Charge-switch derivatization of fatty acid esters of hydroxy fatty acids via gas-phase ion/ion reactions

Abstract

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with anti-diabetic and anti-inflammatory effects. Identification of FAHFAs is challenging due to both the relatively low abundance of these metabolites in most biological samples and the significant structural diversity arising from the co-occurrence of numerous regioisomers. Ultimately, development of sensitive analytical techniques that enable rapid and unambiguous identification of FAHFAs is integral to understanding their diverse physiological functions in health and disease. While a battery of mass spectrometry (MS) based methods for complex lipid analysis has been developed, FAHFA identification presents specific challenges to conventional approaches. Notably, while the MS² product ion spectra of [FAHFA − H]⁻ anions afford the assignment of fatty acid (FA) and hydroxy fatty acid (HFA) constituents, FAHFA regioisomers are usually indistinguishable by this approach. Here, we report the development of a novel MS-based technique employing charge inversion ion/ion reactions with tris-phenanthroline magnesium complex dications, Mg(Phen)₃²⁺, to selectively and efficiently derivatize [FAHFA − H]⁻ anions in the gas phase, yielding fixed-charge cations. Subsequent activation of [FAHFA − H + MgPhen₂]⁺ cations yield product ions that facilitate the assignment of FA and HFA constituents, pinpoints unsaturation sites within the FA moiety, and elucidates ester linkage regiochemistry. Collectively, the presented approach represents a rapid, entirely gas-phase method for near-complete FAHFA structural elucidation and confident isomer discrimination without the requirement for authentic FAHFA standards.

Graphical Abstract

1. Introduction

Fatty acid esters of hydroxy fatty acids (FAHFAs), also referred to as (O-acyl)-hydroxy fatty acids (OAHFAs), are a newly discovered category of lipids found in mammals and plants.[1, 2] Branched FAHFAs are important signaling molecules with potential anti-inflammatory and anti-diabetic effects and have thus received considerable attention as potential therapeutic agents for type 2 diabetes and inflammatory diseases like obesity.[1] FAHFAs are regulated by GLUT4, a major insulin-regulated glucose transporter that serves as a key regulator of whole-body glucose homeostasis, and are suggested to improve glucose tolerance, stimulate insulin secretion, and enhance glucose transport.[1, 3, 4] FAHFAs are found in mammalian serum and tissues as non-esterified (i.e., free) molecules at low concentrations [1], and according to recent studies, FAHFAs can also be incorporated into complex lipid classes like triacylglycerols and sphingolipids.[5, 6] Interestingly, the concentrations of FAHFA-containing triacylglycerols (FAHFA-TGs) in adipose tissue were reported to be 100-fold greater than that of non-esterified FAHFAs, suggesting FAHFA-TGs may serve as a major reservoir of these lipids.[5] In general, FAHFA structure is defined by a fatty acid (FA) esterified to the alcohol of a hydroxy fatty acid (HFA). A large number of distinct FAHFA structural variants arise from modifications to FA and HFA composition, including variations in aliphatic chain length, carbon-carbon double bond number and position, and hydroxylation site. Furthermore, a specific category of isomeric FAHFAs that differ only in the position of the ester linkage along the n-HFA backbone, referred to as n-FAHFA isomers, have been detected in human adipose tissue and serum, noting that n indicates the ester position relative to the HFA carboxyl carbon.[1] Importantly, regulation of n-FAHFA isomers levels under insulin-resistant conditions is both tissue-specific and isomer-specific.[1] Consequently, it is imperative to develop analytical tools that provide unambiguous FAHFA structural identification, as the evolution of such technologies is integral to the advancement of biomedical sciences, including the development of new strategies for the treatment and diagnosis of metabolic and inflammatory disorders (e.g., type 2 diabetes and obesity).

