Fused-core Columns Research Articles

Fast analysis of capsaicinoids in Naga Jolokia extracts (Capsicum chinense) by high-performance liquid chromatography using fused core columns

A rapid high-performance liquid chromatography method with a C18 reverse-phase fused-core column has been developed for the determination and quantification of the main capsaicinoids (nornordihydrocapsaicin, nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin) present in Naga Jolokia peppers. A fused-core Kinetex™ C18 column (50×2.1mm i.d.; 2.6μm) was used for the analysis. The chromatographic separation was obtained with a gradient method in which the mobile phase was water (0.1% acetic acid) as solvent A and acetonitrile (0.1% acetic acid) as solvent B. The separation of all compounds was achieved in less than 3min with a total analysis time (sample-to-sample) of 10min. The robustness of the method was evaluated. The method showed excellent repeatability and intermediate precision expressed as coefficient of variance of less than 2%. The developed method was employed for the quantification of the major capsaicinoids present in different peppers and commercial products containing chilli peppers.

A Rapid Method for the Analysis of Paralytic Shellfish Toxins Utilizing Standard Pressure HPLC: Refinement of AOAC 2005.06.

Superficially porous column technologies have previously been shown to provide faster chromatographic analysis of toxin oxidation products when analyzing shellfish for paralytic shellfish toxins. While sub 3 μm fused core columns have facilitated enhanced method performance, including significantly lower analysis times and lower LOD, they were also found to last for only a few hundred injections before pressure increases rendered them unusable with standard HPLC. Recently 5 μm superficially porous columns have become commercially available. In this study, a 5 μm fused core column was used to develop a fast chromatographic method for the analysis of paralytic shellfish toxins, with performance characteristics and column lifetime being assessed. The 5 μm column was found to be able to perform approximately 3000 injections without significant increases in back pressure or reduction in performance. Data generated using the column were found to be equivalent to that determined using current HPLC column technologies for both screening and quantitation methods. Furthermore, an increase in sensitivity for all toxins tested under the routine monitoring program for British waters was observed and the overall run time of the analysis halved. Overall, the 5 μm fused core column provided a significant increase in sample throughput, a reduction in mobile phase consumption, and an increase in method sensitivity.

An on-line SPE–HPLC method for effective sample preconcentration and determination of fenoxycarb and cis, trans-permethrin in surface waters

A new on-line SPE–HPLC method using fused-core columns for on-line solid phase extraction and large volume sample injection for increasing the sensitivity of detection was developed for the determination of insecticides fenoxycarb and cis-, trans-permethrin in surface waters. The separation was carried out on fused-core column Phenyl–Hexyl (100×4.6mm), particle size 2.7µm with mobile phase acetonitrile:water in gradient mode at flow rate 1.0mLmin−1, column temperature 45°C. Large volume sample injection (1500µL) to the extraction dimension using short precolumn Ascentis Express RP C-18 (5×4.6mm); fused-core particle size 2.7µm allowed effective sample preconcentration and efficient ballast sample matrix removal. The washing mobile phase consisting of a mixture of acetonitrile:water; 30:70, (v/v) was pumped at flow rate of 0.5mLmin−1 through the extraction precolumn to the waste. Time of the valve switch for transferring the preconcentrated sample zone from the extraction to the separation column was set at 3rd min. Elution of preconcentrated insecticides from the extraction precolumn and separation on the analytical column was performed in gradient mode. Linear gradient elution started from 40% of acetonitrile at time of valve switch from SPE column (3rd min) to 95% of acetonitrile at 7th min. Synthetic dye sudan I was chosen as an internal standard. UV detection at wavelength 225nm was used and the method reached the limits of detection (LOD) at ngmL−1 levels for both insecticides. The method showing on-line sample pretreatment and preconcentration with highly sensitive determination of insecticides was applied for monitoring of fenoxycarb and both permethrin isomers in different surface water samples in Czech Republic. The time of whole analysis including on-line extraction, interferences removal, chromatography separation and system equilibration was less than 8min.

