Ultra-high Performance Supercritical Fluid Chromatography Research Articles

Matrix effects in ultra-high performance supercritical fluid chromatography-mass spectrometry analysis of Vitamin E in plasma: the effect of sample preparation and data processing

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both “bind and elute” and “interference removal” modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ- tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to – 72% for α-tocopherol and from -77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels, as a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standard (SIL-IS) was used for the compensation. The significant differences between individual concentration levels and compounds were observed, even if the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. For the selected example of vitamin E, solid-phase extraction was least affected by matrix effects when used in interference removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.

Analysis of vitamin D and its metabolites in biological samples – Part I: Optimization and comparison of UHPSFC-MS/MS and UHPLC-MS/MS methods

Fat-soluble vitamin D is an essential bioactive compound important for human health. Insufficient vitamin D levels can result not only in bone disease but also in other disorders, such as cancer, metabolic disorders, and diseases related to poor immune function. The current methods commonly used for vitamin D analysis are often applied to determine the levels of the most abundant metabolite in plasma, i.e., 25-OH-D2/D3. These methods do not consider the presence of other hydroxylated and esterified metabolites, including isomers and epimers, which are typically found in low concentrations.In this study, we developed a fast and selective ultra-high performance supercritical fluid chromatography (UHPSFC) method using a 150 mm long 1-amino anthracene (1-AA) column and a mobile phase consisting of carbon dioxide and methanol/isopropanol (1/1, v/v) mixed with 8 % water. After thorough optimization of column temperature and back pressure, the separation of four vitamin D3 esters, vitamin D3 and D2, and eight mono- and di-hydroxylated metabolites, including three groups of isomers, was achieved in 10 min. Two ion sources, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization optimized within this study, were compared in tandem mass spectrometry (MS/MS) detection. No significant sensitivity differences were observed. Subsequently, the same 1-AA column chemistry was examined in ultra-high performance liquid chromatography (UHPLC) as the stationary phase that could hypothetically bring different selectivity in the separation of vitamin D and its metabolites. However, this hypothesis was rejected, and C18 was used as a stationary phase in the final optimized UHPLC-MS/MS method. Despite detailed optimization, the final 15 min UHPLC method was not able to separate di-hydroxylated isomers of vitamin D3, while it enabled better resolution of esterified forms compared to UHPSFC.Optimized methods provided similar repeatability of retention times and peak areas, with RSD < 2 % and 10 %, respectively. The lowest limits of quantification were in the range of 1.2 – 4.9 ng/mL for UHPSFC-APCI-MS/MS, while for UHPLC-APCI-MS/MS, they were typically in the range of 2.6 – 9.6 ng/mL. Based on the obtained results, the UHPSFC-APCI-MS/MS method was the most promising approach for fast, selective, and sensitive analysis that could be applied in the analysis of biological samples with emphasis on the separation of both hydroxylated and esterified metabolites, including isomeric forms.

Analysis of vitamin D and its metabolites in biological samples – Part II: Optimization of a sample preparation method for liver tissue

Extraction of vitamin D, including its hydroxylated and esterified metabolites, from soft tissues such as the liver is challenging due to the lipophilic character of matrix and analytes that are expected in very low concentration levels.In this study, we aimed at the optimization of two-step extraction using solid–liquid extraction as the first step, followed by solid-phase extraction. Various solvents, including ethanol, acetonitrile, methanol, acetone, heptane, and heptane with isopropanol, were investigated to isolate vitamin D compounds from liver tissue in the first step. Acetone was finally selected as the most suitable solvent for the solid–liquid extraction, with the highest recovery in the range of 67 – 98% for polar hydroxylated forms and 3 – 28% for lipophilic vitamin D and esters. Two solid phase extraction (SPE) based on the (i) “bind and elute strategy” and (ii) “removal strategy” using hydrophilic-lipophilic balanced SPE sorbent were optimized as a proceeding step for acetone extracts to increase the method selectivity. Finally, two optimized methods, combining solid–liquid extraction and individual SPE strategy, were examined in terms of sensitivity, recovery, matrix effect, accuracy, and precision.The limits of quantification were in the range of 1 – 10 ng/mL and 3 – 20 ng/mL analyzed by ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography hyphenated a with tandem mass spectrometer, respectively. The absolute recovery determined for the “bind and elute strategy” protocol was in the range of 3 – 24 %. Nevertheless, this method was free of matrix effects, which were determined to be in the 73 – 120 % range. On the contrary, the “removal strategy” approach provided higher recovery values for all compounds (47 – 123 %), but the results for nonpolar vitamin D and esters were strongly affected by signal suppression (matrix effects 3 – 51 %). Both methods fulfilled the criteria for accuracy and precision requested by the European Medicine Agency Guideline on Bioanalysis.“Removal strategy” SPE with decreased manual intervention and lower solvent consumption was finally applied to mouse liver tissue to determine vitamin D and its hydroxylated and esterified metabolites for the first time. The results, i.e., vitamin D esters detected in liver tissue, supported the notion that esters of vitamin D can be stored in lipophilic tissues to release vitamin D.

