Assisted Active Learning for Model-Based Method Development in Liquid Chromatography.

In recent decades, there has been a growing interest in fully automated methods for tackling complex optimization problems across various fields. Active learning (AL) and its variant, assisted active learning (AAL), incorporating guidance or assistance from external sources into the learning process, play key roles in this automation by enabling the autonomous selection of optimal experimental conditions to efficiently explore the problem space. These approaches are particularly valuable in situations wherein experimentation is costly or time-consuming. This study explores the application of AAL in model-based method development (MD) for liquid chromatography (LC) by using Bayesian statistics to incorporate historical data and analyte information for the generation of initial retention models. The process involves updating the model parameters based on new experiments, coupled with an active data selection method to choose the most informative experiment to run in a subsequent step. This iterative process balances model exploitation and experimental exploration until a satisfactory separation is achieved. The effectiveness of this approach is demonstrated via two practical examples, resulting in optimized separations in a limited number of experiments by optimizing the gradient slope. It is shown that the ability of AAL to leverage past knowledge and compound information to improve accuracy and reduce experimental runs offers a flexible alternative approach to fixed design methods.

Mass spectrometry-based identification of ortho-, meta- and para-isomers using infrared ion spectroscopy.

Distinguishing positional isomers, such as compounds having different substitution patterns on an aromatic ring, presents a significant challenge for mass spectrometric analyses and is a frequently encountered difficulty in, for example, drug metabolism research. In contrast to mass spectrometry, IR spectroscopy is a well-known and powerful tool in the distinction of ortho-, meta- and para-isomers, but is not applicable to low-abundance compounds in complex mixtures such as often targeted in bioanalytical studies. Here, we demonstrate the use of infrared ion spectroscopy (IRIS) as a novel method that facilitates the differentiation between positional isomers of disubstituted phenyl-containing compounds and that can be applied in mass spectrometry-based complex mixture analysis. By analyzing different substitution patterns over several sets of isomeric compounds, we show that IRIS is able to consistently probe the diagnostic CH out-of-plane vibrations that are sensitive to positional isomerism. We show that these modes are largely independent of the chemical functionality contained in the ring substituents and of the type of ionization. We also show that IRIS spectra often identify the positional isomer directly, even in the absence of reference spectra obtained from physical standards or from computational prediction. We foresee that this method will be generally applicable to the identification of disubstituted phenyl-containing compounds.

Selective drug metabolite trace analysis by very high-volume injections and heartcut two-dimensional (2D)-ultrahigh performance liquid chromatography (UHPLC).

The application of two-dimensional liquid chromatography (2D-LC) is gradually growing also in the area of metabolite profiling and identification. The current contribution describes a heartcut 2D-UHPLC configuration that is applied in support of drug metabolism studies in development. The setup applies four LC columns: two analytical UHPLC columns to perform the first and second dimension separations, which are both preceded by a short HPLC column operated as trapping column. The first HPLC column allows a significant online preconcentration by large volume injection. The second short HPLC column is placed between the first and second dimension columns and enables the selection of orthogonal conditions in the second dimension independent of the first dimension making the heartcutting 2D approach more generic. The value of the setup was demonstrated with selective ultraviolet chromatograms obtained for the two major hydroxylated metabolites of atorvastatin separating them from a very high biological background, originating from an injection of 4 mL feces extract, by heartcut 2D-LC. In a second application, the main metabolite of imipramine was baseline separated from some minor metabolites that were co-eluting in the first dimension, allowing accurate and sensitive quantification. A quantification limit in the attogram/mL range was achieved thanks to the injection of 200 mL diluted urine, corresponding to 100 mL urine on column.

Combined Liquid Chromatography-Infrared Ion Spectroscopy for Identification of Regioisomeric Drug Metabolites.

High-performance liquid chromatography was used in combination with infrared ion spectroscopy for the identification of positional isomers of hydroxy-atorvastatins, the primary metabolites of the drug atorvastatin. The results demonstrate the direct applicability of infrared ion spectroscopy in the field of drug metabolism and, more generally, its promising role in state-of-the-art analytical laboratories for the identification of small molecules buried in complex mixtures. In combination with chromatographic separation, infrared spectroscopy of mass-selected ions provides a promising new route for the identification of the molecular structures of unknown m/z peaks in a mass spectrum. We demonstrate that currently existing experimental protocols allow the measurement of an IR spectrum from less than 10 ng of sample obtained in a collected HPLC fraction.

HPLC 2015.

