Use of Gas Chromatography-Mass Spectrometry Techniques (GC-MS, GC-MS/MS and GC-QTOF) for the Characterization of Photooxidation and Autoxidation Products of Lipids of Autotrophic Organisms in Environmental Samples

This paper reviews applications of gas chromatography-mass spectrometry techniques for the characterization of photooxidation and autoxidation products of lipids of senescent phototrophic organisms. Particular attention is given to: (i) the selection of oxidation products that are sufficiently stable under environmental conditions and specific to each lipid class and degradation route; (ii) the description of electron ionization mass fragmentation of trimethylsilyl derivatives of these compounds; and (iii) the use of specific fragment ions for monitoring the oxidation of the main unsaturated lipid components of phototrophs. The techniques best geared for this task were gas chromatography-quadrupole-time of flight to monitor fragment ions with very high resolution and accuracy, and gas chromatography-tandem mass spectrometry to monitor very selective transitions in multiple reaction monitoring mode. The extent of the degradation processes can only be estimated if the oxidation products are unaffected by fast secondary oxidation reactions, as it is notably the case of ∆5-sterols, monounsaturated fatty acids, chlorophyll phytyl side-chain, and di- and triterpenoids. In contrast, the primary degradation products of highly branched isoprenoid alkenes possessing more than one trisubstituted double bond, alkenones, carotenoids and polyunsaturated fatty acids, appear to be too unstable with respect to secondary oxidation or other reactions to serve for quantification in environmental samples.


Introduction
Phototrophic organisms (mainly green plants, algae, cyanobacteria and some protists) carry out photosynthesis that is, conversion of sunlight energy, carbon dioxide and water into organic materials. Due to the generation of highly reactive oxygen species (ROS) during photosynthetic electron transport, these organisms are particularly sensitive to oxidative damages [1]. Lipids (hydrocarbons, pigments, terpenoids, free fatty acids, acylglycerides, phospholipids, galactolipids, cutins, suberins and waxes [2]) are important components of phototrophic organisms, accounting for 16-26% of organic content in phytoplankton [3] and up to 45% in the green alga Botryococcus Braunii [4]. The relative stability and specificity of lipids makes them popular tracers of the origin of organic matter in environmental samples [5][6][7]. Their abiotic oxidation products can be also very useful for estimating present or past photooxidative and autoxidative alterations in specific phototrophic organisms [8,9].
The most common chromatographic methods for lipid analysis are gas chromatography (GC), and high-performance liquid chromatography (HPLC) coupled with mass spectrometers (MS). GC-based analytical procedures require analytes that are volatile and thermally stable. In practice, this means that GC-based analysis of the oxidation products of mixtures of complex and simple lipids with such techniques demands a chemical Due to the presence of chlorophyll, which is a very efficient photosensitizer [27,28], visible light-induced photosensitized processes act intensively during the senescence of autotrophic organisms. In healthy cells, the excited singlet state of chlorophyll ( 1 Chl) formed after absorption of a quantum of light energy, leads predominantly to the characteristic fast photosynthesis reactions [27]. However, a small proportion of 1 Chl undergoes intersystem crossing (ISC) to form the longer live triplet state ( 3 Chl) [28], which is not only itself potentially damaging in type I reactions [28] but can also generate ROS and, in particular, singlet oxygen ( 1 O 2 ) by reacting with ground state oxygen ( 3 O 2 ) (type II processes). As a defense against oxidative damage, there are many antioxidant compounds (e.g., carotenoids and vitamin E) and enzymes (e.g., superoxide dismutase and catalase) that operate in chloroplasts [27,29].
As fast photosynthesis reactions are clearly not operative in senescent phototrophic organisms, potentially damaging 3 Chl and 1 O 2 [30] are produced at an accelerated rate exceeding the quenching capacity of the photoprotective system and thus damage the membranes (photodynamic effect [31]). 1 O 2 readily oxidizes cellular components of senes-cent autotrophic organisms such as unsaturated lipids (including ∆ 5 -sterols, unsaturated fatty acids, chlorophyll phytyl side-chain, carotenoids and alkenes), proteins, and nucleic acids [32]. The rate of reaction of 1 O 2 with olefins is controlled by the degree of substitution and the configuration (cis-or trans-) of the double bond [33], with highly-substituted and cisdouble bonds being the more reactive. Type II photosensitized oxidation of unsaturated lipids affords allylic hydroperoxides (for reviews see [8,9]).

