In-tube solid-phase microextraction directly coupled to tandem mass spectrometry for anandamide and 2-arachidonoylglycerol determination in rat brain samples from an animal model of Parkinson's disease.

To evaluate the endocannabinoid system in an animal model of Parkinson's disease, in-tube solid-phase microextraction (in-tube SPME) was directly coupled to a tandem mass spectrometry (MS/MS) system for determination of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples. In-tube SPME-which consisted of a microtube of restricted access material (RAM) with a hydrophilic diol external surface and a hydrophobic octyl inner surface-efficiently excluded (up to 95%) macromolecules from the biological samples and selectively pre-concentrated the analytes. In-tube SPME parameters, such as sample volume, mobile phases, flow rate, and pre-concentration time, were evaluated to improve the extraction efficiency and throughput performance. The selectivity of the in-tube SPME and MS/MS (MRM mode) techniques allowed them to be directly coupled online, which dismissed the need for the chromatographic separation step. The in-tube SPME-MS/MS method was validated and shown to be linear from 6.0 to 30.0 ng mL-1 for AEA and from 10.0 to 100.0 ng mL-1 for 2-AG; the intra- and inter-assay accuracy and precision were lower than 15%. Parallelism between the calibration curves constructed in the matrix and aqueous solution confirmed that there was no matrix effect. The method allowed endogenous concentrations of AEA and 2-AG to be determined in rat brain striatum from unilaterally 6-hydroxydopamine-lesioned animals. The concentrations of these endocannabinoids in striatum ipsilateral and contralateral to the lesion differed significantly (p<0.001).

Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

A micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry to determine anandamide and 2-arachidonoylglycerol in rat brain samples.

A simple and reliable method was developed and validated to determine the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples by micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (SALLLE/UHPLC-MS/MS). The SALLE parameters (brain homogenate volume, salting-out agent, salt concentration, salt solution volume, organic solvent, organic solvent volume, and centrifugation temperature) were optimized to improve sensitivity and selectivity of the method. The SALLE/UHPLC-MS/MS method presented linear ranges from 2.00 to 20.00 ng mL-1 for AEA and from 0.300 to 10.00 µg mL-1 for 2-AG, no significant matrix effect, and inter- and intra-assay precision and accuracy with CV and RSE values lower than 15%, respectively. This innovative method was successfully applied to determine AEA and 2-AG in brain hemispheres from a 6-OHDA animal model of Parkinson's disease (PD).

Determination of anandamide in cerebrospinal fluid samples by disposable pipette extraction and ultra-high performance liquid chromatography tandem mass spectrometry.

This work describes the development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method that uses disposable pipette extraction (DPX-UHPLC-MS/MS) to determine the endocannabinoid anandamide (AEA) in cerebrospinal fluid samples (CSF). The DPX parameters sorption equilibrium time, sample volume, number of draw-eject cycles, washing solvent volume, and elution solvent volume were optimized by design of experiments (DOE) techniques. The simple DPX protocol proposed herein required a reduced amount of CSF sample and organic solvent. The DPX-UHPLC-MS/MS method presented linear range from 0.10 ng mL-1 (LLOQ) to 3.0 ng mL-1, inter- and intra-assay accuracy with EPR values varying from -8.2% to 9.6%, inter- and intra-assay precision with CV values ranging from 1.3% to 14.8% (except for the LLOQ), and no significant matrix effect. The innovative DPX-UHPLC-MS/MS method was successfully applied to determine AEA in CSF samples from Parkinson's disease (PD) patients and should therefore be used in clinical studies.

Monoraphidium sp. HDMA-20 is a new potential source of α-linolenic acid and eicosatetraenoic acid.

