Lucie Nováková

A review of current trends and advances in modern bio-analytical methods: chromatography and sample preparation.

Any bio-analytical method includes several steps, all of them being important in order to achieve reliable results. The first step is taking aliquots of samples for the analysis, followed by the extraction procedure and sample clean-up, chromatographic analysis and detection. Chromatographic methods, particularly liquid chromatography, are the methods of choice in bio-analytical laboratories. Current trends in fast liquid chromatographic separations involve monolith technology, fused core columns, high temperature liquid chromatography and ultra-high performance liquid chromatography (UHPLC). UHPLC has recently become a wide-spread analytical technique in many laboratories which focus on fast and sensitive bio-analytical assays. The key advantages of UHPLC are the increased speed of analysis, higher separation efficiency and resolution, higher sensitivity and much lower solvent consumption as compared to other analytical approaches. This is all enabled by specially designed instruments and sub-2-microne particle packed analytical columns. There is a great contrast between ultra-fast chromatographic analysis and conventional sample preparation, which remains highly labor-intensive and time-consuming. Conventional sample preparation techniques including SPE, solid phase extraction; LLE, liquid-liquid extraction; PP, protein precipitation and many modern approaches (RAM, restricted access material; MIP, molecularly imprinted polymers; SPME, solid phase microextraction; LLME, liquid-liquid microextraction; MEPS, microextraction by packed sorbent and many others) have also been featured as fundamental and critical step of bio-analytical methods.

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.

Modern analytical supercritical fluid chromatography using columns packed with sub-2 μm particles: a tutorial.

This tutorial provides an overview of the possibilities, limitations and analytical conditions of modern analytical supercritical fluid chromatography (SFC) using columns packed with sub-2 μm particles. In particular, it gives a detailed overview of commercially available modern SFC instrumentation and the detectors that can be employed (UV, MS, ELSD, FID, etc.). Some advice on the choice of the stationary phase dimensions and chemistries, the nature of the mobile phase (choice of organic modifier and additives) and its flow rate as well as the backpressure and temperature are also provided. Finally, several groups of potentially problematic compounds, including lipophilic compounds, hydrophilic substances and basic drugs, are discussed in detail. All these families of analytes can be resolved with SFC but require specific analytical conditions.

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).

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.

Challenges in the development of bioanalytical liquid chromatography–mass spectrometry method with emphasis on fast analysis

The development of bioanalytical methods has become more and more challenging over the past years due to very demanding requirements in terms of method reliability, sensitivity, speed of analysis and sample throughput. LC-MS/MS has established itself as a method of choice for routine analysis of biological materials. A development of such method consists of several steps including sample preparation and clean-up step, efficient chromatographic separation, sensitive and selective detection of analytes in complex matrices, a choice of convenient data processing and calibration approach and finally method validation. Each of these steps has its own constraints and challenges, which are discussed in detail in this review. Novel and modern approaches in sample preparation, chromatography and detection are especially emphasized. Attention is paid to proper calibration approach and matrix effects that can seriously affect method accuracy and precision.

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.

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.