Ultra-high Performance Supercritical Fluid Research Articles

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.

Fast Optimization of Supercritical Fluid Chromatography-Mass Spectrometry Interfacing Using Prediction Equations.

The effect of makeup solvent composition in ultrahigh-performance supercritical fluid chromatography-triple quadrupole mass spectrometry using electrospray ionization was studied using a set of 91 compounds, 3 stationary phases, and 2 organic modifiers of the mobile phase. The 24 tested makeup solvents included pure alcohols and methanol in combination with commonly used additives such as water, formic and acetic acid, ammonia, and ammonia salts with varying molarity. The behavioral trends for different makeup solvent additives were established in the first step. Subsequently, the correlations between physicochemical properties and the MS responses were calculated using the Pearson correlation test and matrix plots. The regression analysis was performed using five descriptors: molecular weight, pKa, log P, number of hydrogen donors/acceptors, and the MS responses obtained with methanol as the makeup solvent. The resulting regression equations had a high prediction rate calculated as R2-predicted coefficient, especially when 10 mmol/L ammonium in methanol was used as an organic modifier of the mobile phase in positive mode. The trueness of these equations was tested via the comparison between experimental and predicted responses expressed as R2. Values of R2 > 0.8 were found for 88% of the proposed equations. Thus, the MS response could be measured using only one makeup solvent and the responses of other makeup solvents could be easily estimated. The suitability and applicability of determined regression equations was confirmed by the analysis of 13 blind probes, i.e., compounds not included in the original set of analytes. Moreover, the predicted and experimental responses followed the same increasing/decreasing trend enabling one to predict makeup solvent compositions leading to the highest sensitivity.

Determination of 22 alternative plasticizers in wrap film by solid phase extraction and ultra-high performance supercritical fluid chromatography-tandem mass spectrometry

A rapid, accurate and novel analytical method based on ultra-high performance supercritical fluid chromatography-tandem mass spectrometry for determination of 22 alternative plasticizers in wrap film was developed. Instrumental analysis and sample preparation procedures were systematically optimized. The targets were separated on Torus 1-AA column (100 mm × 3 mm, 1.7 µm). Mobile phase A was supercritical carbon dioxide, and mobile phase B was ethanol/methanol (7:3, v/v) containing 0.1% formic acid and 0.5 mM ammonium acetate. Gradient elution was performed. The analytes were extracted by 10 mL n-hexane/dichloromethane (1:1, v/v), and further purified on silica solid phase extraction cartridges. The analytes were quantified by ultra-high performance supercritical fluid chromatography-tandem mass spectrometry with electrospray ionization source, and detection was performed on multiple reaction monitoring mode. Two commercially available isotopically-labelled internal standards were used for quantification calibration, and analytes were divided into two groups according to the more appropriate internal standards (chemistry similarity, closeness of retention time).Method validation was performed in terms of recovery, repeatability, linearity, sensitivity and matrix effect. Linearity was assessed using matrix-matched standard calibration. Satisfactory linearity (r2 ≥ 0.995), intra-day precision (RSDs ≤ 9.6%), inter-day precision (RSDs ≤ 10.9%), recovery (75.6–124.5%) as well as good selectivity was observed. The limits of detection were 0.04–10 µg/kg, while the limits of quantification were 1.0–50 µg/kg. Most targets did not show significant matrix effect. Validation results verified that the proposed method was efficient, rapid and sensitive. Eventually it was successfully applied to food wrap film analysis, and results indicated that DEHA, ATBC, DBA and TnBP were the most frequently detected plasticizers in wrap film samples,which was worthy of attention.

Simultaneous Determination of Vitamin D and Its Hydroxylated and Esterified Metabolites by Ultrahigh-Performance Supercritical Fluid Chromatography–Tandem Mass Spectrometry

In this study, an analytical method has been developed that, for the first time, allows simultaneous determination of vitamin D2 and vitamin D3 along with their hydroxylated and esterified forms. A group of 12 vitamin D analogues including vitamin D2 and vitamin D3, seven hydroxylated metabolites, and three ester forms were separated in a single 8.0 min run using ultrahigh-performance supercritical fluid chromatography coupled with triple quadrupole tandem mass spectrometry. Electrospray ionization and atmospheric pressure chemical ionization were investigated as ion sources, of which the latter showed a higher ionization efficiency. Chromatographic conditions were thoroughly evaluated by a step-by-step method, whereas an experimental design was applied for the optimization of the ionization parameters. Calibration and repeatability studies were carried out to validate the instrumental methodology showing determination coefficients higher than 0.9992 and good intra- and interday precision with relative standard deviations for areas and retention times lower than 10 and 2.1%, respectively, for all target analytes. Limits of quantification were below 3.03 μg/L for all compounds. The methodology was then validated and applied for the evaluation of human plasma samples in order to demonstrate its applicability to the analysis of vitamin D analogues in biological samples. Samples of five individuals were analyzed. Results show that linoleate-D3, vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 24,25-dihydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 could be detected in most samples, while the two latter also were quantified in all analyzed samples.