BACKGROUND: ω-3 polyunsaturated fatty acids (PUFAs) are synthesized from α-Linolenic acid (ALA, C18:3ω3) and play important roles in anti-inflammatory and antioxidant responses in mammal cells. ALA is an essential fatty acid which cannot be produced within the human body and must be acquired through diet. The purpose of this study was to evaluate the potential of a novel microalgal strain (HDMA-20) as a source of ω-3 PUFAs including ALA and eicosatetraenoic acid (ETA, C20:4ω3). METHOD: Phylogenetic Neighbor-Joining analysis based on 18S ribosomal DNA sequence was used to identify the microalga strain HDMA-20. Autotrophic condition was chosen to cultivate HDMA-20 to reduce the cultivation cost. GC-MS was used to determine the fatty acid composition of HDMA-20 lipid. RESULTS: A microalgal strain (HDMA-20) from Lake Chengfeng (Daqing, Heilongjiang province, China) was found to accumulate high content of ω-3 PUFAs (63.4% of total lipid), with ALA and eicosatetraenoic acid (ETA, C20:4ω3) accounting for 35.4 and 9.6% of total lipid, respectively. Phylogenetic analysis based on 18S ribosomal DNA sequences suggested that the HDMA-20 belonged to genus Monoraphidium (Selenastraceae, Sphaeropleales) and its 18S rDNA sequence information turned out to be new molecular record of Monoraphidium species. The biomass productivity and lipid content of HDMA-20 were also investigated under autotrophic condition. The biomass productivity of HDMA-20 reached 36.3 mg L- 1 day- 1, and the lipid contents was 22.6% of dry weight. CONCLUSION: HDMA-20 not only represent an additional source of ALA, but also a totally new source of ETA. The high content of ω-3 PUFAs, especially ALA, of HDMA-20, makes it suitable as a source of nutrition supplements for human health. In addition, HDMA-20 exhibited good properties in growth and lipid accumulation, implying its potential for cost-effective ω-3 PUFAs production in future.

Preparation of High Purity Δ5-Olefinic Acids from Pine Nut Oil via Repeated Lipase-Catalyzed Esterification.

Δ5-Olefinic acids have been characterized in gymnosperm plants and have been reported to have several biological health benefits. Δ5-Olefinic acids from pine nut oil were effectively concentrated by repeated lipase-catalyzed esterification. The pine nut oil contained three major Δ5-olefinic acids, namely taxoleic acid (C18:2 Δ5,9), pinolenic acid (C18:3 Δ5,9,12), and sciadonic acid (C20:3 Δ5,11,14). The fatty acids present in pine nut oil were selectively esterified with ethanol using Lipozyme RM IM from Rhizomucor miehei as a biocatalyst. The Δ5-olefinic acids were concentrated in the unesterified fatty acid fraction. The optimum molar ratio of the substrates (fatty acid:ethanol), temperature, the enzyme loading, and the reaction time were 1:7, 25°C, 5% of total substrate weight, and 6 h, respectively. There was no significant effect in the concentration of Δ5-olefinic acids when water was added in the reaction mixture. The same protocol and optimum conditions were employed for two times repeated lipase-catalyzed esterifications. In first lipase-catalyzed esterification, the Δ5-olefinic acids content in the pine nut oil increased from 17 mol% to 51 mol% with a yield of 40 mol%. In a second lipase-catalyzed esterification, with the Δ5-olefinic acids-concentrated fatty acids obtained from the first reaction as the substrate, the Δ5-olefinic acids content increased to 86 mol% with a yield of 15 mol%. Finally, a maximum Δ5-olefinic acids content of ca. 96 mol% with a yield of 6 mol% was obtained via a third lipase-catalyzed esterification.

Determination of 2-Arachidonoylglycerol by µSPE-LC-MS/MS.

LC-MS/MS is a powerful analytical technique that provides unequivocal identification and reliable quantification of the analytes, using Selected Reaction Monitoring or Multi Reaction Monitoring acquisition mode.2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, which plays a major role in a wide variety of physiological and pathological processes. Analysis of 2-AG by means of LC-MS/MS allows the detection of very low concentrations in biological samples. Here, we describe how to determine 2-AG levels in tiny samples of tissues and plasma through LC-MS/MS, by using very quick and easy to perform extraction procedures, with reduced solvent consumption.

Collaborating with Alexander Scriabine and the Miles Institute for Preclinical Pharmacology.

This article represents a timely opportunity to express my affection, admiration and gratitude to Professor David Triggle. David was my Ph.D. advisor as well as a key consultant in the 1980s and early 1990s for research programs at Miles Institute for Preclinical Pharmacology in West Haven, CT, the U.S. research operation of Bayer AG, in the areas of Ca(2+) and K(+) channel ligands. The binding methodology developed in his laboratory was used to search for an endogenous ligand for L-type Ca(2+) channels. We did not find the substance that we were searching for, a genetically-determined, competitive inhibitor for the 1,4-dihydropyridine binding site, but instead isolated the endogenous ligand for the brain's own marijuana, anandamide. Devane, Mechoulam and coworkers first discovered that this compound was the endogenous ligand for delta-9-tetrahydrocannabinol, the active substance in cannabis. The endogenous endocannabinoid system is now the target of many exciting new approaches to drug discovery.

Isolation of two Δ5 polymethylene interrupted fatty acids from Podocarpus falcatus by countercurrent chromatography.

