Comparison of experimental and simulated separation performance in capillary tube-in-manifold devices.

A metal tube-in-manifold packed bed capillary column device, designed to overcome common limitations associated with capillary LC separations, is described. Experimental results of initial packing tests with sub-3 µm core-shell particles demonstrated efficiencies greater than 47,000 plates/m for a separation performed using the column device. Computational fluid dynamics (CFD) modeling of the multicomponent separation used for this work was validated against experimental LC results and the optimized model was able to effectively predict component peak retention times. However, the accuracy of predicted efficiencies requires further refinement. The tube-in-manifold design demonstrates that packed capillary columns with cylindrical cross-sectional channel geometry and ultrahigh pressure, low dead volume fluidic connections are achievable.

Compact photometric detector integrated with separation microchip for potential portable liquid chromatography system.

In recent years, miniaturized analytical instruments have been developing to meet the needs of portable and rapid analysis. The key of miniaturized analytical equipment is the miniaturization and integration of functional modules. This paper aims to develop a miniaturized photometric detector and separation microfluidic chip for a liquid chromatography (LC) system. The detector uses a light-emitting diode to emit ultraviolet light, which is collimated by an internal double lens. A Z-shaped flow cell with a long optical path is designed and fabricated in the separation microfluidic chip with a three-layer structure, which provides a tubing-free connection between the separation and detection unit. Detector performance is evaluated using hemoglobin (Hb) samples, with an upper limit of detection linearity (95 %) of 0.345 AU and stray light level as low as 0.08 %. Additionally, the microchip channel can be filled with cation exchange resin and C18 particles. Finally, an ion LC system and a reversed-phase LC system were constructed based on the miniaturized photometric detector and two microchips with different packed columns, respectively, and were successfully used in the separation and detection of two metabolic markers (glycated hemoglobin or bilirubin). The results of this study are expected to facilitate the development of a portable LC system and their application in community health services and family health management of chronic diseases.

NiFe-LDH/nylon 6 composite electrospun on polypropylene membrane: A new extractive device development for porous membrane protected micro-solid-phase extraction of organophosphate pesticides from fresh fruit juice samples coupled with liquid chromatography tandem mass analysis.

A unique strategy for developing porous membrane protected micro-solid phase extraction has been provided. An electrospun composite was fabricated on the sheet of membrane. To this end, NiFe-layered double hydroxide/Nylon 6 composite nanofibers were coated on a polypropylene membrane sheet followed by folding into a pocket shape, which were then utilized as a novel extractive device to extract of organophosphorus pesticides from fresh fruit juice samples prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The fabricated hybrid composites were successfully characterized. The effective parameters on extraction performance were investigated. LODs were 0.020-0.065 ng mL-1. Excellent linearity (R2≥0.996) was observed between 0.05 and 100.0 ng mL-1. RSDs% were in the range of 3.1-5.8% (intra-day, n = 3) and 2.6-5.5% (inter-day, n = 3×3). Satisfactory related recovery values within the acceptable range of 90.7-111.2% with RSDs% below 6.7% were achieved for the analysis of real samples.

New liquid chromatography columns for highlighting the interaction of ligand candidates with humic acid.

Humic acid was the main compound in soil and reduced the availability of some organic compounds in soils. In this work, humic acid was immobilized for the first time on a homemade neutravidin poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary column with a 20 µm i.d. for the screening of potential ligands to humic acid and the evaluation of their molecular recognition mechanism. This homemade humic acid column enabling it to work at very low backpressure (0.60 MPa at 20 nl/min flow rate), had a long lifetime, excellent repeatability, and negligible non-specific binding sites. The performance of this affinity humic acid column was demonstrated by the evaluation of recognition assay for a series of known ligands of humic acid (a series of rodenticide molecules) which is the heart of the fragment-based drug design. In addition, this column was used successfully for highlighting the binding mechanism to humic acid of the severe acute respiratory syndrome coronavirus-2-spike protein. As well this new humic acid miniaturized liquid chromatography column developed in this work could be used in the feature for another solute molecule-humic acid binding studies or for a separative mode.

