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

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 < <i>R</i>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

Comparison of the postprandial oxylipin plasma levels in one subject on a usual and modified diet, respectively.

<p>* indicates p < 0.05 (adjusted for multiple comparisons).</p

Baseline and postprandial response levels significantly different of oxylipins for a subject on usualdiet (A), and on modified diet (B).

<p>Values represent the mean ± SEM (n = 3 for each diet and time point). Brackets indicates significantly different time points: ****p < 0.0001, ***p = 0.0002 (adjusted for multiple comparisons).</p

Oxylipin and endocannabinoid profiles in the postprandial state recapitulated as orthogonal scores (t[1] and to[1]) calculated by OPLS-DA.

<p>Time after challenge meal (in hours) is found next to each sample. The subject displayed different oxylipin and endocannabinoid metabolomes on usual (grey circles) vs modified (black squares) background diet. Model assessment parameters were: 1 predictive and 1 orthogonal component, p-value calculated by CV-ANOVA: 0.033, total systematic variation among the metabolites captured by the model (R2X): 0.463, total systematic variation between the diets captured by the model (R2Y): 0.815, predictive ability of the model (Q2): 0.53.</p

Baseline and postprandial response levels of endocannabinoid significantly different for a subject on usual diet (A), and on modified diet (B).

<p>Values represent the mean ± SEM (n = 3 for each diet and time point. ****p < 0.0001, ***p = 0.0002, **p = 0.005 (adjusted for multiple comparisons).</p

Comparison of the postprandial response in NAGly (eCB) plasma levels in one subject on usual (vegan) and modified (vegetarian) diet, respectively.

<p>*p < 0.05 (adjusted for multiple comparisons).</p

Average levels (nM)±SEM of compounds in the oxylipin metabolome of human plasma at fasting (baseline) and in the fasting and postprandial state (all samples) in a subject on her usual or modified diet.

<p>AA – Arachidonic acid (20:4n6)); LA – Linoleic acid (18:2n6); DHA – Docosahexaenoic acid (22:4n6); EPA – Eicosapentaenoic acid (20:5n3); ND – Not detected.</p><p>Average levels (nM)±SEM of compounds in the oxylipin metabolome of human plasma at fasting (baseline) and in the fasting and postprandial state (all samples) in a subject on her usual or modified diet.</p

Mass spectrometry parameters for multiple reaction monitoring transitions [Retention Time (RT), Cell Accelerator Voltage (CA), Collision Energy (CE)], linearity, Limit of Quantification (LOQ), and Limit of Detection (LOD) for the analyzed compounds (Fragmentor Voltage: 380 V for all compounds).

<p>Mass spectrometry parameters for multiple reaction monitoring transitions [Retention Time (RT), Cell Accelerator Voltage (CA), Collision Energy (CE)], linearity, Limit of Quantification (LOQ), and Limit of Detection (LOD) for the analyzed compounds (Fragmentor Voltage: 380 V for all compounds).</p

MRM chromatograms of each analyte analyzed in a standard solution mixture (A) and separation of critical pairs of isomers (B).

<p>MRM chromatograms of each analyte analyzed in a standard solution mixture (A) and separation of critical pairs of isomers (B).</p

Average levels (nM)±SEM of compounds in the oxylipin metabolome of human plasma at fasting (baseline) and in the fasting and postprandial state (all samples) in a subject on her usual or modified diet.

<p>AA – Arachidonic acid (20:4n6)); LA – Linoleic acid (18:2n6); DHA – Docosahexaenoic acid (22:4n6); EPA – Eicosapentaenoic acid (20:5n3); ND – Not detected.</p><p>Average levels (nM)±SEM of compounds in the oxylipin metabolome of human plasma at fasting (baseline) and in the fasting and postprandial state (all samples) in a subject on her usual or modified diet.</p