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.

Rapid profiling of cytokinins using supercritical fluid chromatography coupled with tandem mass spectrometry

BackgroundThe determination of plant hormones is still a very challenging analytical discipline, mainly due to their low concentration in complex plant matrices. Therefore, the involvement of very sensitive high-throughput techniques is required. Cytokinins (CKs) are semi-polar basic plant hormones regulating plant growth and development. Modern methods for CK determination are currently based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which enables the separation of CK isomeric forms occurring endogenously in plants. Here, ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry (UHPSFC-MS/MS) was used for the simultaneous determination of 37 CK metabolites. ResultsThe chromatographic conditions were tested on three different columns with various retention mechanisms. Hybrid silica modified with 2-picolylamine was selected as the stationary phase. Several parameters such as column temperature, back pressure regulation, mobile phase composition and make-up solvent were investigated to achieve efficient separation of CK isomers and reasonable sensitivity. Compared to UHPLC-MS/MS, a 9-min chromatographic analysis using a mobile phase of supercritical CO2 and 5 mM ammonia in methanol represents a three-fold acceleration of total run time. The quantification limit of UHPSFC-MS/MS method was in the range of 0.03–0.19 fmol per injection and the method validation showed high accuracy and precision (below 15 % for most analytes). The method was finally applied to the complex plant matrix of the model plant Arabidopsis thaliana and the obtained profiles of CK metabolites were compared with the results from the conventional UHPLC-MS/MS method. SignificanceThe presented work offers a novel approach for quantification of endogenous CKs in plants. Compared to the conventional UHPLC-MS/MS, the total run time is shorter and the matrix effect lower for the key CK metabolites. This approach opens the opportunity to utilize UHPSFC-MS/MS instrumentation for targeted plant hormonomics including other plant hormone families.

Determination of amphetamine enantiomers in urine by conductive vial electromembrane extraction and ultra-high performance supercritical fluid chromatography tandem mass spectrometry.

Separation and quantification of amphetamine enantiomers are commonly used to distinguish between consumption of prescription amphetamine (mostly S-amphetamine) and illicit forms of the drug (racemate). In this study, electromembrane extraction with prototype conductive vials was combined with ultra-high performance supercritical fluid chromatography (UHPSFC-MS/MS) to quantify R- and S-amphetamine in urine. Amphetamine was extracted from 100 μL urine, diluted with 25 μL internal standard solution and 175 μL 130 mM formic acid, across a supported liquid membrane (SLM) consisting of 9μL of a 1:1(w/w) mixture of 2-nitrophenyloctyl ether (NPOE) and bis(2-ethylhexyl)phosphite (DEHPi) into an acceptor phase containing 300 μL 130 mM formic acid. The extraction was facilitated by the application of 30 V for 15 min. Enantiomeric separation was achieved using UHPSFC-MS/MS with a chiral stationary phase. The calibration range was 50-10,000 ng/mL for each enantiomer. The between-assay CV was ≤5%, within-assay CV ≤ 1.5%, and bias within ±2%. Recoveries were 83%-90% (CV ≤ 6%), and internal standard corrected matrix effects were 99-105 (CV ≤ 2%). The matrix effects ranged from 96% to 98% (CV ≤ 8%) when not corrected by the internal standard. The EME method was compared with a chiral routine method that employed liquid-liquid extraction (LLE) for sample preparation. Assay results were in agreement with the routine method, and the mean deviation between methods was 3%, ranging from -21% to 31%. Finally, sample preparation greenness was assessed using the AGREEprep tool, which resulted in a greenness score of 0.54 for conductive vial EME, opposed to 0.47 for semi-automated 96-well LLE.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of four target flavonoids components in citrus samples

