Qualitative analysis of omega-3 fatty acid oxidation by desorption electrospray ionization-mass spectrometry (DESI-MS)

https://doi.org/10.1016/j.ijms.2014.07.037Get rights and content

Highlights

  • DESI-MS in negative ion mode detected oxidation of deprotonated omega-3 fatty acids, which happens mostly through peroxidations and keto formations.

  • The oxidation of omega-3 fatty acids is detected by DESI and ESI-MS in positive ion mode in the form of alkali metal adducts.

  • The oxidation of the omega-3 fatty acids was due to the aging of the samples, since oxidation increased over time.

  • The oxidation products of the omega-3 fatty acids were detected and compared using DESI, ESI, and MALDI-MS.

  • DHA and EPA as well as some possible oxidation products were identified from the surface of a dog kibble with DESI-MS.

Abstract

The oxidation of omega-3 fatty acids, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha-linolenic acid (ALA), was studied by MS and tandem mass spectrometry (MS/MS) using a custom-designed DESI source, which was coupled to an ion trap (IT)-mass spectrometer. DESI-MS showed that oxidation due to aging occurred most readily by peroxidations and keto formations of the omega-3 fatty acids, which are the expected oxidation pathways for these molecules. The oxidized products detected were compared between DESI-MS and ESI-MS after a three-week period of sample aging at the room temperature and ambient conditions. The mass spectra of the omega-3 fatty acids obtained by these two techniques in negative ion mode were similar, while oxidation of omega-3 fatty acids was observable with DESI-MS in positive ion mode only after prolonged aging of the samples. The peroxidation products of fatty acids, similar to those observed by DESI and ESI-MS, were also detected by MALDI-MS. Additionally, a commercial dog kibble was analyzed by DESI-MS. DHA, EPA, and some possible oxidation products were identified from the surface of this food sample using DESI-MS and MS/MS. The results demonstrate that DESI-MS can be used efficiently in negative ion mode to monitor oxidation of fatty acids due to their aging both in pure forms and on the surfaces of solid food samples.

Introduction

Several ionization techniques have been used successfully for MS analyses of samples at ambient conditions such as desorption electrospray ionization (DESI), direct analysis in real time (DART), desorption atmospheric pressure chemical ionization (DAPCI), and desorption atmospheric pressure photoionization (DAPPI) [1], [2], [3], [4]. DESI-mass spectrometry (DESI-MS) is an advantageous ionization technique for ambient MS analyses because it requires minimal sample preparation and it can be applied for the analysis of solid samples [5]. DESI-MS has been used to analyze a wide array of compounds such as peptides and proteins [2], [5], lipids [6], [7], [8], [9], [10], [11], traces of explosives and chemical warfare agents [12], [13], and polymers [14]. The technique has also been used in forensic applications for the analyses of illicit drugs, inks, and gunshot residues [3]. However, there are some disadvantages of DESI-MS; for example, high flow rates of the nebulizing gas are needed and samples that are easily oxidized may undergo oxidation readily. Fortunately, the extent of oxidation can be minimized under certain instrumental and environmental parameters [15].

DESI-MS has been used in numerous studies that were focused on analyses of different types of lipid samples. For example, phospholipids and sphingolipids were analyzed by DESI-MS in positive and negative ion modes, and the fragmentation patterns and adduct formations were determined [6]. Another study looked at different types of edible oils, such as sunflower and olive oil, to determine which triglycerides were present in them [7]. Most of the peaks observed were due to ammonium adducts, and very little protonated, sodiated, or potassiated ions were observed. The triglycerides in different brands of chocolate were determined in this study [7]. DESI-MS was used to study location of double bonds in unsaturated lipids [16]. Lipid analyses have also been performed on intact bacteria using DESI-MS. Both Gram-positive and Gram-negative bacteria of seven different bacterial species were analyzed, and distinct DESI-MS spectra of lipids were recorded for each of the species [8].

Another aspect that makes DESI-MS a versatile ionization technique for the analysis of lipids, such as omega-3 fatty acids, is its ability to image tissues and solid samples [9], [10], [11]. An important study determined how instrument conditions and other analytical variables affected DESI-MS signal obtained from a tissue sample [9]. DESI-MS detected how brain tissue samples degrade over time, and alpha-linolenic acid (ALA) was a component of a fatty acid mixture that was analyzed in this study [9]. Docosahexaenoic acid (DHA) and phospholipids were also identified by DESI-MS imaging of rat brain tissue sections [10]. Other tissue samples, such as rat spinal cords, porcine adrenal glands, and outer human lens, were analyzed by DESI-MS imaging, and several phospholipids were localized in tissue sections [10]. Different fatty acids and fatty acid dimers were identified from the spinal cords, including DHA [11]. Additionally, DHA and eicosapentanoic acid (EPA) were analyzed in fish oil capsules [4].

