Ultra-high Performance Supercritical Fluid Research Articles

An environmentally friendly and green method for separation and determination of eight phenolic acids in raw and processed Tussilagofarfara L. by ultra-high performance supercritical fluid chromatography

Phenolic acid as an important active ingredient of traditional Chinese medicine, have received extensive attention due to its various biological activities and pharmacological effects. However, the presence of isomers makes the separation of phenolic acids challenging. In the present study an environmentally friendly, sustainable, lower cost, and green that reduce amounts of organic solventsultra-performance convergence chromatography (UPC2) method was developed for separation and quantification eight phenolic acids. Ultra-high performance supercritical fluid chromatography combines the advantages of supercritical fluid chromatography and ultra high performance liquid chromatography technologies with shorter analysis time, higher resolution and better environmental compatibility. The effects of different chromatographic column, co-solvent, column temperature and back pressure were studied. The BEH column was provided the most suitable separation withgradient elution in 20 min using CO2 as the mobile phase and methanol/acetonitrile (70/30, v/v) with 2% TFA as the co-solvent at a flow rate of 0.4 mL/min. A better and more symmetrical peak shape were obtained. Subsequently, the established method was applied to the quantification of phenolic acids in raw and processed Tussilago farfara L. and validated according to FDA guidelines, showed good precision, accuracy, repeatability and linearity in the concentration range. Our findings confirm that the method could be used as an efficient method for exploring phenolic acids of the quality and separation of traditional Chinese medicine.

Simultaneous determination of 17 bisphenols in polycarbonate by ultra-high performance supercritical fluid chromatography with tandem mass spectrometry.

A novel method was developed for the first time for the determination of 17 bisphenols by ultra-high performance supercritical fluid chromatography with tandem mass spectrometry. Under the optimal conditions, 17 bisphenols were separated successfully on a high density diol column in 9min using methanol and carbon dioxide as mobile phase. 0.02% ammonium hydroxide/methanol v/v was used as the post-column compensation solvent to improve response of mass spectrometry. Linear relations of matrix-matched calibration curve were favorable over the selected concentration range of 1-100μg/kg with correlation coefficients greater than 0.9981. The method limit of detection and limit of quantitation were 0.1-0.5μg/kg and 0.5-2.5μg/kg, respectively. The average recoveries at three spiked levels in polycarbonate were in the range of 81.8-114.5%. Intra-day and inter-day precisions for six replicates were below 15.0%. This method was successfully applied to determine bisphenols in polycarbonate.

Comprehensive analysis and quality assessment of Herba Epimedii from multiple botanical origins based on ultra-high performance supercritical fluid chromatography coupled with quadrupole time-of-flight mass spectrometry and photodiode array detector

Herbal medicines (HMs) usually consist of complex matrices of phytochemical compounds responsible for their efficacy. Supercritical fluid chromatography (SFC) and ultra-high performance SFC (UHPSFC) are efficient techniques that can be advantageously used for the analysis of complex HMs. Here, the comprehensive UHPSFC methods were established for separation of similar flavonoids, chemical profiling and differentiation, and quality assessment of Epimedium species (EPs). An efficient separation of 12 polar flavonoids was enabled in UHPSFC with an important additive, oxalic acid. Then, 51 flavonoids were characterized, and 28 potential markers enabling the differentiation of E. wushanense from other four species were discovered by UHPSFC-QTOF/MS combined pattern recognition multivariate statistical analysis. Additionally, the UHPSFC-PDA was developed and validated for simultaneous quantification of 7 predominant flavonoids in five EPs, and remarkable variation in their contents was observed. This integrated UHPSFC approach has potential applications in systematic analysis and quality evaluation of HMs containing hydrophilic flavonoids.

