During drug development, chromatography is frequently used for purity and stability testing of both drug substance and drug product. Reversed phase liquid chromatography (RPLC) is one of the most widely used methodologies due to its wide scope of application. In the later stages of drug development, the specified impurities and degradation products that define the critical quality attribute of the final API, also known as Key Predictive Sample Set (KPSS), are usually well defined and controlled. At this point, a method review enables selecting the most appropriate technique which should be the one providing optimal robustness (ICH-Q14[1]), with the support of Quality by Design (QbD) approaches. Supercritical Fluid Chromatography (SFC) is a preferred technique for its proven diversity in selectivity. The adoption of a technique which presents the most favourable environmental impact, such as, but not limited to, SFC, is also becoming increasingly important as laboratories strive to reduce carbon footprint. Re-developing a method requires high resource-demands in terms of staff, materials, and time. Any step of the process that can be automated can facilitate this approach, speeding up the delivery of the method whilst preserving robustness. In this article we describe how an SFC method was developed for the purity profiling of a late-stage oncology candidate, taking advantage of the superior selectivity of SFC towards structurally similar analytes, owed to the high orthogonality with R2 as low as 0.014 towards the KPSS. We describe two approaches to automate the method development. Firstly, a multifactorial design of experiments (DoE) and secondly, an optimization via a Bayesian algorithm, which was completed in one night, highlighting the potential and limitations, with an insight into the robustness. Both methods achieved baseline separation with varying levels of automation embedded into the process and a large reduction of the resource demands when compared to traditional optimisation methods. Finally, we describe the beneficial environmental impact that implementing SFC methods can yield, with a calculated green score reduced to a value between 17 and 30 % compared to RPLC, depending on the number of runs per sequence.

This work introduces a novel microfluidic backpressure pressure control developed for chip-based supercritical fluid chromatography (chipSFC). The presented on-chip pressure control mechanism involves the post-column addition of a viscous make-up stream, which enables pressure regulation within the range of 73 to 130 bar range. In contrast to approaches using mechanical backpressure regulators, this chip-based make-up-assisted pressure regulation offers a wear-free alternative that functions entirely through fluidic means and contributes minimally to extra column volume. It prevents phase separation of the supercritical mobile phase and, therefore, expands the analytical scope of chipSFC to detection systems with an ambient pressure interface. This was demonstrated by a proof-of-principle experiment, where a model mixture was separated within 30 s and detected using atmospheric pressure ionisation mass spectrometry.

Herein, an improved subtraction model was proposed to characterise the polar stationary phases in supercritical fluid chromatography (SFC). Fifteen stationary phases were selected, including two types of aromatic columns, Waters Torus and Viridis series columns, as well as silica and amino columns. Ethylbenzene and Torus 1-AA were defined as the reference solute and column, respectively. Identifying the interaction with the maximum contribution to retention in SFC separation and using it as the initial term is a key step in modelling. The dipole, or induced dipole interaction (θ\'P), replaced the hydrophobic interaction (η\'H) as the starting term. The improved model was expressed as logα=η\'H+β\'A+α\'B+κ\'C+θ\'P+ε\'E+σ\'S, where the term ε\'E indicated that anion exchange interaction was intentionally supplemented. A 7-step modelling process, including bidirectional fitting and residual analysis, was proposed. The obtained column parameters had reasonable physical significance, with the adjusted determination coefficient (R2adj) greater than 0.999 and the standard error (SE) less than 0.029. Methodological validation was further performed using the other four columns and 12 solutes that were not involved in the modelling. The result revealed good predictions of solutes\' retention, as demonstrated by R2adj from 0.9923 to 0.9979 and SE from 0.0636 to 0.1088. This study indicated the feasibility of using the improved subtraction model to characterise polar stationary phases in SFC, with the most crucial being the determination of an initial term, followed by the addition of a new descriptor and the selection of an appropriate reference column. The study expanded the application scope of the subtraction model in SFC, which will help gain an in-depth understanding of the SFC separation mechanism.

In this study, water was used as an additive in the methanol-modified carbon dioxide-based eluent for the elution of some basic organic compounds from a hybrid silica column via supercritical fluid chromatography (SFC). The experiments were applied to sulfonamides, propranolol, and other organic nitrogen compounds involving aromatic rings from different classes of amine, pyrimidine, and purine with different pKa values (the pKa values for the studied analytes range from 4.6 to 10.4). The results revealed different responses to the different percentages of water addition. Adding 1~2% of water to the modifier (methanol) led to a positive effect manifested by more symmetrical peak shapes and reduced retention times for most compounds. The key factor for this improvement in the properties of chromatographic peaks is due to the adsorption of water on the silanol groups of the stationary phase, consequently resembling the phenomena observed in hydrophilic interaction liquid chromatography (HILIC). Moreover, the availability of hydrogen bond acceptor and donor sites in the analyte structure is an important factor to be considered when adding water as an additive to the modifier for improving the chromatographic peaks. However, introducing water in an amount higher than 3% resulted in perturbed chromatographic signals. It was also found that water as an additive alone could not successfully elute propranolol from the hybrid silica column with an acceptable peak shape; thus, the addition of a strong base such as amine salts was also necessary. The proposed use of a particular amount of water in the mobile phase could have a positive effect compared to the same mobile phase without water, improving the chromatographic peak properties of the elution of some basic organic compounds from the hybrid silica column.

