Supercritical Fluid Chromatography Research Articles

Supercritical fluid chromatography with fluorescence detection for tocochromanol profiling in oils and seed ethanol extracts: A comparative study with NPLC

This study focused on the development of a method designed to simultaneously separate four tocopherol (T), four tocotrienol (T3) homologues and plastochromanol-8 (PC-8) using supercritical fluid chromatography (SFC) with fluorescence detection (FLD). We examined the selectivity and resolution behaviour of tocochromanols on eight different stationary phases and investigated the retention factor of PC-8 (kPC-8) as a function of mobile phase composition, as well as the injection volume’s impact on peak shape. The final SFC-FLD method developed involved a separation with two amino (NH2) columns connected in series (4.6 mm ID; 3 μm dp, 150 mm and 250 mm in length, respectively), at a flow rate of 2.5 mL/min, thermostated at 40 °C and a back-pressure regulator value of 10.0 MPa, injection volume of 10 μL and a gradient comprising of A:CO2 and B:MeOH. The sensitivity of SFC-DAD, SFC-FLD, NPLC-DAD and NPLC-FLD were compared and found to be 58.6–160.9, 5.9–51.9, 19.7–63.5 and 0.42–4.3 pmol, respectively. The SFC-FLD method developed was successfully applied to the analysis of seed ethanol extracts and plant oils and found to be suitable for the routine analysis. Furthermore, the SFC approach serves as a sustainable alternative compared to NPLC, due to the reduced human and environmental exposure and consumption of hazardous solvents.

Chiral separation of terbutaline by supercritical fluid chromatography with peak purity determination by UPLC–MS and modeling for chiral recognition mechanism

AbstractBACKGROUNDTerbutaline is the drug of choice for asthma patients but it exists in racemic mixture. (R)‐(−)‐terbutaline is 200 times more active than (S)‐(+)‐terbutaline yet it is not advisable to prescribe racemic mixture due to certain adverse effects of (S)‐(+)‐terbutaline. Therefore, a fast, effective and reproducible separation method is necessary.RESULTSChiral separation was achieved on Chiralpak‐IE and Chiralpak‐IG columns (250 × 4.6 mm, 5 μm) using carbon dioxide‐methanol (CO2‐MeOH, 60:40) with a 0.2% triethylamine mobile phase. The flow was 1.0 mL min−1 with detection at 223 nm using a photodiode array (PDA) detector. The values of retention, separation and resolution factors were in the ranges 1.88–2.38, 1.14–1.26 and 0.91–1.17, respectively; with best separation with Chiralpak‐IE. The tailing factors and number of theoretical plates were in the range of 1.0–1.23 and 487–3699. The purity of the separated peaks was determined by ultra‐performance liquid chromatography–mass spectrometry (UPLC–MS); indicating 100% purity of the peaks. The chiral recognition was determined by modeling with binding affinities −5.0 and −6.0 for S‐ and R‐enantiomers, respectively; indicating S‐enantiomer elution first followed by R‐enantiomers. The major forces responsible for the chiral resolution were hydrogen bonding and π‐π interactions.CONCLUSIONOwing to the great demand for optically active pure drugs and high economic pressure on analytical techniques, the chiral separation of terbutaline was achieved via inexpensive supercritical fluid chromatography. The reported method may be used to prepare optically active pure terbutaline drugs (R‐enantiomers) at a pilot scale. © 2024 Society of Chemical Industry (SCI).

Retention modeling of therapeutic peptides in sub‐/supercritical fluid chromatography

AbstractChromatographic modeling software packages are valuable tools during method optimization steps. These are well established for reversed‐phase applications utilizing retention time (RT) prediction to optimize separations and receive robust methods, which is of high interest for the analysis of pharmaceuticals. In contrast to liquid chromatography, the knowledge of RT prediction in supercritical fluid chromatography is limited to a manageable number of studies.This study uses the software DryLab to predict the RTs of the peptides bacitracin (Bac), colistin, tyrothricin (Tyro), and insulin analogs. Gradient time, column temperature, and the ternary composition (terC) of carbon dioxide, methanol (MeOH), and acetonitrile (ACN) in the gradient elution are varied in a feasibility approach using a neutral (Viridis BEH) and an amino‐derivatized aromatic (Torus 2‐PIC) stationary phase. In the second part, chromatographic optimization is performed in silico through gradient adjustments to optimize the separation of the fingerprint of Bac. The final gradient method utilizes a Viridis BEH column (100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of ACN/MeOH/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v). In addition, changes in the retention order of Tyro compounds with the proportion of the terC in combination with a Torus Diol column are investigated.

