A validated, rapid UPLC-MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples.

A recent clinical trial assessing human autonomic cardiovascular regulation applied pacemaker channel inhibition with ivabradine, norepinephrine transporter blockade with reboxetine, and beta-adrenoreceptor blockade with metoprolol. To verify patient adherence, we developed and validated a fast UPLC-MS/MS assay measuring all three compounds simultaneously. Deuterium-labeled drugs, d3-ivabradine, d5-reboxetine and d7-metoprolol, served as internal standards. Sample preparation of 200µL human plasma consisted of a single liquid-liquid extraction step by means of ethyl acetate. Chromatographic separation was performed on a 50-mm long BEH C18 column with gradient elution using a mixture of water and methanol each containing 2mM ammonium acetate over 4.5min. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode. Characteristic product ions resulting from collision-induced dissociation of unlabeled and deuterium-labeled drugs with argon were used for quantification in the selected-reaction monitoring mode. We validated the method according to the European Medicines Agency (EMA) guideline on bioanalytical method validation over the range from 1ng/mL to 500ng/mL for all three analytes. Linear responses with correlation coefficients>0.99 over that range were acquired. The LOQ value was 1ng/mL for each drug. Regulatory criteria for accuracy (80-120%) and precision (RSD<15%) were met for all drugs. The internal standard-normalized matrix factor was close to 1 for low and high analyte concentrations. We successfully measured ivabradine, reboxetine, and metoprolol concentrations in 107 human plasma samples from a clinical trial. Quality control samples processed in parallel confirmed the method's reliability in a clinical setting.

Changes of blood endocannabinoids during anaesthesia: a special case for fatty acid amide hydrolase inhibition by propofol?

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: • Available data from animal studies suggest that the narcotic drug propofol interacts with the endocannabinoid system. Inhibition of enzymatic degradation of anandamide could explain some of the characteristics of propofol. Direct measurements have not been reported yet in humans. WHAT THIS STUDY ADDS: • Propofol does not change the time course of anandamide plasma concentrations during anaesthesia. Furthermore, propofol does not inhibit fatty acid amide hydrolase activity ex vivo or in vitro. Thus, specific characteristics of the narcotic drug propofol cannot be explained by peripheral inhibition of anandamide degradation in humans. AIMS: The aim of our study was to describe the time course of endocannabinoids during different anaesthesia protocols in more detail, and to challenge the hypothesis that propofol acts as a FAAH inhibitor. METHODS: Endocannabinoids were measured during the first hour of anaesthesia in 14 women and 14 men undergoing general anaesthesia with propofol and in 14 women and 14 men receiving thiopental/sevoflurane. We also incubated whole human blood samples ex vivo with propofol and the known FAAH inhibitor oloxa and determined FAAH enzyme kinetics. RESULTS: Plasma anandamide decreased similarly with propofol and thiopental/sevoflurane anaesthesia, and reached a nadir after 10 min. Areas under the curve for anandamide (mean and 95% CI) were 53.3 (47.4, 59.2) nmol l(-1) 60 min with propofol and 48.5 (43.1, 53.8) nmol l(-1) 60 min with thiopental/sevoflurane (P= NS). Anandamide and propofol plasma concentrations were not correlated at any time point. Ex vivo FAAH activity was not inhibited by propofol. Enzyme kinetics (mean ± SD) of recombinant human FAAH were K(m) = 16.9 ± 8.8 µmol l(-1) and V(max) = 44.6 ± 15.8 nmol mg(-1) min(-1) FAAH without, and K(m) = 16.6 ± 4.0 µmol l(-1) and V(max) = 44.0 ± 7.6 nmol mg( 1 ) min(-1) FAAH with 50 µmol l(-1) propofol (P= NS for both). CONCLUSIONS: Our findings challenge the idea that propofol anaesthesia and also propofol addiction are directly mediated by FAAH inhibition, but we cannot exclude other indirect actions on cannabinoid receptors.

Targeted stable-isotope dilution GC-MS/MS analysis of the endocannabinoid anandamide and other fatty acid ethanol amides in human plasma.

Anandamide (arachidonoyl ethanol amide, AEA) is an endocannabinoid, acting on CB1 and CB2 receptors. Elevated plasma AEA concentrations in humans have been associated amongst others with obesity, psychological disorders and miscarriage. The occurrence in human plasma of ethanol amides of other unsaturated and saturated fatty acids, including oleic acid and palmitic acid, has also been reported. Most data available on anandamide and other fatty acid ethanol amides (FAEA) until now have been generated by using the LC-MS/MS methodology. Here, we describe a stable-isotope dilution GC-MS/MS method for the quantitative determination of AEA, oleic acid ethanol amide (OEA) and palmitic acid ethanol amide (PEA) in human plasma using their stable-isotope labeled analogs as internal standards. Other FAEA were found in plasma and their concentration was estimated. The present method involves a single solvent extraction of FAEA and their internal standards from plasma (50-1000 microl) with toluene, derivatization to the pentafluorobenzamide pentafluoropropionyl derivatives (FAEA-PFBz-PFP), and simultaneous quantification by selected reaction monitoring of the carboxylate anions produced by collision-induced dissociation of the parent ions [M-PFBz](-). The present method was fully validated for anandamide. Thus, accuracy and imprecision of the method were within the range of 100+/-20% and less than 20%, respectively, in the range investigated (0-4 nM). Mean overall recovery was 90+/-3%. The LOQ and LOD values of the method were determined to be 0.25 nM of added AEA in plasma samples and 400 amol of injected AEA-PFBz-PFP derivative, respectively. In plasma of 16 healthy individuals AEA concentration was measured to be 1.35+/-0.32 nM. This finding is concordant to literature AEA plasma concentrations as measured by LC-MS/MS. The plasma concentrations of OEA, PEA and other FAEA are higher than that of AEA. This GC-MS/MS method is straightforward, accurate, precise, highly specific for FAEA and useful in basic and clinical research.