Potential of atmospheric pressure ionization sources for the analysis of free fatty acids in clinical and biological samples by gas chromatography-mass spectrometry

Abstract

Because of the central role of fatty acids in biological systems, their accurate quantification is still important. However, the impact of the complex matrix of biologically and clinically relevant samples such as plasma, serum, or cells makes the analysis still challenging, especially, when free non-esterified fatty acids have to be quantified. Here we developed and characterized a novel GC–MS method using pentafluorobenzyl bromide as a derivatization agent and compared different ionization techniques such as atmospheric pressure chemical ionization (APCI), atmospheric pressure chemical photoionization (APPI), and negative ion chemical ionization (NICI). The GC-APCI-MS showed the lowest limits of detection from 30 to 300 nM for a broad range of fatty acids and a similar response for various fatty acids from a chain length of 10 to 20 carbon atoms. This allows the number of internal standards necessary for accurate quantification to be reduced. Moreover, the use of pentafluorobenzyl bromide allows the direct derivatization of free fatty acids making them accessible for GC–MS analysis without labor-intense sample pretreatment.

Unraveling the Double Bond Position of Fatty Acids by GC-MS Using Electron Capture APCI and In-Source Fragmentation Patterns

“…The LOD and LOQ values are in the range of 0.1–1 μM for the intact FA ion and around 10 μM for the regiospecific in-source fragment ions (Table ). This is slightly higher compared to results obtained previously using a triple quadrupole mass spectrometer in pseudo-MRM mode . The reason for this is due to the loss of intensity for the characteristic [FA-PFB] − ion and the difference in the mass analyzer; however, the linearity is not affected.…”

“…Both have also been reported in cuttlefish and tuna. , Moreover, quantification can be done using the [FA-PFB] − ion for FAs that are not included as standards in the calibration curve. Here similar FAs can be used for quantification as the response for many FAs is rather similar when ionized by APCI in negative mode …”

“…Then, 600 μL of methyl tert -butyl ether (MTBE), 100 μL of methanol, and 170 μL of water were added, the sample was vortexed for 2 min, and the upper layer was collected. The aqueous phase was washed with 600 μL of MTBE, and the organic layers were combined and dried at 30 °C using a vacuum evaporator …”

“…The dried lipid extracts were suspended in 100 μL of diisopropyl­ethylamine:dichloro­methane (1:9; v/v) and mixed with 100 μL of 2,3,4,5,6-pentafluorobenzyl bromide previously diluted with dichloromethane (1:9; v/v). The samples were incubated for 60 min at 50 °C, dried at 30 °C using a vacuum evaporator, and redissolved in 300 μL of methanol . Fish oil samples were diluted 1:20 or 1:200 in methanol prior to the analysis.…”

“…Excess consumption of ω-6 FAs has been linked to various mental illnesses such as depression, severe mood swings, bipolar disorder, and attention-deficit/hyperactivity disorder (ADHD). , Moreover, the ratio of regioisomeric FAs might also serve as an indicator of multiple sclerosis or cancer. , Thereby, analytical strategies are necessary to identify and determine the FA regioisomers. However, their close structural similarities complicate the chromatographic separation, leading to coelutions of the regioisomers and making the identification of the double bond position challenging …”