AbstractA new method for the analysis of four target flavonoids in two kinds of citrus samples by ultra-high performance supercritical fluid chromatography (UHPSFC) method was developed. Main variables affecting the UHPSFC separation were optimized, and under the optimized conditions the four target compounds (tangeretin, nobiletin, hesperetin and naringenin) can be separated within 10 min. The UHPSFC method allowed the determination of the four target compounds in the diluted stock solutions with limit of detection (LOD) ranging from 1.08 to 2.28 μg mL−1, and limit of quantification (LOQ) ranging from 1.45 to 4.52 μg mL−1, respectively. The coefficients of determination (R2) of the calibration curves were higher than 0.9950. The recoveries of the four target compounds at three different concentrations were in the range of 82.4–117.6%. The validation results demonstrated that the proposed method is simple, accurate, time-saving and environment friendly, and it is applicable to a variety of complex samples such as medicine-food dual purpose herbs and functional foods.

Separation of pyrrolizidine alkaloids in different Senecio species using ultra-high performance supercritical fluid chromatography

Different Senecio species, especially S. inaequidens – a neophyte native to South Africa – have widely spread across Europe and now are found worldwide. The entire genus is known to contain toxic pyrrolizidine alkaloids (PAs), which renders them a possible health hazard to humans and livestock. As they can enter the food chain or occur as contaminants in herbal crops and phytopharmaceutical formulations (e.g. teas), efficient and straightforward assays for their qualitative and quantitative analysis are in high demand. Different techniques have been used for this purpose, most commonly HPLC or GC. As the analysis of PAs is a challenging task, alternative methodologies like ultra-high performance SFC (UHPSFC) may offer an additional benefit in terms of their separation efficiency and orthogonal selectivity. In this study an UHPSFC approach for the simultaneous determination of six PAs (free bases as well as N-oxides) is presented, which achieved the baseline separation of all standard compounds in seven min. Optimal separation was carried out in gradient mode on a Torus™ DEA column with 0.05% ammonia in methanol as modifier. The column temperature was 25 °C, ABPR 1900 psi and flow rate 1.1 mL/min, with a detection wavelength of 215 nm. The assay was validated and fulfilled all ICH criteria exhibiting good linearity (R2 ≥ 0.9994), precision (inter-day variance ≤ 3.67%, intra-day variance ≤ 3.92%) and recovery rates (96.3–104.1%), with detection limits typical for SFC-PDA (≤ 4.24 µg/mL). Furthermore, it could conveniently be coupled to MS-detection, which increased the sensitivity significantly. To confirm practical suitability of the method, different Senecio samples were analyzed, indicating a high qualitative as well as quantitative difference in their PA profile (e.g. total amounts of PA between 0.09 and 4.63 mg/g).

Robust and high-throughput lipidomic quantitation of human blood samples using flow injection analysis with tandem mass spectrometry for clinical use

Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles.

An accurate and reliable analytical strategy for simultaneous determination of target furanocoumarins and flavonoids in cosmetic and pharmaceutical samples by ultra-high performance supercritical fluid chromatography

Furanocoumarins and flavonoids have various important biological activities and wide application. In the present study, a rapid and reliable supercritical fluid chromatography method was proposed for the separation of 10 target components including 8 furanocoumarins and 2 flavonoids. After detailed condition optimization, the 10 target compounds can be baseline separated on a Trefoil CEL1 (3.0 mm × 150 mm, 2.5 µm) column using gradient elution. A 0.07% (v/v) trifluoroacetic acid in ethanol was determined to be the most proper mobile phase for the separation of target compounds. The column temperature, back pressure, flow rate were set at 36 ℃, 2000 psi, 1.0 mL min−1 to 1.4 mL min−1, respectively. The ten target compounds were analyzed within 24 min using the optimized conditions. Under the optimized conditions, all the target compounds showed good linearity with linear correlation coefficients higher than 0.995, and satisfactory recovery in the range of 83.52–112.92%. All these results showed that the developed ultra-high performance supercritical fluid chromatography method was reliable and effective. Finally, the application of the developed method to cosmetic, Psoraleae fructus and Angelicae dahuricae radix samples were presented. The results highlight the applicability of the ultra-high performance supercritical fluid chromatography method to the analysis of interested compounds in pharmaceutical and cosmetic samples.