Ultrahigh-performance supercritical fluid chromatography – mass spectrometry for the qualitative analysis of metabolites covering a large polarity range

The applicability of ultrahigh-performance supercritical fluid chromatography coupled with mass spectrometry (UHPSFC/MS) for the qualitative analysis of metabolites with a wide polarity range (log P: −3.89–18.95) was evaluated using a representative set of 78 standards belonging to nucleosides, biogenic amines, carbohydrates, amino acids, and lipids. The effects of the gradient shape and the percentage of water (1, 2, and 5%) were investigated on the Viridis BEH column. The screening of eight stationary phases was performed for columns with different interaction sites, such as hydrogen bonding, hydrophobic, π-π, or anionic exchange type interactions. The highest number of compounds (67) of the set studied was detected on the Torus Diol column, which provided a resolution parameter of 39. The DEA column had the second best performance with 58 detected standards and the resolution parameter of 54. The overall performance of other parameters, such as selectivity, peak height, peak area, retention time stability, asymmetry factor, and mass accuracy, led to the selection of the Diol column for the final method. The comparison of additives showed that ammonium acetate gave a superior sensitivity over ammonium formate. Moreover, the influence of the ion source on the ionization efficiency was studied by employing atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The results proved the complementarity of both ionization techniques, but also the superior ionization capacity of the ESI source in the negative ion mode, for which 53% of the analytes were detected compared to only 7% for the APCI source. Finally, optimized analytical conditions were applied to the analysis of a pooled human plasma sample. 44 compounds from the preselected set were detected in human plasma using ESI-UHPSFC/MS in MSE mode considering both ionization modes.

Fast analysis of derivatives of polycyclic aromatic hydrocarbons in soil by ultra-high performance supercritical fluid chromatography after supercritical fluid extraction enrichment

An efficient and environment-friendly method for simultaneous determination of 13 typical derivatives of polycyclic aromatic hydrocarbon (PAH) in petroleum-polluted soil with nitro-, oxy- and alkyl-functional group was developed using supercritical fluid extraction (SFE) followed by ultra-high performance supercritical fluid chromatography (UHPSFC). Parameters of UHPSFC, including type of stationary phase and mobile phase modifiers, gradient elution process, backpressure, column temperature, and the flow rate of mobile phase, were systematically optimized, achieving a fast separation within 4.2 min. Limits of detection (LOD) were 0.005–0.1 μg mL−1 or 0.1–2.0 ng g−1, respectively, with a good repeatability (RSD<5.0%). Before UHPSFC-PDA analysis, the PAH-derivatives in soil samples were effectively enriched in 15.0 min using SFE with an online carbon nanotubes (CNTs) collection trap. The soil samples were analyzed by the proposed method and the results were verified by GC-MS. Thus, SFE equipped with an online CNTs trap followed by UHPSFC-PDA analysis, which only consumed about 2.0 mL organic solvent for a whole run, has been demonstrated to be an efficient way for screening and quantitative analysis of trace-level PAH-derivatives in soil samples.

Plasma lipidomic profiles of kidney, breast and prostate cancer patients differ from healthy controls

Early detection of cancer is one of the unmet needs in clinical medicine. Peripheral blood analysis is a preferred method for efficient population screening, because blood collection is well embedded in clinical practice and minimally invasive for patients. Lipids are important biomolecules, and variations in lipid concentrations can reflect pathological disorders. Lipidomic profiling of human plasma by the coupling of ultrahigh-performance supercritical fluid chromatography and mass spectrometry is investigated with the aim to distinguish patients with breast, kidney, and prostate cancers from healthy controls. The mean sensitivity, specificity, and accuracy of the lipid profiling approach were 85%, 95%, and 92% for kidney cancer; 91%, 97%, and 94% for breast cancer; and 87%, 95%, and 92% for prostate cancer. No association of statistical models with tumor stage is observed. The statistically most significant lipid species for the differentiation of cancer types studied are CE 16:0, Cer 42:1, LPC 18:2, PC 36:2, PC 36:3, SM 32:1, and SM 41:1 These seven lipids represent a potential biomarker panel for kidney, breast, and prostate cancer screening, but a further verification step in a prospective study has to be performed to verify clinical utility.

