Development and Validation of a Sensitive UPLC-ESI-MS/MS Method for the Simultaneous Quantification of 15 Endocannabinoids and Related Compounds in Milk and Other Biofluids

Abstract

The endocannabinoid (eCB) system has gained an increasing interest over the past decades since the discovery of anandamide and 2-arachidonoyl glycerol (2-AG). These, and structurally related compounds, are associated with a wide variety of physiological processes. For instance, eCB levels in milk have been associated with infants' feeding and sleeping behavior. A method based on ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was developed and validated for the simultaneous quantification of 15 eCBs and related compounds, including both fatty acid amides and glycerols. Linearity (0.9845 < R(2) < 1), limit of detection and quantification (0.52-293 pg on column), inter- and intraday accuracy (>70%) and precision (CV < 15%), stability, and recovery (in milk and plasma) were established in accordance to the U.S. Food and Drug Administration guidelines. The method was successfully applied to bovine and elk milk revealing species-specific eCB profiles, with significant different levels of 2-AG, 2-linoleoyl glycerol, docosahexaenoyl ethanolamide, palmitoyl ethanolamide, and oleoyl ethanolamide. Furthermore, stearoyl ethanolamide and docosatetraenoyl ethanolamide were only detected in elk milk. In summary, our UPLC-ESI-MS/MS method may be used for quantification of eCBs and related compounds in different biofluids and applied to investigations of the role of these emerging compounds in various physiological processes.

Validation of a tandem mass spectrometry method using combined extraction of 37 oxylipins and 14 endocannabinoid-related compounds including prostamides from biological matrices

“…Our previous methods for profiling the OxL [24] and eCB [25] metabolome separately provided a starting point for further advancement to include a broader range of metabolites, e.g. prostamides, and to increase the sensitivity, with the goal of obtaining a more clinically relevant coverage of the bioactive lipids using smaller sample volumes.…”

“…SPE protocols previously reported for separate extraction of compounds in the eCB [25] and OxL [1,15,24] metabolome were combined and adapted to fit in one single protocol. Briefly, compounds were extracted using Waters Oasis HLB cartridges (60 mg of sorbent, 30 m particle size).…”

“…In a first approach, internal standards for both the OxL and eCB metabolomes were extracted together using the SPE protocol described above. In the second approach, OxL and eCB were extracted separately using our previously reported methods [24,25].…”

“…For profiling the eCB metabolome, including the prostamides, the mobile phase was changed from the previous used methanol buffer (10 mM CH 3 COONH 4 ) [25] to the one employed for OxL analysis. This allowed elimination of an extra cleaning step of the column to remove the buffer in between different batches.…”

Identification of the oleic acid ethanolamide (OEA) isomer cis-vaccenic acid ethanolamide (VEA) as a highly abundant 18:1 fatty acid ethanolamide in blood plasma from rats and humans

“…Recently, methods for the quantification of endocanna binoids and endocannabinoid-related compounds like FAEA including OEA have been developed to simultaneously quantify endocannabinoids and related compounds in milk and other biofluids [24], in human blood cells and human blood plasma [25,26], or in other human bio-matrices [27] by ultrahigh-performance liquid chromatography (UHPLC)-ESI-MS/MS. Since structural differences in FAEAs can lead to different physiological effects, we consider it important to analyze OEA and its C18:1 position-and configuration isomers by comparing the respective pure compounds with those found in blood plasma from rats and humans using UHPLC-ESI-MS/MS.…”