Mass spectrometry (MS) has played a pivotal role in the structural elucidation of lipids.[7–9] Even in contemporary MS-based lipidomics workflows, however, there remain significant challenges associated with lipid structural identification. Currently, two main mass spectrometric strategies for FAHFA analysis have been established.[10] In the first approach, FAHFA molecular structures are first separated via liquid chromatography (LC) prior to admission to a tandem mass spectrometer. While LC-tandem mass spectrometry (MS/MS) can provide increased isomeric resolution, FAHFA structural identifications using this strategy are often reliant on comparison of retention times to authentic lipid standards, which, to date, are limited.[1, 11, 12] Notably, Ma et al. developed an in silico MS/MS library that extends the coverage of FAHFA molecules in LC-MS/MS studies, aiding in the identification of unknown molecular structures when external reference standard compounds are not available.[13] While many LC-MS/MS approaches for FAHFA identification have been described and successfully used for FAHFA analysis [1, 11], direct infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS), or so-called “shotgun lipidomics”, has emerged as a sensitive and powerful approach for lipid identification and quantification.[14–16] Due to the presence of a carboxylic acid moiety, FAHFAs can be ionized by direct infusion negative polarity ESI, forming abundant singly deprotonated FAHFA anions, denoted [FAHFA − H]⁻. From accurate mass measurements (i.e., observed mass-to-charge ratio) alone, FAHFA sum composition (i.e., the total number of carbons and degree of unsaturation) can be obtained. Subsequent interrogation of [FAHFA − H]⁻ ions via low-energy collision induced dissociation (CID) facilitates the assignment of constituent FA and HFA. Explicitly, the [FA − H]⁻ and [HFA − H]⁻ product ions are readily observed in resulting [FAHFA − H]⁻ CID spectra, and in turn, isomeric FAHFAs differing in aliphatic chain length combinations can be discerned.[1, 17] Nevertheless, significant challenges remain for unambiguous FAHFA identification when utilizing conventional shotgun approaches, particularly in distinguishing n-FAHFA isomers that vary only in ester regiochemistry. For instance, the MS² product ion spectra of isomeric [n-FAHFA − H]⁻ ions are remarkably similar, characterized by variations only in product ion relative abundances related to the site of esterification along the HFA backbone. Thus, MS/MS alone is often insufficient to distinguish n-FAHFA isomers.[17] Notably, Yore and coworkers exploited low mass product ions observed using a quadrupole-time of flight mass spectrometer to distinguish FAHFA isomers.[1] However, these diagnostic product ions are not consistently observed across various mass spectrometric platforms and studies, precluding more widespread application of this approach.[13, 17] Deploying a systematic MSⁿ approach, Marshall et al.[17] showed that MS³ of [HFA − H]⁻ ions can reveal the esterification site in FAHFA, as diagnostic product ions indicative of ester position were observed.[17] Although, in some cases, significant overlap in predicted diagnostic product ions arising from unique FAHFA isomeric precursors limits FAHFA identification in this manner. Altogether, unambiguous identification of FAHFA isomers continues to be a difficult task when employing low-energy CID alone.

Often in combination with CID, shotgun and LC-MS strategies employing bespoke ion activation or wet-chemical derivatization have enjoyed success for lipid structural identification by permitting the distinction of lipid isomers such as those varying in carbon-carbon double bond position, stereochemical configuration, or acyl chain branching.[9] To our knowledge, only two of these methods have been applied in the study of FAHFAs. The first approach exploits a combination of CID and ozone-induced dissociation (OzID) to pinpoint ester position along the HFA backbone, achieving near-complete FAHFA structural elucidation.[17] In brief, the [n-FAHFA − H]⁻ precursor anion dissociates to form a dehydrated HFA product ion, denoted [n-HFA − H − H₂O]⁻. Subsequent ion/molecule reactions between the [n-HFA − H − H₂O]⁻ product ion and ozone vapor confirms that this unsaturated product ion is composed of two isomeric alkene structures. Importantly, the carbon-carbon double-bond positions of the two isomeric unsaturated FA ions depend on the location of the fatty acyl ester, and in turn, the dehydrated n-HFA anion is indicative of the n-FAHFA isomer. Specifically, ozonolysis of the MS² [n-HFA − H − H₂O]⁻ product ion population reveals that this anion population is comprised of two distinct, isomeric alkene product ions that contain carbon-carbon double bonds at C_n and C_n+1 positions, respectively. Despite successes, ion-molecule reactions between dehydrated HFA anions and ozone molecules are slow and reaction rates are dependent on the alkene position relative to the carboxylate moiety, impacting sensitivity. In an alternate approach, Han and coworkers employed solution-based charge inversion of FAHFA with N-[4-(aminomethyl)phenyl]pyridinium (AMPP) derivatization giving rise to a fixed-charge cation.[18] Upon collisional activation, the [AMPP-FAHFA]⁺ cation fragments to form product ions indicative of ester position albeit at low abundance. In general, CID of AMPP derivatized FAHFA provide some insight into the structure of the HFA but little, or no, information on the bonding in the FA portion.[19] Additionally, like all traditional derivatization, this approach is reliant on wet-chemical modification, requiring additional sample manipulation prior to analysis and is susceptible to matrix effects in complex lipid extracts. It would thus be desirable to exploit the benefits of charge-inversion derivatization of FAHFAs but to enhance both the efficiency and speed of derivatization by exploiting entirely gas phase chemistries within the mass spectrometer.

