Fused-core Particles Research Articles

Determination of psilocybin and psilocin content in multiple Psilocybe cubensis mushroom strains using liquid chromatography – tandem mass spectrometry

A method for clinical potency determination of psilocybin and psilocin in hallucinogenic mushroom species Psilocybe cubensis was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Five strains of dried, intact mushrooms were obtained and analyzed: Blue Meanie, Creeper, B-Plus, Texas Yellow, and Thai Cubensis. An extraction protocol was developed; this included an evaluation of sample milling technique, extraction solvents, and recovery/stability. Reversed phase chromatography on fused-core particle phases was developed for the determination of the two analytes using internal standard calibration with deuterated isotopologues of each analyte. The separation takes less than 5 min. Matrix effects were investigated by comparing signal response of calibration samples in neat solution and several mushroom matrices; no significant matrix effects were observed. The limit of detection for psilocybin was 1.5 ng/mL (1.5 pg on-column; 300 ng/g mushroom) and for psilocin was 0.15 ng/mL (0.15 pg on-column; 30 ng/g mushroom) using a Shimadzu LCMS-8050 triple quadrupole mass spectrometer. Assessment of the accuracy and precision of the method indicated percent error and RSD were <6 % at all concentration levels. Three whole, intact mushrooms from each strain were analyzed individually to obtain average content differences both between strains and between mushrooms of the same strain. From most to least potent, the study found that the average total psilocybin and psilocin concentrations for the Creeper, Blue Meanie, B+, Texas Yellow, and Thai Cubensis strains were 1.36, 1.221, 1.134, 1.103, and 0.879 % (w/w), respectively. A subset of these mushrooms was also tested in a separate non-affiliated laboratory, and the results were comparable between the two laboratories. Results from the secondary laboratory showed improved precision when multiple mushrooms were homogenized together, prior to extraction.

Ultra high efficiency/low pressure supercritical fluid chromatography (UHE/LP-SFC) for triglyceride analysis: Identification, quantification, and classification of vegetableoils.

Triglycerides of vegetable oils have been extensively studied. Non-aqueous reversed-phase liquid chromatography and silver-ion chromatography are most frequently used to achieve their separation. In previous works, we presented the use of supercritical fluid chromatography with long columns (75cm) packed with fused-core particles to provide ultra-high-performance separations, with a low-toxicity fluid (carbon dioxide) compared to the usual liquid-phase methods. In the present paper, we describe the quantification of triglycerides with supercritical fluid chromatography and evaporative light-scattering detection. Thanks to the isocratic elution mode, this quantification can be simplified, assuming (a) identical response coefficients for compounds having a close structure, (as only triglycerides are quantified), and (b) constancy of the response coefficient along the analysis (no elution gradient). Therefore, the relative concentrations of triglycerides were easily assessed. Only one calibration curve for one reference compound (in this case triolein) was required. The resulting relative concentrations are in good accordance with the numerous publications available. Relative quantification with UV detection at 210nm is also proposed, facilitated by the very low UV absorption of carbon dioxide and with a calibration curve taking account of the variation of UV response according to double bond number. Nineteen vegetable oils are compared. The identification of triglycerides was carried out based on previous knowledge of these oils, but also with the help of a Goiffon retention diagram, based on the relationship between the logarithm of retention factor and the total double bond number. Finally, cluster analyses were computed, based on evaporative light-scattering detection or UV quantification data. They allow a quick comparison of the triglyceride content between the oils, in the goal to exchange one by the other for certain applications, or to compare a new oil to well-known ones.

Development And Validation of a LC-MS Compatible Method for Quantification of Degradation Impurities of Clofazimine Using UHPLC

