Characterization of Wax Esters by Electrospray Ionization Tandem Mass Spectrometry: Double Bond Effect and Unusual Product Ions

Abstract

A series of different types of wax esters (represented by RCOOR′) were systematically studied by using electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (MS/MS) along with pseudo MS3 (in-source dissociation combined with MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer. The tandem mass spectra patterns resulting from dissociation of ammonium/proton adducts of these wax esters were influenced by the wax ester type and the collision energy applied. The product ions [RCOOH2]+, [RCO]+ and [RCO – H2O]+ that have been reported previously were detected; however, different primary product ions were demonstrated for the three wax ester types including: 1) [RCOOH2]+ for saturated wax esters, 2) [RCOOH2]+, [RCO]+ and [RCO – H2O]+ for unsaturated wax esters containing only one double bond in the fatty acid moiety or with one additional double bond in the fatty alcohol moiety, and 3) [RCOOH2]+ and [RCO]+ for unsaturated wax esters containing a double bond in the fatty alcohol moiety alone. Other fragments included [R′]+ and several series of product ions for all types of wax esters. Interestingly, unusual product ions were detected, such as neutral molecule (including water, methanol and ammonia) adducts of [RCOOH2]+ ions for all types of wax esters and [R′ – 2H]+ ions for unsaturated fatty acyl-containing wax esters. The patterns of tandem mass spectra for different types of wax esters will inform future identification and quantification approaches of wax esters in biological samples as supported by a preliminary study of quantification of isomeric wax esters in human meibomian gland secretions.

Compositional Analysis of Wax Esters in Human Meibomian Gland Secretions by Direct Infusion Electrospray Ionization Mass Spectrometry

“…1). [13] The relative amount of the WE isomers were determined from the corresponding set of diagnostic product ions, assuming the summed intensities of these ions was proportional to the total product ions and in turn the amount of the corresponding WE. The diagnostic ions differed depending on the WE type: [RCOOH 2 ] + ion for saturated WE, three ions including [RCOOH 2 ] + , [RCO] + and [RCO – H 2 O] + for WE with unsaturated FA components only and for di-unsaturated WE with unsaturated FA and FAl components, and two ions including [RCOOH 2 ] + and [RCO] + for WE with unsaturated FAl components only.…”

“…The diagnostic ions differed depending on the WE type: [RCOOH 2 ] + ion for saturated WE, three ions including [RCOOH 2 ] + , [RCO] + and [RCO – H 2 O] + for WE with unsaturated FA components only and for di-unsaturated WE with unsaturated FA and FAl components, and two ions including [RCOOH 2 ] + and [RCO] + for WE with unsaturated FAl components only. [13] To obtain more accurate quantitation of WE, two types of corrections were performed for these diagnostic product ions. These two corrections were: 1) monoisotopic diagnostic product ion peak of saturated WE (P s, m ) interfered with by the diagnostic product ions of the third isotopic peak of the corresponding monounsaturated WE (P mu, m+2 ); and 2) different ratios of the summed intensities for these diagnostic product ions to the total product ions.…”

“…[13] For the study presented here, we improved the method by including corrections for peak overlapping and normalization based on product ion type, resulting in a more detailed and accurate profile of WE in human meibum.…”

Meibomian glands, meibum, and meibogenesis

“…More than a hundred major individual lipid species that belong to a dozen or more lipid classes were found in meibum of humans and many mammals and described in detail (Butovich et al, 2007b) (Butovich et al, 2007a) (Butovich, 2008, 2009a, 2010a; Butovich et al, 2012a; Butovich et al, 2011; Butovich et al, 2012b; Butovich et al, 2009; Chen et al, 2010; Lam et al, 2011) (Saville et al, 2011) (Campbell et al, 2011) (Brown et al, 2013) (Lam et al, 2014c) (Mori et al, 2014). Despite the continuing search for progressively minor new members of the already identified families of meibomian lipids (Chen et al, 2010; Chen et al, 2013, 2015), major components of meibum, and their average abundances, are now known (Table 1). It seems that further efforts of this nature, without addressing specific unanswered questions, would produce progressively less new information due to the universal law of diminishing returns.…”

“…3. For example, individual unsaturated extremely long chain WE found in human meibum were originally characterized in ((Butovich et al, 2007b); (Butovich et al, 2007a)), followed by several publications by independent groups ((Chen et al, 2010); (Chen et al, 2015); (Lam et al, 2011)), while saturated WE were characterized in [(Butovich et al, 2012a; Chen et al, 2015)]. Individual human meibomian CE, also of extremely long chain nature, were described in (Butovich, 2009a, 2010a), and, later, in (Chen et al, 2010; Chen et al, 2013).…”