Recently, we demonstrated the structural elucidation of lipids utilizing gas-phase charge inversion ion/ion chemistry.[20–22] Charge inversion ion/ion reactions can provide a significant reduction in chemical noise associated with mass spectra derived from complex mixtures, including lipid extracts, translating to lower detection limits and enhanced sensitivity.[23, 24] In particular, [FA − H]⁻ ions originating from direct negative nESI of non-esterified (i.e., free) FA or released from complex lipid precursor anions by ion-trap CID are derivatized within the mass spectrometer. To do so, [FA − H]⁻ anions are simultaneously stored with tris-phenanthroline magnesium complex dications, denoted [MgPhen₃]²⁺, undergoing a charge inversion ion/ion reaction to yield abundant [FA − H + MgPhen]⁺ complex cations. Subsequent interrogation of charge inverted FA complex cations permits unambiguous isomeric differentiation and the assignment of carbon-carbon double bond positions(s) in unsaturated FA. Explicitly, monounsaturated and diunsaturated FA are identified by direct spectral interpretation, exploiting reproducible fragmentation patterns, while automated matching to a library developed from authentic FA reference standards permits polyunsaturated FA identification. Herein, we deploy gas-phase charge inversion ion/ion reactions to achieve near-complete FAHFA structure elucidation. Conducted entirely in the gas-phase, this strategy permits the identification of ester position and localization of unsaturation sites in FAHFAs without recourse to condensed phase derivatization or separation. Collectively, while also facilitating high-level FAHFA structural characterization on chromatographically relevant timescales, the method supports de novo identification FAHFA molecular structures, in the absence of authentic commercial standards, thereby extending the range of confident FAHFA identification.

2. Experimental

HPLC-grade methanol was purchased from Fisher Scientific (Pittsburgh, PA). Magnesium chloride and 1,10-phenanthroline (Phen) were purchased from MilliporeSigma (St. Louis, MO). All lipid standards were purchased from Cayman Chemical, Co. (Ann Arbor, MI). Solutions of FAHFA standards were prepared in methanol to a final concentration of 1 μM. Magnesium chloride and 1,10-phenanthroline were combined in methanolic solution to a final concentration of 20 μM.

2.1. Mass Spectrometry

All experiments were conducted on a modified Sciex QTRAP 4000 hybrid triple quadrupole/linear ion trap mass spectrometer (SCIEX, Concord, ON, Canada).[25] Instrument modifications are illustrated with Supporting Information Figure S1. Alternately pulsed nanoelectrospray ionization (nESI) emitters permit the sequential injection of lipid anions and reagent dications.[26] Prior to entry into the high-pressure collision cell q2, lipid anions and reagent dications are mass selected in transit in Q1. Next, reagent dications and FAHFA anions were mutually stored for 500 ms. Product cations resulting from the charge inversion ion/ion reaction were then transferred to the low-pressure linear ion trap (LIT), Q3. In Q3, MSⁿ analysis of charge inverted product ions was performed using single frequency resonance excitation (q = 0.2). Mass analysis was conducted using mass-selective axial ejection (MSAE).[27]

2.2. Nomenclature

Lipid nomenclature is based on general literature recommendations by Liebisch et al.[28] To describe fatty acyl structure, we employ the X:Y shorthand notation where X and Y represent the total number of carbons and the degree of unsaturation, respectively (i.e., the 18:1 fatty acyl has 18 carbons and 1 double bond). Proven double bond position is specified after the degree of unsaturation within parentheses, and if known, double bond geometry is labeled as Z for cis and E for trans. For brevity, palmitic acid (FA 16:0) and palmitoleic acid (FA 16:1(9Z)) are referred as PA and PO, respectively, while HSA indicates any hydroxystearic acid (FA 18:0_OH). Proven hydroxylation position in HSA structures are indicated in the front of the HFA abbreviation. For example, 10-HSA indicates hydroxstearic acid with a hydroxyl group at C10. To describe FAHFA structure, we adopt the specific nomenclature proposed by Marshall et al.[17] and Ma et al.[13] Briefly, FAHFA are described using the X₁:Y₁/X₂:Y₂ notation, where the FA and HFA are identified as X₁:Y₁ and X₂:Y₂, respectively. To define the site of esterification, the FAHFA X₁:Y₁-(n-O-X₂:Y₂) notation was adopted herein, in which n indicates the ester position relative to the carboxyl carbon of the HFA. Lastly, the n-FAHFA abbreviations are also utilized for convenience. To illustrate, the FAHFA 16:0-(10-O-18:0) can be represented as 10-PAHSA. Table S1 in the Supplementary Information details the specific n-FAHFA abbreviations used in this study.