Purification of drug degradation products supported by analytical and preparative supercritical fluid chromatography

A stressed degradation (oxidation) was employed to produce metabolites from an active pharmaceutical ingredient (API) with large molecular weight (about 900 g/mol). An analytical chromatographic method was desired to compare the products generated by different degradation methods while a multi-gram-scale preparative chromatographic method was necessary to purify the produced metabolites. Supercritical fluid chromatography (SFC) was selected for both tasks as no other chromatographic method had achieved the resolution of the API and metabolites (two isomeric mono-oxide species and one di-oxide). First, an analytical-scale method was developed with ultra-high performance supercritical fluid chromatography (UHPSFC). Achiral stationary phases containing sub-2 μm fully porous particles or sub-3 μm superficially porous particles, and chiral phases containing 3 and 5 μm fully porous particles were selected for a first screening with gradient elution (carbon dioxide – methanol containing additives). The stationary phase providing the most promising results was ACQUITY Torus 2-PIC (100 x 3 mm, 1.7 μm, Waters). A central composite design (CCD) was conducted to optimize the gradient program and oven temperature. Final gradient conditions were as follows: 50–70% methanol in 3.8 min with oven temperature set at 36 °C, back-pressure set at 11 MPa and flow-rate at 0.8 mL/min. The optimized method was employed to analyze samples obtained with different degradation conditions. Then the method was adapted and transferred to preparative-scale SFC on a 5 μm-particles Torus 2-PIC stationary phase (150 x 30 mm). The method was modified to comprise an isocratic step followed by a gradient, favoring peak shape of the last eluting compound and minimal volume of collected fractions. Batch injections in gradient mode were carried out to purify six grams of crude product.

Hyphenation of ultra-high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 2. Study of chromatographic and mass spectrometry parameters for the analysis of natural non-polar compounds

An analytical method based on Ultra-High-Performance Sub/Supercritical Fluid Chromatography (UHPSFC) coupled with High-Resolution Mass Spectrometry (HR-MS) equipped with an Atmospheric Pressure Chemical Ionisation source (APCI-Q-TOF-HRMS) was developed for the screening of various compounds in oily samples. The hyphenation was achieved using a hybrid UHPSFC system for the vegetable oil analysis, mainly composed of fatty acids, diacylglycerols, triacylglycerols but containing also some minor bioactive compounds. No split was used with this ionisation source in this optimized configuration, allowing the introduction of all compounds in the mass spectrometer, ensuring a better sensitivity. This configuration is preserving peak shapes and efficiency because the dead volume of this interface is optimized by the supplier for the hyphenation with open cell detectors such as Evaporative Light Scattering Detector (ELSD) or MS detectors. The influence of analytical conditions (UHPSFC and APCI parameters) on MS response was studied to understand the behaviour of analytes in UHPSFC-APCI-MS. The tested parameters were: the corona discharge value, the nebulising gas pressure, the drying gas flow rate, MS source temperature and the mobile phase flow rate (at constant modifier percentage). A factorial experimental design was carried out for this study, which displayed the major and negative role of increased nebulising gas pressure. The factorial design was renewed with the four remaining parameters, allowing to enlighten and explain their different effects on MS responses. Finally, some vegetable oils were analysed by UHPSFC-APCI-HRMS with these optimal conditions to determine the chemical structures of unknown compounds from oily samples.

Differential metabolism of clinically-relevant progestogens in cell lines and tissue: Implications for biological mechanisms

