Comprehensive Single-Platform Lipidomic/Metabolomic Analysis Using Supercritical Fluid Chromatography-Mass Spectrometry.

Supercritical fluid chromatography (SFC) is a rapidly expanding technique in the analysis of nonpolar to moderately polar substances and, more recently, also in the analysis of compounds with higher polarity. Herein, we demonstrate a proof of concept for the application of a commercial SFC instrument with electrospray ionization-mass spectrometry (MS) detection as a platform for the comprehensive analysis of metabolites with the full range of polarities, from nonpolar lipids up to highly polar metabolites. The developed single-platform SFC-MS lipidomic/metabolomic method is based on two consecutive injections of lipid and polar metabolite extracts from biphase methyl tert-butyl ether extraction using a diol column and two different gradient programs of methanol-water-ammonium formate modifier. Detailed development of the method focused mainly on the pressure limits of the system, the long-term repeatability of results, and the chromatographic performance, including optimization of the flow rate program, modifier composition and gradient, and injection solvent selection. The developed method enabled fast and comprehensive analysis of lipids and polar metabolites from plasma within a 24 min cycle with two injections using a simple analytical platform based on a single instrument, column, and mobile phase. Finally, the results from SFC-MS analysis of polar metabolites were compared with widely established liquid chromatography MS analysis in metabolomics. The comparison showed different separation selectivity of metabolites using both methods and overall lower sensitivity of the SFC-MS due to the higher flow rate and worse chromatographic performance.

Direct Chiral Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Monoacylglycerol and Diacylglycerol Isomers for the Study of Lipase-Catalyzed Hydrolysis of Triacylglycerols.

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn-1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn-1 position of prochiral sn-1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

Enantiomeric separation of bupropion by liquid chromatography on derivatized cyclofructan chiral stationary phase.

High-performance liquid chromatography (HPLC) is an ideal tool for enantiomeric separations of different drugs. In this study, the direct enantioseparation of bupropion, an atypical antidepressant structurally related to cathinone, was explored by using five chiral columns, including three based on derivatized cyclofructans, macrocyclic glycopeptide teicoplanin, and an immobilized amylose derivative under multimodal elution conditions. Baseline enantioseparation was obtained on the LarihcShell CF6-RN column, with derivatized cyclofructan 6, in the polar organic mode. The effects of the mobile-phase composition, type and content of major components, the nature and the amount of mobile-phase additives, and the column temperature on the retention, selectivity, and resolution were investigated to optimize enantioseparation. The calibration curve was linear in the range of 10-125 µg/ml for each enantiomer. The limits of detection and quantification were 0.1 and 0.3 µg/ml for both enantiomers of bupropion. The chiral recognition was controlled especially by H bonds, π-π, dipole-dipole interactions, and steric effects. Finally, the developed method was applied to the determination of bupropion in the commercially available drug.

Ultrahigh-performance supercritical fluid chromatography for intraclass separation of lipids: Investigation of general principles.

Powerful chromatographic techniques are required for lipidomic analyses due to the extreme complexity of natural lipidomes. In the past few years, ultrahigh-performance supercritical fluid chromatography (UHPSFC) has proven to be a good alternative to conventional LC methods for comprehensive lipidomic analysis. The goal of this work was to study UHPSFC intraclass separation of lipids according to the fatty acyl composition. The effects of column chemistry, mobile phase composition and gradient, flow rate, back pressure, temperature, and column coupling on intraclass separation of lipids were widely investigated and discussed. In general, UHPSFC exhibited interclass selectivity together with intraclass separation of lipids according to their total number of double bonds and acyl chain lengths. Moreover, separations of diacylglycerol and lysophosphatidylcholines regioisomers were achieved in some cases. The nature of the stationary phase showed the most prominent effect on UHPSFC intraclass selectivity, while other chromatographic conditions were used for partial improvement in resolution of lipid species. An octadecyl-based stationary phase showed excellent separation of nonpolar lipid species, including triacylglycerol isobars; however, it provided poor peak shapes and limited retention time reproducibility for polar lipids. Diol- and 1-aminoanthracene-based columns provided the best inter- and intraclass resolution of most lipids. The main benefit for UHPSFC separation of complex lipid samples is the combination of the acyl chain/double bond intraclass separation of lipids with excellent lipid class selectivity, which can facilitate mass spectrometry detection and quantitation of trace species without ion suppression effects.

Enantioselective separation of zopiclone on immobilized polysaccharide chiral stationary phase by HPLC: Method development and validation.

New, sensitive and rapid chiral HPLC method for enantioseparation and determination of widely used hypnotic drug, zopiclone, was developed. Enantioseparation was achieved on the immobilized amylose based Lux i-Amylose 1 column in polar organic mode in less than 7 min. The effects of the mobile phase composition, type and content of major components as well as acid/base ratio and column temperature on the retention and enantioseparation were investigated. The mobile phase consisted of acetonitril and methanol with small addition of triethylamine and acetic acid with a flow rate of 1 mL min-1. Calibration curves of both zopiclone enantiomers were linear over the concentration range of 5-125 µg mL-1. The limits of detection and quantification for (R)-zopiclone were 5 and 15 ng mL-1 and for (S)-zopiclone were 7 and 21 ng mL-1, respectively. It was demonstrated that the proposed method was selective, precise and robust. Finally, the validated method was applied to the determination of zopiclone enantiomers in the commercial tablets.

Enantioselective potential of teicoplanin- and vancomycin-based superficially porous particles-packed columns for supercritical fluid chromatography.

Application of the superficially porous particles (SPPs) grafted with chiral selectors can substantially improve resolution in chromatographic techniques. In this work, we carried out a deeper study on supercritical fluid chromatography systems with 2.7 µm SPPs bonded with teicoplanin and vancomycin. Fast separations of the majority of enantiomers of phytoalexins, substituted tryptophans, and ketamine derivatives, as representatives of important biologically active and structurally diverse chiral compounds have been achieved. The chromatographic behavior of the structurally different analytes served to characterize these separation systems. The influence of separation conditions, namely mobile phase composition, i.e. type of co-solvent and additive on retention, enantioselective resolution and enantioselectivity was examined. The success rate of baseline and partial separations in individual groups of compounds differed with the chiral stationary phase and also with mobile phase composition. The best, baseline separations for the phytoalexins were achieved on the TeicoShell column using methanol as a co-solvent and trifluoroacetic acid as an additive if used. Mostly partial separations were achieved on the vancomycin-based column for all groups of analytes. Complementary separation behavior of these CSPs was confirmed for the majority of the chiral compounds examined in this work.

Fast enantioseparation of indole phytoalexins in additive free supercritical fluid chromatography.

A series of chiral indole phytoalexins with potential anticancer and antimicrobial activity were enantioseparated in supercritical fluid chromatography. Two polysaccharide-based chiral stationary phases composed of tris-(3,5-dimethylphenylcarbamate) derivatives of amylose or cellulose coated on 2.5 µm silica particles were successfully used. The influences of the polysaccharide backbone, co-solvent type and co-solvent content in the mobile phase on retention, enantioselectivity and enantioresolution of indole phytoalexins were investigated. Fast baseline separations were achieved for 26 from 27 tested compounds. Amylose-based chiral stationary phase provided higher number of baseline resolutions of the indole phytoalexins than the cellulose-based one. However, certain complementary enantioresolution results towards the studied compounds were observed between the investigated columns. The relationship between structure of the indole phytoalexins and their chromatographic behavior in supercritical fluid chromatography was discussed.