On-line hyphenation of solid-phase extraction to chromatographic separation of sulfonamides with fused-core columns in sequential injection chromatography

On-line sample pretreatment (clean-up and analyte preconcentration) is for the first time coupled to sequential injection chromatography. The approach combines anion-exchange solid-phase extraction and the highly effective pentafluorophenylpropyl (F5) fused-core particle column for separation of eight sulfonamide antibiotics with similar structures (sulfathiazole, sulfanilamide, sulfacetamide, sulfadiazine, sulfamerazine, sulfadimidine, sulfamethoxazole and sulfadimethoxine). The stationary phase was selected after a critical comparison of the performance achieved by three fused-core reversed phase columns (Ascentis® Express RP-Amide, Phenyl-Hexyl, and F5) and two monolithic columns (Chromolith® High Resolution RP-18 and CN). Acetonitrile and acetate buffer pH 5.0 at 0.60mLmin−1 were used as mobile phase to perform the separations before spectrophotometric detection. The first mobile phase was successfully used as eluent from SPE column ensuring transfer of a narrow zone to the chromatographic column. Enrichment factors up to 39.2 were achieved with a 500µL sample volume. The developed procedure showed analysis time <10.5min, resolutions >1.83 with peak symmetry ≤1.52, LODs between 4.9 and 27µgL−1, linear response ranges from 30.0 to 1000.0µgL−1 (r2>0.996) and RSDs of peak heights <2.9% (n=6) at a 100µgL−1 level and enabled the screening control of freshwater samples contaminated at the 100µgL−1 level. The proposed approach expanded the analytical potentiality of SIC and avoided the time-consuming batch sample pretreatment step, thus minimizing risks of sample contamination and analyte losses.

On-line SPE–UHPLC method using fused core columns for extraction and separation of nine illegal dyes in chilli-containing spices

The presented work describes the development of a simple, fast and effective on-line SPE–UHPLC–UV/vis method using fused core particle columns for extraction, separation and quantitative analysis of the nine illegal dyes, most frequently found in chilli-containing spices. The red dyes Sudan I–IV, Sudan Red 7B, Sudan Red G, Sudan Orange G, Para Red, and Methyl Red were separated and analyzed in less than 9min without labor-consuming pretreatment procedure. The chromatographic separation was performed on Ascentis Express RP-Amide column with gradient elution using mixture of acetonitrile and water, as a mobile phase at a flow rate of 1.0mLmin−1 and 55°C of temperature. As SPE sorbent for cleanup and pre-concentration of illegal dyes short guard fused core column Ascentis Express F5 was used. The applicability of proposed method was proven for three different chilli-containing commercial samples. Recoveries for all compounds were between 90% and 108% and relative standard deviation ranged from 1% to 4% for within- and from 2% to 6% for between-day. Limits of detection showed lower values than required by European Union regulations and were in the range of 3.3–10.3µgL−1 for standard solutions, 5.6–235.6µgkg−1 for chilli-containing spices.

New method for the determination of carbamate and pyrethroid insecticides in water samples using on-line SPE fused core column chromatography

A new HPLC column-switching method using large volume sample injection and fused-core columns for on-line solid phase extraction have been developed for the determination of the following carbamates and pyrethroids: aldicarb, carbaryl, pirimicarb, carbofuran, kadethrin, flumethrin, fenpropathrin, fenoxycarb, tau-fluvalinate and fenvalerate, in surface water samples. Sudan I was used as internal standard. The proposed method was performed using 100µl sample injection followed by an on-line solid phase extraction procedure and finally the compounds were identified and quantified by liquid chromatography with ultraviolet detection. The separation was carried out on C-18 reversed phase column based on fused-core particle technology. The influence of the injected sample volume, the variables affecting to SPE process and the conditions for the separation on an analytical column, were studied and optimized. The limits of detection ranged from 5.5 to 8.9µgL−1, and limits of quantification from 18.4 to 29.7µgL−1, while inter- and intra-day variability was under 15%. This new analytical procedure was satisfactorily applied for the determination of these organic pollutants in surface water samples located in Czech Republic. Concentration levels were found for some of these pollutants up to 26.11µgL−1 in the river Elbe and up to 34.53µgL−1 in the closed lakes samples.