Steroid hormones regulate a variety of physiological processes, including reproductive function, and are widely used in hormonal therapy. Synthetic progestogens, or progestins, were designed to mimic progesterone (P4) for use in contraception and hormonal replacement therapy in women. Medroxyprogesterone acetate (MPA) and norethisterone (NET) are the most widely used injectable contraceptives in the developing world, while other progestins such as levonorgestrel (LNG), etonogestrel (ETG) and nestorone (NES) are used in or being developed for other forms of contraception. As concerns remain about the most appropriate choice of progestin and dosage, and the associated side-effects, the mechanisms and biological effects of progestins are frequently investigated in various in vitro mammalian cell line and tissue models. However, whether progestogens are differentially metabolised in different cell types in vivo or in vitro is unknown. For nine mammalian cell lines commonly used to investigate progestogen mechanisms of action, we developed and validated an ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) protocol for simultaneously quantifying the metabolism of the above-mentioned steroids. We show for the first time that, while 50–100% of P4 was metabolised within 24 h in all cell lines, the metabolism of the progestins is progestin- and cell line-specific. We also show that MPA and NET are significantly metabolised in human cervical tissue, but to a lesser extent than P4. Taken together, our findings suggest that differential progestogen metabolism may play a role in cell-specific therapeutic and side-effects. Relative affinities for binding to steroid receptors as well as potencies, efficacies and biocharacters for transcriptional activity of progestins, relative to P4, are most frequently determined using some of the cell lines investigated. Our results, however, suggest that differential metabolism of progestins and P4 may confound these results. In particular, metabolism may under-estimate the receptor-mediated intrinsic in vitro binding and dose-response values and predicted endogenous physiological effects of P4.

Enantiospecific UPC2 SFC-MS method for separation and quantification of R & S-Eliglustat tartrate in presence of its stereo isomers and degradation impurities

Abstract Eliglustat tartrate is an oral inhibitor of glucosylceramide synthase used in the treatment of Gaucher’s disease. This drug is administered as enantio-specific trans-(R,R) isomer and there is a possibility to have three more potential stereo isomers due to two chiral centers, owing to the toxicological difference between stereoisomers chiral method with high sensitivity is required to detect low level contaminations. A method for enantiomeric separation and quantification of trans-(R,R) Eliglustat tartrate in presence of other isomers trans-(S,S) Eliglustat, cis-(R,S) Eliglustat, cis-(S,R) Eliglustat was developed and validated using ultra-high performance supercritical fluid chromatography with tandem mass spectrometry (UPC2 SFC-MS) which is termed as a green chromatographic technique. Chiral separation was developed using Chiralcel OX-3 (4.6 * 150 mm) 3 µm column which has 4 chloro, 3 methyl phenyl carbamate as chiral selector, with a flow rate of 3 g/min using CO2 and 0.5% diethyl amine in ethanol: methanol (1:1) as a co-solvent. The effect on the enantioseparation by varying chiral additives, gradient conditions and content of organic modifier were investigated. The method has good linearity with concentration ranges from 0.25 to 7.5 µg mL−1. The detection limit was 0.08 µg mL−1 and quantification limit was 0.25 µg mL−1. Forced degradation studies were performed and all the degradation impurities were separated from product peak, the molecular weight of impurities were identified by using UPC2-Mass spectrometer. The method was validated in line to ICH guidelines in terms of linearity, accuracy, precision, LOQ, LOD and robustness.

Detection and Quantitation of ACCUTRACE S10, a New Fiscal Marker Used in Low-Duty Fuels, Using a Novel Ultrahigh-Performance Supercritical Fluid Chromatography–Mass Spectrometry Approach

A new ultrahigh-performance supercritical fluid chromatography–mass spectrometry (UHPSFC–MS) method has been developed using electrospray ionization (ESI) to detect and quantify a new fiscal fuel marker, ACCUTRACE S10, that was introduced into fuel in the U.K. and Ireland from April 1, 2015. S10 is synthesized by the Dow Chemical Company and is used as a replacement for ultraviolet–visible (UV–vis) fuel markers, such as quinizarin, Euromarker, and Solvent Red 24. It is UV–invisible, is doped at a low level (2.5 ppm), and was designed for detection using modern gas chromatography–mass spectrometry (GC–MS) instrumentation. All currently proposed methods for the determination of the ACCUTRACE S10 fuel marker in diesel fuel (automotive diesel oil, with current specifications of EN 590 and ASTM D975) use GC–MS, and two-dimensional (2D) GC–MS is necessary to detect the marker at tank dilutions. However, the lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for S10 are severely compromised...

