-65; fax: +34-968-36-47-65. E-mail address: [email protected] (F.G. Carmona). 0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 1 9 2 - 7

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1. Introduction Lipoxygenases (LOXs) (linoleate: oxygen oxidoreductase, EC 1.13.11.12) form a family of non-heme iron-containing fatty acids dioxygenases which catalyse the regio- and stereospecific dioxygenation of polyunsaturated fatty acids (PUFA) which contain a (1Z,4Z)-pentadiene system to produce their corresponding hydroperoxide fatty acids with Z,E-diene conjugation [1,2]. These enzymes are widely distributed throughout the plant and animal kingdoms [3–7]. In animals, the different LOXs are named according to their positional specificity for the dioxygenation of arachidonic acid (AA): 15-, 12- or 5-LOX, depending on whether they attach the hydroperoxide function to carbon atom 15, 12, or 5 of AA, respectively. As AA is a minor PUFA in the plant kingdom, plant LOXs are generally classified according to their positional specificity for linoleic acid (LA) oxygenation. This substrate is oxygenated at either

carbon atom 9 (9-LOX) or carbon atom 13 (13-LOX) (Fig. 1). The regiospecificity of LOX product formation depends on both the positional specificity of the initial hydrogen removal and the positional specificity of the subsequent oxygen addition [8]. Depending on the number of double bonds of the substrate, one or more possibilities exist for hydrogen removal. If the initial hydrogen removal takes place at one methylene carbon atom, Cm , two positional isomers can be formed. If the mesomeric free radical formed reacts with oxygen at the carbon nearest to the carboxylic group of the fatty acid, the dioxygen is inserted at carbon Cm−2 . Such a reaction is designated (−2), while a shift of the radical electron towards the methyl end of the fatty acid introduces oxygen at carbon Cm+2 and it is designated as a (+2) reaction [2] (Fig. 1). Although, most LOXs catalyze the formation of one particular regioisomer, it has become apparent that several LOXs exhibit a dual or even multiple positional specificity [9–12].

Fig. 1. Mechanism of the dioxygenation reaction catalyzed by lipoxygenases with linoleic acid as substrate. Small arrows indicate electron shifts during the (−2) radical rearrangement of the fatty acid.

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It has been demonstrated that LOXs stereoselectively remove one of the two hydrogen atoms from the prochiral center, Cm , of the pentadiene system [13–15]. Which hydrogen is removed depends on the stereospecificity of the enzyme. The configuration of the prochiral and chiral centers can be specified by the D/L system (Fischer convention) [8,16], or most frequently, using the R/S system [17]. Sometimes the D/L and the R/S assignments are both given: e.g. 15Ls-HPETE, 5Ds-HETE etc. More information on the stereochemistry of LOX reactions is given in [8]. Although, most plant and animal LOXs catalyze the production of hydroperoxides that have the S absolute configuration, LOXs forming R configuration products are also widespread, especially among aquatic invertebrates [18–23] and plants [24]. Fatty acid hydroperoxides, the primary products of LOX, are very reactive molecules which are rapidly transformed into various families of physiologically active compounds, the oxylipins. In plants, these hydroperoxides serve as a substrate for several

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enzymes like hydroperoxide lyase, peroxygenase, hydroperoxide reductase [25], divinylether synthase [26] or allene oxide synthase [27]. Fig. 2 summarizes the major metabolic routes for the metabolism of 13-HPOT [25,28]. All these pathways start with the oxygenation of free linolenic acid which is converted to (9Z,11E,15Z,13S)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13S-HPOT). Allene oxide synthase catalyzes the conversion of 13-HPOT producing a very unstable allene oxide, followed either by its enzymatic cyclization to 12-oxo-phytodienoic acid [29], the precursor of jasmonic acid, or by its spontaneous hydrolysis to a mixture of ␣- and ␥-ketols and racemic derivatives of 12-oxo-phytodienoic acid [30,31]. Hydroperoxide lyase cleaves the HPOT molecule producing short-chain aldehydes and ␻-keto fatty acids, [32–35]. In the peroxygenase pathway intramolecular oxygen transfer converts fatty acid hydroxides to epoxy or hydroxy PUFAs [36]. The reduction of hydroperoxides to their corresponding hydroperoxides is also catalyzed by hydroperoxide reductase. Divinyl

Fig. 2. Metabolic routes for 13-LOX-dependent catabolism of ␣-linolenic acid in plants.

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ether synthase converts fatty acid hydroperoxides into cytotoxic divinyl ethers [26,37]. Finally, lipoxygenase itself can catalyze the synthesis of keto PUFAs [38]. In contrast to the rather well studied products of the 13-LOX reaction, 9-LOX products have only recently become the focus of attention [37,39–41]. In animals, numerous biological activities are ascribed to individual HETEs and HPETEs, and the weight of evidence now suggests that these products act as discrete signaling molecules [6]. The oxidation of AA by LOX is the source of highly active bio-regulators, particularly of leukotrienes (LT) and lipoxins [42,43]. The synthesis of such products is one of the most firmly established functions of LOXs [6]. The complete characterization of LOX products is essential for establishing the physiological role of this enzyme since the biological activity of the LOX products and their ability to initiate the synthesis of different signaling molecules are determined by the positional and enantiomeric nature of the hydroperoxides produced. For example, a sequence of transformations involved in the development of starfish oocytes to mature egg is initiated by 8R-HETE, although its enantiomer, 8S-HETE, is ineffective [19]. Since the early seventies, different methods to determine the positional specificity of LOX products have been proposed. In this review, we will concentrate on the analysis of the primary reaction products of LOX using linoleic, linolenic or AA as substrates. The analysis of metabolites (LT, lipoxines . . . ) synthesized by other enzymes using the primary products of LOX as substrates has been reviewed by other authors [44–47].