The lipids of gymnosperms frequently feature unusual polyunsaturated fatty acids (PUFAs) such as sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) showing a first double bond on C-5 which is separated from the next double bond by five methylene units. Compared to "classic" fatty acids, these fatty acids are not easily commercially available and their prices are quite high. For this reason, we wished to isolate those fatty acids from the seed oil of Podocarpus falcatus by countercurrent chromatography (CCC) after conversion of the fatty acids to methyl esters (FAMEs). The contribution of sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) in the unfractionated sample was 10% and 6% respectively, while oleic acid (18:1Δ9) and linoleic acid (18:2Δ9,12) were the major fatty acids. After a first CCC run with FAMEs from Podocarpus falcatus, fractions enriched in the target compounds were chosen for subsequent isolation by means of two subsequent CCC runs. Initially, 13mg of juniperonic acid was recovered with a purity of 92% according to analysis by gas chromatography with mass spectrometry (GC/MS). Further purification of this fraction yielded 2.7mg with a purity of 99% according to GC/MS. The isolation of sciadonic acid was hampered by high amounts of linoleic acid with the same equivalent chain length in suitable fractions of the first CCC separation. After an enrichment step by CCC, the critical pair sciadonic acid and linoleic acid was finally separated as free fatty acids. After this step, 4.4mg of sciadonic acid was recovered with 99% purity. The methodology could also be applied to isolate larger amounts of those fatty acids or for the isolation of other minor fatty acids.

High-throughput salting-out assisted liquid-liquid extraction with acetonitrile for the determination of anandamide in plasma of hemodialysis patients with liquid chromatography tandem mass spectrometry.

Anandamide (AEA) is an endocannabinoid present in human plasma that is associated with several physiological functions and disease states. However, low AEA plasma levels pose challenges in terms of analytical characterization. Classical liquid-based lipid extraction and solid-phase extraction require complicated procedures and the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here a high-throughput salting-out assisted liquid-liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of AEA in human plasma has been developed and validated. The seamless interface of SALLE and LC-MS eliminated the drying-down step, only 100 µL of plasma is required and minimal volumes of organic solvent are used. Good reproducibility, accuracy and precision were demonstrated during the method validation. The method is linear up to 10 ng/mL with a lower limit of quantitation of 0.1 ng/mL for AEA, the accuracy for AEA was from 93.3 to 96.7% and the precision was <8.57%. This new methodology was successfully applied to analysis of clinical samples from maintenance hemodialysis patients.

Truffles contain endocannabinoid metabolic enzymes and anandamide.

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.

Identification of endocannabinoid system-modulating N-alkylamides from Heliopsis helianthoides var. scabra and Lepidium meyenii.

The discovery of the interaction of plant-derived N-alkylamides (NAAs) and the mammalian endocannabinoid system (ECS) and the existence of a plant endogenous N-acylethanolamine signaling system have led to the re-evaluation of this group of compounds. Herein, the isolation of seven NAAs and the assessment of their effects on major protein targets in the ECS network are reported. Four NAAs, octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid isobutylamide (1), octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid 2'-methylbutylamide (2), hexadeca-2E,4E,9Z-triene-12,14-diynoic acid isobutylamide (3), and hexadeca-2E,4E,9,12-tetraenoic acid 2'-methylbutylamide (4), were identified from Heliopsis helianthoides var. scabra. Compounds 2-4 are new natural products, while 1 was isolated for the first time from this species. The previously described macamides, N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (5), N-benzyl-(9Z,12Z,15Z)-octadecatrienamide (6), and N-benzyl-(9Z,12Z)-octadecadienamide (7), were isolated from Lepidium meyenii (Maca). N-Methylbutylamide 4 and N-benzylamide 7 showed submicromolar and selective binding affinities for the cannabinoid CB1 receptor (Ki values of 0.31 and 0.48 µM, respectively). Notably, compound 7 also exhibited weak fatty acid amide hydrolase (FAAH) inhibition (IC50 = 4 µM) and a potent inhibition of anandamide cellular uptake (IC50 = 0.67 µM) that was stronger than the inhibition obtained with the controls OMDM-2 and UCM707. The pronounced ECS polypharmacology of compound 7 highlights the potential involvement of the arachidonoyl-mimicking 9Z,12Z double-bond system in the linoleoyl group for the overall cannabimimetic action of NAAs. This study provides additional strong evidence of the endocannabinoid substrate mimicking of plant-derived NAAs and uncovers a direct and indirect cannabimimetic action of the Peruvian Maca root.

[A new fluorescent analogue for the investigation of anandamide transport in cell cultures].