Free Radical Oxidation (Autoxidation)
Due to spin restriction [34], the unpaired electrons of ground-state triplet molecular oxygen 3 O 2 can only interact with unpaired electrons of organic radicals, which drive autoxidation reactions. Autoxidation involves free-radical-mediated oxidation chain reactions, which can be divided into three steps: chain initiation, propagation, and termination [35]. Initiation of autoxidation requires initiators that are able to produce radicals by removing an electron to the substrate molecule or breaking a covalent bond. The most common initiators are heat, light, redox-active metal ions undergoing one-electron transfer (e.g., Fe 2+ , Co 2+ , Fe 3+ , Cu 2+ , Mn 2+ , Zn 2+ , Mg 2+ , V 2+ ), and certain enzymes (lipoxygenases). The propagation step involves a succession of reactions in which each radical produced in one reaction is consumed in the next [36]. It generally proceeds via: (i) hydrogen atom abstraction from tertiary, allylic or α to oxygen positions, and (ii) addition of peroxyl radicals to double bonds. Termination results from reactions of radicals affording non-radical products. In senescent phototrophic cells, initiation of autoxidation processes is generally attributed to the cleavage (induced by heat, light, metals or enzymes) of hydroperoxides resulting from type II photosensitized oxidation of cellular components to hydroxyl, peroxyl and alkoxyl radicals [37,38].

Characterization of the Oxidation Products of Lipids
This chapter briefly describes the mechanisms of photooxidation and autoxidation of the main unsaturated lipids of phototrophic organisms. A focus is given to the selection of oxidation products sufficiently stable and specific to act as tracers of these processes in environmental samples (such as: phytodetritus, particulate matter, marine and lacustrine sediments and soil), as well as to the mechanisms of fragmentation of TMS derivatives of these compounds during electron ionization. Some application examples of these tracers are also shown. Note that accurate masses of the different fragment ions formed are given, which makes them amenable to use in GC-QTOF analyses, while the corresponding unit masses can still be used in GC-MS/MS or classical GC-MS analyses.
Note that the loss of a methyl radical by the molecular ion of TMS derivatives of phytol and isophytol also affords a fragment ion at m/z 353.3235. Monitoring ions at m/z 353.3235 and 245.1388 thus allows simultaneous characterization and quantification of phytol and its main photooxidation and autoxidation products in natural samples (see example given in Figure 1). Note that the loss of a methyl radical by the molecular ion of TMS derivatives of phytol and isophytol also affords a fragment ion at m/z 353.3235. Monitoring ions at m/z 353.3235 and 245.1388 thus allows simultaneous characterization and quantification of phytol and its main photooxidation and autoxidation products in natural samples (see example given in Figure 1).

RO
Specific fragment ions [M − 143.0887] + , for which Table 1 gives accurate masses for the ∆ 4 -stera-3β,6β-diols of the more common sterols, thus emerged as very useful for the monitoring of ∆ 5 -sterol photooxidation and autoxidation in phototrophic organisms. An example of their application is given in Figure 2.  Table 1 gives accurate masses for the ∆ 4 -stera-3β,6β-diols of the more common sterols, thus emerged as very useful for the monitoring of ∆ 5 -sterol photooxidation and autoxidation in phototrophic organisms. An example of their application is given in Figure 2.

Unsaturated Fatty Acids
Type II photosensitized oxidation and autoxidation rates of unsaturated fatty acids logically increase with their number of double bonds [51,52]. Unfortunately, oxidation products of the more reactive polyunsaturated fatty acids (PUFAs) are not sufficiently stable under environmental conditions to be used as tracers of these degradation processes in situ. Note that isoprostanoids (cyclopentane-containing oxylipins) resulting from autoxidation of C18, C20 and C22 PUFAs are often used as biomarkers for in vivo oxidative stress in animals and plants [53]. These compounds could be detected in higher plants and

