Comparison of triple quadrupole, hybrid linear ion trap triple quadrupole, time‐of‐flight and LTQ‐Orbitrap mass spectrometers in drug discovery phase metabolite screening and identification in vitro – amitriptyline and verapamil as model compounds

Abstract

Liquid chromatography in combination with mass spectrometry (LC/MS) is a superior analytical technique for metabolite profiling and identification studies performed in drug discovery and development laboratories. In the early phase of drug discovery the analytical approach should be both time- and cost-effective, thus providing as much data as possible with only one visit to the laboratory, without the need for further experiments. Recent developments in mass spectrometers have created a situation where many different mass spectrometers are available for the task, each with their specific strengths and drawbacks. We compared the metabolite screening properties of four main types of mass spectrometers used in analytical laboratories, considering both the ability to detect the metabolites and provide structural information, as well as the issues related to time consumption in laboratory and thereafter in data processing. Human liver microsomal incubations with amitriptyline and verapamil were used as test samples, and early-phase 'one lab visit only' approaches were used with all instruments. In total, 28 amitriptyline and 69 verapamil metabolites were found and tentatively identified. Time-of-flight mass spectrometry (TOFMS) was the only approach detecting all of them, shown to be the most suitable instrument for elucidating as comprehensive metabolite profile as possible leading also to lowest overall time consumption together with the LTQ-Orbitrap approach. The latter however suffered from lower detection sensitivity and false negatives, and due to slow data acquisition rate required slower chromatography. Approaches with triple quadrupole mass spectrometry (QqQ) and hybrid linear ion trap triple quadrupole mass spectrometry (Q-Trap) provided the highest amount of fragment ion data for structural elucidation, but, in addition to being unable to produce very high-important accurate mass data, they suffered from many false negatives, and especially with the QqQ, from very high overall time consumption.

Découverte fortuite d’une intoxication aiguë par vérapamil forme à libération prolongée : intérêt du criblage toxicologique

“…Après administration par voie orale, le vérapamil est absorbé à plus de 90 % et subit un fort effet de premier passage hépatique avec une biodisponibilité de 10 à 20 %. Les 2 principales voies métaboliques sont la N-déméthylation (40 %) et la N-déalkylation (33 %) [3,18]. Les pourcentages de vérapamil et norvérapamil retrouvés dans les urines sont très faibles : respectivement 2 % et 1 % [19].…”

“…L'identification est effectuée par comparaison des masses de l'ion parent et des fragments avec les données de la littérature [3,12,13]. Les 4 métabolites O-déméthylés (B) présentent la même masse d'ion parent et le même temps de rétention ; ils ne peuvent pas être différenciés.…”

“…Il subit un métabolisme hépatique intense avec une biodisponibilité de 10 à 20 % après une administration orale unique [1,2]. Pas moins de 69 métabolites du vérapamil ont été identifiés [3], dont le norvérapamil, métabolite actif pouvant être retrouvé à des concentrations sériques voisines voire supérieures à celles du vérapamil [2,4]. En cas d'intoxication, les demi-vies d'élimination du vérapamil et du norvérapamil sont augmentées [5,6].…”

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…Another study compared QQQ, linear IT (QTrap), TOF and Orbitrap instruments for identification of microsomal metabolites of verapamil and amitriptyline [41]. Only TOF found all 28 amitriptyline and 69 verapamil metabolites; both expected and unexpected.…”

Driving efficiency in a high-throughput metabolic stability assay through a generic high-resolution accurate mass method and automated data mining

“…However, it is still under debate whether the scan speed of Orbitrap instruments is sufficient to quantify sharp peaks eluted by U-HPLC. It has been a concern that the Orbitrap may suffer from slow data acquisition, thus lowering detection sensitivity, compared with TOF instruments (Rousu et al, 2010). In the presents study we conducted a comprehensive examination of the speed, sensitivity and selectivity of Exactive in conjunction with U-HPLC for metabolic stability assays.…”