High-performance Supercritical Fluid Chromatography Research Articles

Circulating adrenal and gonadal steroid hormones heterogeneity in active young males and the contribution of 11-oxy androgens

The classical androgens, testosterone and dihydrotestosterone, together with dehydroepiandrosterone, the precusrsor to all androgens, are generally included in diagnostic steroid evaluations of androgen excess and deficiency disorders and monitored in androgen replacement and androgen suppressive therapies. The C11-oxy androgens also contribute to androgen excess disorders and are still often excluded from clinical and research-based steroids analysis. The contribution of the C11-oxy androgens to the androgen pool has not been considered in androgen deficiency. An exploratory investigation into circulating adrenal and gonadal steroid hormones in men was undertaken as neither the classical androgens nor the C11-oxy androgens have been evaluated in the context of concurrent measurement of all adrenal steroid hormones. Serum androgens, mineralocorticoids, glucocorticoids, progesterones and androgens were assessed in 70 healthy young men using ultra high performance supercritical fluid chromatography and tandem mass spectrometry. Testosterone, 24.5 nmol/L was the most prominent androgen detected in all participants while dihydrotestosterone, 1.23 nmol/L, was only detected in 25% of the participants. The 11-oxy androgens were present in most of the participants with 11-hydroxyandrostenedione, 3.37 nmol, in 98.5%, 11-ketoandrostenedione 0.764 in 77%, 11-hydroxytestosterone, 0.567 in 96% and 11-ketotestosterone: 0.440 in 63%. A third of the participants with normal testosterone and comparable 11-ketotestosterone, had significantly lower dehydroepiandrosterone (p < 0.001). In these males 11-hydroxyandrostenedione (p < 0.001), 11-ketoandrostenedione (p < 0.01) and 11-hydroxytestosterone (p < 0.006) were decreased. Glucocorticoids were also lower: cortisol (p < 0.001), corticosterone (p < 0.001), cortisone (p < 0.006) 11-dehydrocorticosterone (p < 0.001) as well as cortisol:cortisone (p < 0.001). The presence of dehydroepiandrosterone was associated with 16-hydroxyprogesterone (p < 0.001), which was also significantly lower. Adrenal and gonadal steroid analysis showed unexpected steroid heterogeneity in normal young men. Testosterone constitutes 78% of the circulating free androgens with the 11-oxy androgens abundantly present in all participants significantly contributing 22%. In addition, a subset of men were identified with low circulating dehydroepiandrosterone who showed altered adrenal steroids with decreased glucocorticoids and decreased C11-oxy androgens. Analysis of the classical and 11-oxy androgens with the additional measurement of dehydroepiandrosterone and 16-hydroxyprogesterone may allow better diagnostic accuracy in androgen excess or deficiency.

High-performance supercritical fluid chromatography hyphenated to high-resolution quadrupole-time of flight hybrid mass spectrometry (SFC-HR-QTOF-MS/MS) for the determination of cannabinoids in edible products

A growing number of papers suggest the positive action of cannabinoids in the treatment of several diseases, which is ascribed to the ability of phytocannabinoids to affect the human endocannabinoid system. In particular, this activity is characteristic of CBD, a non-psychoactive variation of 9-THC. It initiated a trend in the manufacturing of edible and cosmetic products containing CBD that can be easily acquired in drugstores. However, the composition of such products has rarely been tested. Hence, there is a need for data regarding their safety. Considering the possibility of cannabinoid interconversions as well as the complexity and diversity of edible products’ matrix, their analysis requires a suitable preparation and cleanup procedure. In this paper, the perspective of the application of Supercritical Fluid Chromatography (SFC) coupled to High-Resolution (HR) Quadrupole-Time of Flight (Q-TOF) tandem mass spectrometry (MS/MS) with electrospray ionization (ESI) in quality control of cannabis-containing food products was examined. The focus is on six neutral (CBD, 9-THC, CBC, CBG, CBN, and THCV) and three acidic (CBDA, THCA, and CBGA) cannabinoids. The study covers the thorough optimization of conditions for chromatographic separation with the use of the supercritical CO2 (scCO2)-based mobile phase and the working parameters of MS/MS detection with the elucidation of fragmentation patterns. Thanks to the utilization of scCO2, a short analysis time of 17 min., high intensity and small width of peaks, as well as low consumption of toxic organic solvent have been achieved at the 2-EP column with a 5 μm grain size. To reach such performance in LC, the utilization of more expensive columns packed with sub-2 μm particles is needed. In the case of the solvent matrix, the LOD was equal to 8 ng mL−1 for CBDA, THCA, and CBGA, 15 ng mL−1 for CBG and THCV, and 21 ng mL−1 for THC, CBD, CBC, and CBN. In the case of tea, an LOD of 25 ng mL−1 for acidic and 75 ng mL−1 for neutral cannabinoids has been obtained. The accuracy and precision were within the ± 15 % tolerance limit. Finally, the devised protocol has been applied for cannabinoid determination in hemp tea infusions.

