Adela Sevillano-Cabeza

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).

Urine polyamines determination using dansyl chloride derivatization in solid-phase extraction cartridges and HPLC

The derivatization of biogenic amines such as putrescine, cadaverine, spermidine and spermine with dansyl chloride in solid phase extraction cartridges is described. Different types of filling materials were tested in order to have the highest retention of the different analytes. The best results were obtained by using C18 cartridges. The optimal conditions were: amine solution buffered at pH 12, 2 mM dansyl chloride (acetone-bicarbonate solution 20 mM (pH 9-9.5), 2 + 3 v/v) as reagent concentration, room temperature and 30 min reaction time. The developed procedure was applied to the determination of these polyamines in urine samples from healthy controls and cancer patients using HPLC with 1,7-diaminoheptane as internal standard. The concentrations ranged from 0.5 to 5 micrograms mL-1 and the detection limits were 10 ng mL-1 for all polyamines. By concentrating the urine extracts, the detection limits were improved down to 2 ng mL-1. The accuracy and the precision of the method were tested. The proposed dansylation method is advantageous with respect to solution dansylation. It improves the total analysis time, avoids high temperatures that can affect the thermal stability of the derivatives and could make possible the automation of the procedure.

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).

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.

Derivatization of amines in solid-phase extraction supports with 9-fluorenylmethyl chloroformate for liquid chromatography

Abstract A new method based on reaction in solid-phase extraction cartridges with 9-fluorenylmethyl chloroformate is described for improved derivatization of amines prior to liquid chromatography. The effects of the reaction conditions, the kinetics of the reaction and the kind of packing material on the derivatization have been evaluated. The reliability of the described approach has been tested by derivatizing propylamine, diethylamine, diethanolamine, glycine, glutamic acid, p-aminobenzoic acid and β-phenylethylamine. Reaction yields compared with those of the analogous solution derivatizations were in the 44–169% range, and satisfactory linearity was achieved at concentrations of the amine-FMOC derivatives in the 5–100 μg/ml interval. As an illustrative example of application to real samples, a procedure for the determination of β-phenylethylamine in urine (at 0.01–1.0 μg/ml levels) using C18 solid-phase extraction cartridges and spectrophotometric detection is described. Analyte responses after cleanup plus derivatization were (91 ± 5)%, the limit of detection being 0.1 ng/ml at 254 nm.

Amphetamine and methamphetamine determination in urine by reversed-phase high-performance liquid chromatography with simultaneous sample clean-up and derivatization with 1,2-naphthoquinone 4-sulphonate on solid-phase cartridges

A liquid-solid procedure is proposed for sample clean-up and derivatization of amphetamine and methamphetamine in urine samples. The reagent was 1,2-naphthoquinone 4-sulphonate, and a commercial C18 packing cartridge was used. The samples derivatized at room temperature were chromatographed on a 5-microns Hypersil ODS (250 x 4 mm I.D.) with an elution gradient of acetonitrile-water containing propylamine. Under these conditions, the amines were eluted with short retention times. The procedure was used to determine amphetamine, or methamphetamine with its metabolite amphetamine, in spiked urine samples. The detection limit (at a signal-to-noise ratio of 3) for amphetamine (0.1 microgram/ml) was similar to that obtained with liquid-liquid derivatization and to those obtained with immobilized reagents on a polymeric solid support. The detection limit for methamphetamine (0.4 microgram/ml) was higher than with the liquid-liquid procedure because of the lower reactivity on the cartridge. The precision and accuracy of the method were also studied.

Determination of amphetamine and related compounds in urine using on-line derivatization in octadecyl silica columns with 9-fluorenylmethyl chloroformate and liquid chromatography

A method for the determination of amphetamine and related compounds in urine based on on-line derivatization with 9-fluorenylmethyl chloroformate (FMOC) and high-performance liquid chromatography is described. Derivatization is performed in a 20 x 2.1 mm I.D. column packed with a Hypersil ODS C18, 30 micron stationary phase, which is also used for sample clean-up and enrichment of the analytes. Next, the derivatized analytes are transferred to a LiChrospher 100 RP-C18 (5 micron, 125 x 4 mm I.D.) analytical column for their separation and quantification, using reversed-phase conditions and fluorescence detection. The described assay was applied to the determination of norephedrine, ephedrine, pseudoephedrine, amphetamine, phenylpropylamine and methamphetamine at concentrations of 0.5-10.0 micrograms/ml. Analyte conversions were about 55-96% of those obtained by the off-line derivatization mode under similar conditions, resulting in limits of detection in the 5-25 ng/ml range.

Amphetamine and Methamphetamine Determinations in Biological Samples by High Performance Liquid Chromatography. A Review

Abstract This critical review shows the different high performance liquid chromatography methods proposed for amphetamine and methamphetamine determinations. It is directed mainly towards sample clean up and derivatizations steps, because of their significance in such determinations.