Estudo do potencial antiviral sobre o Sars-Cov-2 dos compostos identificados no extrato bruto das folhas de Tetradenia riparia / Study of antiviral potential on Sars-Cov-2 of compounds identified in Tetradenia riparia leaves crude extract / Estudio del potencial antiviral sobre el Sars-Cov-2 de los componentes identificados en el extracto crudo de las hojas de Tetradenia riparia

A COVID-19 surgiu de forma repentina, acometendo milhões de pessoas e causando muitas mortes no mundo todo. Diante disso, torna-se necessário a busca de substâncias bioativas com propriedades antivirais. No Brasil, a espécie Tetradenia riparia foi inserida como planta ornamental exótica, com aroma intenso e agradável, sendo cultivada em parques, jardins, residenciais e hortos. O objetivo deste estudo foi identificar compostos presentes no extrato bruto das folhas de Tetradenia riparia com interesse antiviral. O extrato bruto das folhas secas foi obtido por maceração dinâmica por esgotamento do solvente e após, concentrado em evaporador rotativo. A composição química do extrato bruto foi analisada por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas de alta resolução (UHPLC-ESI/qTOF). Foram identificados 31 compostos que foram investigados por meio de levantamento bibliográfico quanto ao seu potencial anti- SARS-CoV-2. Os compostos rosmanol, procianidina, cianidina, betulina, ácido betulínico e o ácido sagerínico, apresentaram potencial atividade antiviral sobre o SARS-CoV-2. Esta investigação é promissora, indicando possivelmente que no extrato bruto das folhas de T. ripária existem compostos que podem combater o SARS-CoV-2. Neste sentido, estudos de ancoramento molecular (docking) e análises in silico sobre a proteína Mpro do vírus devem ser realizadas corroborando desta forma a ação dos compostos identificados.

COVID-19 appeared suddenly, affecting millions of people and causing many deaths worldwide. Therefore, it is necessary to search for bioactive substances with antiviral properties. In Brazil, Tetradenia riparia was inserted as an exotic ornamental plant, with an intense and pleasant aroma, cultivated in parks, residential and vegetable gardens. This study aimed to identify compounds present in the crude extract of Tetradenia riparia leaves with antiviral interest. The crude extract of the dried leaves was obtained by dynamic maceration with solvent exhaustion and then concentrated in a rotary evaporator. The chemical composition of the crude extract was analyzed by ultra- performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC- ESI/qTOF). We identified 31 compounds investigated through a literature review for their anti- SARS-CoV-2 potential. The compounds rosmanol, procyanidin, cyanidin, betulin, betulinic acid, and sagerinic acid showed potential antiviral activity against SARS-CoV-2. Therefore, this investigation is promising, possibly indicating that in the crude extract of T. riparia leaves, there are compounds that can fight SARS-CoV-2. In this sense, molecular docking studies and in silico analyzes on the virus Mpro protein must be carried out, thus corroborating the action of the identified compounds.

SARS-CoV-19 ha aparecido repentinamente, afectando a millones de personas y causando muchas muertes en todo el mundo. Por ello, se hace necesaria la búsqueda de sustancias bioactivas con propiedades antivirales. En Brasil, la especie Tetradenia riparia ha sido introducida como planta ornamental exótica, con un aroma intenso y agradable, siendo cultivada en parques, jardines, residencias y centros de jardinería. El objetivo de este estudio fue identificar los compuestos presentes en el extracto crudo de las hojas de Tetradenia riparia con interés antiviral. El extracto crudo de las hojas secas se obtuvo por maceración dinámica por agotamiento del disolvente y después, se concentró en el evaporador rotatorio. La composición química del extracto crudo se analizó mediante cromatografía líquida de ultra rendimiento acoplada a espectrometría de masas de alto rendimiento (UHPLC-ESI/qTOF). Se identificaron 31 compuestos y se investigó su potencial anti-SARS-CoV-2 mediante un estudio bibliográfico. Los compuestos rosmanol, procianidina, cianidina, betulina, ácido betulínico y ácido sagerínico, mostraron una potencial actividad antiviral sobre el SARS-CoV-2. Esta investigación es prometedora, pues posiblemente indica que en el extracto crudo de las hojas de T. riparia hay compuestos que pueden combatir el SARS-CoV-2. En este sentido, deben realizarse estudios de docking y análisis in silico sobre la proteína Mpro del virus para corroborar la acción de los compuestos identificados.

Quantitative structure retention relationship (QSRR) modelling for Analytes' retention prediction in LC-HRMS by applying different Machine Learning algorithms and evaluating their performance.

