Petr Chocholouš

Fast simultaneous spectrophotometric determination of naphazoline nitrate and methylparaben by sequential injection chromatography

Fast simultaneous determination of naphazoline nitrate and methylparaben in pharmaceuticals using separation method based on a novel reversed-phase sequential injection chromatography (SIC) is described in this contribution as an alternative to classical HPLC. A Chromolithtrade mark Flash RP-18e (25mmx4.6mm) column (Merck((R)), Germany) and a FIAlab((R)) 3000 system (USA) with a six-port selection valve and 5.0ml syringe pump were used for sequential injection chromatographic separations in our study. The mobile phase used was methanol/water (40:65, v/v), pH 5.2 adjusted with triethylamine 0.8mulml(-1) and acetic acid, at flow rate 0.9mlmin(-1). UV detection provided by DAD detector and two wavelengths were simultaneously monitored for increasing sensitivity of determination. Detector was set up at 220nm for naphazoline nitrate and 256nm for methylparaben and ethylparaben (IS). There is no necessity to use pre-adjustment of sample of nasal drops (only dilution with mobile phase) so the time of the whole analysis is very short. The validation parameters have shown good results: linearity of determination for both components (naphazoline nitrate and methylparaben), correlation coefficient >0.999; repeatability of determination (R.S.D.) in the range 0.5-1.6% at three different concentration levels, detection limits 0.02mugml(-1) (naphazoline nitrate) and 0.20mugml(-1) (methylparaben and ethylparaben), and recovery from the pharmaceutical preparations in the range 100.06-102.55%. The chromatographic resolution between peaks of compounds was more than 4.0 and analysis time was less than 4min under the optimal conditions. The advantages and drawbacks of SIC against classical HPLC are discussed showing that SIC can be an advantageous alternative in many cases.

Ultra-fast separation of estrogen steroids using subcritical fluid chromatography on sub-2-micron particles

Estrogen steroids, represented by estradiol and its related substances, include both structurally very close and simultaneously different analogs. Their separation still remains an analytical challenge. Subcritical fluid chromatography (SbFC) on sub-2-micron particles was found to be an appropriate tool to obtain fast and efficient separation of nine target analytes. Among the four tested stationary phases charged hybrid modified with PFP (pentafluorophenyl) moiety was found to be the most convenient providing the fastest separation within 1.6 min using quick gradient elution with carbon dioxide and methanol as an organic modifier. However, complete separation was obtained also on other tested phases including bare hybrid stationary phase, hybrid stationary phase modified with 2-EP (2-ethylpyridine) and also C18, which is less typical in SbFC. The baseline separation on the latter columns was achieved by means of a temperature increase, a change in organic modifier type and gradient time increase respectively. Quantitative performance was evaluated at optimized conditions and method validation was accomplished. Excellent repeatability of both retention times (RSD<0.15%) and peak areas (RSD<1%) was observed. The method was linear in the range of 1.0-1000.0 μg/ml for all steroids with the lowest calibration point being an LOQ, except for Δ-derivatives, that provided better sensitivity and thus LOQ of 0.5 μg/ml. The sensitivity was sufficient for the analysis of real samples although it was still five times lower compared to UHPLC-UV experiments.

Enhanced capabilities of separation in Sequential Injection Chromatography--fused-core particle column and its comparison with narrow-bore monolithic column.

In the Sequential Injection Chromatography (SIC) only monolithic columns for chromatographic separations have been used so far. This article presents the first use of fused-core particle packed column in an attempt to extend of the chromatographic capabilities of the SIC system. A new fused-core particle column (2.7 μm) Ascentis(®) Express C18 (Supelco™ Analytical) 30 mm × 4.6 mm brings high separation efficiency within flow rates and pressures comparable to monolithic column Chromolith(®) Performance RP-18e 100-3 (Merck(®)) 100 mm × 3 mm. Both columns matches the conditions of the commercially produced SIC system - SIChrom™ (8-port high-pressure selection valve and medium-pressure Sapphire™ syringe pump with 4 mL reservoir - maximal work pressure 1000 PSI) (FIAlab(®), USA). The system was tested by the separation of four estrogens with similar structure and an internal standard - ethylparaben. The mobile phase composed of acetonitrile/water (40/60 (v/v)) was pumped isocratic at flow rate 0.48 mL min(-1). Spectrophotometric detection was performed at wavelength of 225 nm and injected volume of sample solutions was 10 μL. The chromatographic characteristics of both columns were compared. Obtained results and conclusions have shown that both fused-core particle column and longer narrow shaped monolithic column bring benefits into the SIC method.