Unsaturated Fatty Acids
Type II photosensitized oxidation and autoxidation rates of unsaturated fatty acids logically increase with their number of double bonds [51,52]. Unfortunately, oxidation products of the more reactive polyunsaturated fatty acids (PUFAs) are not sufficiently stable under environmental conditions to be used as tracers of these degradation processes in situ. Note that isoprostanoids (cyclopentane-containing oxylipins) resulting from autoxidation of C 18 , C 20 and C 22 PUFAs are often used as biomarkers for in vivo oxidative stress in animals and plants [53]. These compounds could be detected in higher plants and algae by using GC-MS in negative-ion chemical ionization (NICI) mode [53,54]. However, in the literature there are no reports of such compounds in environmental samples.
In contrast, oxidation products of monounsaturated fatty acids (MUFAs) are sufficiently stable for use as tracers of type-II photosensitized oxidation and autoxidation processes in situ [10]. During type II photosensitized oxidation of MUFAs, attack by 1 O 2 of the two ethylenic carbon atoms of the double bond leads to the formation of two transallylic hydroperoxides [52,55], which subsequently undergo stereoselective radical allylic rearrangement to afford two other isomers with a trans-double bond [56] (Scheme 5). Note that if type II photosensitized oxidation of MUFAs involves UV radiation, then four corresponding cis-allylic hydroperoxides also get produced [57] (Scheme 5). In contrast, free radical oxidation of these compounds affords only two cis-allylic hydroperoxides (corresponding to the oxidation of the two allylic positions of MUFAs) in addition to the four trans-isomeric hydroperoxides [56] (Scheme 5). Consequently, in senescent autotrophic organisms a dominance of these two cis-isomers (among the four cis-isomers) points to the involvement of autoxidation processes, while a dominance of all four cis-isomers points to UV-induced photodegradation.

Free radical oxidation
Tracers of MUFA autoxidation and UV-induced photooxidation Under EI, TMS derivatives of isomeric allylic hydroxy acids resulting from photooxidation and autoxidation of MUFAs and subsequent NaBH4 reduction undergo α-cleavage at their TMS ether group. Cleavage acts on the saturated side of the molecule (as the vinylic position of the double bond hinders cleavage on the other side) and affords stable Under EI, TMS derivatives of isomeric allylic hydroxy acids resulting from photooxidation and autoxidation of MUFAs and subsequent NaBH 4 reduction undergo α-cleavage at their TMS ether group. Cleavage acts on the saturated side of the molecule (as the vinylic position of the double bond hinders cleavage on the other side) and affords stable and specific fragment ions (Scheme 6) that are dependent on the carbon atom number and double-bond position of the MUFA considered [11,15]. The fragment ions resulting from α-cleavage of silylated oxidation products of the more common MUFAs are listed in Table 2. GC-QTOF allows a clean characterization and quantification of TMS derivatives of MUFA oxidation products in autotrophic organisms and environmental samples. Figure  3 gives some examples of the technique application showing typical profiles of visible light-induced, (visible + UV) light-induced and autoxidative degradation products. Scheme 6. Examples of EI fragmentations of TMS derivatives of MUFA oxidation products. Table 2. Accurate masses of the main fragment ions produced during EI fragmentation of silylated allylic hydroxy acids resulting from NaBH 4 -reduction of photo-and autoxidation products of some common MUFAs.

MUFAs
( GC-QTOF allows a clean characterization and quantification of TMS derivatives of MUFA oxidation products in autotrophic organisms and environmental samples.  MRM analyses of TMS derivatives of MUFA oxidation products involve intense and selective transitions from the ions resulting from α-cleavage (precursor ions) to the fragment ion at m/z 129 (product ion) (Scheme 6). Note that this transition is more efficient with precursor ions containing the terminal methyl group than with precursor ions containing the TMS ester group, which can easily lose neutral TMSOH molecules (Scheme 6, Figure 4). Figure 5 gives an example of how MRM analyses can be applied. MRM analyses of TMS derivatives of MUFA oxidation products involve intense and selective transitions from the ions resulting from α-cleavage (precursor ions) to the fragment ion at m/z 129 (product ion) (Scheme 6). Note that this transition is more efficient with precursor ions containing the terminal methyl group than with precursor ions containing the TMS ester group, which can easily lose neutral TMSOH molecules (Scheme 6, Figure 4). Figure 5 gives an example of how MRM analyses can be applied.

Pentacyclic Triterpenes
Pentacyclic triterpenes and their derivatives, which are widely found in angiospe [58], are divided into three main classes, that is, lupanes, oleananes and ursanes.

Lupanes
Type-II photooxidation and autoxidation of lupanes have so far only been stu for betulin [59], but the results obtained can be extended to lupeol or betulinic acid main triterpenoids with betulin of the lupane group). 1 O2 reacts slowly with the C2

Pentacyclic Triterpenes
Pentacyclic triterpenes and their derivatives, which are widely found in angiosperms [58], are divided into three main classes, that is, lupanes, oleananes and ursanes.