Ultrahigh-Performance Supercritical Fluid Chromatography-Multimodal Ionization-Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts.

Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) was employed for the analysis of 17 volatile terpenes on a porous graphitic carbon column within 7.5 min, followed by analysis on short diol column where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A multimodal ionization source combining electrospray and atmospheric pressure chemical ionization (ESCi) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final UHPSFC-ESI/ESCi-MS/MS two-injection approach were determined, and it was applied to the analysis of Eucalyptus sp. extracts prepared by supercritical fluid extraction. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.

HPLC and SFC Enantioseparation of (±)-Trans-β-Lactam Ureas on Immobilized Polysaccharide-Based Chiral Stationary Phases—The Introduction of Dimethyl Carbonate as an Organic Modifier in SFC

A series of nine racemic trans-β-lactam ureas were analyzed for enantiomer separation by high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). The separations were performed on three immobilized polysaccharide-based chiral analytical columns (CHIRAL ART Amylose-SA, CHIRAL ART Cellulose-SB and CHIRAL ART Cellulose-SC). In HPLC mode, a normal-phase consisting of n-hexane/2-PrOH (90/10, v/v), a polar organic mobile phase consisting of 100% MeOH or 100% EtOH, and a non-standard mobile phase consisting of 100% dimethyl carbonate (DMC) were investigated. In SFC mode, the mobile phases CO2/alcohol (80/20, v/v) and CO2/DMC/alcohol (MeOH or EtOH; 70/24/6, v/v/v or 60/32/8, v/v/v) were investigated. The best achieved enantioseparation of trans-β-lactam ureas was obtained with an Amylose-SA column. We have shown that the green solvent dimethyl carbonate (DMC) can be efficiently used as a mobile phase in HPLC mode as well as in SFC mode along with the addition of polar organic modifiers (MeOH or EtOH).

ICH Q14-inspired novel approach to establish an SFC-based purity method for carbamazepine.

The proposed ICH Q14 guideline "Analytical procedure development" describes science and risk-based approaches for development and maintenance of analytical procedures suitable for the assessment of the quality of drug substances and drug products. As a case study, the systematic development and validation of a supercritical fluid chromatography (SFC)-based purity method for carbamazepine is presented. Systematic analytical quality by design (AQbD) principles were applied using the software package Fusion QbD to the method development approach. The relationship between chromatographic parameters and the responses of interest were examined to improve the reliability of the method by understanding, reducing, and controlling sources of variability. Method performance qualification in terms of method robustness was finally carried out with the parameters that were classified as critical after method development and a validation study met previously set acceptance criteria. The developed SFC purity method for carbamazepine demonstrated readiness as a viable alternative to the official HPLC method published in the Ph.Eur. with improved peak resolution, improved peak symmetry, and faster analysis times (3min vs. 80 min for the official method). Its inherent reliability illustrates the superiority of AQbD in method development and application for drug quality assurance.

Synthesis and evaluation of aromatic stationary phases based on linear solvation energy relationship model for expanded application in supercritical fluid chromatography

In the last decade, the separation application based on aromatic stationary phases has been demonstrated in supercritical fluid chromatography (SFC). In this paper, four aromatic stationary phases involving aniline (S-aniline), 1-aminonaphthalene (S-1-ami-naph), 1-aminoanthracene (S-1-ami-anth) and 1-aminopyrene (S-1-ami-py) were synthesized based on full porous particles (FPP) silica, which were not end-capped for providing extra electrostatic interaction. Retention mechanism of these phases in SFC was investigated using a linear solvation energy relationship (LSER) model. The aromatic stationary phases with five positive parameters (a, b, s, e and d+) can provide hydrogen bonding, π-π, dipole-dipole and cation exchange interactions, which belong to the moderate polar phases. The LSER results obtained using routine test solutes demonstrated that the aforementioned interactions of four aromatic stationary phases were influenced by the type and bonding density of the ligand, but to a certain extent. Furthermore, the LSER data verified that the S-1-ami-anth column based on full porous particles silica had higher cation exchange capacity (d+ value), compared to the commercialized 1-AA column (based on the ethylene-bridged hybrid particles). The relationship between the d+ value and SFC additive was quantitatively proved so as to regulate electrostatic interaction reasonably. This value was greatly increased by phosphoric acid, slightly increased by trifluoroacetic acid and formic acid, but significantly reduced by ammonium formate and diethylamine. Taking the S-1-ami-naph column as an example, better peek shape of the flavonoids was obtained after the addition of 0.1 % phosphoric acid in MeOH while isoquinoline alkaloids were eluted successfully within 11 min after adding 0.1 % diethylamine in MeOH. Combined with the unique π-π interaction and controllable electrostatic interaction, the aromatic stationary phases in this study have been proven to have expandable application potential in SFC separation.

