A comprehensive quantification method for eicosanoids and related compounds by using liquid chromatography/mass spectrometry with high speed continuous ionization polarity switching

https://doi.org/10.1016/j.jchromb.2015.05.015Get rights and content

Highlights

  • Quantification of eicosanoids and related metabolites by UHPLC–MS/MS.

  • Method for simultaneous quantification of 137 metabolites.

  • Fast continuous polarity switching for metabolites ionized in different polarity.

  • Method was successfully applied to analysis of mouse tissues.

Abstract

Fatty acids and related metabolites, comprising several hundreds of molecular species, are an important target in disease metabolomics, as they are involved in various mammalian pathologies and physiologies. Selected reaction monitoring (SRM) analysis, which is capable of monitoring hundreds of compounds in a single run, has been widely used for comprehensive quantification. However, it is difficult to monitor a large number of compounds with different ionization polarity, as polarity switching requires a sub-second period per cycle in classical mass spectrometers. In the present study, we developed and evaluated a comprehensive quantification method for eicosanoids and related compounds by using LC/MS with high-speed continuous ionization polarity switching. The new method employs a fast (30 ms/cycle) continuous ionization polarity switching, and differentiates 137 targets either by chromatography or by SRM transition. Polarity switching did not affect the lower limits of quantification, which ranged similarly from 0.5 to 200 pg on column. Lipid extracts from mouse tissues were analyzed by this method, and 65 targets were quantitatively detected in the brain, including 6 compounds analyzed in the positive ion mode. We demonstrated that a fast continuous ionization polarity switching enables the quantification of a wide variety of lipid mediator species without compromising the sensitivity and reliability.

Introduction

Fatty acids are one of the essential components of mammalian organisms. They are esterified as building blocks in various lipids such as neutral lipids (fats), membrane phospholipids, and cholesteryl esters. Fatty acids in the triglycerides are mobilized by lipases, and they serve as energy source in the tissues through β-oxidation. Fatty acids in membrane phospholipids affect their physicochemical properties, and those esterified in cholesterol are critical for cholesterol homeostasis. In addition to these housekeeping roles, a number of fatty acids and related metabolites are known to function as signaling molecules that mediate a variety of physiological and pathological processes through their binding to receptors on target cells.

Eicosanoids such as prostaglandins (PG), thromboxanes (TX), leukotrienes (LT), and hydroxyeicosatetraenoic acids (HETE) are a class of lipid mediators synthesized from dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA), and eicosapentaenoic acid (EPA), and are known to play various pathological and physiological roles in vivo [1], [2]. To date, relations between eicosanoid production and diseases have been extensively studied using disease models in animals or samples obtained from humans, providing insights into their roles in pathogenesis [3], [4], [5]. Recent studies have shed light on the physiological functions of the compounds that were recognized as byproducts or inactive metabolites, such as 12-hydroxy-heptadecatrienoic acid (12-HHT) or 15-keto-PGE2 [6], [7]. Non-enzymatic oxidation by endogenous reactive oxygen species converts AA to isoprostanes such as 8-iso-PGF2α, known as biomarkers of oxidative stress [8], [9]. There is an increasing interest in the physiological or pathophysiological functions of omega-3 fatty acids such as EPA, docosahexaenoic acid (DHA), and α-linolenic acid as well as their metabolites such as resolvins and protectins [10]. Omega-6 linoleic acid causes hydroxy​octadecadienoic acids (HODEs), non-eicosanoid lipid mediator associated with atherosclerosis [11], [12]. The biological functions of arachidonoyl ethanolamide (AEA) and its metabolites such as PG-ethanolamides are also of interest [13]. More than 500 fatty acid related metabolites have been listed in the Lipid MAPS database (http://www.lipidmaps.org/), warranting a method capable of monitoring several hundreds of lipid metabolites, in order to understand the pathogenic mechanisms associated with these metabolites, as well as facilitate lipid biomarker discovery.