2. Determination of the stereospecificity of lipoxygenase products by HPLC One of the most widely used techniques in the analysis of LOX products is high performance liquid chromatography (HPLC), both in reversed-phase and straight-phase. The popularity of this technique arises, in part, from the fact that most LOX metabolites carry chromophores which permit their measurement by photometry, including photodiode array detection without any previous derivatization [44].

2.1. RP-HPLC Reversed-phase HPLC (RP-HPLC) is a useful technique for analyzing LOX products, either as the sole chromatographic step [48–53] or followed by straight-phase HPLC (SP-HPLC) (see Section 2.2.1). Different stationary-phases have been used to separate LOX products, with octadecylsilyl (ODS) being one of the most widely used. Both isocratic and gradient elution separations have been described. 2.1.1. Isocratic elution The isocratic elution mode, mostly used in the analysis of lipid hydroperoxides, presents several advantages over gradient elution: lower solvent consumption, absence of baseline drift and ghost peaks, better reproducibility of retention times, shorter overall analysis times and less sophisticated and expensive equipment. Different isocratic elution systems have been proposed, which can be classified into three main groups: (a) ACN-based systems. (b) Methanol-based systems. (c) Tetrahydrofurane-based systems. In each of these groups, different percentages of eluents and adsorbents have been employed (see Table 1). To maintain the LOX products (fatty acid hydroperoxides or hydroxides) in their protonated state, a small percentage of acetic acid (AcH) is added to the eluents.

Table 1 Analysis of LOX products by RP-HPLC using different elution systems Composition

References

ACN/H2 O ACN/H2 O/AcH ACN/H2 O/HCl ACN/MeOH/H2 O/fosforic acid ACN/MeOH/H2 O/AcH ACN/MeOH/H2 O/AcH + EDTA ACN/MeOH/H2 O/TFA + triethylamine MeOH/H2 O/AcH MeOH/H2 O/AcH + triethylamine MeOH/H2 O/TFA + triethylamine THF/MeOH/H2 O/AcH THF/MeOH/0.1 M phosphate buffer pH 7.0

[59] [23,53,58] [57] [56] [54,55,60] [54] [56] [50,54,61–68,86–91] [70,71] [69,72] [7,48,49,51,52,74–76,92] [77]

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Fig. 3. RP-HPLC analysis of 13- and 9-HPOD using different mobile-phases and a column Inertsil-2-ODS (GL Science Inc., 5 ␮m particles, 4.6 mm × 150 mm). (A) ACN/H2 O (73:27), pH 3.0; (B) MeOH/H2 O (96:4); (C) THF/MeOH/H2 O/AcH (25:30:45:0.1); (D) THF/MeOH/phosphate buffer (25:30:45) at different phosphate concentrations: (i) 0.1 M; (ii) 50 mM; (iii) 22.2 mM; (iv) control with water. The product samples were eluted at a flow rate of 1 ml/min.

2.1.1.1. Acetonitrile-based systems. Two different mixtures have been reported, one consisting of acetonitrile (ACN) plus water, and frequently a small percentage of acid (Fig. 3A), and the other containing ACN, methanol, water and acid (Table 1). The acid can be AcH [23,54,55], phosphoric acid [56], TFA [56] or HCl [57]. Using ACN/water (50:50 v/v, pH 4.5) and a Nova Pak C18 , Tieman and coworkers

[53] obtained good separation of 8-HETE from 8-ketoeicosatetraenoic acid (8-KETE) but in general the separations reported with this system are far from good. Fig. 3A shows the coelution of 9- and 13-HPOD using ACN/H2 O (73:27) pH 3.0. This method was used by [58] to separate different metabolites of LA but the resolution obtained was rather poor. Coelution of isomers using similar systems has been reported