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.

Efficient amidation of weak amines: synthesis, chiral separation by SFC, and antimicrobial activity of N-(9,10-dioxo-9,10-dihydroanthracen-1-yl) carboxamide.

An effective and straightforward method for the synthesis of 1-aminoanthracene-9,10-dione carboxamides by coupling a weakly reactive amine, 1-aminoanthracene-9,10-dione, and sterically hindered carboxylic acids was achieved using COMU as the coupling agent. Furthermore, making use of the advantages associated with the super-critical fluid chromatography (SFC) technique, a simplified and straightforward method for the chiral separation of optically active amide derivatives from the impurities associated with the reaction mixture, in a single step, was demonstrated. The antimicrobial activity of selected 1-aminoanthracene-9,10-dione carboxamides was studied. Advanced NMR and other spectral techniques were used for the thorough characterization of all the compounds. This study provides a general and simplified method for coupling a weak amine with a sterically hindered acid using COMU as a coupling agent, and demonstrates the separation of optically pure compounds from reaction related impurities in a single step using SFC, and identification of amide derivatives of 1-aminoanthracene-9,10-dione as potential antimicrobial agents.

Efikasnost superkritične fluidne hromatografije u kreiranju ekološki prihvatljivih rešenja u farmaceutskoj analizi

Initially employed primarily at a preparative scale for enantiomer separation of chiral drug candidates, Supercritical Fluid Chromatography (SFC) is nowadays extensively used in the analytical mode. Recent advances in SFC separation science have emphasized its potential for modern and environmentally friendly pharmaceutical analysis. The aim of this review is to provide a deeper insight into the main fundamental and practical aspects of the SFC technique in order to familiarize readers with its versatile nature and efficiency in creating sustainable chromatographic solutions. All considerations are made primarily in the context of the most widely used mode of operation - achiral SFC. In addition, recent applications of this promising technique are presented at the end of the article to further promote its use in pharmaceutical analytical practice.

Resolving phytosterols in microalgae using offline two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Sterols are unsaponifiable lipids resulting from plant metabolism that exhibit interesting bioactive properties. Microalgae are a major source of specific phytosterols, most of which are still not fully characterized. The similarity in sterol structures and the existence of positional isomers make the separation of phytosterols challenging. A method was developed based on an offline two-dimensional (2D) system, reversed-phase liquid chromatography (RPLC)-supercritical fluid chromatography (SFC)/quadrupole time-of-flight (Q-ToF) mass spectrometry, for the identification of sterols in microalgae. Subsequent positive-mode MS/MS was used to confirm the identified phytosterols. The 2D chromatogram exhibited a pattern related to the positions of the double bonds, which were confirmed by standard injection, enabling structural elucidation. The analysis of the unsaponifiable fraction of two algae, namely Scenedesmus obliquus, a freshwater microalgae, and Padina pavonica, a marine macroalgae, highlighted the ability of the method to distinguish a large number of sterol isomers.

Ordered mesoporous silica microspheres for supercritical fluid chromatography.

Herein, silica microspheres with ordered mesopores are synthesized and applied as a stationary phase for supercritical fluid chromatography (SFC). The excellent particle monodispersity and pore orderliness coupled with the rapid analytes diffusion of the supercritical fluid lead to an ultra-high column efficiency of 340 000 plate per m.

