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. 2013 Jan 15;432(2):74-81.
doi: 10.1016/j.ab.2012.09.031. Epub 2012 Oct 5.

Quantitative LC-MS/MS analysis of arachidonoyl amino acids in mouse brain with treatment of FAAH inhibitor

Bingnan Han  1 , Rachel Wright, Aaron M Kirchhoff, Julia A Chester, Bruce R Cooper, Vincent Jo Davisson, Eric Barker
Affiliations

Quantitative LC-MS/MS analysis of arachidonoyl amino acids in mouse brain with treatment of FAAH inhibitor

Bingnan Han et al. Anal Biochem. .

Abstract

An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/μl for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), γ-aminobutyric acid (NAGABA), and glycine (NAGly); 0.7-90 pg/μl for AEA-d(0)/d(8); and 7.5-950 pg/μl for 2-AG was determined with R(2) values of 0.99. Also the effects of the FAAH inhibitor URB 597 on the endogenous levels of these analytes were investigated. AEA and NASer brain levels exhibit a dose-dependent increase after systemic administration of URB 597, whereas NAGly and NAGABA were significantly decreased after treatment. NAAla and 2-AG were not altered after URB 597 treatment. The potential benefit of establishing this assay extends beyond the quantification of the Ara-AAs along with AEA and 2-AG in mouse brain, to reveal a variety of pharmacological effects and physiological roles of these analytes.

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Figures

Fig. 1
Fig. 1
Chemical structures of selected analytes and selected precursor and product m/z values with appropriate retention times, fragment voltages, and collision voltages used to identify and quantify analytes, using MRM with LC–MS/MS.
Fig. 2
Fig. 2
Product ion mass spectrum of each targeted analyte, with the chosen precursor ion indicated by [M+H]+ and the product ion indicated by the ion mass.
Fig. 3
Fig. 3
Representative MRM chromatograms of the analytes detected in mouse brain and corresponding synthetic standards. (A) All analyte standards, 12 pg of each arachidonoyl amino acid, 20 pg of AEA-d0/d8, 300 pg of 2-AG, and 50 pg of NAGly-d8 on column. (B) All analytes measured, including ISs, in a representative control mouse brain. 4 pg of NASer, 11 pg of NAGABA, 11.3 pg of NAAla, 14.5 pg of NAGly, 32 pg of NAGly-d8, 814.8 pg of 2-AG, 195.8 pg of AEA-d8, and 10.4 pg of AEA-d0 on column. Each chromatogram has been normalized to display the peak of maximum intensity within the time window. LLOQ: AEA, 3.5 pg; 2-AG, 38.0 pg; NAGly, 1.9 pg; NASer, 1.9 pg; NAGABA, 1.0 pg; and NAAla, 1.9 pg on column. LOD: AEA, 0.5 pg; 2-AG, 7.6 pg; NAGly, 0.5 pg; NASer, 0.5 pg; NAGABA, 0.4 pg; and NAAla, 0.7 pg on column.
Fig. 4
Fig. 4
Dose response of endogenous levels of AEA, NAGly, and NAGABA in mouse brains with single or double injections of the FAAH inhibitor URB 597 (n = 3/group). (A) ANOVA with Dunnett’s test revealed a significant effect of AEA level (**p < 0.01) from the animals that received two injections at doses of 1.0 and 3.0 mg/kg, compared to the vehicle control animals. (B and C) ANOVA with Dunnett’s test revealed that URB 597 treatments had significant effects on the levels of NAGly and NAGABA (**p < 0.01) across all three doses. (D, E, and F) Comparison of represented extracted chromatograms of AEA, NAGly, and NAGABA in mouse brain with treatments of URB 597 across three different doses.
Fig. 5
Fig. 5
Dose response of endogenous levels of NASer, NAAla, and 2-AG in mouse brains with single treatment of the FAAH inhibitor URB 597 and with two accumulated treatments (n = 3/group). (A) ANOVA with Dunnett’s test revealed a significant effect of NASer level (*p < 0.05) in the animals that received two injections at a dose of 3.0 mg/kg, compared to the vehicle control animals. (B and C) ANOVA with Dunnett’s test revealed that URB 597 treatments had no significant effect on the levels of NAAla and 2-AG across all three doses.
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