Ultra-high-performance supercritical fluid chromatography-mass spectrometry for the analysis of organic contaminants in sediments.

A nontarget screening method was developed based on D-optimal designs for ultra-high performance supercritical fluid chromatography with positive and negative electrospray ionization mode mass spectrometry. A mixture of organic contaminants such as pesticides, steroids, surfactants, phenolic and fatty acids, and polycyclic aromatic hydrocarbon derivatives, was used for the optimization. An aprotic mixture of dichloromethane and acetone [3:1] performed overall best as the injection solvent. The highest peak capacities (n) were accomplished at the shallowest gradient (1%B/min), ammonium formate (n = 378 in negative ionization mode), or ammonium acetate (n = 327in positive ionization mode) in methanol as the modifier. Capillary voltage, make-up solvent flow rate, water, and additive concentration were the most significant factors for improving peak intensity: higher peak intensities were obtained at lower additive concentrations (5mM ammonium formate), and with 5% water in positive ionization mode. Conversely, water had detrimental effects in negative ionization mode. The optimized method was used to quantify organic contaminants in 17 freshwater sediment samples from Copenhagen, Denmark. Out of 50 monitored contaminants, 35 were detected in at least one sample. Further, the method has a potential for target and nontarget screening analysis of organic contaminants in solid matrices.

Supercritical fluids in analysis of cannabinoids in various Cannabis products

We developed a fast, selective, and sensitive method for the determination of various neutral and acidic phytocannabinoids with an emphasis on the separation of structurally related compounds. Optimized ultra-high performance supercritical fluid chromatography (UHPSFC) allowed the separation of 2 groups of structural isomers, including isomers of m/z 357: cannabidiolic and Δ9-tetrahydrocannabinolic acid, and isomers of m/z 315: cannabichromene, Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, cannabicyclol, and cannabidiol only in mere 3.5 min followed by 1.5 min equlibration. The 2-ethylpyridine functionalized stationary phase and gradient elution using mobile phase comprising carbon dioxide and methanol: acetonitrile (25:75) + 5% water mixture were selected after the optimization. Tandem mass spectrometry (MS/MS) with electrospray ionization in positive and negative modes with methanol + 5% water as a make-up solvent provided adequate selectivity and sensitivity needed for analysis of phytocannabinoids in complex matrices. The limits of quantification were in the range 0.01–0.5 ng/mL for most of the monitored cannabinoids. The optimized UHPSFC-MS/MS method was then used for the determination of cannabinoids in various products, such as dietary supplements, nutraceuticals, and cosmetics. Solvent extraction methods were optimized for the cosmetic and nutraceutical products with the accuracy in the range 80.4–120.6% and precision 0.5–18.9%. To extract cannabinoids from the herbal infusion matrix, supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) methods were developed using environmentally friendly solvents water, ethanol, and carbon dioxide. The detailed optimization of extraction solvent composition, temperature, and pressure was carried out with the emphasis on avoiding the thermal degradation of cannabinoids. Optimized SFE and PLE methods were compared and applied to different herbal infusions to confirm declared cannabinoids content.

Optimization and validation of a fast supercritical fluid chromatography tandem mass spectrometry method for the quantitative determination of a large set of PFASs in food matrices and human milk