Lupanes
Type-II photooxidation and autoxidation of lupanes have so far only been studied for betulin [59], but the results obtained can be extended to lupeol or betulinic acid (the main triterpenoids with betulin of the lupane group). 1 O2 reacts slowly with the C20-C29

Pentacyclic Triterpenes
Pentacyclic triterpenes and their derivatives, which are widely found in angiosperms [58], are divided into three main classes, that is, lupanes, oleananes and ursanes.
The EI mass spectra of the TMS derivatives of lup-20(30)-ene-3β,28,29-triol and lupan-20-one-3β,28-diol exhibit intense fragment ions at m/z 481.3860 and m/z 395.3305, respectively, whose formation involves elimination of a neutral molecule of TMSOH and subsequent loss of the CH2OTMS group borne by the carbon 28 [59] (Scheme 8). These fragment ions make good candidates for monitoring type-II photosensitized oxidation and autoxidation of betulin, respectively, in environmental samples. As the formation of these ions involves the loss of the group borne by carbon 28, they can be also used as In contrast, the autoxidation of betulin mainly involves peroxyl radical addition to the C 20 -C 29 double bond and mainly affords a diperoxide that is unaffected by NaBH 4 reduction and converted to stable lupan-20-one-3β,28-diol during hot GC injection [59] (Scheme 7). Lupan-20-one-3β,28-diol, lupan-20-one-3β-ol (arising from lupeol) and lupan-20-one-3β-ol-28-oic acid (arising from betulinic acid) can be used as specific tracers of the autoxidation of lupanes in angiosperms [59,60].
The EI mass spectra of the TMS derivatives of lup-20(30)-ene-3β,28,29-triol and lupan-20-one-3β,28-diol exhibit intense fragment ions at m/z 481.3860 and m/z 395.3305, respectively, whose formation involves elimination of a neutral molecule of TMSOH and subsequent loss of the CH 2 OTMS group borne by the carbon 28 [59] (Scheme 8). These fragment ions make good candidates for monitoring type-II photosensitized oxidation and autoxidation of betulin, respectively, in environmental samples. As the formation of these ions involves the loss of the group borne by carbon 28, they can be also used as tracers of the oxidation of lupeol and betulinic acid. Figure 6 gives an example of the specific fragment ion at m/z 395.3305 applied for monitoring lupane autoxidation in environmental samples. tracers of the oxidation of lupeol and betulinic acid. Figure 6 gives an example of the specific fragment ion at m/z 395.3305 applied for monitoring lupane autoxidation in environmental samples.

Ursanes and Oleanes
Studies on type II photosensitized oxidation and autoxidation of ursanes and oleanes have mainly focused on α-and β-amyrins [61]. α-and β-amyrins were found to be totally unaffected during photodegradation experiments, due to steric hindrance preventing 1 O2 reaction with their double bond [61]. Autoxidation of amyrins mainly involves hydrogen