A novel application of Onyx™ monolithic column for simultaneous determination of salicylic acid and triamcinolone acetonide by sequential injection chromatography

A novel and fast simultaneous determination of triamcinolone acetonide (TCA) and salicylic acid (SA) in topical pharmaceutical formulations by sequential injection chromatography (SIC) as an alternative to classical high performance liquid chromatography (HPLC) has been developed. A recently introduced Onyxtrade mark monolithic C18 (50mmx4.6mm, Phenomenex((R))) with 5mm monolithic precolumn were used for the first time for creating sequential injection chromatography system based on a FIAlab((R)) 3000 with a six-port selection valve and 5.0mL syringe pump in study. The mobile phase used was acetonitrile/water (35:65, v/v), pH 3.3 adjusted with acetic acid at flow rate 0.9mLmin(-1). UV detection provided by fibre-optic DAD detector was set up at 240nm. Propylparaben was chosen as suitable internal standard (IS). There is only simple pre-adjustment of the sample of topical solution (dilution with mobile phase) so the analysis is not uselessly elongated. Parameters of the method showed good linearity in wide range, correlation coefficient >0.999; system precision (relative standard deviation, R.S.D.) in the range 0.45-1.95% at three different concentration levels, detection limits (3sigma) 1.00mugmL(-1) (salicylic acid), 0.66mugmL(-1) (triamcinolone acetonide) and 0.33mugmL(-1) (propylparaben) and recovery from the pharmaceutical preparations in the range 97.50-98.94%. The chromatographic resolution between peaks of compounds was more than 4.5 and analysis time was 5.1min under the optimal conditions. The advantages of sequential injection chromatography against classical HPLC are discussed and showing that SIC can be a method of option in many cases.

Advantages of core-shell particle columns in Sequential Injection Chromatography for determination of phenolic acids.

Currently, for Sequential Injection Chromatography (SIC), only reversed phase C18 columns have been used for chromatographic separations. This article presents the first use of three different stationary phases: three core-shell particle-packed reversed phase columns in flow systems. The aim of this work was to extend the chromatographic capabilities of the SIC system. Despite the particle-packed columns reaching system pressures of ≤ 610 PSI, their conditions matched those of a commercially produced and optimised SIC system (SIChrom™ (FIAlab(®), USA)) with a 8-port high-pressure selection valve and medium-pressure Sapphire™ syringe pump with a 4 mL reservoir and maximum system pressure of ≤ 1000 PSI. The selectivity of each of the tested columns, Ascentis(®) Express RP-Amide, Ascentis(®) Express Phenyl-Hexyl and Ascentis(®) Express C18 (30 mm × 4.6mm, core-shell particle size 2.7 μm), was compared by their ability to separate seven phenolic acids that are secondary metabolite substances widely distributed in plants. The separations of all of the components were performed by isocratic elution using binary mobile phases composed of acetonitrile and 0.065% phosphoric acid at pH 2.4 (a specific ratio was used for each column) at a flow-rate of 0.60 mL/min. The volume of the mobile phase was 3.8 mL for each separation. The injection volume of the sample was 10 μL for each separation. The UV detection wavelengths were set to 250, 280 and 325 nm. The RP-Amide column provided the highest chromatographic resolution and allowed for complete baseline separation of protocatechuic, syringic, vanillic, ferulic, sinapinic, p-coumaric and o-coumaric acids. The Phenyl-Hexyl and C18 columns were unable to completely separate the tested mixture, syringic and vanillic acid and ferulic and sinapinic acids could not be separated from one another. The analytical parameters were a LOD of 0.3 mg L(-1), a LOQ of 1.0 mg L(-1), a calibration range of 1.0-50.0 (100.0) mg L(-1) (r>0.997) and a system precision of 10 mg L(-1) with a RSD ≤ 1.65%. The high performance of the chromatography process with the RP-Amide column under optimised conditions was highlighted and well documented (HETP values ≤ 10 μm, peak symmetry ≤ 1.33, resolution ≥ 1.87 and time for one analysis <8.0 min). The results of these experiments confirmed the benefits of extending chromatographic selectivity using core-shell particle column technology in a SIC manifold.

Simple automated generation of gradient elution conditions in sequential injection chromatography using monolithic column.

The paper deals with the concept of simple automated creation of gradient profile of the mobile phase for gradient-elution sequential injection chromatography (GE-SIC). The feasibility and merits of this concept are demonstrated on the separation and simultaneous assay of indomethacin as active principle and of its two degradation products (5-methoxy-2-methylindoleacetic acid and 4-chloro-benzoic acid) in a topical pharmaceutical formulation. The GE-SIC separation was performed with a FIAlab(®) 3000 SIC set-up (USA) equipped with an Onyx™ Monolithic C18 (25 mm × 4.6mm, Phenomenex(®)) column, a six-port selection valve, a 5-mL syringe pump and a fiber-optics UV CCD detector. Ketoprofen was used as an internal standard (IS). The gradient elution was achieved by automated reproducible mixing of acetonitrile and aqueous 0.2% phosphoric acid in the holding coil of the SIC system. Different profiles of the gradient elution were tested. The optimal gradient using two mobile phases 30:70 and 50:50 of acetonitrile/0.2% phosphoric acid (v/v) was achieved under the optimum flow rate 1.2 mL min(-1). The chromatographic resolution R between the peaks of all solutes (including the IS) was >2.00. The repeatability of retention times was characterized by the RSD values 0.18-0.30% (n=6). Net separation time was 3.5 min and the mobile phase consumption was 4.5 mL for a single GE-SIC assay. The figures of merit of the novel GE-SIC method compared well with those of conventional HPLC.

