Eicosanoids are oxidized arachidonate-derived lipids products generated by lipoxygenase, cyclo-oxygenase and cytochrome P450. Their bioactivity is mediated via receptor-dependent mechanisms and they are involved in a diverse array of processes in both health and disease. For many years, GC–MS was the method of choice for eicosanoid analysis, but, more recently, the availability of high-sensitivity electrospray LC (liquid chromatography)–MS/MS (tandem MS) has provided a new approach for quantification, while minimizing sample preparation. The present review summarizes the various methods available for routine quantification of eicosanoids, focusing ultimately on their analysis using a hybrid Q-Trap mass spectrometer.
Eicosanoids are lipid peroxidation products of 20-carbon (eicosa-) polyunsaturated fatty acids, including PGs (prostaglandins)/prostanoids, TXs (thromboxanes), LTs (leukotrienes), epoxides and hydro(peroxy) fatty acids (or oxylipins). They are generated in biological systems by three separate enzyme families, LOX (lipoxygenase), COX (cyclo-oxygenase) and CYP (cytochrome P450), which catalyse lipid peroxidation in a highly regulated manner generating stereo- and regio-specific products (Figure 1). Their expression is highly tissue-localized and varies with inflammatory activation state. Primary products of COX, LOX and CYP are metabolized, depending on cell type, into secondary eicosanoids and their metabolites, some of which are also potently bioactive. A wide variety of eicosanoids, including the classical prostanoids and a complex array of isomers (the isoprostanes), can be formed through non-enzymatic oxidation, and are often used as markers of oxidative stress in vivo.
The families of eicosanoid-generating enzymes
Measurement of eicosanoids
Owing to the chemical and biological complexity of eicosanoids, measurements of their concentrations must be carefully validated. Although there are several approaches for determining these mediators, there are pitfalls that need to be avoided. The assay should be sensitive, specific and accurate. Sometimes, stable metabolites of eicosanoids are measured, rather than unstable precursors, as this will give a more accurate estimate of whole-body/tissue generation. For example, 6-oxo-PGF1α and 2,3-dinor-TXB2, excreted at high concentrations into the urine are used to estimate total systemic PGI2 and TX in humans or mice [1,2]. In this case, an additional advantage is that samples are obtained non-invasively. Although LTE4 can be used as a marker of cysteinyl LT synthesis in vivo, there are currently no other LOX metabolites which are routinely used for systemic determination of this pathway in vivo.
Several excellent reviews have been written on the analysis of eicosanoids. Instead of replicating previous information, we will direct the reader to them where relevant. In particular, the excellent Prostaglandins and Related Substances: a Practical Approach edited by Benedetto et al. [2a] is recommended. For ESI (electrospray ionization)–MS/MS (tandem MS), a review by Murphy et al.  provides a comprehensive list of spectra and MRM (multiple reaction monitoring) transitions for all eicosanoid classes . http://www.lipidmaps.org is the website of the Lipid Metabolites and Pathways Strategy [NIH (National Institutes of Health) Glue Grant], which regularly updates its database with useful information for eicosanoid research, particularly product ion spectra, structures and links to suppliers.
Eicosanoids are unstable and may be generated during sample handling and processing. Also, activation of platelets during venepuncture can cause generation of TXA2. Extraction of eicosanoids from solid tissue may require homogenization, a process that itself can activate eicosanoid synthesis. It is worthwhile considering the inclusion of inhibitors and other drugs to minimize artefactual eicosanoid formation. These include indomethacin (10 μM), EDTA (calcium chelator), DTPA (diethylenetriaminepenta-acetic acid) (iron chelator, 100 μM) and BHT (butylated hydroxytoluene) (antioxidant, 100 μM), depending on the pathway to be measured. Eicosanoid decomposition during extraction can occur. A number of eicosanoids can be hydrolysed by acid. For example, PGE2 can be converted into PGA2, and many epoxides are readily hydrolysed to their diols by treatment with dilute acids used during extractions. Oxidation is also a problem and double-bond rearrangements may also occur. Sample losses due to inactivation can be accounted for by the use of suitable internal standards (see below).