For the first time a new fluorescent analogue of anadamide incorporating BODIPY®-FL-fluorophore, attached to arachidonic acid via 2,2'-(ethylenedioxy)-bis(ethylenediamine), was prepared. Using rat glioma C6 cells it was demonstrated that the fluorescent analogue is a substrate of the cellular anandamide uptake system (Km 4.5 ± 0.9 µM, Vmax 20 ± 1 amol/(min x cell)).

Actual ratios of triacylglycerol positional isomers and enantiomers comprising saturated fatty acids and highly unsaturated fatty acids in fishes and marine mammals.

It has been previously shown that the positional isomers of triacylglycerol (TAG) containing palmitic acid (P) and highly unsaturated fatty acids (HUFAs) such as DHA (D) and EPA (E) vary between fishes and marine mammals. However, it has not yet been understood why in marine mammals HUFAs are located only at the α position when two palmitic acid chains combine, and not in fishes. In order to gain further understanding of the biosynthetic pathways involved in the formation of these asymmetric TAGs, we investigated whether the HUFA in the TAG of marine mammals exists predominantly at the sn-1 or sn-3 position. We examined the TAG positional isomers and enantiomers in marine organisms in detail. As a result, while PDP and PEP were not detected, sn-PPD and sn-PPE were found in abundance in marine mammals. For fishes, on the other hand, PDP, PEP, sn-PPD, and sn-PPE were all identified. In the case of TAGs that contain two HUFAs and one palmitic acid, marine mammals were rich in DPD and EPE whereas fishes were rich in sn-PDD and sn-PEE.

Determination of the two major endocannabinoids in human plasma by µ-SPE followed by HPLC-MS/MS.

Endocannabinoids (ECs) are endogenous compounds that interact with type-1 and type-2 cannabinoid receptors (CB(1) and CB(2)), as well as non-cannabinoid receptors. The multitude of roles attributed to ECs makes them an emerging target of pharmacotherapy for a number of disparate diseases. Here a high-throughput bioanalytical method based on micro SPE (µ-SPE) followed by LC-MS/MS analysis for the simultaneous determination of the two major endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) in human plasma is presented. The chromatographic conditions obtained with the fused-core column allowed a good separation in 10 min also of the AG isomers. A very simple and reliable extraction has been optimised by means of C18-modified tips: it requires only 100 µL of plasma and allows the use of minimal volumes of organic solvent. The present method allows a rapid and effective clean-up, which also minimises the isomerisation of 2-AG. The whole procedure has been validated following the FDA guidelines for bioanalytical methods validation: the satisfactory recovery values, the negligible matrix effect and the good values of accuracy and reproducibility make it a simple and high-throughput analytical tool for clinical and biochemical studies on endocannabinoid signaling in humans.

Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction.

Analysis of the endocannabinoid (EC) system's key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC-MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform-methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60-80%). Based on these favourable toluene properties, we developed and validated a UPLC-MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC-MS/MS method was cross-validated with a previously described fully-validated GC-MS/MS method for AEA in human plasma. A close correlation (r(2)=0.821) was observed between the results obtained from UPLC-MS/MS (y) and GC-MS/MS (x) methods (y=0.01+0.85x). The UPLC-MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1 mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC-MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212 pM corresponding to 12.3, 14.5 and 9.9 pmol/mmol creatinine, respectively.

Fast ultrasound-assisted extraction of polar (phenols) and nonpolar (lipids) fractions in Heterotheca inuloides Cass.

INTRODUCTION: Heterotheca inuloides Cass., also known as "arnica", is used in traditional medicine in Mexico. OBJECTIVE: Development of fast methods for the extraction of lipidic and phenolic fractions from arnica plants and their subsequent characterization. METHODOLOGY: Ultrasound was applied to accelerate extraction of the target compounds from this plant and reduce the use of organic solvents as compared with conventional methods. Gas chromatography-ion trap mass spectrometry and liquid chromatography with diode-array detection were used for the characterization of the lipidic and phenolic fractions, respectively. RESULTS: Under optimal extraction conditions, 9 and 55 min were necessary to complete extraction of the lipidic and phenolic fractions, respectively. The fatty acids present at the highest concentrations in H. inuloides were eicosatetraenoic n3 (24.6 µg/g), cis-9-hexadecenoic n7 (23.1 µg/g), exacosanoic (22.7 µg/g) and cis-9-octadecenoic acid (21.3 µg/g), while the rest were in the range 7.6-1.3 µg/g. The most concentrated phenols were guaiacol (41.5 µg/g), catechin (38.7 µg/g), ellagic acid (35.9 µg/g), carbolic acid (24.2 µg/g) and p-coumaric acid (19.5 µg/g), while the rest were in the range 5.1-0.4 µg/g. CONCLUSION: Ultrasound reduces the time necessary to complete the extraction 160 and 26 times, the extraction volume 2.5 and 4 times, and increases the extraction efficiency 5 and 3 times for lipidic and phenolic fractions, respectively, in comparison with conventional extraction methods. In addition, the characterization of the lipidic and phenolic fractions constitutes a first approach to the H. inuloides metabolome.