Ultra High-Performance Supercritical Fluid Chromatography for the Quantitation of Diterpene Resin Acids in Norway Spruce Samples.

Picea abies (L.) H. Karst. (Pinaceae) is native to Northern, Central and Eastern Europe. The fast-growing tree reaches up to 50 m in height, has modest nutritional requirements and depends on sufficient water supply. The conifer, commonly called Norway spruce, produces exudates which are traditionally used to treat skin wounds in Northern European countries. Major bioactive constituents of the conifer oleoresin are diterpene resin acids (DRAs) of the abietane and the pimarane type. To assure consistent pharmaceutical quality of Norway spruce balm and commercial products thereof, an analytical method for the quantitation of DRAs is the prerequisite. However, high structural similarity among DRAs and their poor UV absorption makes chromatographic separation and detection challenging: Conventional liquid chromatography systems often fail to achieve sufficient separation, moreover, they are not sustainable. Gas chromatography on the other hand requires time-consuming derivatization prior to unacceptably long analyses (>60 min). These drawbacks prompted the development of the first validated supercritical fluid-based protocol for the separation and quantitation of eight DRAs, i.e., pimaric acid (1), sandaracopimaric acid (2), palustric acid (3), isopimaric acid (4), levopimaric acid (5), abietic acid (6), dehydroabietic acid (7), and neoabietic acid (8). By using an ultra high-performance supercritical fluid chromatography (UHPSFC) device hyphenated to a quadrupole mass detector, the DRAs were separated in less than 20 min on a Torus 2-Picolylamin (2-PIC) column (3.0 mm × 100 mm; 1.7 µm particle size) applying supercritical CO2 and ethanol as mobile phase. Regarding selectivity, accuracy (recovery rates: 87–108%), intermediate precision (between 6.6 and 11.1%), and linearity (R2 ≥ 0.99; linear between 0.75 μg/ml and 2.5 mg/ml), results were obtained in line with ICH guidelines. The lowest limit of detection (LOD) was at 0.75 μg/ml (7) and the lowest limit of quantitation (LOQ) at 2 μg/ml (8). As application examples, 22 Norway spruce balm samples and five commercial products were analyzed. The here presented protocol not only simplifies and shortens the analytical workflow, but also reduces the amount of organic solvent waste by about two thirds compared to conventional liquid chromatographic set-ups. These advantages qualify this fast and efficient method as an ideal tool for an environmentally friendly quality control of traditionally used wound-healing Norway spruce balm products.

Recent Trends in Chiral Separation-A Collective Paradigm of Selected Chiral Impurities

Chiral separation plays a very important role in the modern pharmaceutical analysis and will continue in upcoming years. Separation and identification of chiral impurities are indispensable. According to ICH guidelines, only the active enantiomer of the drug has to be marketed, so there is a focus on separation of the inactive enantiomer which acts as a chiral impurity. The impurities present in the enantiomers also pose various toxic adverse effects on bioavailability and efficacy, hence the need to separate these impurities will forever be trending. This review primarily focuses on the separation techniques like Capillary Electrophoresis (CE), High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and Supercritical Fluid Chromatography (SFC) followed by the year-wise trend in the separation of selected chiral impurities. In the coming years, researchers should work on using ultra-fast, selective, and sensitive methods for the effective separation of chiral impurities.

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.

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.

Comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones.

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite-5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended.

Hyphenation of ultra high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 1. Study of the coupling parameters for the analysis of natural non-polar compounds

An analytical method based on Ultra-High-Performance Supercritical Fluid Chromatography (UHPSFC) coupled with Atmospheric Pressure Chemical Ionization − High-resolution mass spectrometry (APCI-Q-TOF-HRMS) was developed for compounds screening from oily samples. The hyphenation was made using a commercial UHPLC device coupled to a CO2 pump in order to perform the chromatographic analysis. An adaptation of the injection system for compressible fluids was accomplished for this coupling: this modification of the injection sequence was achieved to prevent unusual variations of the injected volume related to the use of a compressible fluid. UHPSFC-HRMS hyphenation was optimized to enhance the response of the varied compounds from a seed extract (anthraquinones, free fatty acids, diacylglycerols, hydroxylated triacylglycerols and triacylglycerols). No split was used prior to the APCI ionization source, allowing introducing all the compounds in the spectrometer, ensuring a better sensitivity for minor compounds. The effects of a mechanical make-up (T-piece) added before this ionization source was discussed in terms of standard deviation of response, response intensity and fragmentation percentage. The location of the T-piece with regards to the backpressure regulator (BPR), the flow rate and the nature of the make-up solvent were studied. Results show that the effects of the studied parameters depend on the nature of the compounds, whereas the make-up addition favours the robustness of the mass response (quantitative aspect).

