Variation in the Relative Isomer Abundance of Synthetic and Biologically Derived Phosphatidylethanols and Its Consequences for Reliable Quantification

Abstract

Phosphatidylethanol (PEth) in human blood samples is a marker for alcohol usage. Typically, PEth is detected by reversed-phase liquid chromatography coupled with negative ion tandem mass spectrometry, investigating the fatty acyl anions released from the precursor ion upon collision-induced dissociation (CID). It has been established that in other classes of asymmetric glycerophospholipids, the unimolecular fragmentation upon CID is biased depending on the relative position (known as sn-position) of each fatty acyl chain on the glycerol backbone. As such, the use of product ions in selected-reaction-monitoring (SRM) transitions could be prone to variability if more than one regioisomer is present in either the reference materials or the sample. Here, we have investigated the regioisomeric purity of three reference materials supplied by different vendors, labeled as PEth 16:0/18:1. Using CID coupled with ozone-induced dissociation, the regioisomeric purity (% 16:0 at sn-1) was determined to be 76, 80 and 99%. The parallel investigation of the negative ion CID mass spectra of standards revealed differences in product ion ratios for both fatty acyl chain product ions and ketene neutral loss product ions. Furthermore, investigation of the product ion abundances in CID spectra of PEth within authentic blood samples appears to indicate a limited natural variation in isomer populations between samples, with the cannonical, PEth 16:0/18:1 (16:0 at sn-1) predominant in all cases. Different reference material isomer distributions led to variation in fully automated quantification of PEth in 56 authentic dried blood spot (DBS) samples when a single quantifier ion was used. Our results suggest caution in ensuring that the regioisomeric compositions of reference materials are well-matched with those of the authentic blood samples.

Identification of Phosphatidylcholine Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions

“…PC36:1 then consists of four sn-positional isomers:PC18:0/18:1, PC18:1/18:0, PC20:1/16:0, and PC16:0/20:1. Determination of the relative contributions of these four isomers to the overall PC36:1 ion signal would require generation of calibration curves similar those used with PC34:1, which is dependent on the availability, affordability, and purity69 of synthetic lipid standards. Nonetheless, the identification of multiple isomers represents an improvement over the sum-composition identification of PC34:1.…”

Isomer-Resolved Imaging of Prostate Cancer Tissues Reveals Specific Lipid Unsaturation Profiles Associated With Lymphocytes and Abnormal Prostate Epithelia

“…Quantitative accuracy is reliant on the calibrant standards being structurally identical to the analyte ( 35 ). To generate the calibration ( Figure 3B ), high regiopurity PC standards ( i.e.…”

“…Quantitative accuracy is reliant on the calibrant standards being structurally identical to the analyte (35). To generate the calibration (Figure 3B), high regiopurity PC standards (i.e., the sn-2 unsaturated PC 16:0/18:1n-7 and PC 16:0/18:1n-9) were analyzed as these sn-2 regioisomers are more commonly found in mammalian cells (typically above 90%) (16).…”

Perspective on Emerging Mass Spectrometry Technologies for Comprehensive Lipid Structural Elucidation

“…However, identification of sn -position via this method should be performed with caution, as the relative abundances of fragment ions formed from cleavage at the sn -1 and sn -2 positions can also depend on other factors, such as fatty acid chain length and degree of saturation. Additionally, mixtures of multiple positional isomers are likely representative of biological samples, further complicating this interpretation . Nonetheless, this methodology has been particularly useful in the imaging mass spectrometry analysis of PCs in rat brain tissue (Figure ).…”

“…Additionally, mixtures of multiple positional isomers are likely representative of biological samples, further complicating this interpretation. 139 Nonetheless, this methodology has been particularly useful in the imaging mass spectrometry analysis of PCs in rat brain tissue (Figure 4). 140 While PC anions subjected to CID readily produce fragment ions indicative of the identity and sn-position of the fatty acyl chains in the lipid, 58 ionization suppression from more acidic endogenous glycerophospholipids generally prevents the detection of PC lipids directly from tissue in negative ion mode MALDI imaging mass spectrometry.…”