Background:Clofazimine has antibacterial and leprostatic properties, which has its use in Multidrug Therapy (MDT) of leprosy. As per the FDA guidance for industry, each NDA and ANDA must include the analytical procedures necessary to ensure the identity, strength, quality, purity, and potency of the drug substance and drug product. However, it was noticed that no stability indicating method is available in the literature for the estimation of degradation impurities of Clofazimine.Objective:Objective of the proposed work is to develop and validate a rapid, specific, linear, robust, accurate and sensitive Ultra High-Performance Liquid Chromatography (UHPLC) method with LC-MS compatible mobile phase for the quantification of degradation impurities of Clofazimine in a pharmaceutical dosage form (topical gel 0.5% w/w).Methods:Ultra High-Performance Liquid Chromatography equipped with PDA and Tunable UV (TUV) detector at a wavelength of 284 nm, stationary phase with a fused core particle technology, LC-MS compatible mobile phase was employed in this study. Gradient elution was employed for ensuring the selectivity of degradation impurities and clofazimine. This method was validated in accordance with ICH Q2 guidelines. This is the first reported Ultra High-Performance Liquid Chromatography method for estimation of degradation impurities of clofazimine.Results:The method showed good linearity over the range of 0.25 -1.5μg/ml of clofazimine. All the validation parameters were within the acceptance criteria. The product is found to degrade in the acid and peroxide degradation condition. The major degradant impurities are eluted at relative retention times of 0.35, 0.89 and 0.95. The developed method successfully separated the degradation products of clofazimine and able to quantitate accurately in its formulation.Conclusion:To date, there is no UHPLC method for determination of degradation impurities of clofazimine. in pharmaceutical dosage forms. Being a specific, linear, accurate and robust method, this would help in determining the chemical stability of drug product during the product development as well as in the shelf life of the drug product.

Identification and quantitation of related substances of super potent steroid in its topical combination drug product with vitamin D3 analogue

Clobetasol propionate is an internationally accepted common name for 21-chloro-9-fluoro-11beta,17-dihydroxy-16beta-methylpregna-1,4-diene-3,20-dione 17-propionate. The combination of calcipotriol (vitamin D3 analogue) and clobetasol propionate (super potent steroid) is being used as a topical formulation for the treatment of psoriasis from several years. In this work an ultra-high performance liquid chromatography equipped with photodiode array detector and a mass compatible mobile phase in a gradient elution is employed for the separation of five related substances in presence of calcipotriol and its two impurities with a last eluting impurity in less than 11 min. A simple and efficient sample extraction procedure was developed to achieve the highest sensitivity as of today with limit of detection (LOD) and limit of quantitation (LOQ) of 0.03 % and 0.10 % respectively as required by industry for drug product of clobetasol propionate and its combination products. Stationary phase with fused core particle technology is employed for the separation of impurities. Precision of the method is found to be less than 1.0 % RSD. The correlation coefficient is > 0.999. Accuracy of method is ranged from 93.3 % to 108.0 %. This is the first reported UHPLC method for the estimation of five related substances of clobetasol propionate in its combination product with calcipotriol.

Ultrasonic-Assisted Sol–Gel Synthesis of Core–Shell Silica Particles for High-Performance Liquid Chromatography

Core–shell particles (CSPs), also known as superficially porous particles or fused-core particles, consisting of a solid core and a porous shell, have emerged as an advanced technology in high-performance liquid chromatography (HPLC). We report here an ultrasonic-assisted sol–gel method for the fabrication of CSPs with uniform mesoporous silica shell using tetraethoxysilane as the silica source and dodecylamine as a catalyst, template and porogen agent in a methanol–water solution. Instead of stirring, sonication was adopted to guarantee the particles monodispersity and uniformity. The shell thickness between 300 and 700 nm could be obtained by adjusting the sonication time and the volume ratio of methanol–water. Pore sizes from 3.4 to 8.5 nm were tuned by hydrothermal treatment. The C18-bonded 2.7 μm CSPs with a shell thickness of ~ 500 nm showed some advantages over a commercial 1.7 μm BEH column, and successfully applied for the analyses of a Chinese medicine and peptides. The CSPs could be a promising candidate as HPLC packing materials.

