R. Herráez-Hernández

Column-switching techniques for high-performance liquid chromatography of drugs in biological samples

In recent years, an increasing number of publications have demonstrated the potential of column-switching techniques for the chromatographic separation, determination and preparative isolation of analytes from biological matrices. Column-switching systems greatly facilitate drug analysis, by on-line sample clean-up and trace enrichment, or by improving the analytical separative process. In this paper, the main applications of column-switching techniques to drug analysis in biological samples, are reviewed.

New micromethod combining miniaturized matrix solid-phase dispersion and in-tube in-valve solid-phase microextraction for estimating polycyclic aromatic hydrocarbons in bivalves.

Miniaturized matrix solid-phase dispersion (MSPD) was developed for the extraction of common polycyclic aromatic hydrocarbons (PAHs) from bivalve samples (100mg, dry weight). Additional clean-up and analyte enrichment was accomplished by in-tube solid-phase microextraction (SPME). For this purpose the extracts collected after MSPD were diluted with water and injected into a capillary column coated with the extractive phase. This capillary column was connected to the analytical column by means of a switching valve. Separation and quantification of the PAHs were carried out using a monolithic LC column and fluorescence detection. Since the in-tube SPME device allowed the processing of large volumes of the extracts (2.0 mL) excellent sensitivity was achieved, thus making solvent evaporation operations unnecessary. The overall recoveries ranged from 10% to 28% for the studied compounds. The relative standard deviation (RSD) ranged from 2% to 10% for intra-day variation (n=3), and the limits of detection (LODs) were < or =0.6 ng/g (dry weight). The proposed procedure was very simple and rapid (total analysis time was approximately 20 min), and the consumption of organic solvents and extractive phases was drastically reduced. The reliability of the proposed MSPD/in-tube SPME method was tested by analysing several bivalves (mussels and tellins) as well as a standard reference material (SRM).

Solid-phase extraction techniques for assay of diuretics in human urine samples

Abstract Solid-phase extraction techniques were evaluated for the treatment of urine samples in the analysis of diuretics before injection into an HP-Hypersyl ODS-C18 column. Six different reversed-phase extraction columns were tested, and the results obtained are compared with those obtained in a classical liquid-liquid extraction with ethyl acetate. The solid-phase extraction procedures are the best overall choice for all the diuretics tested, due to their versatility, the minor time-consuming, and the good recovery percentages obtained. C18 and C8 packings give the highest recoveries for a majority of the diuretics studied. However, CH or PH columns, due to their greater selectivity, can be used if the elution of the matrix is not complete in the washing solution. This could be more suitable.

In-tube solid-phase microextraction coupled by in valve mode to capillary LC-DAD: Improving detectability to multiresidue organic pollutants analysis in several whole waters.

A simple and fast capillary chromatographic method has been developed to identify and quantify organic pollutants at sub-ppb levels in real water samples. The major groups of pesticides (organic halogens, organic phosphorous, and organic nitrogen compounds), some hydrocarbons (polycyclic aromatic hydrocarbons), phthalates and some phenols such as phenol and bisphenol A (endocrine disruptors) were included in this study. The procedure was based on coupling, in-tube solid-phase microextraction (IT-SPME) by using a conventional GC capillary column (95% methyl-5% phenyl substituted backbone, 80cmx0.32mm i.d., 3microm film thickness) in the injection valve to capillary liquid chromatography with diode array detection. A comparative study between the IT-SPME manifold and a column-switching device using a C(18) column (35mmx0.5mm i.d., 5microm particle size) has been performed. The IT-SPME procedure was optimal, it allows reaching limits of detection (LODs) between 0.008 and 0.2microg/L. No matrix effect was found and recoveries between 70 and 116% were obtained. The precision of the method was good, and the achieved intra- and inter-day variation coefficients were between 2 and 30%. This procedure has been applied to the screening analysis of 28 compounds in whole waters from several points of the Mediterranean coast (Valencia Community, Spain).

Chromatographic separation of chlorthalidone enantiomers using β-cyclodextrins as chiral additives

Different beta-cyclodextrins have been tested as chiral additives in the mobile phase for the chromatographic analysis of chlorthalidone enantiomers in a C18 LiChrospher (125 x 4 mm I.D.) column. The effect on enantioresolution of different parameters was studied: composition of the mobile phase (percentage of organic solvent, type of buffer and pH), mobile phase flow-rate, and type and concentration of beta-cyclodextrin. A 25:75 mixture of methanol and 0.1 M phosphate buffer, pH 4, containing 2% triethylamine (v/v), and 12.5 mM beta-cyclodextrin, at a flow-rate of 0.8 ml/min, was found to be the best option for the resolution of chlorthalidone enantiomers. Under such conditions, linear calibration curves were obtained in the 0.5-20-microg/ml interval using UV detection at 230 nm. The limit of detection for both isomers was 50 ng/ml. The utility of the described assay has been tested by analyzing chlorthalidone in different pharmaceutical preparations. Examples of application to biological samples are also given.