3. Results and Discussion

3.1. Charge inversion ion/ion strategies to elucidate saturated FAHFA structures

As described above, efforts to discriminate n-FAHFA isomers using conventional approaches have been limited, largely due to the formation of identical product ions arising from multiple isomeric precursors.[17] To achieve unambiguous FAHFA structural elucidation, we employ a charge-switch derivatization approach based on gas phase ion/ion reactions. In this approach, singly deprotonated FAHFA anions are charge-inverted with tris-phenanthroline magnesium complex reagent dications, as indicated in Scheme 1. To conduct this experiment, [10-PAHSA − H]⁻ (m/z 537.5) anions were simultaneously stored with [MgPhen₃]²⁺ reagent dications in the high-pressure collision cell q2 and permitted to react. The results of the ion/ion reaction are provided in Figure 1a. The [10-PAHSA − H + MgPhen₂]⁺ complex cation, detected at m/z 921.6, represents the dominant product ion, while a minor [10-PAHSA − H + MgPhen]⁺ complex cation (m/z 741.5) was also observed (Figure 1a). Importantly, the complex cation is observed at a signal level that is roughly a factor of three greater than that of the [10-PAHSA − H]⁻ ion in negative ion mode when using the same ion multiplier voltage but opposite conversion dynode voltages (Figure S2). We attribute the signal enhancement achieved via charge inversion to the difference in overall gain for detection for each polarity under these conditions. Following the ion/ion reaction, the resulting charge-inverted product ions were energetically transferred from q2 to Q3, giving rise to a ‘beam type’ (BT) CID spectrum. At lower collision energies, BT CID results in the loss of a single phenanthroline ligand, generating the [10-PAHSA − H + MgPhen]⁺ complex cation (m/z 741.5) (Figure S3). As collision energies are increased, abundant [PA − H + MgPhen]⁺ (m/z 459.3) and [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3) product ions were observed (Figure 1b). Note that the [10-HSA − H − H₂O + MgPhen]⁺ product ion observed at m/z 485.3 can also be represented as an unsaturated FA 18:1 complex cation (i.e., [18:1 − H + MgPhen]⁺). Importantly, the BT CID spectrum shown in Figure 1b enables assignment of HFA and FA composition, as the [PA − H + MgPhen]⁺ (m/z 459.3) and [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3) ions were readily observed. The ion-trap CID spectrum of the mass-selected m/z 741.5 ion is provided in Figure S4 and is in good agreement with the BT CID spectrum shown in Figure 1b.

Scheme 1.

Direct charge inversion of the [10-PAHSA − H]⁻ precursor ion via ion/ion reactions with [MgPhen₃]²⁺ reagent dications followed by BT CID, generating the [10-HSA − H − H₂O + MgPhen]⁺ and [PA − H + MgPhen]⁺ complex cations.

Figure 1.

Illustration of charge inversion ion/ion workflow used to identify site of esterification in 10-PAHSA. (a) Product ion spectrum resulting from mutual storage ion/ion reaction between [10-PAHSA − H]⁻ anions (m/z 537.5) and [MgPhen₃]²⁺ reagent dications. (b) Beam type CID spectrum post-ion/ion reaction. (c) Ion trap CID spectrum of mass-selected [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3).

Next, the complex cation of m/z 485.3 was mass-selected in the LIT with a 1 m/z window and subsequently interrogated via ion-trap CID. The CID spectrum of [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3) is displayed in Figure 1c. Consistent with the observations reported by Marshall et al.,[17] the data suggest that the dehydrated 10-HSA complex cation population is comprised of two isomeric monounsaturated FAs with carbon-carbon double bonds at C9=C10 and C10=C11. Indeed, the formation of a pair of alkenes upon dissociation of [PAHSA + MgPhen]⁺ cations was independently verified by measuring the OzID mass spectra of the [HSA − H − H₂O + MgPhen]⁺ product ion population. These data, acquired from solution-formed [n-PAHSA + MgPhen]⁺ cations, confirm that the positions of the carbon-carbon double bonds formed upon dissociation flank the n-position of the ester moiety (Figure S5). Consequently, the presence of FA 18:1(9) and 18:1(10) confirms the hydroxylation position in the [10-HSA − H − H₂O + MgPhen]⁺ complex cation, and in turn, establishes ester regiochemistry in the original PAHSA precursor ion. Importantly, the double bond positions in the isomeric alkene product ions are dependent on site of hydroxylation in the HFA constituent, and therefore, relate to the position of the ester linkage along the HFA backbone. From Figure 1c, the product ions related to dissociation of [18:1(9) − H + MgPhen]⁺ and [18:1(10) − H + MgPhen]⁺ complex cations are denoted with the green (●) and red (●) circles, respectively. As previously described[20, 22], the site of unsaturation in monounsaturated FA can be pinpointed using charge inversion ion/ion chemistry as [FA − H + MgPhen]⁺ complex cations fragment to generate reproducible spectral patterns indicative of double bond location. Briefly, to localize the double bond in monounsaturated FA, a characteristic spectral gap dependent on carbon-carbon double bond position is exploited. The spectral gap arises from dramatic suppression in product ion abundance related to product ions representing carbon-carbon double bond cleavage and carbon-carbon cleavages vinylic to the double bond. Flanking the spectral gap, carbon-carbon cleavage distal (i.e., on the methyl side) and allylic to the double bond yields a product ion doublet composed of a charge remote fragment ion and a terminally saturated product ion. This product ion doublet represents the two heaviest product ions (i.e., highest m/z) in the fragmentation pattern and serves as a key diagnostic marker for double bond position. In the case of 10-PAHSA, the pair of product ions observed at m/z 385.2 and 387.2 serve as diagnostic product ions for FA 18:1(9), while the product ion pair detected at m/z 399.2 and 401.2 indicate the 18:1(10) isomer (Figure 1c). Moreover, in the absence of spectral magnification in Figure 1c, the product ion at m/z 345.2 serves as a marker for hydroxylation position in the HFA ion. Specifically, carbon-carbon cleavage proximal (i.e., on the carboxyl side) and allylic to the C10=C11 double bond in the FA 18:1(10) isomer predominately contributes to the abundant product ion detected at m/z 345.2, though minor contributions to this product ion population are provided by C8–C9 cleavage in the FA 18:1(9) isomer. While the two sets of high-mass product ion doublets confirm the presence of two FA 18:1 isomers, the characteristic product ion at m/z 345.2 serves as a key indicator of hydroxylation position, easily facilitating the identification of original ester position in the 10-PAHSA precursor ion.