A powerful two-dimensional chromatography method for the non-target analysis of depolymerised lignin

BackgroundLignin is an abundant natural polymer obtained as a by-product from the fractionation of lignocellulosic biomass. In the name of a circular economy, lignin should be valorised into valuable chemicals or biomaterials and utilised instead of petrochemicals. For the development of efficient valorisation processes, the structural characterisation of lignin can be highly beneficial. However, this is an arduous task, as the isolated (and sometimes processed) lignin mainly consists of various neutral monomers but also oligomers. In addition, the material contains isomers, which can be especially problematic to separate and identify. ResultsWe present a powerful off-line comprehensive two-dimensional (2D) chromatography method combining liquid chromatography (LC), supercritical fluid chromatography (SFC), and high-resolution mass spectrometry for the non-target analysis of depolymerised lignin. The implementation of a 1-aminoanthracene column in the second dimension enabled a class separation of potential lignin monomers, dimers, trimers, and tetramers with an additional separation based on the number of hydroxyl groups and steric effects. The pentafluorophenyl column in the first dimension additionally improved the separation based on hydrophobicity. The comparison of off-line 2D LC × SFC to 1D SFC showed that besides the overall improved performance, the first method is also superior for the separation of isomers. Advanced data analysis methods (MS-DIAL, SIRIUS, and Feature-Based Molecular Network) were integrated into the non-target workflow to rapidly visualise and study the detected compounds, which proved to be especially beneficial for the characterisation of the separated isomers. SignificanceThe method yielded the first 2D LC plot demonstrating a classification of lignin compounds, which can be applied to compare various lignin sources and processing methods. In addition, the technique demonstrated improved separation of compounds, including isomers, which was especially beneficial as 77 % of the detected compounds had at least one isomer in the same lignin sample.

Simultaneously characterization of multiple constituents in Valeriana jatamansi Jones using an online supercritical fluid extraction-high-performance liquid chromatography/supercritical fluid chromatography-mass spectrometry system.

Valeriana jatamansi Jones is a commonly used traditional Chinese medicine, boasting rich effective compositions with versatile chemical structures and wide polarity, including iridoids, chlorogenic acid, and flavonoids. Previous reports indicate that conventional high-performance liquid chromatography (HPLC) analytical methods have proven inefficient performance in comprehensively characterizing components in Valeriana jatamansi. In the present study, a hybrid online analytical platform combining supercritical fluid extraction with both conventional HPLC separation (reverse phase) and supercritical fluid chromatography (normal phase) has been established and validated. This system can provide online extraction with two different chromatographic separation modes to increase separation ability and has been connected to a mass spectrometer to acquire high-resolution mass spectrometry data. Then, the online platform was applied to screening components in Valeriana jatamansi. A total of 117 compounds were identified, including five lignans, 18 organic acids, six flavonoids, and 88 iridoids. Thirty-three compounds were reported from Valeriana jatamansi for the first time. These results enrich our understanding of the components of Valeriana jatamansi and prove that the developed online platform in this study is a robust approach for accelerating working efficiency in comprehensively analyzing complicated samples.

Evaluation of supercritical fluid chromatography coupled to tandem mass spectrometry for the analysis of pesticide residues in grain.

A supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) technique was developed for the rapid and simultaneous detection of nine pesticides (carbendazim, isoprocarb, paclobutrazol, isoprothiolane, flusilazole, quinalphos, piperonylbutoxide, propargite, and bioresmethrin) in rice, wheat, and maize. The cereal samples were extracted with a solution of 0.5% acetic acid in acetonitrile and purified using quick, easy, cheap, effective, rugged, and safe method. The samples were characterized using multi-reaction monitoring and quantified with the external standard method. Excellent linearities (R2 >0.9991) and limits of quantification (0.4-40.0μg/kg) were established for all nine pesticides. Satisfactory pesticide recovery rates (62.2%-107.4%) were obtained at three standard concentrations (50, 100, and 200μg/kg), with relative standard deviations in the range of 2.1%-14.3%. The results confirmed that the proposed method was suitable for the routine detection of these pesticides in grain samples. Compared with high-performance liquid chromatography-MS/MS, the overall test run time and the amount of solvent required were reduced by 66% and 90%, respectively, when SFC-MS/MS was applied. Therefore, the use of SFC-MS/MS permits a shorter run time and affords greater analytical efficiency, such that it is both economical and environmentally sustainable.