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also by other authors [23,59]. The resolution can be improved adding methanol to the system [54,55]. 2.1.1.2. Methanol-based systems. This seems to be the most widely used RP-HPLC system for analyzing the primary products of LOX (Table 1 and Fig. 3B). The system usually consists of methanol/H2 O/AcH mixtures, the percentage of methanol ranging from 70 to 85% and the amount of AcH from 0.01 to 0.1%. This method gives good results for the separation of HETEs [50,61–64], diHETEs [65], hydroxy eicosapentaenoic acid (HEPEs) [66] and hydroxy octadecadienoic acid (HODEs) [67,68] without the need for derivatization or as methyl esters [65]. However, the inability of this system to separate hydroperoxides from the corresponding hydroxides has also been reported [54]. In the example of Fig. 3B, using MeOH/H2 O (96:4), the resolution of 9- and 13-HPOD is also rather poor. Eskra et al. [69] substituted acetic by trifluoroacetic acid (TFA) and obtained good separation of HETEs and LTs using triethylamine as additive. This compound has also been used by other authors [56,69–72] as it improves the separation of LT. 2.1.1.3. Tetrahydrofuran-based systems. The third elution system consists of tetrahydrofuran (THF), methanol, water, AcH (Table 1 and Fig. 3C). This system was used in 1984 to separate diHETEs, triHETEs and LTs (LTB5 , LTD4 , LTC4 , PGB2 ) [73,74]. Since then its use has been extended to the analysis of products of plant LOXs as it allows separation of hydroperoxides, hydroxides and ketodienoic acids in a single run without derivatization [48,49,51,52,75,76]. RP-HPLC with THF-eluents resolves all the HPOD isomers but has the disadvantage that ketodienoic acids and hydroxides overlap, and that not all their isomers are resolved. The separation of 9- and 13-HPOD using THF/MeOH/H2 O/AcH (25:30:45:0.1) is better than the obtained with ACN or methanol-based systems using an Inertsil-2-ODS column, although the retention times have notably increased (Fig. 3). A modification of this method was proposed by our group [77] replacing water by 0.1 M phosphate buffer, pH 7.0 (Fig. 3D, chromatogram i). The addition of buffers to the mobile-phase is a useful strategy frequently used in RP-HPLC to modify the resolution [78]. In this paper [77], we analyzed the influence of chemical–physical factors such as the pH and

the ionic strength of the buffer on the separation of 9- and 13-HPOD. In this way, when the pH of the mobile-phase is close to the pKa of the fatty acid hydroperoxide, the separation of 9- and 13-HPOD is improved [77]. The effect of the ionic strength is illustrated in Fig. 3D, which shows how an increase in the ionic strength of the buffer leads to a considerable displacement of the retention times which are shortened from 42 min (in the absence of salt) to 12 min at 0.1 M phosphate buffer, thus permitting the analysis of a large number of samples in a short period of time. Furthermore, the separation factor between 9and 13-HPOD improves with increasing phosphate concentration. 2.1.2. Gradient elution Gradient elution has been reported by different authors. Abian et al. [79] described the separation of LA metabolites, produced by maize embryos LOX, using water (pH 3.5 with AcH) with an ACN gradient from 30 to 95% in 30 min on a Spherisorb ODS-2 column. A similar gradient was used by Schepky et al. (1997) [80] for the simultaneous analysis of LOX, phospholipase A2 and prostaglandin synthase products. Grechkin et al. [81] reported the RP-HPLC analysis of products of (1 − 14 C) linolenic acid oxidation by potato tuber LOX using a linear gradient between methanol/water/AcH solvent mixture. Several eicosanoids were separated using a linear gradient between ACN/water/AcH and ACN/methanol/water/AcH [82] and similar systems have been reported by [83–85] to separate different AA metabolites. 2.2. SP-HPLC Normal or straight-phase chromatography is generally performed with a non-polar organic mobile-phase and a silica stationary-phase. To analyze LOX products and as has been explained for RP-HPLC, a small percentage of AcH is usually added to the non-polar eluents. When methyl PUFA derivatives are separated this acid is sometimes omitted [57,66,87,93] (Table 2). 2.2.1. Isocratic elution SP-HPLC is generally better than RP-HPLC for separating closely related isomers, although recoveries can be rather low in some cases [94]. Straight-phase

M. P´erez Gilabert, F.G. Carmona / Analytica Chimica Acta 465 (2002) 319–335 Table 2 Analysis of LOX products by SP-HPLC using different elution systems Composition

References

Hexane/2-propanol Hexane/2-propanol/AcH

[57,61,66,86,87,93,98] [10,23,54,55,62,64,66–68, 81,87,89,90,95,96,100,101, 103,104,106,117–119] [97] [91] [103,107,108,120] [110] [111] [111] [121] [72,76,113–116] [112]

Hexane/2-propanol/H2 O Hexane/2-propanol/H2 O/AcH Hexane/EtOH Hexane/EtOH/AcH Heptane/AcH Heptane/EtOH/AcH Hexane/diethyl ether Hexane/diethyl ether/AcH Hexane/acetone

chromatography can be used as the only separation procedure [95–97] or as a second purification step to improve the separation of products that coeluted in a RP-HPLC column [67,68,81,90,91,98,99]. The use of three successively connected columns (Separon SGX CN 7 ␮m) has been described by Grechkin et al. [81] to further purify the oxidation products of linolenic acid after isolation by RP-HPLC. The products isolated by SP-HPLC include HPOD, HODEs [100], HPOTE [101], HPETE [54,64,95, 101,102] and HETE [87] without derivatization or as methyl esters [103,104]. In general, SP-HPLC can be used for the study of all types of eicosanoids except peptidoleukotrienes [105]. Most authors use elution systems consisting of hexane/2-propanol/AcH with the amount of hexane generally being above 95% (Table 2). With these eluents Shimizu et al. [54], used a Nucleosil 50-5 column (Macherey–Nagel, Duren) for the large scale preparation of 5-HPETE which was separated from 5-HETE, while Wu et al. [106] obtained a good separation of HPOD, HODE and keto fatty acids in a single run. Other authors [103,107,108] have separated different methyl HODEs using 0.5% ethanol in hexane, a system originally proposed by Chan and Levett [109]. A similar solvent system acidified with AcH was employed by Bostock et al. [110]. Koskas et al. [111] used heptane instead of hexane and studied the influence of the percentage of ethanol and AcH in the separation of 13-ZE/EE and 9-EZ /EE