An alternate strategy for FAHFA identification based on charge inversion of product ions generated from CID of deprotonated precursors is outlined for 10-PAHSA in Scheme 2. In this approach, direct infusion negative nESI of 10-PAHSA generates abundant singly deprotonated [10-PAHSA − H]⁻ ions (m/z 537.5) with Ion-trap CID of the mass-selected [10-PAHSA − H]⁻ precursor anion (m/z 537.5) in q2 generating the product ion spectrum shown in Figure 2a. CID of the FAHFA precursor anion enables assignment of the FA and HFA constituents at the sum compositional level. In the case of the CID spectrum of [10-PAHSA − H]⁻, the product ions observed at m/z 255.2, 281.2, and 299.3 reflect the [PA − H]⁻, [10-HSA − H − H₂O]⁻, [10-HSA − H]⁻ anions, respectively. Notably, these product ions are characteristic of the [PAHSA − H]⁻ precursor ion, differentiating it from isomeric FAHFAs comprised of different chain length combinations in the HFA and FA constituents (Figure 2a). However, subtle structural features like ester regiochemistry cannot be easily discerned from this spectrum, and in turn, other n-FAHFA isomers are not easily distinguished at the MS² level.[17]

Scheme 2.

Charge inversion of the product ions generated via CID of the [10-PAHSA − H]⁻ precursor ion via ion/ion reactions with [MgPhen₃]²⁺ reagent dications, generating the [10-HSA − H − H₂O + MgPhen]⁺ and [PA − H + MgPhen]⁺ complex cations.

Figure 2.

Demonstration of charge inversion ion/ion reactions for the identification of ester position in FAHFA. (a) CID spectrum of mass selected [10-PAHSA − H]⁻ (m/z 537.5). (b) Mutual storage product ion spectrum resulting from the charge inversion ion/ion reaction between [MgPhen₃]²⁺ reagent diactions and the product anions shown in panel (a). (c) Product ion spectrum generated via energetic transfer (i.e., beam-type CID) of the mutual storage product cations shown in panel (b) from q2 to Q3. (d) CID spectrum of mass-selected [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3). Note that the lightning bolt () signifies the precursor ion subjected to ion trap CID.

Next, all product anions derived from collisional activation of [10-PAHSA − H]⁻ were subjected to ion/ion reaction with [MgPhen₃]²⁺ reagent dications. The resulting product ion spectrum is illustrated with Figure 2b. Dominating the product ion spectrum are the [PA − H + MgPhen₂]⁺ (m/z 639.4), [10-HSA H₂O + MgPhen₂]⁺ (m/z 665.4), and [10-HSA − H + MgPhen₂]⁺ (m/z 638.4) product ions (Figure 2b). Following the ion/ion reaction, the resulting charge-inverted product ions were collisionally activated via BT CID, giving rise to the BT CID spectrum portrayed in Figure 2c. In general, BT CID results in the neutral loss of a single phenanthroline ligand, as the [PA − H + MgPhen]⁺ (m/z 459.3), [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3), [10-HSA − H + MgPhen]⁺ (m/z 503.3) product ions are predominant (Figure 2c). Note that the [10-HSA − H − H₂O + MgPhen]⁺ product ion observed at m/z 485.3 can also be represented as an unsaturated FA 18:1 complex cation (i.e., [18:1 − H + MgPhen]⁺). In the LIT, CID of [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3) generates the product ion spectrum displayed in Figure 2d. Interestingly, we note that the CID spectra in Figures 1c and 2d are identical, and once more, this CID spectrum exhibits evidence for two isomeric FA 18:1 complex cations. Exploiting characteristic product ions reflective of carbon-carbon double bond position, the 18:1(9) and 18:1(10) isomers were identified from Figure 2d. Additionally, as described above, the product ion at m/z 345.2 once more serves as a clear marker for hydroxylation position and, consequently, confirms the original ester location in the 10-PAHSA precursor ion. Moreover, in comparison to the BT CID spectrum shown with Figure 1b, Figure 2c displays an abundant [10-HSA − H + MgPhen]⁺ (m/z 503.3) product ion that was not previously observed following direct charge inversion and collisional activation of the 10-PAHSA ion (c.f. Figures 1b and 2c). Comparing the two distinct charge inversion ion/ion strategies presented herein (i.e., CID of the FAHFA ion followed by charge inversion vs. direct charge inversion of the FAHFA anion), we chose to utilize the direct charge inversion strategy for the analysis of saturated FAHFAs. While both ion/ion approaches afford identification of FA and HFA constituents, the [10-HSA − H − H₂O + MgPhen]⁺ complex cation is more abundant when utilizing direct charge inversion of the 10-PAHSA precursor anion, leading to reduced integration times and increased sensitivity. Thus, all data presented hereafter for saturated FAHFA analysis employs a direct charge inversion approach.