Ursanes and Oleanes
Studies on type II photosensitized oxidation and autoxidation of ursanes and olean have mainly focused on α-and β-amyrins [61]. α-and β-amyrins were found to be total unaffected during photodegradation experiments, due to steric hindrance preventing 1 O reaction with their double bond [61]. Autoxidation of amyrins mainly involves hydroge abstraction and specifically produces 11α-hydroperoxyamyrins [61] (Scheme 9). These hydroperoxides, which appeared to be unaffected by NaBH 4 reduction, are thermally cleaved to the corresponding 11-oxoamyrins during GC or GC-MS analyses using hot injectors (Scheme 9). 11-Oxoamyrins are sufficiently stable and specific to serve as tracers of amyrin autoxidation in senescent angiosperms or environmental samples.
EI fragmentation of TMS derivatives of 11-oxoamyrins was recently studied [62] and found to involve: (i) retro-Diels-Alder cleavage of the unsaturated ring C leading to the formation of a fragment ion at m/z 232.1822, (ii) γ-hydrogen rearrangement of the ionized 11-keto group and subsequent cleavage of the 7-8 bond affording a well-stabilized fragment ion at m/z 273.2213, and (iii) a fragmentation pathway involving loss of the TMS group together with carbon atoms 1, 2 and 3 of the A ring after initial cleavage of the 3-4 bond [25] producing a fragment ion at m/z 383.3308 (Scheme 10). Subsequent fragmentation of the ion at m/z 273.2213 affords a strongly stabilized ion at m/z 135.0804 after migration of the methyl group 27 from carbon 14 to carbon 13 and concerted cleavage of the 13-18 and 15-16 bonds [63] (Scheme 10). ment ion at m/z 273.2213, and (iii) a fragmentation pathway involving loss of the TMS group together with carbon atoms 1, 2 and 3 of the A ring after initial cleavage of the 3-4 bond [25] producing a fragment ion at m/z 383.3308 (Scheme 10). Subsequent fragmentation of the ion at m/z 273.2213 affords a strongly stabilized ion at m/z 135.0804 after migration of the methyl group 27 from carbon 14 to carbon 13 and concerted cleavage of the 13-18 and 15-16 bonds [63] (Scheme 10). Note that after the loss of a methyl radical, a fragment ion at m/z 217.1588 can be formed from the fragment ion at m/z 232.1822 (Scheme 10). This fragmentation, which is more intense in the case of 11-oxo-β-amyrin due to the thermodynamically-favored loss of a methyl radical from the tertiary carbon 20, may be useful for differentiating 11-oxo-amyrins.
Specific fragment ions at m/z 512.4063 [M] +• , 383.3308, 273.2213 and 232.1822 appeared to be useful for GC-QTOF monitoring of TMS derivatives of 11-oxo-amyrins in environmental samples (see example given in Figure 7A). MRM analyses using the transitions m/z 273 → 135 and m/z 232 → 217 also appeared to be well suited to the detection of traces of these compounds (see Figure 7B). of a methyl radical from the tertiary carbon 20, may be useful for differentiating 11-oxoamyrins.
Specific fragment ions at m/z 512.4063 [M] +• , 383.3308, 273.2213 and 232.1822 appeared to be useful for GC-QTOF monitoring of TMS derivatives of 11-oxo-amyrins in environmental samples (see example given in Figure 7A). MRM analyses using the transitions m/z 273 → 135 and m/z 232 → 217 also appeared to be well suited to the detection of traces of these compounds (see Figure 7B).

Highly Branched Isoprenoid (HBI) Alkenes
HBI alkenes (exhibiting 1-6 double bonds) are produced by some marine and fre water diatoms belonging to the Berkeleya, Haslea, Navicula, Pleurosigma, Pseudosolenia Rhizosolenia genera [67,68]. During the senescence of these organisms, 1 O2 attack is focu on the lesser sterically-hindered trisubstituted double bonds of these alkenes affordin or 4 allylic hydroperoxides according to the E or Z configuration of the double bond [ As an example, Scheme 13 shows type II photosensitized oxidation of Z and E isomer HBI III, which are ubiquitous throughout the world's oceans [70]. In this case, 1 O2 att acts mainly on the C9-C10 double bond and to a lesser extent to the more sterically-h dered C7-C20 double bond affording 9-and 7-hydroperoxides, respectively, as the m oxidation products. Autoxidation processes also act very quickly on HBI III, produc numerous autoxidation products, but predominantly 9-hydroperoxides resulting fr hydrogen atom abstraction at the allylic carbon 11 (Scheme 13) [71]. Indeed, the m oxidation pathway of this compound involves hydrogen abstraction at the bis-allylic position to afford conjugated dienes, which are particularly prone to peroxyl radical ditions and readily undergo copolymerization with oxygen (Scheme 13). Consequen the 7-alcohol resulting from NaBH4-reduction of the corresponding hydroperoxide co be used as specific tracer of type II photosensitized oxidation of HBI III (Scheme 13). H ever, the reduction products of 9-hydroperoxides will only be indicative of oxidation this specific HBI alkene. Unfortunately, in the case of HBI alkenes (such as HBI III) p sessing several trisubstituted double bonds, photooxidation and autoxidation produ are unable to accumulate due to the involvement of fast secondary oxidation reacti [71]. All these tracers can thus only serve to give qualitative indications.

