Measuring the Content of Endocannabinoid-Like Compounds in Biological Fluids: A Critical Overview of Sample Preparation Methodologies

Abstract

Different mass spectrometric techniques have been used over the past decade to quantify endocannabinoids (eCBs) and related lipids. Even with the level of molecular fingerprinting accuracy of an instrument like the most advanced triple quadrupole mass spectrometer, if one is not getting the most optimized sample to the detector in a way that this improved technology can be of use, then advancements can be stymied. Here, our focus is on review and discussion of sample preparation methodologies used to isolate the eCB anandamide and its close congeners N-acyl ethanolamines and structural congeners (i.e., lipo amino acids, lipoamines, N-acyl amides) in biological fluids. Most of our focus will be on the analysis of these lipids in plasma/serum, but we will also discuss how the same techniques can be used for the analysis of saliva and breast milk.

Keywords: Acyl amino acids; Endocannabinoids; HPLC; Lipidomics; Lipoamino acids; Mass spectrometry; Solid phase extraction.

Figure 1.

Examples of N-acyl ethanolamines (NAEs).

Figure 2.

Examples of Lipoamines (a.k.a, lipo amino acids, N-acyl amides).

Fig. 3. Comparisons of AEA method used on plasma between Sciex API 7500, Sciex API 6000, and API 3000.

A) Data are from equivalent amounts of NIST human plasma (100μl) dissolved in methanol, supernatant dried, and reconstituted in 50 μl in 50:50 water: methanol and 50 μl injected on a C18 Phenomenex analytical column. The pink line represents the output from the API 7500 and the blue line is the API 6000. B) Data are from a 20 μl injection on a Zorbex C18 column from 25 μl of plasma partially purified on C18 solid phase extraction column and eluted in 1.5ml 100% methanol. The identical parent and fragment pairs [H+]348/62 in positive ion mode were used for analysis in each instrument.

Figure 4.. Change in percent recovery of lipid analytes in plasma as a function of initial methanolic extraction solution.

See text for methodological and analytical details.