Ultra-high performance supercritical fluid chromatography in impurity control: Searching for generic screening approach

In this study, 63 compounds measured as 10 individual mixtures containing in each case an active pharmaceutical ingredient and its relevant impurities, and additional set of 7 basic beta blockers were analyzed using ultra-high performance supercritical fluid chromatography with UV and mass spectrometry detection. The separations were accomplished using 8 different stationary phases (diol, diethylamine, 2-picolylamine, 1-aminoanthracene, BEH 2-ethylpyridine, BEH, CSH pentafluorophenyl, and HSS C18 SB), 6 modifiers (methanol, ethanol, isopropanol, methanol/acetonitrile, methanol/ethanol, ethanol/acetonitrile) and 5 additives in methanol (0.1% formic acid, 10 mmol/L ammonium formate, 10 mmol/L ammonium acetate, 0.4% ammonium hydroxide, and 2% water).The resulting chromatograms were evaluated using 6 selected parameters: number of eluted peaks, number of separated peaks, resolution between active pharmaceutical ingredient and following impurity, peak symmetry, peak width at 50% of the peak height, and selectivity. Volatile additives such as ammonium formate, ammonium acetate, and especially ammonium hydroxide provided overall generic approach with very good chromatographic performance. Combined modifier methanol/acetonitrile (1:1) was important for separations of some critical pairs in case of neutral compounds. Diol stationary phase proved its suitability for wide range of applications when the separations of all mixtures were satisfactory on this column, except for vardenafil and beta blockers mixture. HSS C18 SB stationary phase offered unique complementary selectivity especially for analysis of structurally close compounds and/or isomers.

Determination of Five Retinol Isomers in Animal Livers Using Ultra-High Performance Supercritical Fluid Chromatography

A novel and efficient ultra-high performance supercritical fluid chromatography method was developed for the quantitative analysis of five retinol isomers in animal livers. The separation of the five retinol isomers was carried out using an Acquity UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 µm) with acetonitrile as a co-solvent. By optimizing the columns, gradient program, co-solvent, column temperature and backpressure, the five retinol isomers and the internal standard 11-cis-13,14-dihydroretinol were successfully separated within 20 min. Samples were saponified and extracted by solid-supported liquid–liquid extraction using a diatomaceous earth cartridge. Comparing with the traditional liquid–liquid extraction, the extraction enables the reduction of time-consuming and laborious procedures. This method used 11-cis-13,14-dihydroretinol as an internal standard to improve the precision and accuracy of quantitative analysis. The correlation coefficients (r2) of the calibration curves were all above 0.999, the limits of detection for the five retinol isomers were in the range of 0.10–0.20 µg mL− 1, and the limits of quantification were in the range of 0.33–0.66 µg mL− 1. The mean recoveries were from 92.5 to 102.5%. The interday and intraday relative standard deviations were within 10%. This method was successfully applied to the determination of retinol isomers in ten raw animal livers and animal liver products (chicken, duck, pig, cattle, and sheep).

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of nine sunscreens in cosmetic samples

A fast and simple utra-high performance supercritical fluid chromatography (UHPSFC) method has been developed for the determination of nine sunscreens (UV filters), namely 2-ethylhexyl-2-hydroxybenzoate (ES), ethylhexyl-methoxycinnamate (EMC), benzophenone-3 (BZ3), octocrylene (OCR), bis-ethylhexyloxyphenol-methoxyphenyl triazine (EMT), butyl-methoxydibenzoyl-methane (BDM), diethylamino-hydroxybenzoyl-hexyl-benzoate (DHB), ethylhexyl-triazone (ET), and diethylhexyl-butamido-triazone (DBT) in cosmetic samples. The separation was achieved with Acquity UPC2 Torus 2-PIC (100 × 3.0 mm, 1.7 μm) column. The influence of key chromatographic parameters on resolution was evaluated. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol containing ammonium acetate, at flow rate 1.2 mL min−1. The back-pressure of the system was set to 150 bar and the temperature to 40 °C. The compounds were determined with diode-array detection at 280 nm, 305 nm and 340 nm depending on absorbance maxima. The proposed UHPSFC method provided separation of the nine target sunscreens within 2.5 min without labor-consuming sample pretreatment procedure. The method was validated according to the concept of the total error and the accuracy profile. The inter-laboratory reproducibility was evaluated between two independent laboratories (in France and Czech Republic). The reliability of the developed method was shown by application to commercial sunscreen-containing cosmetic sample obtained from French market. The measured limits of quantification showed the suitability of the proposed method for determination of UV filters considering the European Union requirements.