High throughput experimentation is a growing and evolving field that allows to execute dozens to several thousands of experiments per day with relatively limited resources. Through miniaturization, typically a high degree of automation and the use of digital data tools, many parallel reactions or experiments at a time can be run in such workflows. High throughput experimentation also requires fast analytical techniques capable of generating critically important analytical data in line with the increased rate of experimentation. As traditional techniques usually do not deliver the speed required, some unique approaches are required to enable workflows to function as designed. This review covers the recent developments (2019-2020) in this field and was intended to give a comprehensive overview of the current \"state-of-the-art.\"

Various new psychoactive substances, such as cathinone and its analogs, possess similar chemical structures. Accurate identification of structurally similar drugs of abuse is important in forensic drug analysis. Chromatographic differentiation is a powerful analytical technique for this purpose. In this study, we applied supercritical fluid chromatography to differentiate ring-substituted regioisomers of six synthetic cathinone analogs (fluoro-α-pyrrolidinovalerophenone, methyl-α-pyrrolidinovalerophenone, methoxy-α-pyrrolidinovalerophenone, fluoro-α-pyrrolidinooctanophenone, fluoropentedrone, and fluorooctedrone). The examined cathinone analogs were weakly retained on the stationary phase (possessing diol functional group) and eluted quickly. We optimized the conditions to retain the examined cathinone analogs to achieve sufficient separation, and found that the types of functional groups on the stationary phase greatly affected the retention and separation. Systematic examination of the chromatographic conditions showed that the two stationary phases possessing anthracene and pentafluorobenzyl groups had good separation capabilities for the examined 2-, 3-, and 4-regioisomers of six cathinone analogs, which had different skeletal structures. Interestingly, the two stationary phases showed different selectivities, and alteration of the elution order was observed. The developed method was validated and its discrimination ability was investigated by measuring mass spectra and absorption spectra. Supercritical fluid chromatography-ultraviolet absorption spectroscopy/mass spectrometry is a powerful analytical technique for differentiation of ring-substituted cathinone analogs.

The packed column supercritical fluid chromatography has risen as a promising alternative separation technique to the conventional liquid chromatography and gas chromatography. Although the packed column supercritical fluid chromatography has many advantages compared to other chromatographic techniques, its separation mechanism is not fully understood due to the complex combination effects of many chromatographic parameters on separation quality and the lacking of global strategies for studying separation mechanisms. This review aims to provide recent information regarding the chromatographic behaviors and the effects of the parameters on the separation, discuss the results, and point out the remaining bottlenecks in the packed column supercritical fluid chromatography retention mechanism studies.

Chromatography is a robust and reliable separation method that can use various stationary phases to separate complex mixtures commonly seen in metabolomics. This review examines the types of chromatography and stationary phases that have been used in targeted or untargeted metabolomics with methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. General considerations for sample pretreatment and separations in metabolomics are considered, along with the various supports and separation formats for chromatography that have been used in such work. The types of liquid chromatography (LC) that have been most extensively used in metabolomics will be examined, such as reversed-phase liquid chromatography and hydrophilic liquid interaction chromatography. In addition, other forms of LC that have been used in more limited applications for metabolomics (e.g., ion-exchange, size-exclusion, and affinity methods) will be discussed to illustrate how these techniques may be utilized for new and future research in this field. Multidimensional LC methods are also discussed, as well as the use of gas chromatography and supercritical fluid chromatography in metabolomics. In addition, the roles of chromatography in NMR- vs. MS-based metabolomics are considered. Applications are given within the field of metabolomics for each type of chromatography, along with potential advantages or limitations of these separation methods.

Methadone has two enantiomers, which exhibit differences in pharmacological effects, with R-methadone being the active and S-methadone the inactive enantiomer. A robust, simple and rapid method for chiral separation of the two enantiomers in serum samples using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MSMS) has been developed and validated. Enantiomeric separation was achieved using a Chiralpak IH-3 column with a mobile phase consisting of CO2 and 30mM ammonium acetate in methanol/water (98/2, v/v). Runtime was 4 minutes. Sample preparation was semi-automated using a Hamilton ML Star robot with protein precipitation, and phospholipid removal was carried out using a Waters OSTRO™ 96-well plate. The calibration range was 50.0-1,500 nM for each enantiomer. The between-assay relative standard deviations were in the range of 1.2-3.6%. Matrix effects ranged from 99% to 115% corrected with internal standard. The method has been implemented in our laboratory and has proven to be a robust and reliable method for determining the ratio of R/S-methadone in authentic patient samples.

A novel silica stationary phase was designed and prepared through thiol-epoxy click chemistry for supercritical fluid chromatography (SFC). The developed stationary phase was characterized by elemental analysis, Fourier transform infrared spectrometry and solid-state 13C/CP MAS NMR spectroscopy. In order to evaluate the chromatographic performance and retention mechanisms of the prepared column, a variety of alkaloids were used, including indoles, isoquinolines, pyrrolidines, piperidines, quinolizidines and organic amines. The stationary phase showed more symmetrical peak shapes and better performance for these compounds compared to the conventional SFC stationary phases. The investigations on the effects of pressure and temperature on retention provided information that the selectivity of the compounds can be improved by changing the density of the supercritical fluids. Moreover, it shows improved separation efficiency of three natural products with alkaloids as the main components at high sample loading. In conclusion, the developed stationary phase could offer flexible selectivity toward alkaloids and complex samples.