A flow injection low-pressure chromatographic system exploiting fused-core columns

The first use of fused-core columns for low-pressure chromatographic separations in a flow injection system.

HPLC column-switching technique for sample preparation and fluorescence determination of propranolol in urine using fused-core columns in both dimensions

A new and fast high-performance liquid chromatography (HPLC) column-switching method using fused-core columns in both dimensions for sample preconcentration and determination of propranolol in human urine has been developed. On-line sample pretreatment and propranolol preconcentration were performed on an Ascentis Express RP-C-18 guard column (5 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water (5:95, v/v) at a flow rate of 2.0 mL min(-1) and at a temperature of 50 °C. Valve switch from pretreatment column to analytical column was set at 4.0 min in a back-flush mode. Separation of propranolol from other endogenous urine compounds was achieved on the fused-core column Ascentis Express RP-Amide (100 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water solution of 0.5% triethylamine, pH adjusted to 4.5 by means of glacial acetic acid (25:75, v/v), at a flow rate of 1.0 mL min(-1) and at a temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 229/338 nm. A volume of 1,500 μL of filtered urine sample solution was injected directly into the column-switching HPLC system. The total analysis time including on-line sample pretreatment was less than 8 min. The experimentally determined limit of detection of the method was found to be 0.015 ng mL(-1).

Liquid chromatography–tandem mass spectrometry

Over the last 20 years LC–MS techniques have advanced dramatically in their sensitivity, specificity, and reliability, mostly because of the development of hyphenated chromatography–mass spectrometry techniques. Currently one of the great challenges in environmental analysis and food safety is evaluation and control of the risks associated with the presence of mixtures of emerging contaminants; this has resulted in swift growth in the use of LC–MS–MS which is, today, the method of choice for determination of many polar and medium-polar classes of contaminants in environmental, biological, and food samples. Much effort has applied to the development of more efficient extraction and clean-up procedures by automation and coupling of sample-preparation and detection systems (for example, on-line SPE or dual LC column systems), application of advanced sorbents (e.g. nanoparticles as sorbents), and use of more environmentally sound approaches, for example solvent-reduced techniques. Moreover, there is a growing demand for high-throughput analysis, because of the growing number of samples, and shortening of analytical run times is often required in laboratories conducting environmental monitoring studies or food quality control. Run times of several tens of minutes are not acceptable, so the emphasis is on achievement of maximum chromatographic resolution in a drastically reduced time by application of fast and high-resolution LC systems (for example ultra-high pressure liquid chromatography (UHPLC), high-temperature liquid chromatography (HTLC), or monolithic and fused-core columns). Another recent trend is the use of hydrophilic interaction chromatography (HILIC) for separation of polar compounds.With regard to mass analysers, hybrid instruments, for example QqTOF, QqLIT, and Orbitrap, are becoming more popular, because of their capabilities in achieving accurate mass measurements and acquiring indispensable qualitative information in the form of full-scan spectra. Another notable trend is a gradual shift from parent compound analysis to analysis of metabolites and transformation products. It is becoming evident that more research is needed to determine breakdown pathways and to evaluate the fate of transformation products formed by biotic and abiotic processes. This issue of “Analytical and Bioanalytical Chemistry” contains selected papers presented at the 8th Annual Workshop on LC–MS–MS—Workshop on Environmental Applications and Food Safety, held on 2–4 July 2012 in Barcelona, Spain, and organized jointly by CSIC (Consejo Superior de Investigaciones Cientificas, Barcelona, Spain), the Catalan Institute for Water Studies (ICRA), Waters Corporation (Manchester, UK), Environment Canada, and the Institut d'Estudis Catalans (Barcelona, Spain), in collaboration with the Spanish Society for Mass Spectrometry (SEEM). In the course of the workshop a total of 35 keynote lectures and 56 posters were presented in three main sections