Considering that omega-3 fatty acids DHA, ALA, and EPA are biologically important lipids, which may inhibit development of neurodegenerative diseases and are often used as food supplements, monitoring of their oxidation due to aging is important [17]. As mentioned above, DESI-MS has been used to detect omega-3 fatty acids in various samples and oxidation of some compounds due to DESI process was studied [15]. Additionally, oxidation of linoleic acid attributed to the effect of the spray solvent was detected by DESI-MS [7]. It was also shown that oxidized EPA ions containing up to six oxygen atoms could be detected by ESI-MS in negative ion mode after allowing the samples to oxidize over several days [18]. However, DESI-MS has not been used for detailed qualitative analyses of aging process of omega-3 fatty acids in free forms and in solid food samples. In stored food products, omega-3 fatty acids inevitably suffer autoxidation leading to rancidity, reduced shelf life, and decreased nutritional quality of food [17]. Therefore, it would be very beneficial to show that a technique such as DESI-MS can be used for the analysis of fatty acid oxidation due to their aging.

It was shown that samples of prescription drugs, such as tamoxifen and reserpine, can be oxidized during the process of DESI, leading mainly to the net addition of one or more oxygen atoms to the sample molecules through peroxidations or alcohol adducts [15]. This autoxidation during DESI was shown to occur through the sample exposure to oxygen and light at ambient conditions or possibly due to analyte interactions with reactive oxygen species (ROS) in the gas phase [15]. However, this autoxidation can be minimized by controlling several different parameters for the experiments. The parameters and conditions that have an effect on the extent of oxidation observed in DESI-MS include: time between the sample deposition and analysis, exposure of the samples to ambient fluorescent lighting, DESI spray voltage, solvent flow rate, high analyte surface concentration, and the addition of redox buffers to the spray solvent [15].

In this work, we demonstrate that DESI-MS can be efficiently used for qualitative analyses of oxidation products of omega-3 fatty acids. The results obtained indicate that DESI-MS in negative ion mode is an advantageous technique compared to ESI-MS and MALDI-MS for the monitoring of oxidation of omega-3 fatty acids in solid samples.

Section snippets

Materials and reagents

HPLC grade acetonitrile (CH3CN) and water, sodium bicarbonate, and sodium acetate were purchased from Fisher Scientific (Pittsburgh, PA). ALA, EPA, and DHA were obtained from Cayman Chemical Company (Ann Arbor, MI). The commercial small dog kibbles were purchased from a local store. Samples were used without further purification. 2,5-Dihydroxybenzoic acid (DHB) and MALDI-MS peptide calibration standard were from Bruker Daltonics (Bremen, Germany), and 6-aza-2-thiothymine (ATT) was from Sigma

Instrument optimization

A DESI-MS source was successfully coupled to an IT-mass spectrometer. Several parameters needed to be optimized in order to increase the ion transmission into the MS. Some of the parameters that were important to optimize were the incident and collection angles, the sprayer tip-to-surface distance, the MS inlet-to-surface distance, the pressure of the nebulizing gas, flow rate of the solvent, and the capillary voltage applied to the solvent. Scheme S1 shows a schematic of the DESI-MS setup with

Conclusions

DESI and ESI-MS were used to study omega-3 fatty acids DHA, ALA, and EPA and their oxidation products. These two techniques yield similar results in negative ion mode, while ESI-MS detected oxidized fatty acids after sample aging more efficiently than DESI-MS in positive ion mode (Table 1). In our experiments, the oxidation of the omega-3 fatty acids was observed with DESI-MS in positive ion mode only after prolonged aging of the sample, which is a disadvantage of the technique in comparison to

Acknowledgements

This work was supported by the research funds from the Mars Global Food Safety Centre, Huairou, Beijing, China and from the University of Toledo (DI). MALDI-MS instrument was acquired using funding provided by an NSF-MRI grant (award #0923184). Authors thank Dr. Eric Findsen and Walter Berger Jr. (University of Toledo), who helped developing some of the components used for DESI-MS setup [19], and Bill Mahn (Thermo Scientific) for technical support.

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