New generation of sequential injection chromatography: Great enhancement of capabilities of separation using flow analysis

Since its inception, sequential injection chromatography (SIC) has evolved through several stages. Key moments including introduction of the novel technique combining sequential injection analysis and monolithic column, the first generation of commercial SIC system employing robust pump, the utilization of columns packed with fused-core particles, the on-line hyphenation of extraction and separation steps in SIC, are now followed by the second generation of commercial SIC system employing stainless steel syringe pump and parts optimized for chromatographic separation. The key developments always mean acceleration of the evolution by opening new avenues and reduction of compromises in automated analytical methods based on the flow analysis. The updates, new features, and prospects of the novel instrument are described and discussed on perspective of the method developed for extraction and separation of selected phenolic acids (gallic, protocatechuic, caffeic, p-coumaric and ferulic). The method hyphenates miniaturized on-line solid phase extraction using strong anion exchange sorbent in commercial cartridge for HPLC (20 × 1 mm) and liquid chromatography using chromatographic column (C18 50 × 4.6 mm, 5 μm particles) packed with fused-core particles in the SIC manifold. The separation in gradient mode used acetonitrile: aqueous formic acid pH 2.0 mobile phase and spectrophotometric detection at 270, 300, and 320 nm. Injected sample volumes were 200 and 500 μL. The performance of the extraction step was characterized by the recovery 94.0–107.8%, enrichment factors about 20 or 50, and the separation by peak capacities 13–34, peak symmetries 1.17–1.64, and resolutions 0.82–3.75). While using a sample volume of 200 μL, our method was characterized by the following validation parameters: LODs of 0.0075–0.03 mg L−1, LOQs of 0.025–0.10 mg L−1, calibration ranges 0.025–2.50 mg L−1 (r > 0.999), repeatability of signal at 0.50 mg L−1of RSD ≤ 1.46% (n = 6), and overall time of analysis 7.1 min. The results including pilot analysis of white and red wines demonstrated the capability of novel SIC instrument to enable fast, selective, and sensitive analysis.

Quantification and In Silico Toxicity Assessment of Tazarotene and its Impurities for a Quality and Safe Drug Product Development.

Tazarotene is internationally accepted common name for ethyl 6-[(4,4-dimethylthiochroman-6-yl)ethynyl]nicotinate. It is a synthetic retinoid used for the topical treatment of mild to moderate plaque psoriasis, acne vulgaris and photo aging. To ensure the quality of drug product and drug substance, a LC-MS compatible UHPLC method was developed for quantification of drug and its related substances. Stationary phase with fused core particle technology is used for the separation of impurities. Limit of quantification and limit of detection of the method are 0.1 and 0.03%, respectively. Precision of the method for Tazarotene and all its related substances is less than 2.2% RSD. The correlation coefficient is >0.999. Accuracy of method is ranged from 95.3% to 107.0%. Application of this method in stability analysis has been demonstrated by analyzing stressed samples. Experimental design is used for the verification of robustness of the method. To ensure the safety, an in silico toxicity of the drug and its related substances were determined using TOPKAT and DEREK toxicity predictions Both UHPLC and in silico methods were validated as per the ICH Q2 and ICH M7 guidelines, which will enable a rapid product development of Tazarotene topical formulations while ensuring the safety and quality of product.

A validated UHPLC method for the determination of caffeoylquinic and di-caffeoylquinic acids in green coffee extracts using an RP-Amide fused-core column

The presented work describes the development and validation of a rapid UHPLC-UV method using a fused core particle column with an RP-Amide stationary phase for the separation and quantitative analysis of caffeoylquinic and di-caffeoylquinic acids in green coffee extracts. Three caffeoylquinic acids (3-caffeoylquinic acid, 4-caffeoylquinic acid, and 5-caffeoylquinic acid) and two di-caffeoylquinic acids (1,3-di-caffeoylquinic acid, and 3,5-di-caffeoylquinic acid) were separated and analyzed in 8 min. That was possible due to the unique selectivity of the RP-Amide stationary phase for the analyzed acids. The retention behavior of all analytes under different compositions of the mobile phase on different columns was evaluated in this study. The optimal chromatographic separation was performed using an Ascentis Express RP-Amide (100 × 2.1 mm) fused-core column with a particle size of 2.7 μm at a temperature of 30 °C. For validation of the newly developed method, acetonitrile was used as mobile phase B and 5% formic acid, filtrated through a 0.22 μm filter, was used as mobile phase A. They were delivered at a flow rate of 0.9 mL min−1 according to the elution gradient program. The detection wavelength was set at 325 nm. A solid-liquid extraction with a solution of methanol and a 5% water solution of formic acid (25 + 75 v/v) using an ultrasonic bath was chosen for the preparation of the available commercial samples of food supplements containing a green coffee extract. Recoveries for all analyzed acids were 98.2–101.0% and the relative standard deviation ranged from 0.3% to 1.4% for intra-day and from 0.3% to 3.0% for inter-day repeatability. The limits of detection were in the range of 0.30–0.53 μg mL−1.

