Use of Cyclic Ion Mobility Spectrometry (cIM)-Mass Spectrometry to Study the Intramolecular Transacylation of Diclofenac Acyl Glucuronide.

1-ß-O-Acyl-glucuronides (AGs) are common metabolites of carboxylic acid-containing xenobiotics, including, e.g., many nonsteroidal anti-inflammatory drugs (NSAIDs). They are of concern to regulatory authorities because of the association of these metabolites with the hepatotoxicity that has resulted in drug withdrawal. One factor in assessing the potential risk posed by AGs is the rate of transacylation of the biosynthetic 1-ß-O-acyl form to the 2-, 3-, and 4-O-acyl isomers. While transacylation can be measured using 1H NMR spectroscopy or liquid chromatography-mass spectrometry (LC-MS), the process can be time consuming and involve significant method development. The separation of these positional isomers by ion mobility spectrometry (IMS) has the potential to allow their rapid analysis, but conventional instruments lacked the resolving power to do this. Prediction of the collision cross section (CCS) using a machine learning model suggested that greater IMS resolution might be of use in this area. Cyclic IMS was evaluated for separating mixtures of isomeric AGs of diclofenac and was compared with a conventional ultraperformance liquid chromatography (UPLC)-MS method as a means for studying transacylation kinetics. The resolution of isomeric AGs was not seen using a conventional traveling wave IMS device; however, separation was seen after several passes around a cyclic IMS. The cyclic IMS enabled the degradation of the 1-ß-O-acyl-isomer to be analyzed much more rapidly than by LC-MS. The ability of cyclic IMS to monitor the rate of AG transacylation at different pH values, without the need for a prior chromatographic separation, should allow high-throughput, real-time, monitoring of these types of reactions.

A structure-activity relationship linking non-planar PCBs to functional deficits of neural crest cells: new roles for connexins.

Migration of neural crest cells (NCC) is a fundamental developmental process, and test methods to identify interfering toxicants have been developed. By examining cell function endpoints, as in the 'migration-inhibition of NCC (cMINC)' assay, a large number of toxicity mechanisms and protein targets can be covered. However, the key events that lead to the adverse effects of a given chemical or group of related compounds are hard to elucidate. To address this issue, we explored here, whether the establishment of two overlapping structure-activity relationships (SAR)-linking chemical structure on the one hand to a phenotypic test outcome, and on the other hand to a mechanistic endpoint-was useful as strategy to identify relevant toxicity mechanisms. For this purpose, we chose polychlorinated biphenyls (PCB) as a large group of related, but still toxicologically and physicochemically diverse structures. We obtained concentration-dependent data for 26 PCBs in the cMINC assay. Moreover, the test chemicals were evaluated by a new high-content imaging method for their effect on cellular re-distribution of connexin43 and for their capacity to inhibit gap junctions. Non-planar PCBs inhibited NCC migration. The potency (1-10 µM) correlated with the number of ortho-chlorine substituents; non-ortho-chloro (planar) PCBs were non-toxic. The toxicity to NCC partially correlated with gap junction inhibition, while it fully correlated (p < 0.0004) with connexin43 cellular re-distribution. Thus, our double-SAR strategy revealed a mechanistic step tightly linked to NCC toxicity of PCBs. Connexin43 patterns in NCC may be explored as a new endpoint relevant to developmental toxicity screening.

From definition to implementation: a cross-industry perspective of past, current and future MIST strategies.

The article describes and discusses the evolution of strategies to characterize metabolites in support of safety studies over the last 40 years, as well as future trends. Approaches to derive qualitative and quantitative information on metabolites are described, with a particular focus on the comparison of options to quantify metabolites in the absence of authentic standards. Current strategies to assess metabolite profiles are summarized into four general approaches and compared against a number of key criteria. Potential future strategies are discussed, including the use of clinical samples as the starting point for metabolite investigations, minimizing the need for animal radiolabelled studies and establishing metabolite safety without radiolabelled studies in animals or human.

Investigation into Small Molecule Isomeric Glucuronide Metabolite Differentiation Using In Silico and Experimental Collision Cross-Section Values.

Identifying isomeric metabolites remains a challenging and time-consuming process with both sensitivity and unambiguous structural assignment typically only achieved through the combined use of LC-MS and NMR. Ion mobility mass spectrometry (IMMS) has the potential to produce timely and accurate data using a single technique to identify drug metabolites, including isomers, without the requirement for in-depth interpretation (cf. MS/MS data) using an automated computational pipeline by comparison of experimental collision cross-section (CCS) values with predicted CCS values. An ion mobility enabled Q-Tof mass spectrometer was used to determine the CCS values of 28 (14 isomeric pairs of) small molecule glucuronide metabolites, which were then compared to two different in silico models; a quantum mechanics (QM) and a machine learning (ML) approach to test these approaches. The difference between CCS values within isomer pairs was also assessed to evaluate if the difference was large enough for unambiguous structural identification through in silico prediction. A good correlation was found between both the QM- and ML-based models and experimentally determined CCS values. The predicted CCS values were found to be similar between ML and QM in silico methods, with the QM model more accurately describing the difference in CCS values between isomer pairs. Of the 14 isomeric pairs, only one (naringenin glucuronides) gave a sufficient difference in CCS values for the QM model to distinguish between the isomers with some level of confidence, with the ML model unable to confidently distinguish the studied isomer pairs. An evaluation of analyte structures was also undertaken to explore any trends or anomalies within the data set.

Application of a tiered approach to the validation of accelerator MS assays.