Chiral separation of ephedrines by liquid chromatography using β-cyclodextrins

β-cyclodextrins (β-CDs) have been evaluated as chiral recognition agents for the enantioseparation of ephedrines by liquid chromatography (LC). The compounds tested were norephedrine, ephedrine, pseudoephedrine, N-methylephedrine and N-methylpseuoephedrine. Two possibilities have been investigated, the employment of a chiral stationary phase consisting of immobilized β-cyclodextrin, and the addition of the chiral selector to the mobile phase. In the latter approach the following chiral agents have been assayed, native β-cyclodextrin (β-CD), methyl-β-cyclodextrin (M-β-CD), carboxyethyl-β-cyclodextrin (CE-β-CD), carboxymethyl-β-cyclodextrin (CM-β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD). The experimental factors affecting the chiral separations have been examined, including the percentage of organic solvent, the ionic strength and pH of the buffer, the type and concentration of chiral selector, and the mobile-phase flow rate. Differences in enantioselectivity displayed by the different methods are discussed.

A new selective method for dimethylamine in water analysis by liquid chromatography using solid-phase microextraction and two-stage derivatization with o-phthalaldialdehyde and 9-fluorenylmethyl chloroformate

A new method is presented for the determination of DMA in water as its 9-fluorenylmethyl chloroformate (FMOC) derivative using solid-phase microextraction (SPME) and liquid chromatography. The method is based on the employment of SPME fibres coated with carbowax-templated resin (CW-TR) for analyte extraction and derivatization. The fibres were successively immersed in the samples, in a solution of o-phthalaldialdehyde and N-acethyl-l-cysteine (OPA-NAC) and finally, in a solution of FMOC. OPA-NAC reacted on the fibre with possible primary aliphatic amines present in the samples, particularly with PA which is a direct interferent in the determination of DMA with FMOC. In such a way, the formation of PA-FMOC during the second stage was prevented, and thus the method was selective for DMA. The proposed procedure was applied to the determination of DMA in the 1.0-10.0mug/mL range. The method provided suitable linearity, accuracy and reproducibility, and limits of detection and quantification of 0.3 and 1.0mug/mL, respectively. The applicability of the method for the determination of DMA in different types of water is shown.

Strategies for the enantiomeric determination of amphetamine and related compounds by liquid chromatography

This paper summarizes recent research on the stereospecific analysis of amphetamine, its analogs and metabolites, by liquid chromatography. The different methods proposed have been evaluated and compared in terms of resolution power, time of analysis, sensitivity, or potential for automation. Chiral derivatization, followed by separation of the diastereomers formed in achiral chromatographic systems, is still the method preferred for the analysis of amphetamines at trace levels, as derivatization also improves analyte detectability. This is the method of choice for the enantiomeric analysis of amphetamines at the low concentrations typically encountered in biological samples. In recent years, special attention has been devoted to the development of alternatives for the automation of the analytical process by integrating the derivatization step into the chromatographic scheme. A promising alternative is the employment of beta-cyclodextrins as chiral selectors, both immobilized on the stationary phase and added to the mobile phase. However, with a few exceptions, beta-cyclodextrins perform better for non-derivatized amphetamines. Therefore, the utility of these selectors in the analysis of biological samples is limited. The reliability of less-used chiral stationary phases (Pirkle type, cellulose based or protein based), as well as methods based on the mathematical treatment of the chromatographic signal, are also discussed.

Liquid chromatographic determination of aliphatic amines in water using solid support assisted derivatization with 9-fluorenylmethyl chloroformate

SummaryA simple and sensitive method has been developed for the liquid chromatographic determination of short-chain aliphatic amines in water. Analytes are retained in solid-phase extraction (SPE) cartridges, and then derivatized by drawing an aliquot of the fluorogeneic reagent 9-fluorenylmethyl chloroformate (FMOC) through the cartridges. After a certain reaction time the derivatives formed are desorbed with acetonitrile. The collected extracts are then chromatographed on a LiChrospher 100 RP18 125 mm×4 mm i.d., 5 μm, column using an acetonitrile-water gradient. The influence of experimental conditions (SPE material, volume of sample, concentration of FMOC, time of reaction and pH) has been investigated. Optimal results have been obtained with C18 SPE cartridges using a sample volume of 5.0 mL. For derivatization, 0.25 mL aliquots of 25 mM FMOC have been used, the reaction time being only 2 min. The method has been applied to the quantification of several aliphatic amines: methylamine, ethylamine, dimethylamine,n-butylamine,n-pentylamine andn-hexylamine. Under the proposed conditions the percentages of analytes retained plus derivatized were of about 54–107% compared to those obtained with direct solution derivatization. The method provided good reproducibility, linearity and accuracy within the 0.050–1.0 mg L−1 concentration range. The limits of detection were in the 0.25–5.0 μg L−1 range. The utility of the described approach has been tested by analysing tap water, river water and industrial waste water.

Development of the H-point standard additions method for coupled liquid chromatography and UV-visible spectrophotometry

Abstract This work establishes the fundamentals of the H-point standard additions method for liquid chromatography for the simultaneous analysis of binary mixtures with overlapped chromatographic peaks. The method was compared with the deconvolution method of peak suppression and the second derivative of elution profiles. Different mixtures of diuretics were satisfactorily resolved.