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HPOD (Table 2). They obtained good results but run times were very long (about 80 min). A mixture of hexane/acetone (95:5 v/v) was described by Gardner [112] to separate methyl HODEs on a Zorbax SIL column. Teng and Smith [113] described another SP-HPLC system for the complete separation of all the HPOD and HODE isomers using hexane/ether eluents (Table 2). The different ketodienoic acid isomers could also be resolved with these methods [114]. Hilbers et al. [116] separated ketodienes, hydroperoxides and hydroxides of LA after methylation using hexane/diethyl-ether/AcH (90:10:0.1 v/v/v). SP-HPLC is especially useful for analyzing phospholipids and their oxidation products. Pérez-Gilabert et al. [97] reported the separation of mono- and dihydroperoxy dilinoleoyl phosphatidylcholine (DL-PC) using a CP-sphere Si column and hexane/2-propanol/ water (4:6:1 v/v/v). In this system, the least polar DL-PC eluted first, followed in order of increasing polarity by monohydroperoxy-DL-PC, and then by dihydroperoxy-DL-PC with two HPOD chains. 2.2.2. Gradient elution Boeynaems et al. [122] proposed a linear solvent gradient from hexane/AcH (100:0.8 v/v) to chloroform/AcH (100:0.8 v/v) on a semipreparative Microporasil column to generate highly purified HETEs. Fürstenberger et al. [123] reported the use of a gradient between solvent A (n-hexane/AcH; 99:1 v/v) and solvent B (n-hexane/2-propanol/AcH; 99:1:1 v/v/v) to separate 8-HPETE from 8-HETE employing a Zorbax SIL column. 2.3. Silver-loaded cation exchange HPLC Although at present, not widely used, silver-loaded cation exchange HPLC (or argentation HPLC) can be used for the analysis of all types of eicosanoids except peptido-LT [105]. Argentation HPLC will separate mixtures of closely related products and can be used to separate isotopically labeled AA metabolites from their unlabeled analogs [124,125]. This method is useful as a second step in the purification of AA metabolites, but the results obtained with complex mixtures can be difficult to interpret, because structurally unrelated products of greatly different polarities (e.g. PGF2␣ and 15-HETE) can have identical

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retention times) [94]. Silver-loaded cation exchange HPLC columns used with straight-phase solvent systems are especially sensitive to the cis or trans configuration of double bonds [126,127]. Hawkins and Brash [66] used argentation chromatography with a Sepralyte SCX 5 ␮m cation exchange HPLC column with a solvent system of hexane/2-propanol/water for the characterization of several hydroxy eicosanoids.

3. Determination of stereospecificity of lipoxygenase products Two principal methods have been used for the chromatographic analysis of the enantiomeric isomerism of LOX products: reaction with resolving agents and use of chiral-phase columns.

used by other authors for the separation of LOX products [133–135]. A commercially available resolving agent, (+)-␣-methoxy-␣-trifluoromethylphenyl acetic acid, was used to resolve the enantiomeric products of LOX by André and Funk [136]. Methyl esters of fatty acid hydroperoxides that had been reduced to the corresponding alcohols were converted into diastereomeric esters of (+)-␣-methoxy␣-trifluoromethylphenyl acetic acid that could be readily resolved by normal-phase HPLC. Derivatization with a chiral reagent presents one possible disadvantage in that the reaction rates of the two enantiomers of the analyte may be unequal [137]. Thus, it should be previously demonstrated that a racemic standard is resolved into two peaks of equal area [16]. 3.2. Use of chiral columns

3.1. Reaction with resolving agents In 1971, Hamberg [128] introduced a method which was based on the gas–liquid chromatographic separation of the diastereomeric fragments of the LOX products obtained after conversion with R-(−)-menthylchloroformate and oxidative ozonolysis. Shimizu et al. [10,54] employed this method to determine the chirality of several potato HPETEs. Van Os et al. [129] reported a similar gas chromatographic procedure in which ozonolysis of the acetylated hydroxy dienoates yields acetylated 2-hydroxy acids. The latter are converted into R-(−)-2-butyl esters and then reacetylated. The enantiomeric composition of these preparations can be determined by capillary gas chromatography. It has also been demonstrated that the stereochemistry of some LOX products can be determined by HPLC of the menthoxycarbonyl derivatives with no need for further derivatization [130,131]. Synthesis of the menthoxycarbonyl derivative is simple and utilizes commercially available reagents; diastereomers may be resolved by SP-HPLC or RP-HPLC [16,20,66,131]. This technique, however, apparently only applies to certain LOX products, since resolution was not reported for several positional isomers such as 9-RS-, 12-RS- and 15-RS-HETEs [130]. Corey et al. [132] employed the isocyanate derived from dehydroabietylamine to resolve the diastereomeric urethanes of 5-HETE, this method was later