Comparative analysis of triglyceride profiles in human milk from Egyptian and Chinese women across lactation stages

Understanding human milk triacylglycerols (TAGs) provides valuable insights for infant formula development. This study utilized ultra-performance supercritical fluid chromatography to investigate how lactation stages and maternal diet affect TAG profiles. 18:1/16:0/18:1 (O/P/O), 16:0/18:1/16:0 (P/O/P), and 18:1/16:0/18:2 (O/P/L) were most abundant in human milk, accounting for 20% of the total TAGs. Furthermore, significant differences in TAG compositions were observed across the three stages of lactation. Also, there are apparent differences in the TAG of Egyptian and Chinese human milk. Egyptian human milk exhibited higher levels of TAGs esterified with three saturated fatty acids (SSS), TAGs esterified with two saturated fatty acids, and one unsaturated fatty acid (S2U), and total palmitic acid compared to Chinese human milk. On the other hand, TAGs with three long-chain fatty acids (LLL) and TAGs with three unsaturated fatty acids (UUU) were at higher levels in Chinese human milk. Understanding the prevalence of these specific TAGs, particularly related to O/P/O, can help in formulating infant formulas that aim to mimic the composition of human milk more accurately.

SFC-MS/MS for orthogonal separation of hydroxylated 17α-methyltestosterone isomers.

Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase-I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC-MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC-MS/MS)methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC-MS[/MS]) methods have a lower separation efficiency than GC-MS methods. Both techniques lack selectivity for hydroxylated 17α-methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometrymethod was developed to separate four hydroxy metabolites of 17α-methyltestosterone (2α-/2β-/4-/6β-hydroxy-17α-methyltestosterone). This project aimed to get a more in-depth look at the metabolization and analysis of 17α-methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6ng/ml at an accuracy of 85-115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β-hydroxy-17α-methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β-hydroxy-17α-methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations.

Highly sensitive determination of multiple pesticide residues in foods by supercritical fluid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry

This experiment aimed to establish a green, simple and highly sensitive method (supercritical fluid chromatography (SFC) coupled with ion mobility quadrupole time-of-flight mass spectrometry (IM-Q-TOF/MS)) for the detection of multiple pesticides in foods. During the experiments, several important SFC parameters, such as stationary phase, modifier, make-up solution, back-temperature and back-pressure were optimized. Here, single-field collision cross section (CCS) values and multifield CCS values of 20 pesticides were examined by IM-Q-TOF/MS as highly specific parameters with excellent experimental precision. In addition, based on accurate mass matching and fragment ion comparison, mass fragments were obtained by IM-Q-TOF/MS, which elucidated the regularities of compound structure and characteristic fragment ions. Under the optimized conditions, satisfactory linearity (R2 ≥ 0.9989) and recoveries (79.60% to 112.97%) were obtained. The intra- and interday precisions were favorable, with RSDs lower than 4.91 and 7.65%, respectively. Additionally, the method showed low limits of detection (0.1-8.8 ng/mL). The proposed method has been successfully applied to the highly sensitive detection of phenylurea herbicide, triazine herbicides, organophosphorus pesticide, pyrethroid insecticide and acaricide in yam and potato.

Supercritical Fluid Chromatography–Mass Spectrometry with Atmospheric Pressure Chemical Ionization: Negatively Charged Ions of Mobile Phase Components

Supercritical Fluid Chromatography–Mass Spectrometry with Atmospheric Pressure Chemical Ionization: Negatively Charged Ions of Mobile Phase Components

Supercritical fluid chromatography is not (only) normal-phase chromatography

Supercritical fluid chromatography (SFC), now using carbon dioxide as a major component of the mobile phase, has been known for over 60 years but still some misunderstandings remain about its capabilities. Amongst them, SFC is often described as a normal-phase chromatographic technique, based on different considerations: polarity of the stationary phase, elution order of the analytes, relative non-polarity of the mobile phase, non-linear retention behaviour, or adsorption retention mechanisms. All of these assumptions are true to a certain extent, and in certain circumstances. But also, all of these assumptions are wrong in different circumstances. In this paper, the criteria to categorize SFC as a normal-phase chromatographic method will be examined individually, considering all knowledge acquired from the early years of its development. Finally, it will appear that the “normal-phase” glass lens is greatly reducing the true extent of SFC's possibilities.