Rapid Simultaneous Separation of Four Oral Antidiabetic Drugs and Quantitative Determination of Glibenclamide Using Conventional and Fused-Core Silica Columns.

Diabetes mellitus is a chronic disease with high and growing prevalence worldwide. Therefore, the development of fast and efficient methods for the QC of antidiabetic drugs is of fundamental importance. Two ultra-fast methods, using a conventional (C18 100 × 2.1 mm, 5 μm fully porous particle) column or a fused-core (C18 100 × 2.1 mm, 2.7 μm fused-core particle) column, were developed for the simultaneous determination of four antidiabetic drugs (chlorpropamide, glibenclamide, gliclazide, and glimepiride). The developed methods were compared in terms of efficiency, speed of analysis, resolution, and peak symmetry. Both methods were validated with respect to selectivity, system suitability, linearity, precision, accuracy, LOD, LOQ, and robustness, using glibenclamide as model. Conventional and fused-core methods were shown to be appropriate for the determination of glibenclamide in tablets; however, the fused-core column presented higher efficiency, detectability, and resolution. Also, it enabled faster analysis, with separation of the four drugs in less than 1 min.

Ultrafast Chiral Chromatography as the Second Dimension in Two-Dimensional Liquid Chromatography Experiments.

Chromatographic separation and analysis of complex mixtures of closely related species is one of the most challenging tasks in modern pharmaceutical analysis. In recent years, two-dimensional liquid chromatography (2D-LC) has become a valuable tool for improving peak capacity and selectivity. However, the relatively slow speed of chiral separations has limited the use of chiral stationary phases (CSPs) as the second dimension in 2D-LC, especially in the comprehensive mode. Realizing that the recent revolution in the field of ultrafast enantioselective chromatography could now provide significantly faster separations, we herein report an investigation into the use of ultrafast chiral chromatography as a second dimension for 2D chromatographic separations. In this study, excellent selectivity, peak shape, and repeatability were achieved by combining achiral and chiral narrow-bore columns (2.1 mm × 100 mm and 2.1 mm × 150 mm, sub-2 and 3 μm) in the first dimension with 4.6 mm × 30 mm and 4.6 mm × 50 mm columns packed with highly efficient chiral selectors (sub-2 μm fully porous and 2.7 μm fused-core particles) in the second dimension, together with the use of 0.1% phosphoric acid/acetonitrile eluents in both dimensions. Multiple achiral × chiral and chiral × chiral 2D-LC examples (single and multiple heart-cutting, high-resolution sampling, and comprehensive) using ultrafast chiral chromatography in the second dimension are successfully applied to the separation and analysis of complex mixtures of closely related pharmaceuticals and synthetic intermediates, including chiral and achiral drugs and metabolites, constitutional isomers, stereoisomers, and organohalogenated species.

High-Throughput Serum 25-Hydroxy Vitamin D Testing with Automated Sample Preparation.

