Petr Solich

Advantages of application of UPLC in pharmaceutical analysis

Ultra Performance Liquid Chromatography (UPLC) is a relatively new technique giving new possibilities in liquid chromatography, especially concerning decrease of time and solvent consumption. UPLC chromatographic system is designed in a special way to withstand high system back-pressures. Special analytical columns UPLC Acquity UPLC BEH C(18) packed with 1.7 microm particles are used in connection with this system. The quality control analyses of four pharmaceutical formulations were transferred from HPLC to UPLC system. The results are compared for Triamcinolon cream containing trimacinolone acetonide, methylparaben, propylparaben and triamcinolone as degradation product, for Hydrocortison cream (hydrocortisone acetate, methylparaben, propylparaben and hydrocortisone degradation product), for Indomethacin gel (indomethacin and its degradation products 4-chlorobenzoic acid and 5-methoxy-2-methylindoleacetic acid) and for Estrogel gel (estradiol, methylparaben, propylparaben and estrone as degradation product). The UPLC system allows shortening analysis time up to nine times comparing to the conventional system using 5 microm particle packed analytical columns. In comparison with 3 microm particle packed analytical columns analysis should be shortened about three times. The negative effect of particle decrease is back-pressure increase about nine times (versus 5 microm) or three times (versus 3 microm), respectively. The separation on UPLC is performed under very high pressures (up to 100MPa is possible in UPLC system), but it has no negative influence on analytical column or other components of chromatographic system. Separation efficiency remains maintained or is even improved. Differences and SST parameters, advantages and disadvantages of UPLC are discussed.

An overview of analytical methodologies for the determination of antibiotics in environmental waters

The widespread occurrence of antibiotics as contaminants in the aquatic environment has increased attention in the last years. The concern over the release of antibiotics into the environment is related primarily to the potential for the development of antimicrobial resistance among microorganisms. This article presents an overview of analytical methodologies for the determination of quinolone (Qs) and fluoroquinolone (FQs), macrolide (MLs), tetracycline (TCs), sulfonamide (SAs) antibiotics and trimethoprim (TMP) in different environmental waters. The analysis of these antibiotics has usually been carried out by high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) and to a lesser extent by ultraviolet (UV) or fluorescence detection (FD). A very important step before LC analysis is sample preparation and extraction leading to elimination of interferences and prevention of matrix effect and preconcentration of target analytes.

Analysis of phenolic compounds by high performance liquid chromatography and ultra performance liquid chromatography

Two novel chromatographic methods both based on utilization of sub-2-micron particle columns were developed for the analysis of phenolic compounds in this work. An HPLC system was equipped with C(18) silica-based analytical column (50 mm x 4.6 mm, 1.8 microm) and a UPLC system with ethylene-bridged hybrid C(18) analytical column (100 mm x 2.1 mm, 1.7 microm). In total 34 phenolic substances were divided into groups of phenolic acids, flavonoids, catechins and coumarins and were analysed in sequence using different gradient methods. System suitability test data, including repeatability of retention time and peak area, mean values of asymmetry factor, resolution, peak capacity and the height equivalent of a theoretical plate were determined for each gradient method by 10 replicate injections. The developed methods were applied in the analysis of real samples (grape wines, teas).

Automated flow-injection spectrophotometric determination of catecholamines (epinephrine and isoproterenol) in pharmaceutical formulations based on ferrous complex formation

A novel automated flow-injection spectrophotometric method for the determination of catecholamines (epinephrine and isoproterenol) has been developed based on the formation of their coloured complexes with Fe(II) in aminoacetic-carbonate buffer pH 8.3 and measuring of the absorbance peaks at the lambda(max) of 530 nm. A fully automated FIA system controlled by home-made software (FIA-MOD) was used for optimising the chemical and manifold parameters and running of routine measurements. The calibration graph was linear in the range of 5-200 mg l(-1) for epinephrine with an RSD of 0.24% (n = 5; c = 150 mg l(-1)) and 10-300 mg(-1) for isoproterenol with an RSD of 0.13% (n = 5; c = 200 mg l(-1)). Measurement throughput was 120 h(-1) ensuring a sample throughput of 40 h(-1) analysed in triplicate. Common excipients for tablets and injections were found not interfering. The proposed method was applied for the assay of various commercial pharmaceutical formulations containing epinephrine and isoproterenol and for the content uniformity test for the isoproterenol tablets. The assay results with RSD 2-4% (n = 3) were comparable with those obtained with the official USP XXIII methods (mean difference 1.9%).

