The Response Surface Optimization of Supercritical CO2 Modified with Ethanol Extraction of p-Anisic Acid from Acacia mearnsii Flowers and Mathematical Modeling of the Mass Transfer.

A widely disseminated native species from Australia, Acacia mearnsii, which is mainly cultivated in Brazil and South Africa, represents a rich source of natural tannins used in the tanning process. Many flowers of the Acacia species are used as sources of compounds of interest for the cosmetic industry, such as phenolic compounds. In this study, supercritical fluid extraction was used to obtain non-volatile compounds from A. mearnsii flowers for the first time. The extract showed antimicrobial activity and the presence of p-anisic acid, a substance with industrial and pharmaceutical applications. The fractionation of the extract was performed using a chromatographic column and the fraction containing p-anisic acid presented better minimum inhibitory concentration (MIC) results than the crude extract. Thus, the extraction process was optimized to maximize the p-anisic acid extraction. The response surface methodology and the Box-Behnken design was used to evaluate the pressure, temperature, the cosolvent, and the influence of the particle size on the extraction process. After the optimization process, the p-anisic acid yield was 2.51% w/w and the extraction curve was plotted as a function of time. The simulation of the extraction process was performed using the three models available in the literature.

Supercritical carbon dioxide extraction of the Oak Silkworm (Antheraea pernyi) Pupal Oil: process optimization and composition determination.

Supercritical carbon dioxide (SC-CO(2)) extraction of oil from oak silkworm pupae was performed in the present research. Response surface methodology (RSM) was applied to optimize the parameters of SC-CO(2) extraction, including extraction pressure, temperature, time and CO(2) flow rate on the yield of oak silkworm pupal oil (OSPO). The optimal extraction condition for oil yield within the experimental range of the variables researched was at 28.03 MPa, 1.83 h, 35.31 °C and 20.26 L/h as flow rate of CO(2). Under this condition, the oil yield was predicted to be 26.18%. The oak silkworm pupal oil contains eight fatty acids, and is rich in unsaturated fatty acids and α-linolenic acid (ALA), accounting for 77.29% and 34.27% in the total oil respectively.

Diffusion coefficients of an extensive set of pharmaceutical compounds in supercritical fluid chromatography over a wide range of mobile phase compositions.

Diffusion data are essential for adequate analysis of the kinetic separation performance of any chromatographic system. Unfortunately, for Supercritical Fluid Chromatography (SFC), very little data is available of the diffusion coefficients in mobile phases typically used in contemporary methods, i.e. with a non-negligible amount of polar modifier such as methanol. In this work, a relative simple method which only requires minor modifications to a standard commercially available SFC instrument is used to determine the diffusion coefficient of an extensive set of pharmaceutical compounds in the range of 10-50 vol% of modifier (methanol) in CO2. By using a traditional SFC column, the solute is first separated from the sample solvent plug, before entering a long capillary, where the band broadening can be linked to its diffusion coefficient using the Taylor-Aris equation. By using two UV-detectors, before and after the capillary, the effect of the dispersion in the column can be eliminated and the true volumetric flow rate determined. It was found that in the investigated range of conditions, the change in mobile phase viscosity in a first approximation allows to predict the variation in diffusion coefficient. Chemical structure and more particularly functional groups can however have a significant effect on the diffusion coefficient.

Development and validation of an ultra-high performance supercritical fluid chromatography-photodiode array detection-mass spectrometry method for the simultaneous determination of 12 compounds in Piper longum L.

An ultra-high performance supercritical fluid chromatography-photodiode array detection-mass spectrometry (UHPSFC-MS) method for quality control of Piper longum L. has been developed and optimized. Hexane/isopropanol (70/30, v/v) was determined as the final injection solvent and methanol as the organic modifier. A design-of-experimental (DoE) approach was used to optimize column temperature, back-pressure and the gradient slope simultaneously using Trefoil CEL1 column. The back-pressure, temperature, flow rate were set at 130 bar, 32.5 °C and 1.0 mL/min, respectively. Positive electrospray ionization was used in the single ion monitoring mode. The 12 analytes were analyzed within 8 min using the optimized conditions. The linearities of the standard calibrations were satisfactory with coefficients of determination (R2) > 0.995. The recovery measured varied from 96.34% to 105.00% with relative standard deviation (RSD) ≤ 4.68%. The method was sensitive, reliable and effective, and successfully applied to simultaneous determination of 12 compounds in 28 batches of P. longum.

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

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

First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities.

Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter-laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within- and between-laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.

Determination of cannabinoid and synthetic cannabinoid metabolites in wastewater by liquid-liquid extraction and ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.