Serum from bar-coded tubes, and then internal standard, are pipetted to 96-well plates with an 8-channel automated liquid handler (ALH). The first precipitation reagent (methanol:ZnSO4) is added and mixed with the 8-channel ALH. A second protein precipitating agent, 1 % formic acid in acetonitrile, is added and mixed with a 96-channel ALH. After a 4-min delay for larger precipitates to settle to the bottom of the plate, the upper 36 % of the precipitate/supernatant mix is transferred with the 96-channel ALH to a Sigma Hybrid SPE(®) plate and vacuumed through for removal of phospholipids and precipitated proteins. The filtrate is collected in a second 96-well plate (collection plate) which is foil-sealed, placed in the autosampler (ALS), and injected into a multiplexed LC-MS/MS system running AB Sciex Cliquid(®) and MPX(®) software. Two Shimadzu LC stacks, with multiplex timing controlled by MPX(®) software, inject alternately to one AB Sciex API-5000 MS/MS using positive atmospheric pressure chemical ionization (APCI) and a 1.87 min water/acetonitrile LC gradient with a 2.1 × 20 mm, 2.7 μm, C18 fused core particle column (Sigma Ascentis Express). LC-MS/MS through put is ~44 samples/h/LC-MS/MS system with dual-LC channel multiplexing. Plate maps are transferred electronically from the ALH and reformatted into LC-MS/MS sample table format using the Data Innovations LLC (DI) Instrument Manager middleware application. Before collection plates are loaded into the ALS, the plate bar code is manually scanned to download the sample table from the DI middleware to the LC-MS/MS. After acquisition-LC-MS/MS data is analyzed with AB Sciex Multiquant(®) software using customized queries, and then results are transferred electronically via a DI interface to the LIS. 2500 samples/day can be extracted by two analysts using four ALHs in 4-6 h. LC-MS/MS analysis of those samples on three dual-channel LC multiplexed LC-MS/MS systems requires 19-21 h and data analysis can be done by two analysts in 4-6 h.

Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine

A liquid chromatography–tandem mass spectrometry method was developed and used to simultaneously detect 19 small peptide hormones prohibited in sport and their main metabolites in urine after solid-phase extraction. Detection was achieved using a triple-quadrupole mass spectrometric detector coupled with an electrospray ionization interface after chromatographic separation with an octadecyl column based on fused-core particle technology. Sample pretreatment was performed by solid-phase extraction. The extraction procedure was optimized by comparison of different sorbents and washing/elution protocols. The best results were obtained using a mixed-mode weak cation exchange sorbent, two washing steps (ultrapurified water and methanol), and elution using 300 mM ammonium formate in 25 % ammonia/methanol (10/90) or 25 % ammonia/10 % formic acid/methanol (8/12/80). The procedure was validated in terms of sensitivity (lower limits of detection: 0.05–2.0 ng/ml depending on the target analyte), specificity, recovery [>60 %, coefficient of variation (CV) <15 % except for TB500 17–23 fragment, AOD9604, and ARA290 for which recovery was <50 %), ion suppression/enhancement (<35 %), robustness, carryover, stability of the target analytes [stable for at least for 2 days (25 °C, 2 weeks (4 °C), 2 months (−20 °C)], and repeatability of retention times (CV <0.1 %) and relative abundances of the selected ion transitions (CV <15 %). The suitability of the method was confirmed by analyzing spiked and excreted urines, the latter collected after intravenous injection of 0.1 mg of GHRP-2.

HPLC Method for Simultaneous Determination of Potential Impurities of Norgestimate and Ethinyl Estradiol in Combination Drug Product Using Sub-2 µm Column

This paper describes a strategic development and validation of stability-indicating high-performance liquid chromatography (HPLC) method for the determination of potential impurities present in highly potent and low dose combination drug product of Norgestimate and Ethinyl estradiol tablets. Effective and faster separation of impurities from the drug products was achieved on a sub-2 µm fused core particle C8 column (150 mm × 4.6 mm i.d.) using water and acetonitrile as mobile phase in gradient pump mode. Flow rate was selected 1.0 mL/min with a detection quantification wavelength of 230 nm. The calibration curve obtained from linearity data (r > 0.995) in the relevant ranges (up to 150% of the expected concentration of the analytes in the test concentration), accuracy (>95% recovery for the impurities of respective analytes), method precision and intermediate precision (RSD < 5.0%), specificity (purity is less than purity threshold for all potential impurities present in respective drug components), and method robustness parameters were evaluated, and system suitability parameters were determined. The validated HPLC method was successfully applied to the quantification of impurities in Norgestimate and Ethinyl estradiol tablets helping to improve quality control and to assure therapeutic efficacy.

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.