Since its introduction into the drug-development arena, accelerator mass spectrometry (coupled with liquid chromatography fractionation) has been used to support a variety of study types. The uses to which the technique has been put include parent and/or metabolite quantification in pharmacokinetic studies, total radioactivity measurement in adsorption, metabolism and excretion studies, and quantitative metabolite profiling. A tiered approach has been applied to the verification of accelerator mass spectrometry assays, dependant on in which type of study and at what stage of drug development they are used. As accelerator mass spectrometry is an absolute detector that can quantify without the use of analyte-related standards, the specific assay verification requirements differ from those for LC-MS/MS assays. This article describes when screening, qualified and validated assay verification procedures should be applied, and suggests what parameters should be assessed in each case.

Ask the experts: automation: part I.

Bioanalysis invited a selection of leading researchers to express their views on automation in the bioanalytical laboratory. The topics discussed include the challenges that the modern bioanalyst faces when integrating automation into existing drug-development processes, the impact of automation and how they envision the modern bioanalytical laboratory changing in the near future. Their enlightening responses provide a valuable insight into the impact of automation and the future of the constantly evolving bioanalytical laboratory.

Conference Report: Drug Metabolism Discussion Group Short Meeting: microsampling--the next big thing. Alderley Park, Macclesfield, UK, 14 March 2012.

On behalf of the Drug Metabolism Discussion Group, Regulatory Bioanalysis AstraZeneca (UK) recently organized and hosted an extremely successful Drug Metabolism Discussion Group Short Meeting on 'microsampling--the next big thing'. This attracted over 140 delegates and a strong line up of presenters of respected scientists within the field. This meeting focused on the impact of taking a reduced sample (5-20 µl) from an animal, or later in the clinic, particularly neonates. The agenda covered the spectrum of microsampling, from capillary plasma microsampling, as championed by Ove Jonsson and Kristian Königsson, through to dried blood spots. The day was split up in to three sections, the morning concentrating on the sampling aspects from animals. A highlight of the first section was the 'poster blitz' where four poster presenters gave a quick overview of their work. This introduced the poster session and created a good atmosphere for general debate between the delegates. The mid-session saw the bioanalytical challenges discussed from the discovery to the preclinical stage. To encourage interaction between the presenters and the audience, a panel discussion was used that led to interesting insights into study design from toxicological and bioanalytical viewpoints. The final session was left to clinical aspects of microsampling and a particularly interesting presentation from Hitesh Pandya from the Pediatric Respiratory Medicine Department (University of Leicester, Leicester, UK). An eloquent and hard-hitting presentation put into perspective the importance of advancements in this field that enables sample to be taken in a noninvasive manner. The meeting was well received with excellent feedback from all concerned.

European Bioanalysis Forum recommendation: scientific validation of quantification by accelerator mass spectrometry.

Accelerator mass spectrometry (AMS) is being used more widely to provide PK data for early decision making or to generate absolute bioavailability data in later phases of development. Presently, there is no clear consensus on the level of the scientific validation required for these assays. The European Bioanalysis Forum (EBF) has conducted two surveys with its members and presented the results at its 4th Open Symposium. With AMS being used for discrete scientific assessment, method establishment of AMS assays should focus on science rather than trying to fit the assay parameters into validation criteria used for Regulated Bioanalysis guidance, and an amount of freedom of execution and interpretation is needed. Hence, the EBF focuses their recommendation on introducing terminology around scientific qualification or validation to be used in relation to AMS. Guidance is given on which parameters should be investigated when a qualified method is required. The recommendations of the EBF for scientific validation are described herein. The scientific validation of AMS assays will be different to that applied for LC-MS/MS assays, and an example is that accuracy and precision limits, as used for ligand-binding assays, would be more appropriate.

AMS method validation for quantitation in pharmacokinetic studies with concomitant extravascular and intravenous administration.

A technique has emerged in the past few years that has enabled a drug's intravenous pharmacokinetics to be readily obtained in humans without having to conduct extensive toxicology studies by this route of administration or expand protracted effort in formulation. The technique involves the intravenous administration of a low dose of (14)C-labelled drug (termed a tracer dose) concomitantly with a non-labelled extravascular dose given at therapeutically levels. Plasma samples collected over time are analysed to determine the total parent drug concentration by LC-MS (which essentially measures that arising from the oral dose) and by LC followed by accelerator mass spectrometry (AMS) to determine the (14)C-drug concentration (i.e., that arising from the intravenous dose). There are currently no published accounts of how the principles of bioanalytical validation might be applied to intravenous studies using AMS as an analytical technique. The authors describe the primary elements of AMS when used with LC separation and how this off-line technique differs from LC-MS. They then discuss how the principles of bioanalytical validation might be applied to determine selectivity, accuracy, precision and stability of methods involving LC followed by AMS analysis.

An AMS method to determine analyte recovery from pharmacokinetic studies with concomitant extravascular and intravenous administration.

The absolute bioavailability, clearance and volume of distribution of a drug can be investigated by administering a very low dose of the (14)C-drug intravenously along with a therapeutic nonlabeled dose by the extravascular route (typically orally). The total drug concentration is measured by an assay such as LC-MS and the (14)C-drug is measured by accelerator MS (AMS). In another article in this issue, a method validation is proposed where AMS was used as the analytical assay. Part of the validation is to assess the recovery of the analyte being measured as this has a direct impact on its quantification. In this article, a method of internal standardisation is described where the UV response of the nonlabeled analyte, spiked in excess into the matrix being analysed, is used for internal standardization. The method allows for the recovery of analyte to be measured in each individual sample being analysed. It is important to know the recovery of a (14)C-labeled analyte when determining its mass concentration from (14)C:(12)C isotopic ratio data using AMS. A method is reported in this article that utilizes the UV response of the nonlabeled drug for internal standardization, so that the recovery for each individual sample analyzed can be ascertained.