For the first time, ultra-high performance supercritical fluid chromatography (UHPSFC) coupled to tandem mass spectrometry has been used to determine cannabinoid and synthetic cannabinoid residues in wastewater. Combined with a downscaled version of the classic liquid-liquid extraction, the proposed method allows for the quantification of Δ9-tetrahydrocannabinol, three of its major metabolites (the monohydroxylated, the dehydroxylated, and the carboxylated species) and four synthetic cannabinoid metabolites (from the JWH-series) at low ng L-1 levels. Limits of quantification are in the 1-59 ng L-1 range, with recovery between 62 and 122% in ultrapure water and between 59 and 138% in wastewater. The applicability of the developed methodology was confirmed by the analysis of real wastewater, where cannabis metabolites could be positively quantified in all the samples analyzed. It is, therefore, a fast and simple alternative to common solid-phase extraction-liquid chromatography-mass spectrometry procedures for the determination of these low polar substances in water. Copyright © 2017 John Wiley & Sons, Ltd.

Rapid analysis of tristyrylphenol ethoxylates in cucumber-field system using supercritical fluid chromatography-tandem mass spectrometry.

Tristyrylphenol ethoxylates (TSPxEOs), nonionic surfactant adjuvants, are widely used to replace alkylphenols in many agricultural formulations. Herein, a rapid and sensitive method based on a modified quick, easy, cheap, effective, rugged, and safe "QuEChERS" and supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method was developed to quantify TSPxEOs in cucumber, leaves, and soil. Following optimization, the tested oligomers were fully separated within 8 min. The matrix-matched calibration standards showed satisfactory linearity with coefficients of determination (R2) ≥ 0.991. The limits of quantification (LOQs: 0.001-0.048 µg kg-1) were excellent for TSPxEOs (x = 6-29). The average recoveries at three (low, medium, and high) fortification levels were ranged between 62.5% and 112.9% with relative standard deviations (RSDs) ≤20.0% for TSPxEOs (x = 6-29) in various matrices. The method was successfully applied to determine TSPxEOs residual levels in field-incurred samples. Conclusively, the developed method is versatile and could be used for monitoring TSPxEOs in food and environmental samples.

Ruggedness and robustness testing.

Due to the strict regulatory requirements, especially in pharmaceutical analysis, analysis results with an acceptable quality should be reported. Thus, a proper validation of the measurement method is required. In this context, ruggedness and robustness testing becomes increasingly more important. In this review, the definitions of ruggedness and robustness are given, followed by a short explanation of the different approaches applied to examine the ruggedness or the robustness of an analytical method. Then, case studies, describing ruggedness or robustness tests of high-performance liquid chromatographic (HPLC), capillary electrophoretic (CE), gas chromatographic (GC), supercritical fluid chromatographic (SFC), and ultra-performance liquid chromatographic (UPLC) assay methods, are critically reviewed and discussed. Mainly publications of the last 10 years are considered.

Validation of direct assay of an aqueous formulation of a drug compound AZY by chiral supercritical fluid chromatography (SFC).

Supercritical fluid chromatography (SFC) is increasingly being recognized as a powerful technique for analysis of pharmaceutical compounds in various dosage forms. Assay of aqueous formulations of research compounds by SFC is, however, a relatively unexplored area primarily due to the potential problems associated with it. This work describes the development of a direct assay of a chiral drug compound AZY in a 100% aqueous formulation by SFC, and its qualification following ICH and FDA validation guidelines on chromatographic methods. The results indicated that SFC has the potential for assaying aqueous formulations of research compounds with high degree of selectivity, accuracy, precision, robustness, sensitivity, and linearity over a wide range of concentrations. This work also confirmed a previous hypothesis that direct formulation assay by SFC approach is applicable to both acidic and basic pharmaceutical compounds with equal degree of success.

Effect of system variables involved in packed column supercritical fluid chromatography of stavudine taken as model analyte using response surface methodology along with study of thermodynamic parameters.

A multifactor optimization technique is successfully applied to study the effect of simultaneously varying the system variables on feasibility of stavudine analysis by packed column supercritical fluid chromatography (PC-SFC). The effect of simultaneously varying the pressure, temperature and modifier concentration was studied to optimize the method in order to obtain excellent chromatographic figures of merit. The method is based on isocratic elution using methanol-modified supercritical carbon dioxide as the mobile phase at the flow rate of 3.0 ml/min through a JASCO Finepak SIL-5, ODS [C(18) (5 microm, 25 cm x 4.6 mm, i.d.)] column support using photodiode array detection. The optimal conditions were determined with the aid of the response surface methodology using 3(3) factorial designs. From the response surface graphs optimum regions were selected to be +1, -1, and +1 for temperature (60 degrees C), pressure (20 MPa) and percent modifier concentration (17.81%, v/v), respectively. Linearity dynamic range was found to be in the range of 2.0-150.0 microg/ml with significantly high value of correlation coefficient. The method was validated for precision, robustness and recovery to assess the viability of the established method. The chromatographic limit of detection and quantitation were 0.80 and 1.50 microg/ml respectively. The method has been successfully used to analyze commercial dosage form to assess the chromatographic performance of SFC system which was found to be 99.91%+/-1.62. The present work briefs the thermodynamic applications of PC-SFC with an emphasis on the results of stavudine. The foremost of such applications is the determination of solute diffusion coefficient in supercritical mobile phase by Taylor-Aris peak broadening technique.