A novel dual-valve sequential injection manifold (DV-SIA) for automated liquid–liquid extraction. Application for the determination of picric acid

A novel dual-valve sequential injection system (DV-SIA) for online liquid-liquid extraction which resolves the main problems of LLE utilization in SIA has been designed. The main idea behind this new design was to construct an SIA system by connecting two independent units, one for aqueous-organic mixture flow and the second specifically for organic phase flow. As a result, the DV-SIA manifold consists of an Extraction unit and a Detection unit. Processing a mixture of aqueous-organic phase in the Extraction unit and a separated organic phase in the Detection unit solves the problems associated with the change of phases having different affinities to the walls of the Teflon tubing used in the SI-system. The developed manifold is a simple, user-friendly and universal system built entirely from commercially available components. The system can be used for a variety of samples and organic solvents and is simple enough to be easily handled by operators less familiar with flow systems. The efficiency of the DV-SIA system is demonstrated by the extraction of picric acid in the form of an ion associate with 2-[2-(4-methoxy-phenylamino)-vinyl]-1,3,3-trimethyl-3H-indolium reagent, with subsequent spectrophotometric detection. The suggested DV-SIA concept can be expected to stimulate new experiments in analytical laboratories and can be applied to the elaboration of procedures for the determination of other compounds extractable by organic solvents. It could thus form a basis for the design of simple, single-purpose commercial instruments used in LLE procedures.

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 0.60 mL min(-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 500 µL sample volume. The developed procedure showed analysis time <10.5 min, resolutions >1.83 with peak symmetry ≤1.52, LODs between 4.9 and 27 µg L(-1), linear response ranges from 30.0 to 1000.0 µg L(-1) (r(2)>0.996) and RSDs of peak heights <2.9% (n=6) at a 100 µg L(-1) level and enabled the screening control of freshwater samples contaminated at the 100 µg L(-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.

New ionophores for vitamin B1 and vitamin B6 potentiometric sensors for multivitaminic control

The construction, evaluation and analytical application of potentiometric sensors sensitive to vitamin B1 and vitamin B6 are reported. The solid contact electrodes were produced using beta-cyclodextrins as ionophores in a carboxylated poly(vinyl chloride) support matrix. Near Nernstian slopes (mV/decade) of 51.7+/-0.8, 60.6+/-0.6 and 61.1+/-1.4, within the intervals (M) of 1.0 x 10(-4) to 1.0 x 10(-1), 5.8 x 10(-5) to 1.0 x 10(-1) and 4.3 x 10(-5) to 1.0 x 10(-1) were obtained, for thiamine and pyridoxine I and II prepared membranes, respectively. A pH operational range of 6.5-8.5 for thiamine and 2-4.5 for pyridoxine electrodes was found. Assessment of selectivity coefficients toward a large number of inorganic cations and organic cations usually present in multivitamin formulations revealed good performance. Analysis of vitamins B1 and B6 in complex multivitamin drugs was achieved with recoveries within the intervals of 95.1-99.6% for thiamine and 95.1-102% for pyridoxine. Furthermore, the results enabled by the proposed procedure revealed good agreement with those provided by HPLC.

Sequential Injection Chromatography with post-column reaction/derivatization for the determination of transition metal cations in natural water samples

In this work, the applicability of Sequential Injection Chromatography for the determination of transition metals in water is evaluated for the separation of copper(II), zinc(II), and iron(II) cations. Separations were performed using a Dionex IonPAC™ guard column (50mm×2mm i.d., 9 µm). Mobile phase composition and post-column reaction were optimized by modified SIMPLEX method with subsequent study of the concentration of each component. The mobile phase consisted of 2,6-pyridinedicarboxylic acid as analyte-selective compound, sodium sulfate, and formic acid/sodium formate buffer. Post-column addition of 4-(2-pyridylazo)resorcinol was carried out for spectrophotometric detection of the analytes׳ complexes at 530nm. Approaches to achieve higher robustness, baseline stability, and detection sensitivity by on-column stacking of the analytes and initial gradient implementation as well as air-cushion pressure damping for post-column reagent addition were studied. The method allowed the rapid separation of copper(II), zinc(II), and iron(II) within 6.5min including pump refilling and aspiration of sample and 1mmol HNO3 for analyte stacking on the separation column. High sensitivity was achieved applying an injection volume of up to 90µL. A signal repeatability of<2% RSD of peak height was found. Analyte recovery evaluated by spiking of different natural water samples was well suited for routine analysis with sub-micromolar limits of detection.