3.2. Differentiation of isomeric n-FAHFAs using direct charge inversion ion/ion strategies

In addition to 10-PAHSA, we examined an array of synthetic n-PAHSA isomers including 13-PAHSA, 12-PAHSA, 9-PAHSA, and 5-PAHSA. Ultimately, isomeric FAHFA differing only in the site of esterification can be discerned using gas-phase charge inversion chemistry. Utilizing the direct charge inversion method outlined above, [n-PAHSA − H]⁻ anions were selectively derivatized in the gas-phase via ion/ion reactions with [MgPhen₃]²⁺ reagent dications, yielding abundant [n-PAHSA − H + MgPhen]⁺ complex cations. Once more, [n-HSA − H − H₂O + MgPhen]⁺ complex cations were obtained from BT CID of charge-inverted n-PAHSA complex cations. We note that the resulting BT CID spectra are insensitive to ester position, as collisional activation of charge-inverted n-PAHSA complex cations yielded nearly identical product ion spectra (Figure S6). Individual CID spectra of the charge inverted dehydrated n-HSA complex cations originating from the 13-PAHSA, 12-PAHSA, 9-PAHSA, and 5-PAHSA precursor ions are provided in Figure 3.

Figure 3.

CID spectra of isomeric [n-HSA − H − H₂O + MgPhen]⁺ complex cations derived from (a) 13-PAHSA, (b) 12-PAHSA, (c) 9-PAHSA, and (d) 5-PAHSA.

Analogous to the results described above with 10-PAHSA, the individual CID spectra of [n-HSA − H − H₂O + MgPhen]⁺ complex cations derived from the various n-PAHSA isomers indicate the presence of two isomeric unsaturated octadecenoic ions. Furthermore, distinct spectral differences confirm that unsaturated [n-HSA − H − H₂O + MgPhen]⁺ complex cations are unique to each n-PAHSA isomer (Figure 3). While the fragmentation highlighted in the grey shading can be rationalized as indicated above, ester position in isomeric n-PAHSA structures are marked by an abundant product ion arising from carbon-carbon cleavage proximal and allylic to the site of unsaturation in the [18:1 − H + MgPhen]⁺ complex cation. Considering 13-PAHSA, the CID spectrum of [13-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3) provided in Figure 3a contains an abundant product ion at m/z 387.1 that can be exploited to assign the site of hydroxylation and thus ester bond position. Paralleling results described above, carbon-carbon cleavage proximal and allylic to the C13=C14 in the [18:1(13) − H + MgPhen]⁺ complex cation is the dominant contributor to the m/z 387.1 product ion population. As there is a dramatic suppression in the relative abundances of product ions observed at m/z values greater than m/z 387.1, the product ion at m/z 387.1 is recognized as a key indicator of hydroxylation position, confirming the original ester location in the 13-PAHSA precursor ion. In general, as the ester position in the original n-PAHSA precursor ion moves closer to the carboxyl carbon of the HFA constituent, the diagnostic product ion shifts lower in m/z. For example, interrogation of the [12-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3) the produces ion at m/z 373.1 indicates the 18:1(12) structure, confirming the original ester position in the 12-PAHSA precursor ion (Figure 3b). In an additional example, the CID spectrum of [18:1 − H + MgPhen]⁺ ions derived from 9-PAHSA again illustrates a distinct fragmentation pattern, with the presence of 9-octadecenoate indicated by the product ion at m/z 331.1 (Figure 3c). Lastly, in the case of 5-PAHSA, the product ion at m/z 275.1 from FA 18:1(5) was observed upon dissociation of [5-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3), as shown in Figure 3d. Collectively, these data demonstrate the utility of charge inversion ion/ion reactions for the structural elucidation of isomeric FAHFA. As alkene position in unsaturated n-HFA complex cations is dependent on the original site of ester linkage along the HFA backbone, the CID spectra of [n-HSA − H − H₂O + MgPhen]⁺ complex cations will reflect differences in n-HSA composition. In turn, interrogation of unsaturated n-HFA complex cations reveals distinct, reproducible spectral patterns that enable the unambiguous assignment of hydroxylation position, and consequently, fatty acyl ester location in the initial FAHFA precursor ion.