A comparison of single quadrupole mass detection and diode array ultraviolet detection interfaced to ultra-high performance supercritical chromatography for the quantitative analysis of synthetic cathinones

A comparison of single quadruple mass spectrometry and diode array-ultraviolet (PDA-UV) detection interfaced to ultra-high performance supercritical fluid chromatography was performed for the quantitative analysis of synthetic cathinones. Synthetic cathinones, also known as “bath salts”, are derived from cathinone, a component of the khat plant. For 15 controlled solutes linearity, repeatability, and limits of detection were determined using both UV and MS detection. Quantitation studies were performed using the above detectors for 19 mixtures of up to 4 of the above compounds with an adulterant to simulate seized samples. MS detection provided approximately an order of magnitude greater linearity range and allowed for two to three orders of magnitude lower limits of detection than UV detection. Both detection techniques exhibited similar results of analysis and comparable repeatability. The latter detection mode which provided significantly high linearity correlation coefficients (0.9994 ≤ R2 ≤ 1.0000) would be preferred for quantitative analysis.

Lipidomics study of the protective effects of isosteviol sodium on stroke rats using ultra high-performance supercritical fluid chromatography coupling with ion-trap and time-of-flight tandem mass spectrometry

Isosteviol sodium (STV-Na) was reported to possess significant protective effects on ischemic stroke in recent years. However, the protective mechanism of STV-Na against stroke was still unclear. In this work, an untargeted lipidomics approach based on the ultra high-performance supercritical fluid chromatography coupling with ion-trap and time-of-flight tandem mass spectrometry (UHSFC-IT-TOF/MS) was employed to investigate the lipid profiles of stroke rats with STV-Na treatment for the first time. The possible mechanism of STV-Na was further elucidated. The UHSFC-IT-TOF/MS-based method achieved a fast separation of various lipids within 9 min with a qualified repeatability. Multivariate statistical analysis was used to show differences in lipid profiles induced by stroke and STV-Na treatment. The results showed a clear separation of the model group and the sham group, with the STV-Na group as well as EDA group located much closer to the sham group than the model group, which was consistent with the results of physiological and pathological assays, indicating the protective effects of STV-Na. Fifteen differential lipids that presented significant differences between the sham group and the model group were screened and identified. With the treatment of STV-Na, 15 differential lipids in stroke rats showed a tendency to the normal levels. Among them, 6 lipids were significantly reversed to the normal levels by STV-Na. The results of pathway analysis suggested the protective effects of STV-Na might be related to the regulation of several metabolic pathways including glycerophospholipid metabolism, arachidonic acid metabolism and sphingolipid metabolism. This work demonstrated that the UHSFC-IT-TOF/MS-based lipidomics profiling method was a useful tool to investigate the protective effects of STV-Na against stroke.

Ultra-high performance supercritical fluid chromatography hyphenated to atmospheric pressure chemical ionization high resolution mass spectrometry for the characterization of fast pyrolysis bio-oils