In metabolomics, retention prediction methods have been developed based on the structural and physicochemical characteristics of analytes. Such methods employ regression models, harnessing machine learning algorithms mapping experimentally derived retention time (tR) analytes with various structural and physicochemical descriptors, known as Quantitative Structure Retention Relationships (QSRR) models. In the present study, QSRR models have been developed by applying four Machine Learning regression algorithms, i.e. Bayesian Ridge Regression (BRidgeR), Extreme Gradient Boosting Regression (XGBR) and Support Vector Regression (SVR) using both linear and non-linear kernels, all tested and compared for their retention prediction ability on experimentally derived and on publicly available chromatographic data, using Molecular Descriptors to describe the physical, chemical or structural properties of molecules. Various configurations of the available datasets, in terms of the highly-correlated features levels (defined as the maximum absolute value of the Pearson's correlation coefficient calculated between any pair of features) they contained, were analyzed in parallel. This is the first study, to the best of our knowledge, of the effect of collinearity on the performance of QSRR predictive models. In the vast majority of cases studied there was no statistically significant difference in the performance of the generated QSRR predictive models among the specified dataset configurations, indicative of the ability of the selected regression algorithms to effectively handle collinearity. In terms of the individual performance of the selected regression algorithms, no pattern was found where one algorithm (or class of algorithms) stood out significantly relative to the others among the study datasets.

Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

Use of micro, capillary, and nano liquid chromatography for forensic analysis.

The use of micro, capillary, and nano liquid chromatography systems for forensic analysis has excellent potential. In a field where sample size is often limited, several studies have presented the viability of capillary columns with microflow and nanoflow, and when using mass spectrometric analysis limits of detection can be improved. Reduction in flow rates result in significant reduction in operating costs. Recent advances in miniaturized liquid chromatography systems also aim at in-laboratory and on-site detection, which have already been applied to forensic drug cases. This critical review will discuss the advantages, disadvantages, and applicability of microflow and nano liquid chromatography. In this regard, included in this article is a discussion of some promising areas not yet applied to forensic research.

Development and validation of an LC-MS/MS method for pharmacokinetic study of lobetyolin in rats

Abstract A simple and selective liquid chromatography tandem with mass spectrometry (LC-MS/ MS) method for quantification of lobetyolin in rat plasma was developed and validated. Chromatographic separation was achieved on a Thermo ODS C18 reversed-phase column using 0.1% aqueous formic acid-methanol (50:50, v/v) in an isocratic elution mode at a flow rate of 0.4 mL.min-1. LC/MS performance was done in a positive ion ESI mode and the MS/MS transitions were monitored at m/z 419.3 [M+Na]+ → m/z 203.1 for lobetyolin and m/z 394.9 [M+Na]+ → m/z 231.9 for IS, respectively. The assay exhibited a linear dynamic range over 1.0-500 ng.mL-1 for lobetyolin in plasma. Both the precision (%RSD) and accuracy (RE%) were within acceptable criteria (<15%). Recoveries ranged from 87.0% to 95.6%, and the matrix effects were from 91.0% to 101.3%. After oral administration, the peak plasma concentration of lobetyolin was obtained as 60.1 ng.mL-1 at 1.0 h. The proposed LC-MS/MS method could be applied to a pharmacokinetic study employing 66 samples from 6 Wistar rats

Development of an immobilized-trypsin reactor coupled to liquid chromatography and tandem mass spectrometry for the analysis of human hemoglobin adducts with sulfur mustard.

Sulfur mustard reacts with blood proteins, such as hemoglobin, to form stable adducts that can be used as long-lived biomarkers of exposure. These adducts can be analyzed by liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS) after an enzymatic digestion step. The objective of this study was to develop trypsin-based immobilized enzyme reactors (IMERs) in order to obtain a faster digestion of hemoglobin than the conventional in-solution digestion. Trypsin IMERs were synthetized by grafting the enzyme on a CNBr-Sepharose gel and the influence of several parameters on the digestion yields, such as the transfer volume between the injection loop and the IMER, the temperature and the digestion time was studied. The repeatability of the digestion on three laboratory-made IMERs was demonstrated for pure hemoglobin and hemoglobin previously exposed to different concentrations of sulfur mustard (RSD inferior to 13% and 21% respectively) and was better than that obtained for in-solution digestions (RSD inferior to 28% and up to 53% respectively). A preferential adduction of sulfur mustard on the histidine residues of hemoglobin was confirmed, for both in-solution and IMER digestion results. On a quantitative point of view, the performances of in-solution and IMER digestions were similar, with the theoretical possibility to detect peptides resulting from the in vitro incubation of hemoglobin in pure water with sulfur mustard at 7.5 ng⋅mL-1. However, digestion on IMER proved to be more repeatable and 32 times faster than in-solution digestion, and a given IMER could be reused at least 60 times.