An Ultra-High Performance Supercritical Fluid Chromatography coupled with tandem Mass Spectrometry analytical method (UHPSFC-MS/MS) was developed for the determination of 34 perfluoroalkylated substances (PFASs) in food-related matrices. Two parameters (i.e. stationary phase and co-solvent) were selected and optimized using a step-by-step method, while a design of experiment (DoE) method using a central composite design (CCD) was implemented to optimize column temperature, mobile phase flow rate, co-solvent concentration and automated back pressure regulator (ABPR). The Torus 2-PIC column was selected along with ammonium acetate AcoNH4 as additive in the co-solvent. DoE optimization of both peak width and resolution enabled validating an optimized model (desirability 0.613) and setting column temperature at 38.7 °C, AcoNH4 concentration at 8 mM, mobile phase flow rate of 1.9 mL/min and ABPR at 1654 psi. The validated resulting method enabled reaching limits of quantification below 0.2 ng/g (w.w.) for 97 % PFASs in accordance with current EU requirements. The strategy was successfully applied to the characterization of a range (n > 30) of food-related matrices (red meat, poultry meat, eggs, fish and breast milk) collected in Algeria in 2019. PFOA and PFBA were observed as the most frequently detected PFASs, i.e. in 96.96 % and 90.9 % of the samples respectively. The highest concentrations were determined in fishery products up to 4.42 ng/g (w.w.) for PFTeDA and 0.75 ng/g (w.w.) for PFOS.

Supercritical fluid chromatography coupled to high-resolution tandem mass spectrometry: an innovative one-run method for the comprehensive assessment of chocolate quality and authenticity.

To assess chocolate quality and authenticity comprehensively, a combination of various analytical procedures is involved, thereby making the process time-consuming and costly. Thus, we investigated the potential of ultra-high performance supercritical fluid chromatography coupled to quadrupole-time of flight mass spectrometry (UHPSFC-QTOF-MS) as an alternative to "classic" methods. By combining hexane and aqueous extracts from sequential extraction, a single 8-min analytical run enabled us (i) to determine cocoa butter equivalents (CBEs) and milk fat content based on the detection of selected triacylglycerols, (ii) to calculate dry non-fat cocoa solids based on determined theobromine and caffeine content, and (iii) to profile contained sugars. To obtain the most comprehensive information about sample composition, the MS method comprised a full MS scan for non-target screening and several time-scheduled targeted MS/MS functions ("parallel reaction monitoring") optimized according to the possible concentration ranges of the analytes. For 40 different chocolate samples, our results and those obtained by using standard methods (LC-UV for non-fat cocoa solids, and GC-FID for CBEs) were in good agreement. Compared to the conventional approach for chocolate quality and authenticity control, the presented SFC-MS method is a fast, cost-effective, and efficient alternative, and only samples suspicious for the presence of CBE should be referred to the standard GC-FID method for exact CBE quantification. In the study, also some challenges offered by SFC-MS have been addressed.

Sub/supercritical fluid chromatography versus liquid chromatography for peptide analysis

The aim of this study was to evaluate the potential of ultra-high performance supercritical fluid chromatography (UHPSFC) for peptide analysis by comparing its analytical performance to several chromatographic approaches based on reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and mixed-mode liquid chromatography. First, the retention behavior of synthetic peptides with 3 to 30 amino acids and different isoelectric points (acid, neutral, and basic) was evaluated. For all the tested conditions (13 peptides in 8 conditions), only 4 results were not exploitable (not retained or not eluted), confirming that all the tested chromatographic conditions can be successfully applied when analyzing a wide range of diverse peptides. Average tailing factor were quite comparable across all chromatographic modes, while the best peak capacity values were obtained under mixed-mode LC conditions. Selectivity for each chromatographic mode was also evaluated for six closely related peptides having minor modifications on their structures. The LC-based chromatographic modes confirmed their superior selectivity over UHPSFC. By contrast, when analyzing short peptides (di- or tripetides), UHPSFC was the only technique allowing to simultaneously separate highly polar and less polar peptides within the same run confirming its unique versatility. In addition, the sensitivity of each chromatographic approach was accessed by for two representative peptides by both UV and MS detection. With UV detection, limit of detection (LOD) values were comparable among the different chromatographic modes, ranging from 0.5 to 2 µg mL−1. However, major differences were found when employing MS detection (LOD values ranged from 0.05 to 5 µg mL−1). The best results were obtained under HILIC conditions, followed by SFC, and finally mixed-mode LC and RPLC modes.

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.

Simultaneous Quantitative Analysis of the Major Bioactive Compounds in Gentianae Radix and its Beverages by UHPSFC-DAD.