Extensive characterization of complex mixtures requires the combination of powerful analytical techniques. A Supercritical Fluid Chromatography (SFC) method was previously developed, for the specific case of fast pyrolysis bio oils, as an alternative to gas chromatography (GC and GC × GC) or liquid chromatography (LC and LC × LC), both separation methods being generally used prior to mass spectrometry (MS) for the characterization of such complex matrices. In this study we investigated the potential of SFC hyphenated to high resolution mass spectrometry (SFC-HRMS) for this characterization using Negative ion Atmospheric Pressure Chemical ionization ((−)APCI) for the ionization source. The interface between SFC and (−)APCI/HRMS was optimized from a mix of model compounds with the objective of maximizing the signal to noise ratio. The main studied parameters included both make-up flow-rate and make-up composition. A methodology for the treatment of APCI/HRMS data is proposed. This latter allowed for the identification of molecular formulae. Both SFC-APCI/HRMS method and data processing method were applied to a mixture of 36 model compounds, first analyzed alone and then spiked in a bio-oil. In both cases, 19 compounds could be detected. Among them 9 could be detected in a fast pyrolysis bio-oil by targeted analysis. The whole procedure was applied to the characterization of a bio-oil using helpful representations such as mass-plots, van Krevelen diagrams and heteroatom class distributions. Finally the results were compared with those obtained with a Fourier Transform ion-cyclotron resonance mass spectrometer (FT-ICR/MS).

Systematic profiling and comparison of the lipidomes from Panax ginseng, P. quinquefolius, and P. notoginseng by ultrahigh performance supercritical fluid chromatography/high-resolution mass spectrometry and ion mobility-derived collision cross section measurement

Lipidomics currently is still confronted with challenges from chromatographic separation and lipids identification. Here we report a lipidomics platform by integrating ultrahigh performance supercritical fluid chromatography/quadrupole time-of-flight mass spectrometry (UHPSFC/QTOF-MS) and collision cross section (CCS) measurement using ion mobility spectroscopy/time-of-flight mass spectrometry (IMS/QTOF-MS), aiming to enhance the profiling performance and identification reliability of lipids. The lipidomes extracted from three congeneric Panax species (P. ginseng, P. quinquefolius, and P. notoginseng) by methyl tert-butyl ether are comprehensively profiled and compared by use of this platform. A potent UHPSFC/QTOF-MS approach was developed on a 1.7-μm particles packed Torus 2-PIC column using CH3OH (in CO2) as a modifier and CH3OH/0.2 mM ammonium acetate as the makeup liquid, enabling well resolution of six lipid subclasses by both positive and negative MSE modes. In contrast to the reversed-phase chromatography, “normal-phase” like elution order and better resolution of polar lipids and some lipid isomers were achieved by UHPSFC separation. Pattern recognition chemometric analysis of 60 batches of Ginseng samples ultimately unveiled 24 lipid markers, of which triacylglycerols were the most important. Aside from the automated MS database searching against HMDB and LIPID MAPS, the application of CCS retrieval or CCS prediction improved lipid identification by reducing the possible hits. In conclusion, this integral platform can significantly improve the chromatographic separation and the reliability of lipids identification in lipidomics studies. It is the first report that systematically compares the lipidomic difference of three reputable Panax species, providing useful information for their quality control in addition to ginsenoside analysis.

Screening of stationary phase selectivities for global lipid profiling by ultrahigh performance supercritical fluid chromatography

The performance of seven sub-2-μm particle packed columns (2-picolylamine, 2-PIC; charged surface hybrid fluoro-phenyl, CSH-FP; high strength silica C18 SB, HSS-C18; diethylamine, DEA; 1-aminoanthracene, 1-AA; high density diol and ethylene bridged hybrid; BEH) was examined for lipid separation in ultra-high performance supercritical fluid chromatography (UHPSFC) coupled to quadrupole time-of-flight mass spectrometry. Based on the results of the column screening a method for profiling of multiple lipid species from the major lipid classes was developed.Stationary phases containing β-hydroxy amines, i.e. 1-AA, DEA and 2-PIC, yielded strong retention and poor peak shapes of zwitterionic lipids with primary amine groups, such as phosphatidylserines, phosphatidylethanolamines and its lyso forms. The BEH and HSS-C18 columns showed strong retention of polar and nonpolar lipids, respectively. The Diol column retained the majority of major lipid classes and also produced symmetric peaks. In addition, this column also produced the highest resolution within and between major lipid classes. An injection solvent composed of methanol:chloroform (1:2, v:v) and the addition of 20 mM ammonium formate in the mobile phase improved chromatographic separation and mass spectrometry detection in comparison to ammonium acetate or absence of additive. Finally, chromatographic and mass spectrometric parameters were optimized for the Diol column using a design of experiments approach. The separation mechanism on the Diol column depended on the lipid functionality and the length and degree of unsaturation of the acyl groups. The developed method could resolve 18 lipid classes and multiple lipids within each class, from blood serum and brain tissue in 11 min.