Fast HPLC Method for Determination of Fenoxycarb and Permethrin in Antiparasitic Veterinary Shampoo Using Fused-Core Column

A new and fast high-performance liquid chromatography (HPLC) method using technology of fused-core columns for separation of fenoxycarb and cis-, trans-permethrin has been developed and used for their determination in antiparasitic veterinary shampoo. Separation of insecticides and internal standard sudan II was achieved on the fused-core column Ascentis Express RP-Amide (100 × 3.0 mm), particle size 2.7 μm, with mobile phase acetonitrile/water (55:45, v/v) at a flow rate of 1.0 mL min−1 and at temperature 60 °C. The detection wavelength of detector was set at 225 nm for both compounds and internal standard sudan II. Under the optimum chromatographic conditions standard calibration curves were measured with good linearity [r 2 = 0.99991 for fenoxycarb, r 2 = 0.99987 for trans-permethrin, and r 2 = 0.99984 for cis-permethrin (n = 8)]. Commercial samples of antiparasitic veterinary shampoo were extracted with ethanol in ultrasound bath for 5 min. A 2-μL sample volume of the filtered solution was directly injected into the HPLC system. Accuracy of the method defined as a mean recovery of insecticides from shampoo matrix was in the range 100.43–103.85 % for both insecticides.

Recent Advances in Chromatography for the Efficient Profiling of Crude Extracts and Isolation of Natural Products

The large chemical space occupied by natural products (NPs) is directly linked to a high variability of their intrinsic physicochemical properties that render their separation, detection and characterization challenging. The analysis of NPs in complex crude extracts requires efficient separation methods. In this respect, high-performance liquid chromatography (HPLC) has been recognized since the early 1980s as the most versatile technique for their profiling directly in crude mixtures without the need for complex sample preparation [1]. HPLC has greatly developed through the years in terms of convenience, speed, choice of column stationary phases, high sensitivity, applicability to a broad variety of sample matrices, and ability to hyphenate the chromatographic methods to spectroscopic detectors. The latest developments of HPLC, including the recent introduction of very pH-stable phases, sub-2-μm particles, monolith and fused core columns, have considerably improved the performances of HPLC. In particular in phytochemical analysis, the recent introduction of Ultra High Pressure Liquid Chromatography (UHPLC) systems operating at very high pressures and using sub-2 μm packing columns have allowed a remarkable decrease in analysis time and increase in peak capacity, sensitivity and reproducibility compared to conventional HPLC [2]. Such developments have a significant impact on the quality of data that can be obtained for metabolite profiling, dereplication studies and metabolomics when efficient spectroscopic detectors such as time–of-flight mass spectrometers (TOF)-MS are hyphenated to UHPLC. On the other hand, a good mastery of the chromatography parameters at the analytical scale enables efficient gradient transfer to the semi-preparative or the preparative scale based on chromatographic calculation. This efficient upscale allows high loading of crude mixture either for rapid at-line biological profiling of crude extracts with sensitive assay and/or de novo structure determination with microflow NMR methods with microgram of NPs. These developments, as well as other new trends in chromatography such as the renewed interest for superfluid critical fluid separation, the use of two dimensional LC or the hyphenation with additional separation efficiency in the gas phase provided by ion mobility in LC-MS hyphenation for the deconvolution of NPs in complex crude plant extracts will be discussed.

Clinical applications of fast liquid chromatography: A review on the analysis of cardiovascular drugs and their metabolites

One of the major challenges facing the medicine today is developing new therapies that enhance human health. To help address these challenges the utilization of analytical technologies and high-throughput automated platforms has been employed; in order to perform more experiments in a shorter time frame with increased data quality. In the last decade various analytical strategies have been established to enhance separation speed and efficiency in liquid chromatography applications. Liquid chromatography is an increasingly important tool for monitoring drugs and their metabolites. Furthermore, liquid chromatography has played an important role in pharmacokinetics and metabolism studies at these drug development stages since its introduction. This paper provides an overview of current trends in fast chromatography for the analysis of cardiovascular drugs and their metabolites in clinical applications. Current trends in fast liquid chromatographic separations involve monolith technologies, fused-core columns, high-temperature liquid chromatography (HTLC) and ultra-high performance liquid chromatography (UHPLC). The high specificity in combination with high sensitivity makes it an attractive complementary method to traditional methodology used for routine applications. The practical aspects of, recent developments in and the present status of fast chromatography for the analysis of biological fluids for therapeutic drug and metabolite monitoring, pharmacokinetic studies and bioequivalence studies are presented.

