Kati S. Hakala

N-in-One Permeability Studies of Heterogeneous Sets of Compounds across Caco-2 Cell Monolayers

AbstractPurpose. The purpose of the study was to evaluate several n-in-one cocktails of heterogeneous compounds to increase the throughput of permeability studies across Caco-2 monolayers, to investigate the reliability and applicability of the method, and to develop fast and sensitive analysis for the compounds. Compounds with potential interactions in efflux and/or active transport were chosen. Methods Permeability experiments with verapamil, propranolol, midazolam, hydroxyzine, timolol, buspirone, procaine, naproxen, ketoprofen, and antipyrine as single compounds and in cocktails of 5-10 compounds were performed at 50 μM concentration both in the apical-to-basolateral and basolateral-to-apical direction. The compounds were quantified by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI/MS/MS). Toxicity tests were performed to determine cellular damage. Results The analytical method was sensitive, accurate, and rapid. The individual permeabilities of compounds in cocktails correlated well with permeabilities as single compounds. No significant interactions between the compounds within the mixtures were observed, except for acidic compounds. The studied mixtures did not show any toxicity. Conclusions The use of n-in-one cocktails is a suitable method to improve the capacity in routine permeability experiments and higher throughput screening of drug candidates, although potential interactions should always be borne in mind. The use of LC-ESI/MS/MS technology provides an excellent tool in fast and accurate analysis of small amounts of heterogeneous compounds.

Direct Analysis of Glucuronides with Liquid Chromatography-Mass Spectrometric Techniques and Methods

Glucuronidation is one of the main phase II metabolic reactions in humans and animals. A variety of analytical techniques and methods have been used for the detection and quantification of glucuronides of both endogenous and xenobiotic compounds from different biological samples of humans and animals. Drug metabolism has been extensively studied with both in vitro and in vivo experiments under various conditions. The purpose of this review is to explore in detail the benefits and drawbacks of different liquid chromatography-mass spectrometric (LC/MS) methods and techniques in detection and identification of all forms of glucuronide conjugates from in vitro, biological, and environmental samples. The entire analytical procedure is covered, from sample treatment, separation, and ionization to qualitative and quantitative analyses. The aim of this review is not to cover every published paper where glucuronides are identified and/or quantified, but rather to focus on special cases where a new analytical approach or technical development has led to a better, more specific, or more comprehensive detection, identification, or quantitation of glucuronide conjugates.

Characterization of metabolites of sibutramine in primary cultures of rat hepatocytes by liquid chromatography-ion trap mass spectrometry.

Liquid chromatography–ion trap mass spectrometry was used for the detection and structural characterization of metabolites of the anti-obesity drug sibutramine. Metabolites were profiled from incubations of sibutramine in primary cultures of rat hepatocytes. In addition, enantioselectivity of sibutramine metabolism was investigated by carrying out separate incubations with (R)- and (S)-sibutramine. As a result, biotransformation profile for sibutramine with rat hepatocytes is proposed. Nineteen metabolites and several of their isomers formed via demethylation, hydroxylation, dehydrogenation, acetylation, attachment of CO2, and glucuronidation were identified in MS2 and MS3 experiments, though the exact position of the functionality, mostly hydroxylation, could not always be determined from the mass spectrometric information. However, clear enantioselective formation was observed for two hydroxyl derivatives and two glucuronide conjugates, indicating that the hydroxyl/glucuronic acid moiety in those structures is close to the chiral center. Most of the metabolites found in this study are new metabolites of sibutramine, which were not previously reported.

Analysis of neonicotinoids from plant material by desorption atmospheric pressure photoionization-mass spectrometry

RATIONALE Neonicotinoids are widely used insecticides which have been shown to affect the memory and learning abilities of honey bees, and are suspected to play a part in the unexplainable, large-scale loss of honey bee colonies. Fast methods, such as ambient mass spectrometry (MS), for their analysis from a variety of matrices are necessary to control the use of forbidden products and study the spreading of insecticides in nature. METHODS The feasibilities of two ambient MS methods, desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPPI), for the analysis of five most used neonicotinoid compounds, thiacloprid, acetamiprid, clothianidin, imidacloprid and thiamethoxam, were tested. In addition, DAPPI was used to analyze fresh rose leaves treated with commercially available thiacloprid insecticide and dried and powdered turnip rape flowers, which had been collected from a field treated with thiacloprid-containing insecticide. RESULTS DAPPI was found to be more sensitive than DESI, with 2-11 times better signal-to-noise ratios, and limits of detection at 0.4-5.0 fmol for the standard compounds. DAPPI was able to detect thiacloprid from the rose leaves even 2.5 months after the treatment and from the turnip rape flower samples collected from a field. The analysis of plant material by DAPPI did not require extraction or other sample preparation. CONCLUSIONS DAPPI was found to be suitable for the fast and direct qualitative analysis of thiacloprid neonicotinoid from plant samples. It shows promise as a fast tool for screening of forbidden insecticides, or studying the distribution of insecticides in nature.