No abstract

Endocannabinoids (ECs), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), modulate a number of physiological processes, including pain, appetite and emotional state. Levels of ECs are tightly controlled by enzymatic biosynthesis and degradation in vivo. However, there is limited knowledge about the enzymes that terminate signaling of the major brain EC, 2-AG. Identification and quantification of 2-AG, 1-AG and arachidonic acid (AA) is important for studying the enzymatic hydrolysis of 2-AG. We have developed a sensitive and specific quantification method for simultaneous determination of 2-AG, 1-AG and AA from mouse brain and adipose tissues by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a simple brain sample preparation method. The separations were carried out based on reversed phase chromatography. Optimization of electrospray ionization conditions established the limits of detection (S/N = 3) at 50, 25 and 65 fmol for 2-AG, 1-AG and AA, respectively. The methods were selective, precise (%R.S.D. < 10%) and sensitive over a range of 0.02-20, 0.01-10 and 0.05-50 ng/mg tissue for 2-AG, 1-AG and AA, respectively. The quantification method was validated with consideration of the matrix effects and the mass spectrometry (MS) responses of the analytes and the deuterium labeled internal standard (IS). The developed methods were applied to study the hydrolysis of 2-AG from mouse brain extracts containing membrane bound monoacylglycerol lipase (MAGL), and to measure the basal levels of 2-AG, 1-AG and AA in mouse brain and adipose tissues.

The use of a hybrid triple quadrupole-linear ion trap (QqQ(LIT)) mass spectrometer system for a comprehensive study of fragmentation mechanisms is described. The anxiolytic drug, buspirone, was chosen as a model compound for this study. With the advent of a QqQ(LIT) instrument, both the traditional quadrupole and the new linear ion trap scans (LIT) could be performed in a single LC run. In the past, a sample had to be run on two different instruments, namely, a triple quadrupole instrument (QqQ) and a 3D ion trap (3D IT) to obtain similar information. With the new QqQ(LIT) technology, collision-induced dissociation (CID) occur in a quadrupole collision cell, q2, and fragment ions are trapped and analyzed in Q3 operated in LIT mode. In this work, high-sensitivity product ion spectra of buspirone were obtained from the one-stage 'Enhanced Product Ion' scan (EPI) with rich product ions and no low mass cut-off. Furthermore, detailed fragmentation pathways were elucidated by further dissociation of each of the fragment ions in the EPI spectrum using MS(3) mode in the same run. The MS(3) scan was performed by incorporating CID in q2, and trapping, cooling, isolation, and resonance-excitation in Q3 when operating in LIT mode. This approach allowed unambiguous assignment of all fragment ions quickly with fewer experiments and easier interpretation than the previous approach. The overall sensitivity for obtaining complete fragment ion data was significantly improved for QqQ(LIT) as compared with that of QqQ and 3D IT mass spectrometers. This is beneficial for structure determination of unknown trace components. The method allowed structure determination of metabolites of buspirone in rat microsomes at 1 microM concentration, which was a 10-fold lower concentration than was needed for QqQ or 3D IT instruments. The QqQ(LIT) instrument provided a simple, rapid, sensitive and powerful approach for structure elucidation of trace components.

No abstract

We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCtheta (IC50=4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCtheta. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC50 value of 0.28 nM for the inhibition of PKCtheta.