Since the mid-1980s, HPLC separation of enantiomers has increasingly depended on chiral stationaryphases. Two types of chiral bonded-phases are used in the separation of LOX products, Pirkle and Chiralcel columns. 3.2.1. Pirkle columns (DNBPG columns) Silica derivatized with the substituent R-(−)-N-(3,5dinitrobenzoyl)-α-phenylglycine (DNBPG) is one of the commonly used chiral-phases developed by W.H. Pirkle. Using these columns, Kühn et al. [103] developed the first general method for analyzing the enantioselectivity of lipid hydroperoxides (Table 3). Although, baseline resolution was not always observed, this method proved to be useful in many cases for establishing whether a product is either a racemic mixture or is predominantly of the R or S configuration [66,138,139]. When these columns are used, it is essential to carry out mixing experiments with synthetic standards for each derivative because of gradual prolongation of the retention time with each successive run [140]. In addition, the resolving power of individual HPLC columns prepared with the DNBPG-phase is variable and the columns tend to lose resolution after extensive use [16]. The conversion of HETE methyl esters to their benzoyl and naphtoyl ester derivatives greatly improved the resolution of HETE enantiomers on the DNBPG column [137]. Although, the results

M. P´erez Gilabert, F.G. Carmona / Analytica Chimica Acta 465 (2002) 319–335 Table 3 Different chiral columns and elution systems employed in the analysis of the stereospecificity of LOX products Column

Eluents

Reference

DNBPG

Hexane/2-propanol/ AcH Hexane/2-propanol

[119]

DNBPG Chiralcel OD Chiralcel Chiralcel Chiralcel Chiralcel Chiralcel Chiralcel

OD OD-R OD-H OB-H OB OB

Chiralcel OC Chiralcel OJ

Hexane/2-propanol/ AcH Hexane/2-propanol MeOH/H2 O/AcH Hexane/2-propanol Hexane/2-propanol Hexane/2-propanol Hexane/2-propanol/ AcH Hexane/2-propanol Hexane/2-propanol

[23,48,66,87,88,103, 108,140] [64,67,68,89,100,141] [23,61,99] [76,116] [121] [142] [62,98] [91] [50,54,60,98] [55]

obtained by derivatizating the hydroxyl group are much better than for underivatized HETEs on the DNBPG columns, this method offers little improvement over direct analysis on Chiralcel columns [16].

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3.2.2. Chiralcel columns Different Daicel polysaccharide type columns for straight and reversed-phase chromatography have been used for the enantiomeric resolution of LOX products (Table 3). The chemical structure of the chiral stationary-phases is shown in Fig. 4. Kitamura et al. [60] reported the complete resolution of 12-RS-HETE methyl ester on a Chiralcel OC column, a column which was later used by other authors (Table 3). The use of Chiralcel OD columns to analyze LOX products is nowadays common and good resolution is obtained. It is frequently observed that the same author might modify the percentage of the eluents slightly, depending on the hydroxides to be separated [64]. A Chiralcel OB column gives complete resolution of the 8-HETE [7,62,98] and 5R/S-HETE enantiomers [64] chromatographed in the form of the methyl ester derivative. Using this column and a solvent of hexane/2-propanol (100:2 v/v) 8-S-HETE methyl ester enantiomer elutes first [16,62,98]. The same order is observed for methyl 12-HETE enantiomers using a Chiralcel OJ and n-hexane/2-propanol (99:1 v/v) [55].

Fig. 4. Chemical structure of the Chiralcel columns reported in the text.

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Martini and Iacazio [121] reported the use of a Chiralcel OD-H column in the near-baseline resolution of methyl esters of various hydroxy derivatives of linoleic, ␣-linolenic and AA. Such a column also permitted the analysis of all trans isomers of HODE, which are generally obtained in very low quantities. The R enantiomer is eluted first in all cases tested by these authors [121]. Reversed-phase Chiralcel columns such as Chiralcel OD-R have also been successfully used in the analysis of several methyl ester hydroxy fatty acid enantiomers [76,116]. The analysis of enantiospecificity using Chiralcel columns requires only simple chemical manipulation, i.e. reduction of the hydroperoxide function and methylation of the carboxylic acid, although several papers report on the analysis of free hydroxides [64,67,68,100,141]. If the enzyme used generates a mixture of regio- and stereoisomeric hydroxy fatty acids, a further purification of individual compounds should be undertaken, since their peaks overlap on the chromatogram and might lead to false attribution. The ease of use of these columns is an advantage which, when added to their good resolution, has made them the method of choice for analyzing racemic hydroperoxides.