Highly Branched Isoprenoid (HBI) Alkenes
HBI alkenes (exhibiting 1-6 double bonds) are produced by some marine and freshwater diatoms belonging to the Berkeleya, Haslea, Navicula, Pleurosigma, Pseudosolenia and Rhizosolenia genera [67,68]. During the senescence of these organisms, 1 O 2 attack is focused on the lesser sterically-hindered trisubstituted double bonds of these alkenes affording 2 or 4 allylic hydroperoxides according to the E or Z configuration of the double bond [69]. As an example, Scheme 13 shows type II photosensitized oxidation of Z and E isomers of HBI III, which are ubiquitous throughout the world's oceans [70]. In this case, 1 O 2 attack acts mainly on the C 9 -C 10 double bond and to a lesser extent to the more sterically-hindered C 7 -C 20 double bond affording 9-and 7-hydroperoxides, respectively, as the major oxidation products. Autoxidation processes also act very quickly on HBI III, producing numerous autoxidation products, but predominantly 9-hydroperoxides resulting from hydrogen atom abstraction at the allylic carbon 11 (Scheme 13) [71]. Indeed, the major oxidation pathway of this compound involves hydrogen abstraction at the bis-allylic C 8 position to afford conjugated dienes, which are particularly prone to peroxyl radical additions and readily undergo copolymerization with oxygen (Scheme 13). Consequently, the 7-alcohol resulting from NaBH 4 -reduction of the corresponding hydroperoxide could be used as specific tracer of type II photosensitized oxidation of HBI III (Scheme 13). However, the reduction products of 9-hydroperoxides will only be indicative of oxidation of this specific HBI alkene. Unfortunately, in the case of HBI alkenes (such as HBI III) possessing several trisubstituted double bonds, photooxidation and autoxidation products are unable to accumulate due to the involvement of fast secondary oxidation reactions [71]. All these tracers can thus only serve to give qualitative indications.  EI mass spectra of the TMS derivatives of the 9-alcohols resulting from HBI III oxidation exhibit an intense fragment ion at m/z 213.1670 corresponding to α-cleavage relative to the TMS ether group [72] (Scheme 14). This fragment ion can readily lose a neutral molecule of TMSOH to give a fragment ion at m/z 123.1170, or undergo a hydrogen transfer with concerted cleavage of the bond between carbon atoms 3 and 4, yielding a fragment ion at m/z 143.0887 (Scheme 14). In the case of the 7-alcohol, α-cleavage relative to the TMS ether group affords two fragment ions at m/z 295.2452 and 321.2610, which are then cleaved in the α position relative to the ionized TMS ether group after hydrogen transfers to give fragment ions at m/z 183.1201 and 181.1044, respectively (Scheme 14).

Alkenones
Alkenones are a class of mono-, di-, tri-, tetra-and penta-unsaturated C 35 -C 40 methyl and ethyl ketones, which are produced by certain haptophytes [74][75][76][77][78]. The unsaturation ratio of C 37 37:3 ] are the concentrations of di-and tri-unsaturated C 37 methyl alkenones, respectively) varies positively with the growth temperature of the alga [79,80] and is thus now routinely used for paleotemperature reconstructions (e.g., [81,82]). Due to the transgeometry of the alkenone double bonds [83], which is poorly reactive with 1 O 2 [33]), alkenones are not affected by type II photosensitized oxidation processes [84,85]. However, they are highly reactive to autoxidation processes [86]. Autoxidation of alkenone double bonds (separated by five or three methylene groups) affords six hydroperoxides as in the case of MUFAs (see Section 3.3). Isomeric alkenediols resulting from NaBH 4 reduction of these oxidation products could make very useful indicators of autoxidative alterations of the unsaturation ratio U K 37 , but unfortunately they fail to accumulate due to the subsequent oxidation of the other double bonds [87]. Note that TMS derivatives of alkene-triols, tetraols or pentaols obtained after NaBH 4 reduction and derivatization of secondary oxidation products of di-, tri-or tetraunsaturated alkenones are too heavy and labile to be analyzed by GC-MS. The characterization of alkenone autoxidation products in sediments or phytodetritus with more adapted analytical techniques constitutes a very important challenge.