3.3. Structure elucidation of unsaturated FAHFAs using charge inversion ion/ion reactions

Lastly, we demonstrate the near-complete structure elucidation of unsaturated FAHFA, pinpointing the sites of unsaturation and hydroxylation in the constituent FA and HFA, respectively. This is illustrated with the analysis of 10-POHSA, a synthetic FAHFA in which PO is esterified to 10-HSA. Using the direct charge inversion approach, singly deprotonated 10-POHSA anions were reacted with [MgPhen₃]²⁺ reagent dications and subsequently collisionally activated via BT CID, giving rise to the product ion spectrum shown in Figure 4a. Two unsaturated product ions were observed at m/z 457.3 and 485.3 indicating the [16:1 − H + MgPhen]⁺ and [18:1 − H + MgPhen]⁺ complex cations, respectively. Mass-selection and ion-trap CID of the product ion at m/z 485.3 from Figure 4a yielded a product ion spectrum portrayed in Figure 4b. Once more, CID of the [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3) complex cation provides product ions arising from the dissociation of two octadecenoic isomers. Specifically, the product ions at m/z 387.1 and 385.1 signify FA 18:1(10), while those at m/z 373.1 and 371.1 indicate the 18:1(9) isomer (Figure 4b). Importantly, the product ion at m/z 345.2 can be exploited identifies the hydroxylation site in the 10-HSA and the original ester position in synthetic 10-POHSA, as formation of this ion occurs via proximal carbon-carbon cleavage allylic to the C10=C11 double bond in the [18:1(10) − H + MgPhen]⁺ complex cation. Next, ion-trap CID of the [16:1 − H + MgPhen]⁺ (m/z 457.3) complex cation from Figure 4b was employed to localize the site of unsaturation in the fatty acyl substituent of 10-POHSA. The product ion spectrum of [16:1 − H + MgPhen]⁺ (m/z 457.3) is provided in Figure 4c. Notably, the CID spectrum of the product ion at m/z 457.3 exhibits evidence for only a single hexadecenoic acid isomer, as a single high-mass product ion doublet was detected at m/z 387.1 and 385.1. Together, these diagnostic ions and the identifiable spectral gap facilitate the assignment of the C9=C10 double bond in the [16:1 − H + MgPhen]⁺ ion consistent with palmitoleic acid. We note that while the C9=C10 double bond in 16:1 was assigned de novo exploiting the reproducible spectral pattern, carbon-carbon double bond positions, particularly in polyunsaturated FAs, can be assigned by automated spectral matching to the previously developed FA library.[²⁰] Taken together the two sets of CID products ions explicitly confirm the structure of both FA and HFA components, as well as the regiochemistry of the FAHFA.

Figure 4.

(a) Product ion spectrum resulting from ion/ion reaction between [10-POHSA]⁻ anions and [MgPhen₃]²⁺ reagent dications and subsequent BT CID. Ion-trap CID spectra of (b) [10-HSA − H − H₂O + MgPhen]⁺ (m/z 485.3) and (c) [16:1 − H + MgPhen]⁺ (m/z 457.3).

While for the 10-POHSA standard the above direct charge inversion analysis is unambiguous, in a complex mixture there exists the potential for dehydrated HFA ions (i.e., [HFA − H − H₂O + MgPhen]⁺) to be isomers of unsaturated FAs. Explicitly, the direct charge inversion strategy is prone to overlapping product ion signals arising from dehydrated HFA and unsaturated FA ions, and in turn, the dehydrated HFA constituent could be misidentified as a FA. Fortunately, this ambiguity to can be removed by implementing the complementary approach, described above, wherein CID of the [FAHFA − H]⁻ ions can be conducted first followed by charge-switch derivatization of the product ions. In q2, CID of the [10-POHSA − H]⁻ anion (m/z 535.5) yields an abundant singly deprotonated 16:1 carboxylate anion at m/z 253.2, reflecting the FA substituent (Figure 5a). Dissociation of the [10-POHSA − H]⁻ precursor ion also generated the [10-HSA − H]⁻ (m/z 299.3) and [10-HSA − H − H₂O]⁻ (m/z 281.2) product ions that readily facilitate identification of the HFA constituent in the unsaturated FAHFA precursor ion. Charge-inversion of the product ions shown in Figure 5a followed by BT CID provides the product ion spectrum illustrated with Figure 5b. Following the ion/ion reaction, BT CID results in the generation of the [16:1 − H + MgPhen]⁺ (m/z 457.3) complex cation, along with charge-inverted product ions pertaining to the HSA constituent. Specifically, the product ion observed at m/z 485.3 corresponds to the [10-HSA − H − H₂O + MgPhen]⁺ complex cation, while the complex cation at m/z 503.3 represents [10-HSA − H + MgPhen]⁺. Subsequent interrogation of the unsaturated complex cation at m/z 485.3 and 457.3 provide identical product ion spectra to those shown in Figure 4b and 4c, respectively. Again, CID of the [10-HSA − H − H₂O + MgPhen]⁺ product ion can be used to establish the original ester position along the HFA backbone, while dissociation of the [16:1 − H + MgPhen]⁺ complex cation facilitates assignment of carbon-carbon double bond position in the unsaturated FA constituent. In total, both charge inversion strategies outlined herein can be utilized to assign ester position in the initial FAHFA precursor ion and localize double bond positions in the unsaturated fatty acyl constituent. However, we note that direct charge inversion of unsaturated FAHFA anions could complicate the task of unambiguous FAHFA structure elucidation, especially if multiple isomers are present in a complex biological sample. Therefore, we find that by performing CID of the unsaturated FAHFA anion prior to charge inversion, the HFA and FA constituents can be confidently identified, and ensuing charge inversion via ion/ion reactions can be employed to pinpoint subtle - yet key - structural features such as the site(s) of unsaturation and hydroxylation.