Quantitative determination of salbutamol sulfate impurities using achiral supercritical fluid chromatography.

In the last years, supercritical fluid chromatography has largely been acknowledged as a singular and performing technique in the field of separation sciences. Recent studies highlighted the interest of SFC for the quality control of pharmaceuticals, especially in the case of the determination of the active pharmaceutical ingredient (API). Nevertheless, quality control requires also the determination of impurities. The objectives of the present work were to (i) demonstrate the interest of SFC as a reference technique for the determination of impurities in salbutamol sulfate API and (ii) to propose an alternative to a reference HPLC method from the European Pharmacopeia (EP) involving ion-pairing reagent. Firstly, a screening was carried out to select the most adequate and selective stationary phase. Secondly, in the context of robust optimization strategy, the method was developed using design space methodology. The separation of salbutamol sulfate and related impurities was achieved in 7min, which is seven times faster than the LC-UV method proposed by European Pharmacopeia (total run time of 50min). Finally, full validation using accuracy profile approach was successfully achieved for the determination of impurities B, D, F and G in salbutamol sulfate raw material. The validated dosing range covered 50 to 150% of the targeted concentration (corresponding to 0.3% concentration level), LODs close to 0.5µg/mL were estimated. The SFC method proposed in this study could be presented as a suitable fast alternative to EP LC method for the quantitative determination of salbutamol impurities.

Comparison of different methods for extraction of Cinnamomi ramulus: yield, chemical composition and in vitro antiviral activities.

Hydrodistillation (HD), supercritical fluid extraction (SFE) and reflux extraction (RE) were applied to obtain Cinnamomi ramulus extracts. The yields, chemical compositions and antiviral activities of the extracts were investigated. Extracts were analysed using gas chromatography-mass spectrometry and the antiviral activities were evaluated using cytopathic effect inhibition assay. HD, SFE and RE afforded 0.376, 1.227 and 5.914% yields, respectively. Cinnamaldehyde (CA), SFE and ethanol extracts exhibited antiviral activities against herpes simplex virus type 1. Moreover, CA and other three extracts had inhibition efficacy against respiratory syncytial virus. The most efficient antiviral activities were obtained with SFE.

Instrument modifications that produced reduced plate heights <2 with sub-2 µm particles and 95% of theoretical efficiency at k=2 in supercritical fluid chromatography.

The concept of peak fidelity was shown to be helpful in modeling tubing and detector cell dimensions. Connection tubing and flow cell variances were modeled to determine appropriate internal ID's, lengths, and volumes. A low dispersion plumbing configuration, based on these calculations, was assembled to replace the standard plumbing and produced the reported results. The modifications made were straightforward using commercially available parts. The full theoretical efficiency of a 3×100 mm column packed with 1.8 µm totally porous particles was achieved for the first time in supercritical fluid chromatography (SFC). Peak fidelity of >0.95 was maintained to below k=2. A reduced plate height as low as 1.87 was measured. Thus, true "ultra high performance" SFC was achieved, with the results a major improvement from all previous SFC reports. Since there were no efficiency losses, none could be attributed to thermal gradients caused by the expansion of the fluid over large pressure drops, under the conditions used. Similarly, changes in diffusion coefficients caused by significant decreases in density during expansion are apparently balanced by the increase in linear velocity, keeping the ratio between the diffusion coefficient and the linear velocity a constant. Changing modifier concentration to change retention was shown to not be a significant problem. All these issues have been a concern in the past. Diffusion coefficients, and viscosity data needs to be collected at high pressures before the actual limits of SFC can be discovered.

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 UPC(2) Torus 2-PIC, Acquity UPC(2) Torus Diol, Acquity UPC(2) Torus DEA and Acquity UPC(2) Torus 1-AA (1.7µm 3.0×100mm), and three chiral columns, including Acquity UPC(2) Trefoil AMY1, Acquity UPC(2) Trefoil CEL1 and Acquity UPC(2) 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.

A closer study of methanol adsorption and its impact on solute retentions in supercritical fluid chromatography.