Anandamide levels in human female reproductive tissues: solid-phase extraction and measurement by ultraperformance liquid chromatography tandem mass spectrometry.

Anandamide (N-arachidonoylethanolamide), a bioactive lipid, is reported to play a role in pregnancy maintenance and parturition. Our aims were to (1) evaluate AEA levels at the human maternal:fetal interface and (2) validate the use of solid-phase extraction of AEA from tissues. AEA was analyzed in cord and maternal blood, amniotic fluid, placenta, and fetal membranes collected during Caesarean section (n=14). Extraction efficiencies were 42 and 36% for the placenta and the fetal membranes, respectively. Tissue AEA was quantified using an isotope-dilution method and UPLC-ESI-MS/MS giving intra- and inter-day variability for tissues spiked with 0.2, 1, and 5pmol/g AEA of less than 12%. Accuracy for these spiked samples was between 95% and 103% for fetal membranes and between 99% and 114% for placenta. Mean AEA concentrations were 2.72 + or - 1.04 pmol/g for placenta and 1.19 + or - 0.68 pmol/g for fetal membranes, and 0.93 + or - 0.28, 0.88 + or - 0.33, 0.77 + or - 0.30, and 0.06 + or - 0.04nM for maternal, umbilical vein, and umbilical artery plasma and amniotic fluid. Higher AEA concentrations were found in placenta compared to fetal membranes (P<0.0001), in umbilical vein compared with umbilical artery (P=0.0015), and in plasma from maternal circulation compared with umbilical artery (P=0.0152). The relevance of these changes in AEA concentrations at the maternal:fetal interface requires further investigation.

A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices.

N-Arachidonoylethanolamine (AEA, anandamide) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and biofluids, in the low nanomolar range, using lipid extraction techniques with organic solvents. These techniques require the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid, and fluid from an ovarian cyst. AEA was detected in serum and plasma from blood isolated from 20 adult women (means+/-standard deviations: 0.68+/-0.29 and 0.64+/-0.28 nM, respectively), from pregnant women at term (1.37+/-0.42 nM), and from umbilical vein (1.26+/-0.33 nM) and umbilical artery (1.14+/-0.35nM), in milk (0.12+/-0.05 nM) and from amniotic (0.03+/-0.02 nM), peritoneal (0.93+/-0.27 nM), follicular (1.17+/-0.51 nM), and ovarian cyst (0.32+/-0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also improved dramatically using SPE (8 and 4 fmol/ml) compared with organic extraction (25 and 18.75 fmol/ml plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and interday variability were comparable, and the mean AEA concentration of pooled plasma samples (1.18 nM, n=15) was identical with the two techniques. Similarly, when 56 plasma samples from laboring and nonlaboring women were analyzed using both techniques, no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from biomatrices to replace the existing liquid extraction methods, with the SPE technique being superior in terms of speed, extraction efficiency, and sample size required.

An optimized GC-MS method detects nanomolar amounts of anandamide in mouse brain.

The endocannabinoids anandamide and 2-arachidonoylglycerol, as well as several anandamide-related N-acylethanolamines, belong to a family of lipid transmitter that regulate fundamental physiological processes, including neurotransmission and neuroinflammation. Their precise quantification in biological matrices can be achieved by gas chromatography-mass spectrometry (GC-MS), but this method typically requires multiple time-consuming purification steps such as solid-phase extraction followed by HPLC. Here we report a novel solid-phase extraction procedure allowing for single-step, and thus higher throughput, purification of endocannabinoids and N-acylethanolamines before GC-MS quantification. We determined the minimal amount of mouse brain tissue required to reliably detect endocannabinoids and N-acylethanolamines when using this approach and provide direct evidence for quantification accuracy by using radioactive and deuterated standards spiked into mouse brain samples. Using this method, we found that mouse brain contains much higher levels of anandamide (>1 nmol/g tissue) than previously reported, whereas levels of 2-arachidonoylglycerol and other N-acylethanolamines are well within the range of previous reports. In addition, we show that mouse brain amounts of endocannabinoids and N-acylethanolamines differ depending on animal gender as well as on whether the tissue was fixed or not. Our study shows that endocannabinoid and N-acylethanolamine levels quantified in mouse brain by GC-MS depend closely on tissue amount and preparation as well as on animal gender and that, depending on such parameters, anandamide levels could be underestimated.