Development and Validation of a LC-MS/MS Technique for the Analysis of Short Chain Fatty Acids in Tissues and Biological Fluids without Derivatisation Using Isotope Labelled Internal Standards.

The gut microbiota is critical to the maintenance of physiological homeostasis and as such is implicated in a range of diseases such as colon cancer, ulcerative colitis, diabetes, cardiovascular diseases, and neurodegenerative diseases. Short chain fatty acids (SCFAs) are key metabolites produced by the gut microbiota from the fermentation of dietary fibre. Here we present a novel, sensitive, and direct LC-MS/MS technique using isotopically labelled internal standards without derivatisation for the analysis of SCFAs in different biological matrices. The technique has significant advantages over the current widely used techniques based on sample derivatization and GC-MS analysis, including fast and simple sample preparation and short LC runtime (10 min). The technique is specific and sensitive for the quantification of acetate, butyrate, isobutyrate, isovalerate, lactate, propionate and valerate. The limits of detection were all 0.001 mM except for acetate which was 0.003 mM. The calibration curves for all the analytes were linear with correlation coefficients r2 > 0.998. The intra- and inter-day precisions in three levels of known concentrations were <12% and <20%, respectively. The quantification accuracy ranged from 92% to 120%. The technique reported here offers a valuable analytical tool for use in studies of SCFA production in the gut and their distribution to host tissues.

Theoretical framework for mixer design for noise reduction and gradient fidelity.

The mixing of two or more solvent streams to deliver a stable and accurate solvent composition is crucial to the performance, repeatability and reproducibility of a liquid chromatographic separation. We provide a theoretical treatment of axial mixing of a sequence of solvent packets with the framework of continuous stirred tank reactors (CSTRs) in series and investigate the tradeoffs presented between the primary goal of mixers (noise reduction) and it's necessary side-effects of gradient deformation and asymmetry. An experimental setup to mimic CSTR conditions was created using a stop-flow setup where the fluid flow was periodically paused and sonicated within pods of a certain volume. The effects of mixer volume relative to the volume of pump strokes and gradient volumes were investigated and discussed. A total mixer volume that is six-fold the pump stroke volume was found to be necessary to achieve sufficient (95%) noise reduction necessary for certain applications. A series of two or more CSTR elements was found to outperform a single CSTR element for larger mixer-to-pump stroke volume ratio in dampening baseline noise. For linear gradients, a gradient volume that is ten times larger than the mixer volume was found to sufficiently maintain gradient fidelity. For very small gradient volumes relative to the mixer volume, deformation of linear gradients was found to be significantly greater than predicted by the analytical solution. Furthermore, the nature of the solvent gradient deformation was asymmetric, with the latter half of the solvent gradient deforming significantly more than the first half. Combining analytically and numerically derived solutions for multiple CSTRs connected in series with experimental data, several suggestions can be made on mixer dimensions and design for a certain pump system and method transfer, given a pump stroke volume and gradient time.

Computational fluid dynamics study of potential solutions to alleviate viscous heating band broadening in 2.1 millimeter liquid chromatography columns.

We report on a numerical simulation study of a number of potential column technology solutions to minimize the plate height contribution (Hvh) originating from the use of ultra-high pressures and their concomitant viscous heating effect. Looking as far as possible into the future of UHPLC, all main results are obtained for the case of a 2500 bar pressure gradient. However, to generalize the result, a correlation is given that can be used to interpolate the results to lower pressures with some 10% accuracy. For the considered case of a 2.1mm column, a liquid flow rate of 0.45 ml/min, an analyte with retention factor k(25°C)=3 and a retention enthalpy chosen such that ΔHR/R= -1000 K, it is found that, in order to keep the global plate height as measured at the column outlet (Hvh,glob,out) below 1 µm, the bed conductivity would need to be raised to λbed=2.4 W/m•K, i.e., 4 times higher than a typical packed bed of fully-porous or core-shell silica particles. An equivalent effect on the band broadening could be obtained if it would be possible to replace the steel column wall with a low conductivity material. In this case, a wall conductivity of 0.25 W/m•K, i.e., 64 times smaller than the conductivity of steel, would be needed to keep Hvh,glob,out below 1 µm. Results are also interpreted based on contour plots of the axial and radial velocity variation of a retained analyte.