Surface excess adsorption isotherms of methanol on a diol silica adsorbent were measured in supercritical fluid chromatography (SFC) using a mixture of methanol and carbon dioxide as mobile phase. The tracer pulse method was used with deuterium labeled methanol as solute and the tracer peaks were detected using APCI-MS over the whole composition range from neat carbon dioxide to neat methanol. The results indicate that a monolayer (4Å) of methanol is formed on the stationary phase. Moreover, the importance of using the set or the actual methanol fractions and volumetric flows in SFC was investigated by measuring the mass flow respective pressure and by calculations of the actual volume fraction of methanol. The result revealed a significant difference between the value set and the actually delivered volumetric methanol flow rate, especially at low modifier fractions. If relying only on the set methanol fraction in the calculations, the methanol layer thickness should in this system be highly overestimated. Finally, retention times for a set of solutes were measured and related to the findings summarized above concerning methanol adsorption. A strongly non-linear relationship between the logarithms of the retention factors and the modifier fraction in the mobile phase was revealed, prior to the established monolayer. At modifier fractions above that required for establishment of the methanol monolayer, this relationship turns linear which explains why the solute retention factors are less sensitive to changes in modifier content in this region.

Design of experiments for enantiomeric separation in supercritical fluid chromatography.

A new chiral melatoninergic ligand, potentially successor of Valdoxan(®), presenting an improved pharmacological profile with regard to agomelatine, was chosen as a probe for a supercritical fluid chromatographic separation carried-out on an amylose tris[(S)-1-α-methylbenzylcarbamate] based stationary phase. The goal of this work was to optimize simultaneously three factors identified to have a significant influence to obtain the best resolution in the shortest analysis time (i.e., retention time of the second eluting enantiomer) for this chiral compound. For this purpose a central circumscribed composite (CCC) design was developed with three factors: the flow-rate, the pressure outlet and the percentage of ethanol to optimize of two responses: shortest analysis time and best resolution. The optimal conditions obtained via the optimizer mode of the software (using the Nelder-Mead method) i.e., CO2/EtOH 86:14 (v:v), 104bar, 3.2mLmin(-1) at 35°C lead to a resolution of 3.27 in less than 6min. These conditions were transposed to a preparative scale where a concentrated methanolic solution of 40mM was injected with a sample loop of 100µL. This step allowed to separate an amount of around 65mg of racemic melatonin ligand in only 3h with impressive yields (97%) and enantiomeric excess (99.5%).

Use of isopycnic plots to understand the role of density in SFC - I. Effect of pressure variation on retention factors.

This paper aims to demonstrate the effect of pressure variations in modifying analyte retention behavior in SFC. There is a general understanding that in SFC increasing pressure decreases the retention factor (k'), and vice versa. What is not clearly discussed or explained in any recent literature is that these variations can be very different at different operating pressures, temperatures and modifier concentrations. It is important to have a clearer understanding on these variabilities during method development and results analysis. In this paper the nature of k' variation with pressure, at different temperatures and modifier concentrations, will be explained with the help of isopycnic plots of CO2 and CO2+methanol mixtures.

Method developments approaches in supercritical fluid chromatography applied to the analysis of cosmetics.

Analyses of complex samples of cosmetics, such as creams or lotions, are generally achieved by HPLC. These analyses are often multistep gradients, due to the presence of compounds with a large range of polarity. For instance, the bioactive compounds may be polar, while the matrix contains lipid components that are rather non-polar, thus cosmetic formulations are usually oil-water emulsions. Supercritical fluid chromatography (SFC) uses mobile phases composed of carbon dioxide and organic co-solvents, allowing for good solubility of both the active compounds and the matrix excipients. Moreover, the classical and well-known properties of these mobile phases yield fast analyses and ensure rapid method development. However, due to the large number of stationary phases available for SFC and to the varied additional parameters acting both on retention and separation factors (co-solvent nature and percentage, temperature, backpressure, flow rate, column dimensions and particle size), a simplified approach can be followed to ensure a fast method development. First, suited stationary phases should be carefully selected for an initial screening, and then the other operating parameters can be limited to the co-solvent nature and percentage, maintaining the oven temperature and back-pressure constant. To describe simple method development guidelines in SFC, three sample applications are discussed in this paper: UV-filters (sunscreens) in sunscreen cream, glyceryl caprylate in eye liner and caffeine in eye serum. Firstly, five stationary phases (ACQUITY UPC(2)) are screened with isocratic elution conditions (10% methanol in carbon dioxide). Complementary of the stationary phases is assessed based on our spider diagram classification which compares a large number of stationary phases based on five molecular interactions. Secondly, the one or two best stationary phases are retained for further optimization of mobile phase composition, with isocratic elution conditions or, when necessary, two-step gradient elution. The developed methods were then applied to real cosmetic samples to assess the method specificity, with regards to matrix interferences, and calibration curves were plotted to evaluate quantification. Besides, depending on the matrix and on the studied compounds, the importance of the detector type, UV or ELSD (evaporative light-scattering detection), and of the particle size of the stationary phase is discussed.

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.