This study presents the first ultra-high performance supercritical fluid chromatography–diode array detector (UHPSFC–DAD) assay for simultaneous quantitation of secoiridoids, iridoids, xanthones, and xanthone glycosides in Gentiana lutea L. Separation was reached within 12 min on an Acquity UPC2 BEH 2-EP column using CO2 and methanol with 5.5% water as mobile phases. Method validation for nine selected marker compounds (gentisin, isogentisin, swertiamarin, sweroside, gentiopicroside, loganic acid, amarogentin, gentioside, and its isomer) confirmed the assay’s sensitivity, linearity, precision, and accuracy. The practical applicability was proven by the analysis of 13 root specimens and 10 commercial liquid preparations (seven liqueurs and three clear spirits). In all root batches, the secoiridoid gentiopicroside dominated (2.1–5.6%) clearly over all other metabolites. In the liqueurs, the metabolite content and distribution were extremely variable: while gentiopicroside was the main compound in four liqueurs, sweroside dominated in one preparation and loganic acid in two others. In contrast, measurable amounts of the metabolites were not detected in any of the examined clear spirits.

Ultrahigh-Performance Supercritical Fluid Chromatography and Detection of Multiple Biogenic Amines in Gentamicin Sulfate: Method Development Using Computer-Assisted Modeling.

In order to solve the problem of difficult separation of various biogenic amines (BAs), which have similar structures or very different polarities, in gentamicin, by conventional liquid chromatography, a new ultrahigh-performance supercritical fluid chromatography (UHPSFC) method was developed. In this method, 10 BAs were derivatized precolumn using dansyl chloride and separated using a UHPSFC system. By computational simulation, complete separation of 10 BAs was successfully achieved. Detection was performed using a photodiode array (PDA) and single-quadrupole mass spectrometry (MS) together with electrospray ionization (ESI). A wide linear range (10-2500 ng/mL) was achieved, with the limits of detection (LODs) between 1.2 and 10.0 ng/mL and the limits of quantification (LOQs) between 5.0 and 25.0 ng/mL. Apart from high sensitivity, this UHPSFC-PDA/ESI-MS detection method also displayed high accuracy, the matrix effect was reduced by an appreciable extent, and the recovery rates of the 10 BAs were between 84.1 and 117.1%. For comparison, high-performance liquid chromatography-tandem mass spectrometry (MS/MS) was also used for the detection of underivatized BAs in gentamicin, showing good linearity and high sensitivity (LODs from 0.05 to 1.00 ng/mL and LOQs from 1.00 to 12.50 ng/mL) for all BAs except for spermine and spermidine. Although single-quadrupole MS is inferior to MS/MS in terms of sensitivity, the UHPSFC method could detect more BAs. It also achieved the quantification limits required for impurity determination, demonstrating a potential strategy to offer a map overview of possible BA presence in fermentation antibiotics.

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.

Highly repeatable and selective ultrahigh-performance supercritical fluid chromatography – Mass spectrometry interclass separation in lipidomic studies

Extensive research has recently established ultrahigh-performance supercritical fluid chromatography – mass spectrometry (UHPSFC/MS) coupling as an excellent tool for the fast and comprehensive characterization of lipids. However, the long-term instability of retention times using UHPSFC needs to be addressed, which is especially undesirable in lipidomic studies involving large clinical cohorts that are based on the comparison of lipid profiles between disease and control samples. In this work, we focused on conducting a detailed evaluation of the UHPSFC interclass separation of total lipid extracts for selective and repeatable lipidomic analysis. The selectivity of 9 chromatographic columns with different chemistry and the effect of particular chromatographic conditions on the lipid class resolution were carefully assessed. The most pronounced influence was found for the solvent type that was used as a modifier, which enabled the lipid class resolution to be finely adjusted. When propan-2-ol was added to methanol, a positive effect was observed for the resolution of mainly polar lipid classes and the intraclass resolution of lipid species was mildly suppressed; these were both favorable for the interclass separation used in this work. The optimized UHPSFC/MS method is based on a diol column and a gradient of methanol – propan-2-ol – water – ammonium formate modifier that provides the resolution of all lipid classes in brain samples within 7 min of analysis. This method has shown excellent retention times repeatability for the 555 injections in lipidomic study with the standard deviations below 0.012 min for all lipids. This demonstrates the ability of the optimized UHPSFC/MS method for highly selective and repeatable lipidomic analysis of biological samples.