Improving LC/MS/MS-based bioanalytical method performance and sensitivity via a hybrid surface barrier to mitigate analyte - Metal surface interactions.

The accurate determination of the pharmacokinetics (PK) of a candidate drug molecule is critical in both drug discovery and development. Over the last 30 years, the sensitivity and selectivity of LC/MS has resulted in it being established as the technology of choice for these studies. However, unwanted chemical interactions between analyte(s) and the metal components in a chromatography system can result in poor peak shape and reduction in signal response, which can adversely affect the analysis of low concentrations of drugs and their metabolites in biological samples. This study evaluated the benefits of employing an inert hybrid surface technology (HST) applied to the metallic components in the LC flow path, column frits and column wall to mitigate these interactions. The results obtained were compared with that of an identical conventional LC for the bioanalysis of two steroid phosphate drugs (dexamethasone phosphate and hydrocortisone phosphate) and an epidermal growth factor receptor (EGFR) inhibitor (gefitinib) in human plasma. The results showed that for the two steroid phosphates, the peak width was reduced by 20%, peak tailing factors reduced by up to 30% and the assay sensitivity improved by factors of 7.5 and 10. This resulted in a significant improvement in the limit of detection. The new LC system also improved the reproducibility of peak integration for gefitinib, thereby reducing assay coefficients of variation (%CV) from greater than 10% to less than 5% at the lower limit of quantification.

On-surface autosampling for liquid chromatography-mass spectrometry.

An on-surface multi-purpose autosampler was built for liquid chromatography-mass spectrometry (LC-MS) based on the autoTLC-MS interface, taking advantage of open-source hard- and software developments as well as 3D printing. Termed autoTLC-LC-MS system, it is introduced for orthogonal hyphenation of normal phase high-performance thin-layer chromatography with reversed phase high-performance LC (HPLC) and high-resolution MS (HRMS). For verification of its functionality, a multi-class antibiotic mixture was applied as a calibration band pattern on an adsorbent layer and detected by the Bacillus subtilis bioassay. This effect-image was uploaded as a template in the updated TLC-MS_manager software. The clicked-on antibiotic zones were sequentially eluted without intervention from the planar counterpart (without bioassay) via a monolithic HPLC column into the HRMS system. For elution of antibiotics of 7 structural classes at 5 different calibration levels, the new on-surface autosampler achieved intra-day precisions of 2.1-14.1%, while inter-day precisions ranged 2.5-16.1% (all n = 3). The new hyphenation offers potential for planar sample clean-up prior to HPLC, concentration of liquid samples, increase of peak capacity and proof of peak purity or isomers. The integrated autoTLC-LC-MS system enabled high sample throughput, efficiency and reproducibility for the first time through fully automated TLC-LC-MS sequence operation. Its contact-closure signal functionality, versatile 3D printed planar sample holder and open-source software made it readily adjustable for new analytical tasks. Undoubtedly, any planar material can be investigated for leachables, such as textiles, foils, papers and other packagings, as well as planar biological samples for ingredients.

Theoretical study of the efficiency of liquid chromatography columns with particle size gradient.

Modern analytical applications of liquid chromatography require more and more efficient columns. In this work, the possibility of utilizing particle size gradient in the chromatographic column was studied by a theoretical approach. In the course of our work three different scenarios of particle size gradients were considered with different shapes (linear, convex and concave). The evolution of bandwidth inside the column was plotted for each scenario. As a reference point, the bandwidth of the uniform column was used, which had the same pressure drop as the non-uniform column. According to our calculations, in isocratic elution mode, the non-uniform column does not offer any advantage compared to the uniform column, regardless the type of the particle size gradient. In gradient elution mode, however, extra band compression occurs was found. For negative particle size gradients, the final physical bandwidth was found to be approximately 1-4 % smaller than for uniform columns. This slight gain in efficiency in terms of bandwidth compression can be expanded to 5-8 % by the optimization of the limiting particle sizes. These optimized results are obtained when the final particle size is approximately 40% of the initial particle diameter.