4. Detectors coupled to HPLC Identification of specific LOX products can be achieved by using HPLC to separate individual species, which are then detected by UV absorbance; the use of dual wavelength monitoring (e.g. 234 and 270 nm) provides selectivity in the detection of compounds carrying conjugated dienes and trienes. Diode array detector is a valuable tool in the identification process since it permits the spectra of the product to be recorded directly [65] and also the detection at multiple wavelengths [82,84]. Quantitation of these products is achieved by comparison with an internal standard, usually PGB2 which enables an assessment of recovery and absolute quantitation [48,74,143,144]. In addition, the measurement of the ratio of PGB2 to 19-hydroxy-PGB2 enables the assessment of sample to sample recovery of the polar LOX products [145]. Detection by UV absorbance is limited to the low nanogram level [146]. 13-HODE has also been used

as internal standard [23,82]. Most of the separation methods reviewed in this paper are based on UV detection. The use of a diode array UV detector connected in series with a highly sensitive light-scattering detector permitted the simultaneous determination by RP-HPLC of different classes of inflammatory cascade metabolites, including LOX products [80]. The analysis of LOX derivatives produced by oxidation of radioactivity-labeled PUFA is widely extended [53,81,99,123,147]. The simultaneous detection of LOX products using a UV absorbance detector and a scintillation detector is especially useful for differentiating between endogenous products and products formed from the exogenous radioactivity-labeled PUFA [62,110,141]. Radioactivity detection has also been used in chiral-phase chromatography (CP-HPLC) [99]. More specific information can be gained by coupling HPLC with post-column chemiluminiscence detection based on luminol or isoluminol oxidation during the reaction of hydroperoxides and cytochrome-C [148,149]. Chemiluminiscence detection has the advantage of high sensitivity and can detect picomoles of hydroperoxides in each lipid class. The post-column chemiluminescence reaction is specific for the hydroperoxy group and it enables the hydroperoxides to be distinguished from the corresponding hydroxy derivatives. Some applications to biological systems have also been reported [150–156]. The post-column reaction of luminol to luminol oxide progresses only under aqueous alkaline conditions [150] and so the number and type of organic solvent used for mobile-phase is relatively limited. Post-column detection of hydroperoxides based on the formation of fluorescent products has also been carried out [157,158]. Ohshima et al. [159] reported the quantitative and qualitative determination of isomeric lipid hydroperoxides with post-column detection using diphenyl-1-pyrenylphosphine (DPPP). Demin [160] reported the derivatization of several 12-LOX products into fluorescent anthryl esters using anthryl diazomethane (ADAM) reagent. These derivatives were analyzed by RP-HPLC with fluorescent detection. Electrochemical (EC) (amperometric) detection in the reductive [161–163] or oxidative mode [164] can be combined with RP-HPLC to identify lipid

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hydroperoxides. Compared with the HPLC chemiluminiscence systems, reductive-mode HPLC–EC is less expensive, easier to operate, and subject to fewer artifacts, since post-column reactions are not involved [163]. On the other hand, hydroperoxides detection limits cited in early HPLC–EC publications [161,162,165] are considerably higher than those reported for chemiluminiscence detection [148,149]. Recently, the evaporative light-scattering detector (ELSD) coupled to HPLC has been used in the analysis of different lipid classes [166–170]. ELSD is a universal detector, useful for any sample less volatile than the mobile-phase. Its response is proportional to sample mass and does not depend on the sample’s chemical structure or optical characteristics. Furthermore, solvents that are unsuitable for UV detection can be used with ELSD. There are few reports on the use of this detector in the analysis of lipid hydroperoxides [171–173]. However, the methods described earlier alone, cannot structurally identify unknown hydroperoxides and are most useful for analyzing particular hydroperoxides with known HPLC retention values. Mass spectrometry is a very powerful technique for identifying compounds, but the mass spectra obtained from mixtures are usually too complex to be helpful. The combination of chromatographic techniques and mass spectrometry offers a wide range of analytical possibilities, which have been extended to the characterization of LOX products. These possibilities are discussed in the later sections.

5. LC/MS The possibility of coupling HPLC on line with a mass spectrometer opened up new possibilities in the separation and structural analysis of intact LOX products. These compounds are frequently involatile and thermally labile, and so must be transferred into the gas-phase under mild conditions. In addition, these labile molecules need to be ionized under conditions which prevent the accumulation of large amounts of internal energy, which would result in extensive ion fragmentation and loss of molecular mass information. Several LC/MS interfaces such as thermospray (TSP), fast atom bombardment (FAB), electrospray (ES), atmospheric pressure chemical ionization (APCI) have

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been used to analyze LOX products. For a general introduction to these techniques see [174–176]. 5.1. Thermospray–HPLC–MS TSP–HPLC–MS has proved to be an effective means of analyzing underivatized oxygenated fatty acid metabolites. This technique, which is almost exclusively operated with reversed-phase systems, provides both component separation and structural information on individual hydroperoxides [79,177,178]. Yergey et al. [177] analyzed oxygenated metabolites of docosahexaenoic acid using HPLC coupled to a TSP interface. The separation was performed on a Du Pont 25 cm × 4.6 mm, 5 ␮m Zorbax ODS column, using a gradient from 75:25 v/v 0.1 M ammonium acetate/ACN to 58:42 v/v in 20 min. The observation that the number of fragments due to losses of water is directly related to the number of oxygenated sites on the fatty acid chain represents an advantage for analyzing oxygenated fatty acid metabolites of unknown structure. TSP–HPLC–MS analysis of 13-HPOD yielded ions at m/z 313 (MH+ ), 295 (MH+ –H2 O) and 279 (MH+ –HOOH) [179]. Abián et al. [79] showed that the use of methoximated derivatives was a useful strategy for enhancing the structural information given by TSP–HPLC–MS. They used a Spherisorb ODS-2 column and a mobile-phase consisting of mixtures of 0.1 and 0.05 M ammonium acetate buffer in methanol, a method which permitted the elucidation of the chemical structure of several LOX products not previously described. The main drawbacks of TSP–HPLC–MS, however, are its lack of sensitivity, low quantitative precision, and its unavailability to most researchers [179]. 5.2. Electrospray 5.2.1. Electrospray–HPLC–MS MacMillan and Murphy [180] presented a simple negative-ion ES–MS method for the direct determination of lipid hydroperoxides and related long-chain keto acids. The technique proved to be very sensitive and permitted the detection of 100 pg of hydroperoxides. The authors analyzed the fragments obtained at various orifice potentials (from 40 to 75 V). All the hydroperoxides analyzed gave carboxylate anions [M–H]− , which were more abundant at low