Determination of eicosanoids in biological samples usually requires extraction into organic solvent. The preferred methods are either liquid-based or solid-phase extraction columns. Solvents can be removed at low temperatures under inert gas when required. In the past, most laboratories used liquid extraction. Efficiency can be influenced by adding salt or altering pH. Indeed, with the pKa of carboxylic acids at approx. 4.5, lower pH enhances efficiency. We use low concentrations of ethanoic (acetic) acid (1–5%), since this acid is relatively volatile. For extraction of eicosanoids from primary human and mouse leucocytes and platelets, we routinely use a hexane/propan-2-ol/ethanoic acid two-step solvent extraction . For assays of LOX metabolites, we sometimes include a reduction step using SnCl2. This converts HpETEs (hydroperoxyeicosatetraenoic acids) into the more stable HETEs (hydroxyeicosatetraenoic acids).
Solid-phase extraction is also widely used for eicosanoid extraction, and is especially useful for large volumes of fluid [5,6]. Columns used include normal-phase (silica), reverse-phase (C18) and possibly ion-exchange. Analytes are eluted from the reverse phase in order of decreasing polarity. Eicosanoids bind to C18 columns, with salts and polar compounds washing off in water, and are then eluted with ethyl acetate. A second clean-up could involve a silica cartridge for removal of neutral lipids, then elution of eicosanoids in methanol. The most specific solid-phase extraction involves antibody binding of the analyte, with the antibody bound to a Sepharose matrix [7,8].
During eicosanoid extraction, losses can be up to 50% or more, depending on analyte and method. Use of internal standards is absolutely essential for quantitative assays using HPLC, GC–MS or LC (liquid chromatography)–MS. Stable isotope-labelled standards are optimal since they behave identically with the analyte of interest in terms of extraction and recovery, and standards are eluted on GC or LC close to the unlabelled analyte. Deuterated internal standards are available only for a small number of eicosanoids and are rather costly. As a compromise, for LC–MS/MS determination of eicosanoids, a stable isotope-labelled standard for each eicosanoid class (e.g. 12-[2H8]HETE for all HETEs or [2H4]PGE2 for all PGs) is included in all samples before extraction. Standard curves are constructed using mixtures of labelled and unlabelled standards for all analytes of interest. The use of unlabelled standards for all other analytes is essential, since different compounds will give different responses in terms of ionization sensitivity, even within the same class of molecule. In addition, they are required for retention time determination for the analytes in the biological sample. The concentration range for detection must also be linear in the standard curve. For example, in our LC–MS/MS system, detection of 12-HETE is linear from approx. 1 to 500 pg on the column. Concentrations measured in biological samples must fall within the linear part of the standard curve, or quantification will be inaccurate.
Immunoassays should be used with caution since they may hugely (even up to 1000-fold) overestimate analyte cross-reaction. This results from the cross-reactivity of the antibodies with other eicosanoids and can be very difficult to eliminate. It is possible to increase the selectivity of the RIA or ELISA by using it after HPLC separation of eicosanoids, where retention time is also used for identification. It is recommended that, if an ELISA is to be employed, it is first validated in the biological samples of interest using a physicochemical method, such as GC–MS or LC–MS/MS. To date, validation of ELISAs has not been extensively carried out, and their use in biological determination of eicosanoids should be approached with caution.
For a small number of eicosanoids, HPLC with UV detection is useful. Compounds containing conjugated diene, triene or tetraene chromophores, such as mono-, di- and tri-hydroxy lipids from the LOX pathway absorb at 235, 269 and 301 nm respectively and possess a characteristic UV profile [5,9–12]. However, sensitivity is low, in the nanogram range. UV detection is of limited use for PGs as they do not absorb light in this region. In general, HPLC–UV assays are only suitable where relatively large amounts of eicosanoids are present in clean biological samples. Plasma contains numerous UV-absorbing compounds at higher levels than the eicosanoids. Radiochemical detection following LC separation can be used, where [14C]arachidonate or [3H]arachidonate is provided as the precursor, and LC retention time is used to determine product. However, this relies on pre-treatment of cells in culture with radiolabelled arachidonate. As mentioned above, HPLC has been combined with RIA, and this can greatly enhance sensitivity [13–15].
GC has long been the method of choice for routine separation of fatty acids and eicosanoids. However, the high cost and technical expertise required for this approach has limited its use, and even now it is still only utilized in a handful of specialized laboratories worldwide for eicosanoid research. For more detail regarding GC–MS of eicosanoids, including derivatization protocols, the reader is directed to Barrow et al. . GC utilizes the partition of the analyte between stationary and gaseous phase. Therefore molecules to be analysed must be volatile and thermally stable at high temperatures. For eicosanoids, this requires derivatization of polar groups. Typical methods include N-acylation, methoximine formation, esterification and trimethylsilyl ether formation (full details are given in ). Typical GC capillary columns consist of fused silica to which is bonded the stationary phase [17,18].