Development of a high-throughput method for the comprehensive lipid analysis in milk using ultra-high performance supercritical fluid chromatography combined with quadrupole time-of-flight mass spectrometry

Milk lipids are one of the most complex materials in nature and are associated with many physiological functions, hence it is important to comprehensively characterize lipids profiles to evaluate the nutritional value of milk. A quick method was developed by ultra-high performance supercritical fluid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (UHPSFC-ESI-QTOF-MS) to analyze the non-polar and polar lipids profiles of cow, goat, buffalo, human milk, and infant formulas in 7 min. All chromatographic conditions were carefully optimized and their effect on the chromatographic behavior of lipid classes and species was discussed. Under optimized conditions, 12 lipid classes (triacylglycerols, diacylglycerols, monoglyceride, fatty acids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, sphingomyelin, lyso-phosphatidylcholine, and lyso-phosphatidylethanolamine) were separated and each class was further separated in single analysis to facilitate the identification. 250 lipid species in real samples were characterized and quantified. This result demonstrates the applicability of the UHPSFC-ESI-QTOF-MS method in the high-throughput and comprehensive lipid analysis of milk, and will hopefully help to provide nutritionists with the lipid distribution in different types of milk, as well as help in the design of more suitable infant formula for babies.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of six compounds in Guizhi Fuling capsule and tablet samples.

A fast and simple ultra-high performance supercritical fluid chromatography method has been developed for the determination of six analytes, namely (paeonol, coumarin, cinnamic alcohol, cinnamic acid, paeoniflorin, and amygdalin) in Guizhi Fuling capsule and tablet samples. The influence of the key chromatographic parameters for the separation purposes was evaluated. The optimal column was Trefoil CEL1 column. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol at flow rate of 1.0mL/min. The back pressure of the system was set to 1.38 × 107 Pa and the temperature to 45°C. The six compounds were separated within 11min by the proposed ultra-high performance supercritical fluid chromatography method with satisfactory resolution. Method validation confirmed that the procedure is accurate with the recovery rates from 87.04 to 104.30%, intraday precision values less than 4.81% and interday precision less than 5.22%, and linear with R2 higher than 0.9967. Therefore, this work provides a simple and novel method for the simultaneous analysis of six compounds in Guizhi Fuling capsule and tablet samples.

Separation of monomeric and dimeric phenolic compounds in lignosulphonate lignin on different stationary phases using ultrahigh-performance supercritical fluid chromatography

Lignin is a promising renewable resource and its valorization could help to reduce our dependency on fossil carbon resources. Especially the production of small molecular weight and economically valuable compounds, such as vanillin, are of interest. A good separation of the sample components is crucial for a confident identification of compounds in complex sample mixtures using for instance mass spectrometry. In this work, the resolving power and selectivity of five different stationary phases for ultrahigh-performance supercritical fluid chromatography were studied for the class separation of lignin monomers (LMs) and dimers (LDs). A separation of LMs and LDs will help to identify such compounds in complex technical lignin samples. It could be shown that stationary phases with both hydrogen-bonding acceptor and donator groups offer high overall resolving power, while π-π-interactions are advantageous for the separation of the two compound classes. An almost complete separation combined with an improved overall resolving power was achieved with the 1-aminoanthracene stationary phase, which offers both hydrogen-bonding interactions and π-π-interactions.

Intra-laboratory comparison of four analytical platforms for lipidomic quantitation using hydrophilic interaction liquid chromatography or supercritical fluid chromatography coupled to quadrupole - time-of-flight mass spectrometry