Analysis of new psychoactive substances in human urine by ultra-high performance supercritical fluid and liquid chromatography: Validation and comparison.

New psychoactive substances represent serious social and health problem as tens of new compounds are detected in Europe annually. They often show structural proximity or even isomerism, which complicates their analysis. Two methods based on ultra high performance supercritical fluid chromatography and ultra high performance liquid chromatography with mass spectrometric detection were validated and compared. A simple dilute-filter-and-shoot protocol utilizing propan-2-ol or methanol for supercritical fluid or liquid chromatography, respectively, was proposed to detect and quantify 15 cathinones and phenethylamines in human urine. Both methods offered fast separation (<3min) and short total analysis time. Precision was well <15% with a few exceptions in liquid chromatography. Limits of detection in urine ranged from 0.01 to 2.3ng/mL, except for cathinone (5ng/mL) in supercritical fluid chromatography. Nevertheless, this technique distinguished all analytes including four pairs of isomers, while liquid chromatography was unable to resolve fluoromethcathinone regioisomers. Concerning matrix effects and recoveries, supercritical fluid chromatography produced more uniform results for different compounds and at different concentration levels. This work demonstrates the performance and reliability of supercritical fluid chromatography and corroborates its applicability as an alternative tool for analysis of new psychoactive substances in biological matrixes.

Enantiomeric separation and quantification of R/S-amphetamine in urine by ultra-high performance supercritical fluid chromatography tandem mass spectrometry

To distinguish between legal and illegal consumption of amphetamine reliable analytical methods for chiral separation of the R- and S-enantiomers of amphetamine in biological specimens are required. In this regard, supercritical fluid chromatography (SFC) has several potential advantages over liquid chromatography, including rapid separation of enantiomers due to low viscosity and high diffusivity of supercritical carbon dioxide, the main component in the SFC mobile phase. A method for enantiomeric separation and quantification of R- and S-amphetamine in urine was developed and validated using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS). Sample preparation prior to UHPSFC-MS/MS analysis was a semi-automatic solid phase extraction method. The UHPSFC-MS/MS method used a Chiralpak AD-3 column with a mobile phase consisting of CO2 and 0.2% cyclohexylamine in 2-propanol. The injection volume was 2 μL and run-time was 6 min. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 50–10,000 ng/mL for each enantiomer. The between-assay relative standard deviations were in the range of 3.7–7.6%. Recovery was 92–93% and matrix effects ranged from 100 to 104% corrected with internal standard. After development and validation, the method has been successfully implemented in routine use at our laboratory for both separation and quantification of R/S-amphetamine, and has proved to be a reliable and useful tool for distinguishing intake of R- and S-amphetamine in authentic patient samples.

Determination of 16 polycyclic aromatic hydrocarbons in tire rubber by ultra-high performance supercritical fluid chromatography combined with atmospheric pressure photoionization-tandem mass spectrometry

A novel and rapid method based on UHPSFC-APPI-MS/MS was developed to simultaneously analyze 16 polycyclic aromatic hydrocarbons.

Development of a new and environmentally-friendly method to evaluate phenolic compounds from Flos Lonicerae Japonicae with ultra-high performance supercritical fluid chromatography (UHPSFC) combined with chemometrics

The chemical variation of 10 batches of Flos Lonicerae Japonicae from different geographical origins was investigated by quantitation of phenolic acids..