Determination of steroid hormones in biological and environmental samples using green microextraction techniques: An overview

Residues of steroid hormones have become a cause for concern because they can affect the biological activity of non-target organisms. Steroid hormones are a potential risk for wildlife and humans through the consumption of contaminated food or water. Their determination requires extraction and clean-up steps, prior to detection, to reach low concentration levels. In recent years, a great effort has been made to develop new analytical methodologies, such as microextraction techniques, that reduce environmental pollution. Researchers have modified old methods to incorporate procedures that use less-hazardous chemicals or that use smaller amounts of them. They are able to do direct analysis using miniaturised equipment and reduced amounts of solvents and wastes. These accomplishments are the main objectives of green analytical chemistry. In this overview, we focus on microextraction techniques for the determination of steroid hormones in biological (e.g., human urine, human serum, fish, shrimp and prawn tissue and milk) and environmental (e.g., wastewaters, surface waters, tap waters, river waters, sewage sludges, marine sediments and river sediments) samples. We comment on the most recent applications in sorptive-microextraction modes, such as solid phase microextraction (SPME) with molecularly imprinted polymers (MIPs), in-tube solid-phase microextraction (IT-SPME), stir-bar sorptive extraction (SBSE) and microextraction in packed sorbent (MEPS). We also describe liquid-phase microextraction (LPME) approaches reported in the literature that are applied to the determination of steroid hormones.

Development and application of UHPLC–MS/MS method for the determination of phenolic compounds in Chamomile flowers and Chamomile tea extracts

UHPLC-MS/MS method using BEH C18 analytical column was developed for the separation and quantitation of 12 phenolic compounds of Chamomile (Matricaria recutita L.). The separation was accomplished using gradient elution with mobile phase consisting of methanol and formic acid 0.1%. ESI in both positive and negative ion mode was optimized with the aim to reach high sensitivity and selectivity for quantitation using SRM experiment. ESI in negative ion mode was found to be more convenient for quantitative analysis of all phenolics except of chlorogenic acid and kaempherol, which demonstrated better results of linearity, accuracy and precision in ESI positive ion mode. The results of method validation confirmed, that developed UHPLC-MS/MS method was convenient and reliable for the determination of phenolic compounds in Chamomile extracts with linearity >0.9982, accuracy within 76.7-126.7% and precision within 2.2-12.7% at three spiked concentration levels. Method sensitivity expressed as LOQ was typically 5-20 nmol/l. Extracts of Chamomile flowers and Chamomile tea were subjected to UHPLC-MS/MS analysis. The most abundant phenolic compounds in both Chamomile flowers and Chamomile tea extracts were chlorogenic acid, umbelliferone, apigenin and apigenin-7-glucoside. In Chamomile tea extracts there was greater abundance of flavonoid glycosides such as rutin or quercitrin, while the aglycone apigenin and its glycoside were present in lower amount.

Ultra high performance liquid chromatography tandem mass spectrometric detection in clinical analysis of simvastatin and atorvastatin.