The lipidomic research is currently devoting considerable effort to the harmonization that should enable the generation of comparable and accurate quantitative lipidomic data across different laboratories and regardless of the mass spectrometry-based platform used. In the present study, we systematically investigate the effects of the experimental setup on quantitative lipidomics data obtained by two lipid class separation approaches, hydrophilic interaction liquid chromatography (HILIC) and ultrahigh-performance supercritical fluid chromatography (UHPSFC), coupled to two different quadrupole – time of flight (QTOF) mass spectrometers from the same vendor. This approach is applied for measurements of 268 human plasma samples of healthy volunteers and renal cell carcinoma patients resulting in four data sets. We investigate and visualize differences among these data sets by multivariate data analysis methods, such as principal component analysis (PCA), orthogonal partial least square discriminant analysis (OPLS-DA), box plots, and logarithmic correlations of molar concentrations of individual lipid species. The results indicate that even measurements in the same laboratory for the same samples using different analytical platforms may yield slight variations in the molar concentrations determined. The normalization to a reference sample with defined lipid concentrations can further diminish these small differences, resulting in highly homogenous molar concentrations of individual lipid species. This strategy indicates a potential approach towards the reporting of comparable quantitative results independent from the quantitative approach and mass spectrometer used, which is important for a wider acceptance of lipidomics data in various biomarker inter-laboratory studies and ring trials.

Ultra-high performance supercritical fluid chromatography method for separation and quantitation of saikosaponins in herbal medicine

Saikosaponins are the main active ingredients of Bupleuri Radix and have been shown to have hepatoprotective, immunomodulatory and anti-viral activities. Among the saikosaponins, saikosaponin a (SSa), saikosaponin b1 (SSb1) and saikosaponin b2 (SSb2) are a group of isomers, which are difficult to separate by HPLC. In this study, a new method for separation and quantitation of saikosaponins was established by using ultra-high performance supercritical fluid chromatography (UHPSFC). A Torus Diol column (100 mm × 3 mm, 1.7 μm) was applied in this study. The mobile phase CO2 (A) was the main solvent with MeOH (B) as co-solvent. The results showed that the five saikosaponins were successfully separated within 22 min through optimization of chromatographic conditions. Besides, the UHPSFC method was applied for the quantitation of saikosaponins in a patent medicine Chaihu Dropping Pills, and demonstrated a good correlation coefficient (R2) ≥ 0.9990 in the range of 0.025 – 0.25 mg/mL. The recoveries of the five saikosaponins at three different concentrations were in the range of 90.23–99.84%. This study indicates that the proposed method has high separation efficiency in analyzing saikosaponins, which provides a new way for the separation and quantitation of saikosaponins in herbal medicines.

High Throughput Semiquantitative UHPSFC-MS/MS Lipid Profiling and Lipid Class Determination.

High throughput and high-resolution lipid analyses are important for many biological model systems and research questions. This comprises both monitoring at the individual lipid species level and broad lipid classes. Here, we present a nontarget semiquantitative lipidomics workflow based on ultrahigh performance supercritical fluid chromatography (UHPSFC)-mass spectrometry (MS). The optimized chromatographic conditions enable the base-line separation of both nonpolar and polar classes in a single 7-minute run. Ionization efficiencies of lipid classes vary 10folds in magnitude and great care must be taken in a direct interpretation of raw data. Therefore, the inclusion of internal standards or experimentally determined Response factors (RF) are highly recommended for the conversion of raw abundances into (semi) quantitative data. We have deliberately developed an algorithm for automatic semiquantification of lipid classes by RF. The workflow was tested and validated using a bovine liver extract with satisfactory results. The RF corrected data provide a more representative relative lipid class determination, but also the interpretation of individual lipid species should be performed on RF corrected data. In addition, semiquantification can be improved by using internal or also external standards when more accurate quantitative data are of interest but this requires validation for all new sample types. The workflow established greatly extends the potential of nontarget UHPSFC–MS/MS based analysis.

A Highly Sensitive Determination of Sesquiterpenes and Curcumin Compounds from Radix Curcumae by Ultra-High Performance Supercritical Fluid Chromatography Coupled with Drift Tube Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry

A highly sensitive method was developed for simultaneously separating and identifying multiple compounds in radix curcumae. It is a challenging work. The determination of these compounds is achieved by the combination supercritical fluid chromatography with drift tube ion mobility quadrupole time-of-flight mass spectrometry. Related parameters are optimized: the RX-SIL column was used as stationary phase, methanol was selected as organic modifier, backpressure 120 bar, back temperature 60 °C, the mobile phase flow rate 1.75 mL/min, the makeup solvent 0.2% FA/MeOH, the make-up pump flow rate 0.7 mL/min. Under the optimal conditions, multipolar compounds were separated. Furthermore, these compounds were identified by the values of collision sectional areas. The established method was verified by related parameters and exhibited good linearity, sensitivity, precision, and accuracy. It could be extended to analyze other curcuminoids and sesquiterpenoids in natural products.