Advances in chromatography: Efficient profiling of crude extracts and isolation of natural products

The large chemical space occupied by natural products (NPs) is directly linked to a high variability of their intrinsic physicochemical properties that render their separation, detection and characterization challenging. The analysis of NPs in complex crude extracts requires efficient separation methods. In this respect, high-performance liquid chromatography (HPLC) has been recognized since the early 1980s as the most versatile technique for their profiling directly in crude mixtures without the need for complex sample preparation.[1] HPLC has greatly developed through the years in terms of convenience, speed, choice of column stationary phases, high sensitivity, applicability to a broad variety of sample matrices, and ability to hyphenate the chromatographic methods to spectroscopic detectors. The latest developments of HPLC, including the recent introduction of very pH-stable phases, sub-2-µm particles, monolith and fused core columns, have considerably improved the performances of HPLC. In particular in phytochemical analysis, the recent introduction of Ultra High Pressure Liquid Chromatography (UHPLC) systems operating at very high pressures and using sub-2µm packing columns have allowed a remarkable decrease in analysis time and increase in peak capacity, sensitivity and reproducibility compared to conventional HPLC.[2] Such developments have a significant impact on the quality of data that can be obtained for metabolite profiling, dereplication studies and metabolomics when efficient spectroscopic detectors such as time-of-flight mass spectrometers (TOF)-MS are hyphenated to UHPLC. On the other hand, a good mastery of the chromatography parameters at the analytical scale enables efficient gradient transfer to the semi-preparative or the preparative scale based on chromatographic calculation. This efficient upscale allows high loading of crude mixture either for rapid at-line biological profiling of crude extracts with sensitive assay and/or de novo structure determination with microflow NMR methods with microgram of NPs. These developments, as well as other new trends in chromatography such as the renewed interest for superfluid critical fluid separation, the use of two dimensional LC or the hyphenation with additional separation efficiency in the gas phase provided by ion mobility in LC-MS hyphenation for the deconvolution of NPs in complex crude plant extracts will be discussed.

Fast Chromatography in The Regulated Bioanalytical Environment: Sub-2-µM Versus Fused-Core Particles

Increased throughput and rapid data turnaround have necessitated the need for faster LC separations in the regulated bioanalytical environment; sub-2-μm and fused-core chromatography are two viable approaches to accomplish this. Sub-2-μm columns offer high efficiency and resolution separations at the cost of high system backpressure and are best suited for preclinical sample analysis, whereas fused-core columns offer slightly lower efficiency and resolution, but are more tolerant to dirty extracts (such as those seen in clinical samples) that may clog the inlet filter. For clinical sample analyses, sub-2-μm columns should be restricted to sample extracts obtained via LLE or SLE, whereas fused-core columns can be used with sample preparations techniques ranging from protein precipitation to SLE.

Chromatographic behavior of the biologically active proline derivative captopril on particulate, monolithic and core–shell narrow bore columns

In the present work, the chromatographic behavior of the proline derivative captopril was studied on three different kinds of stationary phases. Four narrow-bore analytical columns were used in this study including particulate, monolithic, and two fused-core columns. The secondary equilibrium distribution of captopril and the peak shape were investigated at different mobile phase pH values and column temperatures. Under selected chromatographic conditions (pH: 2.5, 50 °C) where a single peak of captopril was obtained the performance of the studied narrow bore columns was compared.