Use of Fast HPLC Multiple Reaction Monitoring Cubed for Endogenous Retinoic Acid Quantification in Complex Matrices

Retinoic acid (RA), an essential active metabolite of vitamin A, controls numerous physiological processes. In addition to the analytical challenges owing to its geometric isomers, low endogenous abundance, and often localized occurrence, nonspecific interferences observed during liquid chromatography (LC) multiple reaction monitoring (MRM) quantification methods have necessitated lengthy chromatography to obtain accurate quantification free of interferences. We report the development and validation of a fast high performance liquid chromatography (HPLC) multiplexing multiple reaction monitoring cubed (MRM(3)) assay for selective and sensitive quantification of endogenous RA from complex matrices. The fast HPLC separation was achieved using an embedded amide C18 column packed with 2.7 μm fused-core particles which provided baseline resolution of endogenous RA isomers (all-trans-RA, 9-cis-RA, 13-cis-RA, and 9,13-di-cis-RA) and demonstrated significant improvements in chromatographic efficiency compared to porous particle stationary phases. Multiplexing technology further enhanced sample throughput by a factor of 2 by synchronizing parallel HPLC systems to a single mass spectrometer. The fast HPLC multiplexing MRM(3) assay demonstrated enhanced selectivity for endogenous RA quantification in complex matrices and had comparable analytical performance to robust, validated LC-MRM methodology for RA quantification. The quantification of endogenous RA using the described assay was validated on a number of mouse tissues, nonhuman primate tissues, and human plasma samples. The combined integration of fast HPLC, MRM(3), and multiplexing yields an analysis workflow for essential low-abundance endogenous metabolites that has enhanced selectivity in complex matrices and increased throughput that will be useful in efficiently interrogating the biological role of RA in larger study populations.

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.

HPLC determination of serum pteridine pattern as biomarkers

Pteridinic derivatives are important biomolecules considered as biomarkers for several diseases, especially in cancer and infectious pathologies. A new fluorimetric-HPLC method for the analysis of nine pteridines in human serum has been reported. Two analytical columns composed by C18 porous and fused core particles were assayed and the results compared. Fused core particle column allows us adequate separation, in only one run and in 15min. Acid precipitation step of the proteins and clean-up process with an Isolute ENV+ (hydroxylated polystyrene-divinylbenzene copolymer) cartridge of the serum samples have been optimized. Analytes were determined by fluorimetric detection, exciting at 272nm and measuring the fluorescence emission at 410nm for isoxanthopterin, at 465nm for xanthopterin, and at 445nm for the analysis of the other pteridines. Detection limits between 0.07 and 0.61ngmL−1 were calculated according to Clayton criterium. Intraday precision varied from 1.2 to 5.3 and interday precision between 1.2 and 7.4, both expressed as RSD (%). External standard and standard addition calibrations were compared in the analysis of serum samples. The pteridine amounts in serum (expressed as ngmL−1±confidence interval) were 3.69±1.78; 1.35±0.24; 0.46±0.14; 0.54±0.24; 0.84±0.55; 2.10±0.51 and 0.23±0.11 for XAN, NEO, MON, ISO, BIO and 6HMPT, respectively, using the external standard method. Comparable results were obtained by the standard addition method. It is noticeable that 7BIO was not detected in the healthy serum samples analyzed.

Degradation study of nitroaromatic explosives 2-diazo-4,6-dinitrophenol and picramic acid using HPLC and UHPLC-ESI-MS/MS

A new and fast HPLC method with UV detection was developed and validated for the simultaneous determination of nitroaromatic explosives diazodinitrophenol (DDNP), the raw material picramic acid (PicA) and three degradation products of DDNP. DDNP and PicA were subjected to forced degradation. DDNP was found to be stable under acidic and basic conditions, while thermal, oxidative and photolytic decomposition resulted in three degradation products. Degradation products were separated from DDNP using Ascentis® Express C18 fused core particle column and subsequently characterised using UHPLC-ESI-MS/MS method. Optimal chromatography was performed using the isocratic elution of mobile phase consisting of acetonitrile–orthophosphoric acid 0.085% 30 : 70 (v/v) pumped at a flow rate of 0.8 mL min−1 at a temperature of 50 °C. System suitability test parameters, linearity and precision of the HPLC method were evaluated. The new HPLC method is suitable for a fast determination of DDNP and degradation products in various samples. Our work provides a basic view on the stability and aspects of degradation process of nitroaromatics DDNP and PicA, and shows the formation of degradation products.