When coupled to MS detection, GC is both highly sensitive and selective for eicosanoid detection. Multiple analytes can be detected in one sample, greatly reducing cost for routine detection once the equipment is in place and operational.
Following derivatization and chromatography, the sample passes into the mass spectrometer and is ionized by EI (electron impact), CI (chemical ionization) (e.g. methane) or EC (electron capture). In EI, the analyte is bombarded by a beam of electrons, removing a low-energy electron. This is converted into a radical cation (M+), which decomposes into fragments. The fragmentation pattern is diagnostic for the analyte. The MS is set to analyse a small number of ions in SIM (selected ion mode), and, for some lipids, amounts as low as 1 pg can be detected.
A major advance in lipid research has been the development of high-sensitivity ESI–MS/MS and Q-trap instruments. Newer instruments are reaching the sensitivity of GC–MS, with detection of many eicosanoids down to 1 pg. The major advantage of GC is that derivatization is not required. This saves time and decreases losses. This technique uses HPLC on the front end to separate eicosanoids. In recent years, microbore (e.g. 1–2 mm or less with flow rates of 50–100 μl/min) columns have become routine. Following separation, samples are introduced into the source of the mass spectrometer and are ionized. For most eicosanoids, a negative ion due to the carboxy group forms easily, giving a strong negative [M-H]− signal. In the positive-ion mode, the protonated [M+H]+ ion can be accompanied by dehydration with accompanying signals at −18 a.m.u. (atomic mass units). Several scanning modes can be utilized for eicosanoid analysis. However, in practice, MRM transitions and product ion spectra are most common. Routine quantification of prostaglandins requires stable isotope-labelled internal standards, as described above. Following ionization, analytes are selected in Q1, then undergo CID (collisionally induced decomposition) in Q2 using inert gas, before passing into Q3. In MRM mode, diagnostic daughter ion fragments are selected by m/z in Q3, before being scanned out to the detector. For example, 12-HETE with m/z 319.2 corresponding to the [M-H]− fragments on CID in Q2, to give several daughter ions, including 179.2 a.m.u. (Figure 2). Other positional isomers, e.g. 5-HETE or 15-HETE, do not generate this ion. The characteristic fragmentation pattern is exploited to ensure selective and specific detection of each lipid. In Figure 3 we show LC–ESI–MS/MS detection of several eicosanoids, in a single run.
LC–MS/MS detection of 12-HETE and its stable isotope-labelled isomer, 12-[2H8]HETE
LC–ESI–MS/MS detection of several HETEs and PG standards
A useful feature of some instruments is the ability to change modes on a millisecond time scale during a run and obtain a product ion spectrum of an analyte. This then provides definitive proof of the compound of interest. On most triple-quadrupole instruments, it is difficult to get good-quality spectra at low concentrations in a biological sample; however, with hybrid instruments, e.g. ESI–Q-Trap, where Q3 can be used as an ion trap, this can be done. In our laboratory using the Applied Biosystems 4000 Q-Trap, high-quality product ion spectra have been obtained as low as 10× the limit of detection for MRM transitions.
Eicosanoids are a diverse family of signalling lipids that are an active focus of research in many fields of biomedicine, plant and animal research. Employment of reliable and cost-effective techniques for their quantification is required for their accurate determination in biological samples. LC–ESI–MS/MS and GC–MS are the gold-standard methods for quantifying and identifying eicosanoids. However setting them up represents a cost and time investment. Unfortunately, this puts them out of reach of many, although prices are decreasing as instruments are being modified further. It is possible to purchase immunoassays off the shelf and run them locally, or collaborate with others who have routine GC or LC assays set up in their laboratories. We would recommend LC–MS/MS or GC–MS where at all possible, unless the alternative assay has been carefully validated for the particular type of biological matrix of interest.
Bioanalysis in Oxidative Stress: A Biochemical Society Focused Meeting held at the University of Exeter, U.K., 2–3 April 2008. Organized and Edited by John Moody (Plymouth, U.K.) and Paul Winyard (Peninsula Medical School, Exeter, U.K.).
atomic mass units
collisionally induced decomposition
multiple reaction monitoring