Application of Fused-Core Particle Column in Two Dimensional Reversed Phase - Reversed Phase LC/MS Analysis of Biological Samples. Impact of Extra-Column Volume

We demonstrate successful applicability of fused–core column in analysis of biological samples (diluted urine and SPE–pre-purified plasma or serum) using 2 dimensional chromatography. Implementation of fused-core material in the second dimension of 2D chromatographic platform allowed improving S/N above 4 fold in comparison to traditional porous C18 column. Sample load onto the first dimension (5 μm) eliminates limitations of sample volume to be injected. The main limitation factor of the fused-core material–low loading capacity was overcome by using 2D chromatography. We emphasize importance of reduction of post-column (extra column) volume in LC/MS applications utilizing fused-core columns.

Modifying conventional high-performance liquid chromatography systems to achieve fast separations with Fused-Core columns: A case study

The theoretical increase in performance from the use of high efficiency columns with conventional HPLC equipment is generally not observed due to the design limitations of such equipment, particularly with respect to extra-column dispersion (ECD). This study examines the impact of ECD from a Waters Alliance 2695 system on the performance of 2.7 μm HALO ® C 18 Fused-Core superficially porous particle columns of various dimensions. The Alliance system was re-configured in different ways to reduce extra-column volume (ECV) and the ECD determined in each case as a function of flow rate up to a maximum of 2 mL/min. The results obtained showed a progressive decrease in ECD as the ECV was reduced, irrespective of the flow rate employed. However, this decrease in ECD was less than theoretically expected for the lower ECV configurations. The inability to reduce the actual extra-column dispersion further was attributed to additional dispersion associated with the design/volume of the auto-injector. This was confirmed by making sample injections with a low dispersion manual injection valve, instead of auto-injection, for the two lowest ECV configurations studied. In each case, the measured and predicted ECD values were in good agreement. The auto-injector module is an integral part of the Alliance 2695 instrument and cannot be easily modified. However, even with autosampler injection, for a 3 mm ID × 100 mm Fused-Core ® column approximately 70% of the maximum plate count (∼84% of the resolution or more) could still be obtained in isocratic separations for solutes with k ≥ ∼4.5 when using the lowest ECV configuration. This study also highlights some of the problems inherent in trying to measure accurately the true extra-column dispersion of a chromatographic system and compares the results obtained to those theoretically predicted. Using this same lowest volume instrument configuration, two real-world pharmaceutical methods were scaled to separations that are ∼3–3.5-fold faster, while still maintaining comparable data quality (resolution and signal-to-noise ratios).

Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0–3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.

Instrumental considerations for the effective operation of short, highly efficient fused-core columns. Investigation of performance at high flow rates and elevated temperatures

Fused core or superficially porous columns offer the advantages of higher efficiency compared with totally porous columns of the same particle size, but similar operating pressures. However, their performance may be adversely affected by extra-column effects that become more significant as the column efficiency increases, and as the diameter of the column is reduced. In this study, we show that 10 cm × 0.46 cm fused-core columns can be used on modified conventional instruments (“microbore systems”) without serious loss in performance, and this approach is at present likely to yield superior results compared with use of 0.21 cm columns (of identical efficiency) on current UHPLC instruments that have minimised extra-column volume. Furthermore, the true efficiency of commercial narrow-bore fused-core columns appears to be reduced compared with those of conventional bore, which may be due to packing difficulties for the former type. The fused-core columns in general gave excellent performance, showing no evidence of an upturn in the Knox plots at high flow velocities and elevated temperatures. Careful control of experimental conditions is necessary to ensure accurate data for these plots.

Tryptic digest analysis by comprehensive reversed phase×two reversed phase liquid chromatography (RP‐LC×2RP‐LC) at different pH's

As an alternative to the classical approach of combining strong cation exchange SCX-LC and RP-LC for the separation of complex proteomic samples, this essay describes the online comprehensive RP-LCxRP-LC separation of BSA and human blood serum. High orthogonality and peak capacity are achieved through the application of a significantly different pH in the two dimensions. The coupling of fused-core columns in series ensures high efficiency in the first dimension, while a previously designed interface with parallel second dimension columns further enhances the separation capability of the comprehensive system.