Simvastatin and atorvastatin belong to the group of hypolipidemic drugs, more exactly to the second generation of inhibitors of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. They induce a significant reduction in total cholesterol, low-density lipoprotein cholesterol and plasma triglycerides, therefore they are widely used in the treatment of hypercholesterolemia even of its severe form-familiar hypercholesterolemia. Simvastatin and atorvastatin as the most widely used statins in clinical treatment and their hydroxy-acid/lactone forms were determined by means of UPLC in connection with triple quadrupole mass spectrometer. Deuterium labeled reference standard compounds were used as internal standards for the quantitation. Separation was performed on Acquity BEH C18 (100 mm x 2.1 mm, 1.7 microm) using gradient elution by mobile phase containing acetonitrile and ammonium acetate pH 4.0, which is convenient in order to prevent interconversion of analytes. ESI in positive mode was used for the ionization of all compounds. Two SRM (selected reaction monitoring) transitions were carefully optimized for each analyte in order to get high sensitivity and selectivity. SPE on Discovery DSC-18 was used as a sample preparation step. Intra-day precision was generally within 10% RSD, while inter-day precision within 15% RSD. Method accuracy expressed as recovery ranged from 75 to 100%. The method was validated with the sensitivity reaching LOQ 0.08-5.46 nmol/l and LOD 0.01-1.80 nmol/l in biological samples. Atorvastatin, simvastatin, its metabolites and hydroxy-acid/lactone forms were monitored in human serum and in lipoprotein fractions (LDL, HDL and VLDL) at patients with end stage renal diseases.

Reversed-phase porous silica rods, an alternative approach to high-performance liquid chromatographic separation using the sequential injection chromatography technique.

A commercially available porous silica rod column was used as a separation tool for the sequential injection analysis (SIA). A porous solid monolithic column showed high performance at a low pressure, allowing sequential injection analysis to be used for the first time for separation in HPLC fashion. In this contribution, we tried to demonstrate a new separation concept with SIA manifold for the simultaneous determination of four different compounds (methylparaben (MP), propylparaben (PP), triamcinolone acetonide (TCA) and internal standard ketoprofen (KP)) in a pharmaceutical triamcinolon cream 0.1% formulation. A Chromolith Flash RP-18e, 25 mm x 4.6 mm column with a 10 mm pre-column (Merck, Germany) and a FIAlab 3000 system (USA) with an 8-port selection valve and 10 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-methanol-water (35:5:65, v/v/v) + 0.05% nonylamine, pH 2.5, flow rate 0.6 ml min(-1). The analysis time was <6 min. A novel sequential injection chromatography (SIC) technique with UV spectrophotometric detection was optimised and validated.

Rapid qualitative and quantitative ultra high performance liquid chromatography method for simultaneous analysis of twenty nine common phenolic compounds of various structures

Twenty nine phenolic compounds comprising nine phenolic acids, sixteen flavonoids (including eight tea catechins, glycosides and aglycones), four coumarins plus caffeine were analysed within 20 min using ultra high performance liquid chromatography (UHPLC) with PDA detection. UHPLC system was equipped with C18 analytical column (100 mm x 2.1mm, 1.7 microm), utilising 0.1% formic acid and methanol mobile phase in the gradient elution mode. The developed method was tested for the system suitability: resolution, asymmetry factor, peak capacity, retention time repeatability and peak area repeatability. The method was fully validated in the terms of linearity (r(2)>0.9990 for all 30 compounds), range (typically 1-100 mg L(-1)), LOD, LOQ, inter/intra-day precision (<3% and <9% respectively) and inter/intra-day accuracy (typically 100+/-10%). Subsequently the method was applied to the identification (spectral information and peak purity calculations were profited) and quantification of phenolic compounds and caffeine present in tea infusions and extracts.

Simultaneous determination of methylparaben, propylparaben, sodium diclofenac and its degradation product in a topical emulgel by reversed-phase liquid chromatography

Abstract A novel reversed-phase liquid chromatographic method with UV spectrophotometric detection was developed and validated for the determination of compounds in topical emulgel. The method describes determination of active component sodium diclofenac, its degradation product 1-(2,6-dichlorophenyl)-indolin-2-one (occurring in formulation after long-term stability tests) and two preservatives presented in the emulgel, methylparaben and propylparaben, using flurbiprofen as an internal standard. The chromatographic separation was performed on a SUPELCO Discovery C18 column; the mobile phase for separation of all the compounds was methanol/phosphate buffer, pH 2.5 (65:35, v/v). The analysis time was