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potentials. Loss of water was also observed for all species. Fragmentation was induced by increasing the orifice voltage. The molecular-ion continued to be observed up to 65–75 V. At this orifice potential other ions, indicative of the hydroperoxide position, were observed. This technique was also used by Iwase et al. [58] who obtained similar results when analyzing 13- and 9-HPOD, keto octadecadienoic acid (KODE) and LA. However, negative-ion ES–MS of fatty acid hydroperoxides and hydroxides is not useful for localizing the double bond or the position of hydroxyl moieties due to double bond migration [181] as has been seen using other methods of MS. 5.2.2. Electrospray–HPLC tandem mass spectrometry The difficulty of extracting structural information at the same time as measuring relative molecular mass is the major inconvenience not only of ES but also of other “soft ionization” techniques, such as negative-ion chemical ionization (NICI), FAB systems or APCI. Normal ions formed in the ion source with these systems can be induced to fragment further by the addition of more internal energy after they leave the source. This activation process has led to the development of the mass spectrometry/mass spectrometry (MS/MS) or tandem mass spectrometry techniques [174]. One of the most frequently used system to induce fragmentation during the analysis of LOX products is collision-induced dissociation (CID), which is achieved by making use of their kinetic energy, gained on acceleration from the ion source. Two collisional activation regimes are used, one at low kinetic energy values corresponding to acceleration of ions by applying 0–100 eV and one at high kinetic energy, corresponding to acceleration at more than 1 keV [174]. ES ionization tandem mass spectrometry is at present one of the most frequently reported LC/MS techniques for analyzing LOX products. The CID of lipid hydroperoxides generates unique and characteristic spectra that share some common features and, more importantly, fragmented-ions that are indicative of the position of the hydroperoxide [180]. Saturated and unsaturated monohydroxy fatty acids and other LOX products were analyzed by Kerwin and Torwik [181] using ES tandem mass spectrometry (ES–MS/MS). The identification of LOX derived fatty acid hydroper-

oxides by positive-ion ES tandem mass spectrometry has been also reported [92]. Product-ion spectra obtained by low-energy CID (7 eV) of [M+NH4 + ] derived from HPODs and HPOTs revealed characteristic fragment patterns which permitted identification of 9- and 13-regioisomers. ES–MS/MS has recently been used to quantify 5-ketoeicosatetraenoic acid (5-keto-ETE) [182,183], a LOX metabolite which could not be analyzed by GC–MS/MS [182], and other eicosanoids [146]. Collisional activation at high kinetic energy, has also been employed in the study of LOX products [184,185]. Griffiths et al. [185] applied this technique to identify different hydroxylated eicosanoids using a hybrid tandem magnetic sector-time-of-flight instrument. Oliw et al. [63,186,187] carried out a qualitative and quantitative analysis of different LOX products using ES ionization-ion trap mass spectra. An ion trap is a mass spectrometer that stores ions in an evacuated cavity by applying appropriate electric fields. Collisional activation can then be repeated on selected fragment-ions in order to explore the fragmentation of this new precursor ion. This group found that MS3 spectra of hydroperoxy fatty acids and MS2 spectra of mono- and dihydroxy fatty acids provided sufficient information for identification [186]. 5.3. Fast atom bombardment The use of FAB as ion source permits the ionization of samples by bombardment with atoms (usually of xenon). Ionization occurs directly from the solution, so the sample does not have to be vaporized first. The coupling of LC with FAB is termed continuous-flow FAB or dynamic FAB. Typically, the eluents employed contain 1–10% of glycerol or some other solvent of relatively low vapor pressure to aid the FAB ionization process. Negative-ion formation by FAB of underivatized LT, prostaglandins and other eicosanoids results in abundant carboxylate anions, and collisional activation and tandem mass spectrometry (MS/MS) of these ions produces structurally significant products [188–190]. Thus, both molecular weight and molecular structural information are obtained. The group of Murphy [191] applied FAB MS/MS analysis to underivatized HODEs, HOTEs and HETEs using low-energy CID with a triple

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quadrupole mass spectrometer. The CID spectra of these compounds yielded abundant carboxylate anions and structurally significant product ions which revealed the position of the hydroxyl substituent in relation to the sites of unsaturation.