Figure 5.

(a) CID spectrum of [10-POHSA]⁻ (m/z 535.5). (b) Product ion spectrum resulting from ion/ion reaction between the [10-POHSA]⁻ product anions shown in (a) and [MgPhen₃]²⁺ reagent dications followed by collisional activation via BT CID.

4. Conclusions

FAHFAs are a newly discovered class of endogenous lipid found in mammalian tissues and serum that have received considerable attention recently due to their anti-inflammatory and anti-diabetic effects. While the general FAHFA structure consists of a FA esterified to the alcohol moiety of a HFA, FAHFAs exhibit extensive structural diversity, including a category of FAHFA isomers differing only in the ester position along the HFA backbone. In this study, we report the use of gas-phase charge inversion reactions to achieve near-complete structure elucidation of FAHFA. Herein, we explored two distinct charge-inversion strategies. In the first approach, the charge inversion of n-FAHFA isomers is followed by subsequent interrogation of charge-inverted FAHFA complex cations. In the second strategy, n-FAHFA isomers ionized in negative ion mode were first collisionally activated via ion-trap CID and then, the resulting product ions were charge inverted via ion/ion reactions with reagent dications. In general, both strategies permit the identification of hydroxylation position along the HFA backbone and can therefore determine the original ester position in the FAHFA precursor ion. Furthermore, using either charge inversion approach, additional carbon-carbon double bond positions in unsaturated FAHFA can be pinpointed exploiting the CID spectra of [FA − H + MgPhen]⁺ complex cations generated via ion/ion reactions. Ultimately, we find that n-FAHFA structure elucidation is limited by the direct charge inversion strategy, particularly regarding the analysis of unsaturated FAHFAs. Therefore, while direct charge inversion offers increased sensitivity, by performing CID of the FAHFA anion prior to charge inversion we readily achieve unambiguous FAHFA structural elucidation and isomeric discrimination.

Representing a key advantage of the presented method, charge inversion ion/ion chemistry overcomes the difficulties of isomeric FAHFA differentiation when commercial standards are not available. Explicitly, reproducible fragmentation patterns of charge inverted unsaturated FA complex cations can be exploited to localize critical subtle FAHFA structural features like ester position and unsaturation sites, affording unambiguous FAHFA molecular structural assignments. In turn, as monounsaturated FA identification relies on direct spectral interpretation, only authentic FA standards are required to identify polyunsaturated FAs, many of which are readily commercially available. Noting that as additional unsaturated HFA standards become accessible, this approach could likely be further extended to the analysis of FAHFA incorporating unsaturated HFA. Moreover, utilizing the developed ion/ion approach, complementary information obtained from both polarities can be exploited, constituting a major advantage of the ion/ion chemistry over wet-chemical derivatization. Lastly, this entirely gas-phase approach represents a rapid, sensitive MS-based method, affording high-level FAHFA structural detail. Though yet to be demonstrated, charge inversion ion/ion reactions are likely compatible with orthogonal modes of separation, as experimental timescales are consistent with chromatographic separations. Therefore, when FAHFAs are present in higher abundance, a direct infusion strategy could be employed. However, in other cases, where FAHFAs are present at low concentrations, prefractionation could be required. For example, following lipid extraction, FAHFAs could be enriched using solid-phase extraction (SPE) prior to chromatographic separation utilizing an LC-MS/MS protocol paralleling that established by Kolar et al.[11] While experiments done herein were not conducted in the context of LC-MS/MS, the current scan function duration is approximately 1.25 s. While current scan rates are already amendable to LC experiments, shorter scan times could be attained by the appropriate tuning of mutual storage and spray conditions. Thus, in conjunction with separation methods prior to MS analysis, ion/ion charge inversion chemistry could not only reduce mixture complexities arising from biological samples but also represent a viable strategy towards achieving complete FAHFA structural elucidation, with the potential to increase the understanding of FAHFA biological functions and their underlying roles in metabolic and inflammatory diseases.

Highlights:

• Selective gas-phase charge switching of anions of fatty acid esters of hydroxyl fatty acids (FAHFAs)

• Charge switching of either the intact FAHFA anion following by collision-induced dissociation (CID) or collision-induced dissociation of FAHFA with charge inversion of the FA or HFA product anions

• Charge switching and CID enables the elucidation of ester linkage regiochemistry.

• Charge switching and CID enables the elucidation of site(s) of unsaturation in the FA moiety.

• Charge switching and CID facilitates the assignment of the FA and HFA consituents of a given FAHFA.