Quantification of fatty acid oxidation products using online high-performance liquid chromatography tandem mass spectrometry

Abstract

Oxidized fatty acids formed via lipid peroxidation are implicated in pathological processes such as inflammation and atherosclerosis. A number of methods may be used to detect specific oxidized fatty acids containing a single or multiple combinations of epoxide, hydroxyl, ketone and hydroperoxide moieties on varying carbon chain lengths from C8 up to C30. Some of these methods are nonspecific and their use in biological systems is fraught with difficulty. Measures of specific-oxidized fatty acid derivatives help in identifying oxidation pathways in pathological processes. We used liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) as efficient, selective and sensitive methods for identifying and analyzing multiple specific fatty acid peroxidation products in human plasma and other biological matrices. We then distilled the essential components of a number of these analyses to provide an efficient protocol by which fatty acid oxidation products and their parent compounds can be determined. In this protocol, addition of synthetic internal standard to the sample, followed by base hydrolysis at elevated temperature, and liquid-liquid phase sample extraction with lighter than water solvents facilitates isolation of the oxidized fatty acid species. These species can be identified and accurately quantified using stable isotope dilution and multiple reaction monitoring. Use of a coupled multiplexed gradient HPLC system on the front end enables high-throughput chromatography and more efficient use of mass spectrometer time.

Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry

“…The data acquisition window is 6 minutes, and began after an initial 4.5 minutes of column eluent diversion to waste. To increase sample throughput, we routinely use 2 multiplexed binary pump HPLC system equipped with separate columns and automated column valve system [10] (Fig. 3), allowing for data acquisition only during a narrower time window from each column.…”

Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples

“…Since a deuterated internal standard is either an analogous lipid metabolite or a molecule with similar chemical characteristics (chemical structure of the eicocanoids can be found in Ref. 2, 21 and 23), both lipid metabolites and internal standards will have similar ion suppression and extraction efficiencies. Thus, internal standards were used to evaluate the matrix effect and the recovery rate of the method.…”

“…The rapid progress of liquid-chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has facilitated the use of this technology for accurate monitoring of eicosanoid metabolites in biological samples [7]. Previous reports include liquid-liquid extraction for the determination of PGE 2 and LTB 4 in plasma using LC-MS [15], the analysis of four kinds of PGs and LTs in cell culture media by LC-MS [16], an on-line two dimensional reverse-phase LC-MS for the simultaneous determine of PGE 2 , PGF 2a and 13,4-dihydro-15-keto PGF 2a [17], and a LC-MS method for the simultaneous determination of twenty-three eicosanoids [18], a UPLC-MS platform that enables profiling of 122 eicosanoids from human whole blood[19], a targeted HPLC-MS/MS analysis platform for 100 oxylipins and 36 oxylipins was detected from 250 uL human plasma in 26 min[20], a LC–MS/MS method for rapid and concomitant quantification of 26 PUFA metabolites from Caco-2 cells [21], a LC–MS/MS for the simultaneous analysis of arachidonic acid and 32 related metabolites in 1 mL human plasma [22], a online HPLC-MS/MS analyzed more than 20 different oxidized fatty acids and their precursors from 200 uL plasma or urine [23], a fast LC–MS/MS method including on-line SPE and LIT fragment confirmation for the profiling of 7 PUFAs and 94 oxidized metabolites from 200 uL plasma [24]. We previously developed a high-throughput lipidomic analysis of eicosanoids using LC-MS to monitor 141 unique species in a single 22 min analysis [25], and applied it to investigate the human plasma lipidome [26,27].…”

Early physiological and biochemical responses of rice seedlings to low concentration of microcystin-LR

“…Measurement of catalase, superoxide dismutase, glutathione, or products of lipid peroxidation, or protein phosphatase bioassays could be used in further assays to study the extension of oxidative stress in rice plants caused by exposure to MC-LR (Asensi et al 1999;Chen et al 2004;Lambert et al 1994;Oh et al 2001;Vinagre et al 2012). In this regard, mass-spectrometry based sensitive methods have been suggested for the identification and quantitation of oxidative stress parameters and oxidation products (Lee and Britz-McKibbin 2009;Levison et al 2013). On the other hand, the lack of response of the two biochemical markers may have been due to a poor capacity of the plant to take up and accumulate the toxin.…”