Cannabis through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatography.

By using the Inverted Chirality Columns Approach (ICCA) we have developed an enantioselective UHPSFC method to determine the enantiomeric excess (ee) of (-)-Δ9-THC in medicinal marijuana (Bedrocan®). The ee was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not negligible, and it is worth a systematic evaluation of bioactivity.

Supercritical fluid chromatography coupled with in-source atmospheric pressure ionization hydrogen/deuterium exchange mass spectrometry for compound speciation

An experimental setup for the speciation of compounds by hydrogen/deuterium exchange (HDX) with atmospheric pressure ionization while performing chromatographic separation is presented. The proposed experimental setup combines the high performance supercritical fluid chromatography (SFC) system that can be readily used as an inlet for mass spectrometry (MS) and atmospheric pressure photo ionization (APPI) or atmospheric pressure chemical ionization (APCI) HDX. This combination overcomes the limitation of an approach using conventional liquid chromatography (LC) by minimizing the amount of deuterium solvents used for separation. In the SFC separation, supercritical CO2 was used as a major component of the mobile phase, and methanol was used as a minor co-solvent. By using deuterated methanol (CH3OD), AP HDX was achieved during SFC separation. To prove the concept, thirty one nitrogen- and/or oxygen-containing standard compounds were analyzed by SFC-AP HDX MS. The compounds were successfully speciated from the obtained SFC-MS spectra. The exchange ions were observed with as low as 1% of CH3OD in the mobile phase, and separation could be performed within approximately 20min using approximately 0.24mL of CH3OD. The results showed that SFC separation and APPI/APCI HDX could be successfully performed using the suggested method.

Assessment of ultra high performance supercritical fluid chromatography as a separation technique for the analysis of seized drugs: Applicability to synthetic cannabinoids

The recent development of modern methods for ultra high performance supercritical fluid chromatography (UHPSFC) has great potential for impacting the analysis of seized drugs. In the separation of synthetic cannabinoids the technique has the potential to produce superior resolution of positional isomers and diastereomers.To demonstrate this potential we have examined the capability of UHPSFC for the analysis of two different groups of synthetic cannabinoids. The first group was a mixture of 22 controlled synthetic cannabinoids, and the second group included JWH018 and nine of its non-controlled positional isomers The clear superiority of UHPSFC over other separation techniques was demonstrated, in that it was capable of near baseline separation of all 10 positional isomers using a chiral column. In total we examined four achiral columns, including Acquity UPC2 Torus 2-PIC, Acquity UPC2 Torus Diol, Acquity UPC2 Torus DEA and Acquity UPC2 Torus 1-AA (1.7μm 3.0×100mm), and three chiral columns, including Acquity UPC2 Trefoil AMY1, Acquity UPC2 Trefoil CEL1 and Acquity UPC2 Trefoil CEL2 (2.5μm 3.0×150mm), using mobile phase compositions that combined carbon dioxide with methanol, acetonitrile, ethanol or isopropanol modifier gradients. Detection was performed using simultaneous PDA UV detection and quadrupole mass spectrometry. The orthogonality of UHPSFC, GC and UHPLC for the analysis of these compounds was demonstrated using principal component analysis. Overall we feel that this new technique should prove useful in the analysis and detection of seized drug samples, and will be a useful addition to the compendium of methods for drug analysis.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. I: Investigation of mobile phase and MS conditions

The conditions for the analysis of selected doping substances by UHPSFC–MS/MS were optimized to ensure suitable peak shapes and maximized MS responses. A representative mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI− modes. The best compromise for all compounds in terms of MS sensitivity and chromatographic performance was obtained when adding 2% water and 10mM ammonium formate in the CO2/MeOH mobile phase. Beside mobile phase, the nature of the make-up solvent added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best candidate as it was able to compensate for the negative effect of 2% water addition in ESI− mode and provided a suitable MS response for all doping agents.Sensitivity of the optimized UHPSFC–MS/MS method was finally assessed and compared to the results obtained in conventional UHPLC–MS/MS. Sensitivity was improved by 5–100-fold in UHPSFC–MS/MS vs. UHPLC–MS/MS for 56% of compounds, while only one compound (bumetanide) offered a significantly higher MS response (4-fold) under UHPLC–MS/MS conditions.In the second paper of this series, the optimal conditions for UHPSFC–MS/MS analysis will be employed to screen >100 doping agents in urine matrix and results will be compared to those obtained by conventional UHPLC–MS/MS.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples

The potential and applicability of UHPSFC–MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC–MS/MS and UHPSFC–MS/MS in both ESI+ and ESI− modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC–MS/MS and for 32% in UHPSFC–MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC–MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.

