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

Concentrations (pM) of AEA, PE, OEA, LEA and 2-AG in the medium following TNFα treatment of DU145 cells.

<p>Concentrations (pM) of AEA, PE, OEA, LEA and 2-AG in the medium following TNFα treatment of DU145 cells.</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

Concentrations (pM) of lipids in the medium following treatment with TNFα and addition of AEA to DU145 cells.

<p>Concentrations (pM) of lipids in the medium following treatment with TNFα and addition of AEA to DU145 cells.</p

Effect of treatment of DU145 cells with TNFα upon mRNA levels of AEA and 2-AG metabolic enzymes.

<p>Effect of treatment of DU145 cells with TNFα upon mRNA levels of AEA and 2-AG metabolic enzymes.</p

Expression of <i>PTGS2</i> (COX2), <i>NAPEPLD</i>, <i>FAAH</i> and <i>NAAA</i> in DU145 and RAW264.7 cells.

<p>Panels A and B. mRNA levels of <i>PTGS2</i> (A) and <i>NAPEPLD</i> (B) measured in DU145 cell lysates at different times after TNFα treatment. The individual data points summarised in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185011#pone.0185011.t002" target="_blank">Table 2</a> are shown for these two proteins. Panels C and D show mRNA for <i>PTGS2</i>, <i>NAPEPLD</i>, <i>FAAH</i> and <i>NAAA</i> in control (V) and TNFα treated (2 h, T) DU145 cells (Panel B), and in control and LPS + IFNγ-treated (24 h, L/I) RAW264.7 cells (Panel C). Data are for 8–9 separate experiments, undertaken concomitantly with the uptake experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185011#pone.0185011.g002" target="_blank">Fig 2</a>. ***P<0.001, **P<0.01, ^NSP>0.05, exact two-sided permutation tests (complete enumeration) for the comparison shown. Panel E shows a Western blot for COX-2 with unstimulated and stimulated DU145 (TNFα) and RAW264.7 (LPS + IFNγ) cells. Human recombinant COX-2 is included as a positive control.</p

Uptake of 100 nM [Ara-³H]AEA into RAW264.7 and DU145 cells.

<p>Panel A-C show data for control and LPS + IFNγ-treated RAW264.7 cells; Panels D-F for control and TNFα-treated DU145 cells. Panels A, D show the time courses for the accumulation of radiolabel 5, 10, 20 and 30 min after addition of 100 nM of [Ara-³H]AEA. Note that the uptake is per well, and not normalised to the protein content (shown in Panels B and E). In Panel C, rates of uptake were determined for each experiment from the individual slopes of each the time course divided by the protein content. Shown are means and 95% confidence intervals, N = 8. Data was analysed using bootstrapped linear models (for details, see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185011#pone.0185011.ref040" target="_blank">40</a>]). For the main effects model, the P values were: LPS + IFNγ, P<0.0001; flurbiprofen, P = 0.89; URB597, P<0.0001. For the interactions model, the P values of the three bivariate interactions and the trivariate interaction were all ~0.9. The time courses for uptake into DU145 cells shown in Panel D could not be used to obtain robust slope replots. In consequence, the difference in uptake (per unit protein) between TNFα-treated and control cells were determined for each time point. The data is shown in Panel F (means and 95% confidence intervals, N = 6–7). A one-way ANOVA not assuming equal variances gave a P value of 0.018. Note that at the 30 min incubation time point, the confidence limits do not straddle zero.</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

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

Metabolism of 100 nM AEA by DU145 cells.

<p>Panels A show the time courses for the hydrolysis of 100 nM [Et-³H]AEA. Note that the values are per well, and not normalised to the protein content (shown in Panel B). In Panel C, rates of hydrolysis were determined for each experiment from the individual slopes (going through the origin) of the first two time points divided by the protein content. Data are means and 95% confidence intervals, N = 9. For the vehicle and flurbiprofen treated cells, the bootstrapped linear main effects model gave P values of TNFα, 0.19; flurbiprofen, 0.62. The interaction model gave a P value for TNFα x flurbiprofen of 0.80. However, a small effect of flurbiprofen can be masked by the large inter-experimental variation. Expressing the effect of flurbiprofen as % of the corresponding control value gave values of: untreated cells, 90 (81–99.5); TNFα- treated cells 87 (77–96) (means and 95% confidence limits, N = 9). Panels D-E: TLC separation of [Ara-³H]AEA, [³H]arachidonic acid (AA), [³H]PGF_2α and [³H]bimtoprost (Bimat) using ethyl acetate: methanol (90:10 v/v) as solvent system. Panel D shows the complete sampling from a single experiment, and Panel E shows the total recovery for three separate experiments over the R_f range shown. In Panel F, cells were incubated with 100 nM [³H]AEA, labelled in the arachidonoyl part of the molecule for 30 min prior to workup and separation by TLC. Shown are means of individual experiments conducted in triplicate for vehicle (V) and TNFα (T)-treated DU145 cells, and for vehicle and LPS + IFNγ-treated (L/I) RAW264.7 cells.</p