6. Analysis of lipoxygenase products by GC/MS The advantages offered by the combination of gas chromatography and mass spectrometry (chromatographic resolution and sensitivity together with structural information on individual components of complex mixtures) justify the important role played by GC/MS in the structure elucidation of LOX products. Modern analytical gas chromatography is conducted in long capillary tubes made of flexible fused silica. The inside wall of these columns is coated with a layer of a non-volatile liquid. Table 4 presents the different columns employed in the analysis of LOX products. Many compounds are not sufficiently volatile to vaporize significantly at room temperature. Therefore, most gas chromatography is conducted at high temperatures where the analytes have a chance of volatilizing. Unfortunately, analysis of intact hydroperoxides by GC/MS generally has not been possible due to the thermal instability of these compounds [192]. To avoid this problem, hydroperoxides may be reduced and the resulting hydroxy analogs analyzed by GC/MS. The reduction of fatty acid hydroperoxides can be carried out with compounds such Table 4 GC columns employed by different groups to analyze TMS ethers methyl esters of different HETEs and HODEs by GC/MS Column

Reference

SE-54 column SPB-1 fused silica capillary column CP-SIL 5 fused silica capillary column DB-1 fused silica capillary column OV-1 fused silica capillary column OV-1 capillary glass column 3% OV-1 2% OV-17 on chromosorb W Methyl silicane capillary column Capillary RSL-150 HP-1 capillary column HP-5MS fused silica capillary column CP-SIL 5 CB

[103] [62,65] [59] [52,72,97,116] [123] [102] [122] [55] [206] [67,89] [23] [58] [76]

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as sodium borohydride [122,142,178], triphenylphosphine [66,123], or SnCl2 [142]. The conversion of fatty acid hydroperoxides into their hydroxide derivatives, although widely used, does not distinguish between hydroperoxides and their corresponding hydroxy analogs in systems where both species are present. Turnipseed [179] reported a method which allows the analysis of fatty acid hydroperoxides by capillary GC/MS without chemical or thermal decomposition. After reduction, the resulting hydroxy acids are converted to volatile derivatives such as pentafluorobenzyl ethers [193], methyl ester or trimethylsilyl ethers [194]. Trimethylsilylation of hydroxyl groups is a frequent derivatization procedure that has been carried out using different reagents: bis(trimethylsilyl)-trifluoroacetamide (BSTFA) in pyridine [23,72,117,122,195]; n-trimethylsilylimidazole [58]; or a mixture of dry pyridine, hexamethyldisilazane and trimethylchlorosilane (5:1:1 v/v/v) [52,76,102]. GC/MS has also been used in the quantification of hydroxy fatty acids [123,193,196,197]. Negative-ion electron capture CI–GC/MS analysis of the pentafluorobenzyl ester TMS ether derivatives of fatty acid hydroxides, provides the most sensitive method for molecular weight determination [198,199]. However, this method does not provide structural information as all regioisomeric HODEs or HETEs show intact carboxylate anions, and even low-energy CID of these carboxylate anions does not yield structurally significant ions [200]. Thus, as regioisomeric hydroxy fatty acids exhibit the same fragment-ion mass, identification of a specific structure can only be based on the GC retention time. On the other hand, electron impact (EI)–GC/MS is a method with lower absolute sensitivity than negative-ion CI but producing structure-specific fragment-ions with high relative intensity [174,201]. Thus, EI–GC/MS analysis has been employed for the determination of hydroxy substituent position. The specific detection of 9- and 13-HODE by capillary column EI–GC/MS is difficult, since the methyl ester–trimethylsilyl ether derivatives of the 9- and 13-isomer exhibit very similar chromatographic properties. In addition, they both show fragment-ions at m/z 225 and 311 and only differ in their relative abundance, 225 being the most abundant fragment mass for 9-HODE [202] and 311 for 13-HODE [203]. Prior reduction of double bonds by hydrogenation creates a much more favorable

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situation. After hydrogenation each isomer is characterized by isomer-specific fragment masses. Furthermore, hydrogenation introduces a difference into the gas chromatographic retention times between these regioisomeric molecules, 9-HODE eluting slightly earlier than 13-HODE. Different catalysts have been used in the hydrogenation of fatty acid hydroxides: PtO2 [72,117,122]; Pt/Al2 O3 [147]; Rh/Al2 O3 rhodium on alumina [23,204] and Pd/CaCO3 [205]. Mass spectra of the methyl ester–trimethylsilyl ether derivatives of several HETEs and of the corresponding hydrogenated compounds is shown in [122].

7. Conclusion Both RP- and SP-HPLC systems have been used to analyze LOXs products. RP-HPLC is predominantly used to separate HPETEs and HETEs and SP-HPLC to analyze HPODs and HPOTs and their corresponding hydroxides. SP-HPLC can be also used to improve the separation of products that coelute in a RP-HPLC column. Chiralcel columns are the method of choice for analyzing racemic hydroperoxides due to the ease of use and the good resolution obtained. Detection of LOX products separated by HPLC is mainly achieved by using UV absorbance or radioactivity detectors. At present, ES ionization tandem mass spectrometry is one of the most frequently reported LC/MS techniques for analyzing LOX products, however is still unavailable to many researchers. GC/MS continues playing an important role in the structure elucidation of LOX products.

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