The HPLC 2015 Conference was held from 21 to 25 June at the International Conference Center in Geneva, Switzerland. The emphasis of the meeting was around fundamental aspects of separations, sample preparation, novel developments and applications and hyphenation with MS. In this conference report, a selection of highlights of the Conference is given, based on the sessions attended by the authors, and focusing on subjects with possible relevance in the field of drug metabolism and bioanalysis. Selected papers from HPLC 2015 will be published in a virtual special issue of the Journal of Chromatography.

High volume injections of biological samples for sensitive metabolite profiling and quantitation.

An online preconcentration approach was developed allowing the injection of very high volumes of biological samples, thereby greatly increasing sensitivity while maintaining LC resolution. The approach was applied to the analysis of radioactive samples from both in vitro and in vivo metabolism studies where typically the concentration of radioactivity given is often limited, while sample volume is usually not. The described online preconcentration approach reduces sample preparation and, therefore, also the risk for degradation and recovery issues often seen with offline preconcentration methods. In addition to facilitating the identification and profiling of low level metabolites within a sample, the described approach also provides robust quantitative analysis of samples derived from a range of biological matrices. The application of this approach is illustrated on real life samples from different matrices and containing drugs and metabolites with a wide variety in polarity, more specifically the analysis of extracts derived from an in vitro hepatocyte incubation, 36mL of blood/acetonitrile (1/1, v/v; 28dpm/mL) and 72mL of urine/methanol (9/1, v/v; 208dpm/mL).

Use of relative 12C/14C isotope ratios to estimate metabolite concentrations in the absence of authentic standards.

BACKGROUND: There is considerable interest in the determination of relative abundances of human metabolites in plasma (and potentially excreta) with reasonable accuracy early on in the drug development process in order to make scientifically sound decisions with regard to the presence of potentially active or toxic disproportionate metabolites. At this point, authentic metabolite standards are generally not available. RESULTS: A new methodology is proposed for the estimation of metabolite concentrations in the absence of authentic standards. A reference sample containing radiolabeled metabolites of interest is produced by incubating the (14)C-labeled drug in vitro, and mixed with a sample to be quantitated containing the unlabeled metabolites. The (12)C/(14)C isotope ratio is measured with high-resolution ESI-MS for each metabolite, and used as a basis for quantitation of the cold metabolite based on the concentration of radioactive metabolite, determined from independent analysis of the radioactive sample with LC-radiochemical detection. The (14)C-labeled metabolite serves as an isotopically labeled internal standard, which corrects for any variations in injection volume, sample preparation, MS intensity drift, matrix effects and/or saturation of electrospray ionization. The approach was validated by the analysis of solutions containing variable amounts of the analyte with a fixed amount of radioactive standard on a QToF Synapt(®) G2 MS system. The same methodology was also successfully applied to first-in-human plasma samples analyzed on a LTQ-Orbitrap(®). CONCLUSION: The metabolite abundances obtained by (12)C/(14)C isotope ratio measurements showed suitable accuracy and precision and were very close to those obtained with matrix mixing. The parent drug concentrations also corresponded well with the bioanalytical results obtained with a validated LC-MS/MS method.

Improved liquid chromatography-Online radioactivity detection for metabolite profiling.

If very-high-pressure liquid chromatography (VHPLC) is to replace conventional HPLC as the ultimate separation tool for metabolism studies in development, coupling it efficiently with online radioactivity detection (RAD) is needed. We describe the successful combination of VHPLC/RAD, facilitated by improvements in online radioactivity detection, as well as in column loading and peak capacity. The sensitivity of (14)C detection was improved by the use of a variable scintillation flow achieved via a simple modification to the classical online radiochemical detection set-up. A modification of the flow-through cell design in which internal diameter of the tubing was reduced further increased the sensitivity and resolution by decreasing peak tailing. The injection of relatively large injection volumes was made possible by the use of columns packed at ultra-high pressure with 2.2 microm particles. Because of the reduced back pressure generated using these larger particle sizes, two 150 mm x 3 mm columns could be coupled, allowing 4-fold larger injection volumes and a 50% increase in theoretical plate number at a similar back pressure compared to a standard 150 mm x 2.1mm Waters UPLC column. The value of the methodology described was demonstrated by the analysis of in vitro and in vivo metabolism samples of (3)H- and (14)C-labeled compounds and compared with conventional radio-HPLC. We have shown that metabolite separation can be achieved with increased efficiency while maintaining a sensitivity comparable to that of conventional HPLC/RAD.