Carotenoids
Carotenoids, which are important antioxidant constituents of thylakoid membranes, play special roles in the protection of tissues against damage caused by light and oxygen [88]. These compounds can very efficiently quench 1 O 2 by energy transfer (quenching), but also by chemical reaction (scavenging) [89]. They are also good scavengers of ROS [90]. The attack of β-carotene by 1 O 2 affords β-carotene-5,8-endoperoxide (Scheme 15) [91]. If this compound is generally considered a useful early signal of 1 O 2 production in plant leaves [92], it may be also formed during autoxidation of β-carotene [89] and is clearly not stable enough to serve as a viable environmental tracer. Unfortunately, the reaction of 1 O 2 and ROS with carotenoids produces oxidation products that are not sufficiently stable and specific (production of similar compounds by enzymatic processes) [89] to be used as unequivocal indicators of type-II photosensitized oxidation or autoxidation of carotenoids in senescent phototrophic organisms and environmental samples. important challenge.

Carotenoids
Carotenoids, which are important antioxidant constituents of thylakoid membranes, play special roles in the protection of tissues against damage caused by light and oxygen [88]. These compounds can very efficiently quench 1 O2 by energy transfer (quenching), but also by chemical reaction (scavenging) [89]. They are also good scavengers of ROS [90]. The attack of β-carotene by 1 O2 affords β-carotene-5,8-endoperoxide (Scheme 15) [91]. If this compound is generally considered a useful early signal of 1 O2 production in plant leaves [92], it may be also formed during autoxidation of β-carotene [89] and is clearly not stable enough to serve as a viable environmental tracer. Unfortunately, the reaction of 1 O2 and ROS with carotenoids produces oxidation products that are not sufficiently stable and specific (production of similar compounds by enzymatic processes) [89] to be used as unequivocal indicators of type-II photosensitized oxidation or autoxidation of carotenoids in senescent phototrophic organisms and environmental samples.

Conclusions
In this review, a focus was given to the selection and characterization of stable and specific tracers of photooxidation and autoxidation of lipid components (chlorophyll phytyl side-chain, ∆ 5 -sterols, MUFAs, pentacyclic triterpenes and dehydroabietic acid) of phototrophs. The author hope that it will contribute to a better consideration of photooxidative and autoxidative processes almost ignored so far in the literature when studying the degradation of autotrophic organisms in marine and terrestrial environments.
The different oxidation products selected could be used as indicators of: (i) oxidative stress of specific phototrophic organisms; (ii) paleoenvironmental changes of the conditions of sedimentation (oxic or anoxic); (iii) abiotic alteration of paleoproxies in oxic environments; (iv) environmental problems related to ozone depletion, and (v) abiotic degradation of permafrost released under the effect of global warming [60].
In the future, a special attention should be given to the detection of oxidation products of alkenones and HBI alkenes (possessing several trisubstituted double bonds) sufficiently stable and specific to act as tracers of oxidative alteration of these proxies in oxic environments (water column of oceans and oxic layer of sediments). MALDI-MS and IM-MS techniques, which allow simultaneous characterization of all molecular species in biological tissues and reduce sample preparation artifacts arising from extensive purification procedures [93], seem to be particularly well-adapted to this task.

Conclusions
In this review, a focus was given to the selection and characterization of stable and specific tracers of photooxidation and autoxidation of lipid components (chlorophyll phytyl side-chain, ∆ 5 -sterols, MUFAs, pentacyclic triterpenes and dehydroabietic acid) of phototrophs. The author hope that it will contribute to a better consideration of photooxidative and autoxidative processes almost ignored so far in the literature when studying the degradation of autotrophic organisms in marine and terrestrial environments.
The different oxidation products selected could be used as indicators of: (i) oxidative stress of specific phototrophic organisms; (ii) paleoenvironmental changes of the conditions of sedimentation (oxic or anoxic); (iii) abiotic alteration of paleoproxies in oxic environments; (iv) environmental problems related to ozone depletion, and (v) abiotic degradation of permafrost released under the effect of global warming [60].
In the future, a special attention should be given to the detection of oxidation products of alkenones and HBI alkenes (possessing several trisubstituted double bonds) sufficiently stable and specific to act as tracers of oxidative alteration of these proxies in oxic environments (water column of oceans and oxic layer of sediments). MALDI-MS and IM-MS techniques, which allow simultaneous characterization of all molecular species in biological tissues and reduce sample preparation artifacts arising from extensive purification procedures [93], seem to be particularly well-adapted to this task.
NICI GC-MS, HPLC-MS and IM-MS techniques should be also used to give evidence of the presence of isoprostanoids resulting from PUFA oxidation in environmental samples. Due to the very high reactivity of PUFA towards photooxidation and autoxidation processes, such compounds could be very sensitive tracers of the early stages of oxidative damages.