Separation of Natural Food Pigments in Saponified and Un-Saponified Paprika Oleoresin by Ultra High Performance Supercritical Fluid Chromatography (UHPSFC)

The natural pigments in paprika were rapidly and efficiently separated by ultra high performance supercritical fluid chromatography. The separation of both un-saponified and saponified mixtures of paprika oleoresin were optimized, with run times of 10.6 min. Three different C18 columns, a cyano, silica and diol column, all 3 × 100 mm, with 1.8 μm particles were compared. The best separation for the un-saponified sample was found with an SB-C18 column, while the saponified samples were best separated on a bare silica, RX-Sil column. A SB-CN column allowed near optimum separation of both the unsaponified, and saponified samples, with similar run times. The best mobile phase was carbon dioxide (CO2) modified with isopropyl alcohol (IPA), with a composition gradient. Fingerprints of several commercial pepper products indicated that one appeared to be colored with artificial dyes, while the color of a chili powder may have been enhanced with a paprika extract. Spectra, using CO2 with IPA as modifier, produced a single maximum at 453 nm, which appears to represent up to a 30 nm solvatochromic shift from the maxima in most organic solvents. Acetonitrile (ACN) as modifier produced spectra with two maxima and a similar solvatochromic shift. These results appear to be the first on saponified paprika oleoresin samples using SFC. It is also the first detailed report on the separation of un-saponified samples. The results are up to six times faster than comparable results by HPLC. It appears that SFC is a viable, superior alternative to HPLC for the analysis of this important commercial product, without using ACN, or chlorinated solvents.

Evaluation and comparison of various separation techniques for the analysis of closely-related compounds of pharmaceutical interest

The aim of the present work was to compare various separation techniques for the fast analysis of closely-related compounds, including structurally-related compounds, positional isomers, diastereoisomers, Z/E isomers. Three analytical techniques were evaluated, namely ultra high performance liquid chromatography (UHPLC), ultra high performance supercritical fluid chromatography (UHPSFC), both with sub-2μm particles, and capillary electrophoresis (CE) using non-aqueous solvents. To fairly compare the three analytical techniques, only two starting conditions for further method development were considered. All the selected mobile phases or background electrolyte were MS-compatible. As expected, CE often provided excellent results for the analysis of basic compounds but it was difficult to find out conditions that could be widely applied. On the other hand, UHPLC and UHPSFC were more generic and the performance was better than CE for the analysis of neutral and acidic compounds. In all cases, the analysis time was systematically lower than 3min. In conclusion, UHPLC was the most versatile strategy for the analysis of closely-related compounds and should be tested in a first instance. UHPSFC and CE approaches offered some drastic changes in selectivity and should be considered a second choice to reach alternative selectivity as they also allow high throughput separations.

Development of the Certified Reference Material of Poly(ethylene glycol) Nonylphenyl Ether by Supercritical Fluid Chromatography

Poly(ethylene glycol) nonylphenyl ether (PEG NPE) is a nonionic surfactant used widely as a detergent or industrial product, but the toxicity of nonylphenol, which is decomposed from PEG NPE, should be controlled to meet regulatory requirements. Under these situations, we plan to investigate the certified reference material in order to be able to exactly determine not only the average molecular weight but also all of the mole fractions of PEG NPE samples through the use of a well-calibrated evaporative light scattering detector coupled with high-performance supercritical fluid chromatography. In addition, experimental uncertainty was determined in accordance with the ISO's Guide to the Expression of Uncertainty in Measurement.

Certification and uncertainty evaluation of the certified reference materials of poly(ethylene glycol) for molecular mass fractions by using supercritical fluid chromatography

Poly(ethylene glycol) (PEG) is a useful water-soluble polymer that has attracted considerable interest in medical and biological science applications as well as in polymer physics. Through the use of a well-calibrated evaporative light-scattering detector coupled with high performance supercritical fluid chromatography, we are able to determine exactly not only the average mass but also all of the molecular mass fractions of PEG samples needed for certified reference materials issued by the National Metrology Institute of Japan. In addition, experimental uncertainty was determined in accordance with the Guide to the expression of uncertainty in measurement (GUM). This reference material can be used to calibrate measuring instruments, to control measurement precision, and to confirm the validity of measurement methods when determining molecular mass distributions and average molecular masses. Especially, it is suitable for calibration against both masses and intensities for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.