Selected reaction monitoring (SRM) is a selective and sensitive detection mode available in triple quadrupole mass spectrometers (TQ–MS), which detects target compound(s) by monitoring a precursor-to-product ‘transition’ of target ions. We have previously reported the simultaneous monitoring method for 13 eicosanoids and platelet activating factor by using the column-switching liquid chromatography (LC)/TQ–MS system [14]. Isoprostanes have also been adopted as targets for comprehensive profiling in the evaluation of stress markers [15], [16]. Recent studies describe methods that analyze around 100 eicosanoid species in a single chromatographic analysis [17], [18], [19], [20], [21].

Although SRM has the potential to monitor several hundreds of compounds simultaneously, chromatographic separation is required for compounds that are not differentiated by SRM transitions. In SRM analysis, there are some trade-offs between the number of targets and the sensitivity, as mentioned in some reports [18], [20]. Recent MS systems offer (so called) segmented- or scheduled-SRM to minimize such trade-offs, where each target compound is monitored in a narrow chromatographic time window set around the compound’s retention time, minimizing the SRM transitions run simultaneously at a certain time point.

Ionization polarity switching during the analysis is a common function of a mass spectrometer for detecting the compounds that are ionized in different polarity, and has been available over the last decade. Most recent instruments are equipped with a fast polarity switching capability of several tens of milliseconds (ms), making it possible to simultaneously monitor compounds that are ionized in different polarity. A quantification of 135 primary metabolites, including sugars and amino acids with continuous polarity switching, has been reported previously [22]. However, in the lipid field, the benefits of the fast polarity switching have not been fully demonstrated. Dumlao et al. reported a method that detects 36 ethanolamides by positive electrospray ionization (ESI) and 141 eicosanoids by negative ESI in a separate run [19]. Recently, the application of positive ESI for SRM quantification of cysteinyl-LTs (cys-LTs) in combination with negative ESI for LTB4 and for 6-trans LTB4 in a single chromatographic analysis was reported [23]; however, this method does not use continuous polarity switching, since the retention times of these targets were quite different.

Here, we report a comprehensive quantitative method for lipid mediators and its biological application by using a high-speed TQ–MS with fast (15 ms) continuous ionization polarity-switching system.

Section snippets

Chemicals

All lipid standards, including 151 target compounds and 12 deuterium-labeled compounds, that were used as internal standards were purchased from Cayman Chemical (Ann Arbor, MI), and were dissolved in methanol and stored at −80 °C. LC/MS-grade acetonitrile, HPLC-grade formic acid, HPLC-grade methanol, and ethanol were purchased from WAKO (Osaka, Japan). Ultrapure water was prepared by using the milli-Q system (Millipore, Billerica, MA).

LC/MS conditions

The LC/MS system consisted of two LC-30AD pumps, an SIL-30AC

Development of continuous ionization polarity switching method

We first examined the electrospray ionization of 151 commercially available fatty acid-related lipids (Fig. 1 and Supplementary Fig. S1) and 12 deuterium-labeled internal standards. Most of the compounds were ionized dominantly or only in the negative ion mode, but 21 compounds, including 2 internal standards, were better ionized in the positive ion mode. CID spectra were analyzed for each compound to determine product ions to be used for SRM (Supplementary Fig. S2). SRM transitions were

Discussion

In the present report, we developed an LC/MS based method for quantification of a large number of lipid mediators using a high speed continuous ionization polarity switching method. To the best of our knowledge, this is the first report on the development of a method employing continuous ionization polarity switching for mediator lipidomics analysis.

A very fast ionization polarity switching capability of the latest MS instrument allowed us to perform as many as 45 positive/negative SRM

Conflict of interest

Department of Lipidomics is funded in part by Shimadzu Corporation, Kyoto, Japan, and Ono Pharmaceutical Co., Ltd., Osaka, Japan.

Acknowledgments

We thank Ms. Ayako Kobayashi for technical support. This work was supported in part by MEXT KAKENHI Grant numbers 25116707 (to Y.K.) and 24229003 (to T.S.), and Takeda Science Foundation (to T.S.).

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