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems.

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.

Novel Fast Chromatography-Tandem Mass Spectrometric Quantitative Approach for the Determination of Plant-Extracted Phytosterols and Tocopherols.

Phytosterols and tocopherols are commonly used in food and pharmaceutical industries for their health benefits. Current analysis methods rely on conventional liquid chromatography, using an analytical column, which can be tedious and time consuming. However, simple, and fast analytical methods can facilitate their qualitative and quantitative analysis. In this study, a fast chromatography-tandem mass spectrometric (FC-MS/MS) method was developed and validated for the quantitative analysis of phytosterols and tocopherols. Omitting chromatography by employing flow injection analysis-mass spectrometry (FIA-MS) failed in the quantification of target analytes due to analyte-to-analyte interferences from phytosterols. These interferences arise from their ambiguous MS fingerprints that would lead to false identification and inaccurate quantification. Therefore, a C18 guard column with a 1.9 µm particle size was employed for FC-MS/MS under isocratic elution using acetonitrile/methanol (99:1 v/v) at a flow rate of 600 µL/min. Analyte-to-analyte interferences were identified and eliminated. The false peaks could then be easily identified due to chromatographic separation. In addition, two internal standards were evaluated, namely cholestanol and deuterated cholesterol. Both internal standards contributed to the observed analyte-to-analyte interferences; however, adequate shift in the retention time for deuterated cholesterol eliminated its interferences and allowed for an accurate quantification. The method is fast (1.3 min) compared to published methods and can distinguish false peaks observed in FIA-MS. Seven analytes were quantified simultaneously, namely brassicasterol, campesterol, stigmasterol, ß-sitosterol, α-tocopherol, δ-tocopherol, and γ-tocopherol. The method was successfully applied in the quantitative analysis of phytosterols and tocopherols present in the unsaponifiable matter of canola oil deodorizer distillate (CODD). ß-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.

Large-scale preparative isolation of bergenin standard substance from Saxifraga atrata using polyamide coupled with MCI GEL® CHP20P as stationary phases in medium pressure chromatography.

In this study, polyamide and MCI GEL® CHP20P were employed as stationary phases in medium pressure chromatography (MPC) for the efficient preparative separation of bergenin from Saxifraga atrata. Ethanol-water, methanol-water, and acetonitrile-water mobile phases all showed good enrichment capacity for bergenin fraction when polyamide was used as a stationary phase. After 5 cycles of polyamide MPC using acetonitrile/water, 1.2 g of bergenin fraction was isolated from 180 g Saxifraga atrata herb. Further purification of this fraction was conducted using MCI GEL® CHP20P styrene-divinylbenzene beads. The bergenin fraction was separated into two fractions, and after three runs of MPC, 714.2 mg of bergenin with purity above 99% was obtained. The results demonstrate that the combination of polyamide and styrene-divinylbenzene MPC can be utilized for preparative isolation of compounds from natural products with high yield and purity.

Column-in-valve designs to minimize extra-column volumes.

We report on the design and performance of in-house built column cartridges that can be directly screwed into the ports of a commercial rotor-stator valve to minimize extra-column band broadening and pressure-drop losses when pursuing ultra-fast separations such as those needed in 2D and 3D-LC separations. Two basic designs were evaluated and were compared with the results obtained with a commercial screw-in column cartridge. The system produces an extra-column band broadening as low as 0.05 to 0.1 µL2 for the employed UV-detector set-up. Despite these very low values, the obtained separation efficiency of the in-house fabricated cartridge columns was very low, corresponding to a reduced minimal plate height around h=7 at the very best, which, for the 1.7 µm particle and 26.4 mm long columns corresponds to a number of theoretical plates of N=2200 under isocratic conditions. A similar poor performance was obtained with a commercial column cartridge with similar dimensions using the same set-up. One possible explanation of the observed performance could be found in the inner diameter of the column cartridges (i.d. =0.75 mm and 1 mm) which, for the employed sub 2-µm particles, falls into a region of column diameters that, according to literature models, is most likely to suffer from inherent packing problems.