Trace-level monitoring of chloroanilines in environmental waters using on-line trace-enrichment and liquid chromatography with UV and electrochemical detection

Summary An on-line procedure is described for the trace-level determination of mono-, di- and methyl-chloroanilines in aqueous samples using selective preconcentration with a cation-exchanger and liquid chromatography with UV and electrochemical detection. Because direct percolation through a cation-exchanger has to be avoided owing to the high content of inorganic anions present in natural waters, a two-step on-line preconcentration was carried out: chloroanilines were first trapped on a precolumn packed with an apolar polymeric sorbent (PRP-1) in their neutral form. Then the PRP-1 precolumn was coupled in series with a second precolumn containing cation exchange material. The chloroanilines were removed from the first precolumn with 3 mL of deionised water: acetonitrile (31) at pH 1 and retained by the cation exchange column. The contents of the cation exchange column were finally desorbed onto the analytical column and eluted with a water: acetonitrile gradient. The combination of selective trace enrichment and sensitive electrochemical detection allows the simultaneous determination of chloroanilines from 150 mL of river water samples with detection limits below 30 ng/l. Identification is confirmed by the selective preconcentration and the two detection modes.     

Off-Line and On-Line Preconcentration Techniques for the Determination of Phenylureas in Freshwaters

Abstract

Phenylurea herbicides are analysed by reversed-phase liquid chromatography using UV detection at 244 nm after a concentration step in order to determine ppb or sub-ppb levels in drinking and river waters. With an average UV detection limit of 5 ng, a 500 ml sample volume is necessary to reach the 10 ppt level for spiked LC grade water samples and enables easy determination of concentrations below the ppb level for river water samples. Off-line and on-line methods are compared for the concentration step. Off-line concentration consists in a liquid sorption on n-octadecyl silica (C18) and elution by a suitable organic solvent. Polar phenylureas have low retention volumes on C18 silica and consequently the length of the concentration column has to be 10 cm to concentrate them at the ppb level from 100 ml of water and longer for lower levels of detection. Nevertheless, we show that increasing the size of the concentration column does not improve the limits of detection because of the numerous interferences also concentrated when percolating high volumes of water. On-line technology can be used only with short precolumns and requires a sorbent with a great retention for phenylureas. The copolymer-based PRP-1 is found to be an excellent sorbent and it is then possible to apply on-line precolumn technology with preconcentration through two precolumns (10 × 21 mm ID) in series, the first one being packed with C18 silica and the other with the PRP-1 polymer. Interfering compounds are then trapped onto the first precolumn acting as a filter and common phenylurea-breakthrough volumes on the PRP-1 precolumn are higher than 500 ml. Knowing the amounts preconcentrated on both precolumns and using UV and electrochemical detection help the identification of phenylureas in river water.     

Properties of low-capacity macroporous anion-exchangers and their use with high-pH eluents for the determination of weak acids in urine

Low-capacity anion-exchangers have been synthesized from the macroporous styrene-divinylbenzene co-polymer Hamilton PRP-1. These anion-exchangers have non-polar adsorbent properties for neutral sample molecules. The dependence of the capacity factor for a polar weak-acid metabolite of the drug polythiazide on the type and concentration of electrolyte, the organic solvent concentration, and pH of aqueous eluents has been studied to characterize the exchangers. As an application, a method has been developed to determine the metabolite in urine at levels as low as 10 ng ml . The method employs the exchangers in both a precolumn, which is used to preconcentrate and clean-up the metabolite from urine, and an analytical column. Eluents containing 0.001-0.01M sodium hydroxide are used to ionize the weak-acid metabolite.     

Use of disposable open tubular ion exchange pre-columns for in-line clean-up of serum and plasma samples prior to capillary electrophoretic analysis of inorganic cations

A simple analytical system using disposable, open-tubular ion exchange clean-up precolumns coupled in-line to capillary electrophoresis for direct injection of biological samples is presented. The clean-up precolumns were prepared from fused silica capillaries by thermally initiated layer-by-layer polymerization of poly(butadiene-maleic acid) (PBMA) directly on the capillary wall. Typically, 6 cm long precolumns with 4-layers of PBMA were used for sample pretreatment. A robust and reproducible coupling between the precolumn (75 μm ID) and the analytical capillary (50 μm ID) was achieved using an inexpensive, commercially available low dead volume union. No extra dispersion of the analyte zones was observed. Proteins and other high molecular weight compounds from biological sample matrices were retained on the cation-exchanger sites of the precolumn, which eliminated their adsorption on analytical capillary walls and ensured stable electroosmotic flow and migration times of target analytes. Unretained small inorganic cations migrated freely into the analytical capillary for separation and detection. Applicability of the sample clean-up procedure was proved by determination of major inorganic cations in blood serum and plasma samples using capillary electrophoresis with contactless conductivity detection. Separations were performed in background electrolyte solution consisting of 15 mM L-arginine, 12.5 mM maleic acid, 3 mM 18-crown-6 at pH 5.5 and repeatabilities of migration times and peak areas were below 1.5% and 7.3%, respectively. Less than 1 μL of biological sample was required for injection.     

Determination of phenoxyacid herbicides in water

Novel clean-up techniques for a polymeric precolumn (PLRP-S) for the subsequent determination of bentazone and eight phenoxy acid herbicides in surface water samples are described. After preconcentration of the components at pH 3 on a 10 x 2 mm I.D. precolumn, the technique consists of a clean-up with 1000 microliters of 0.1 mol/l sodium hydroxide solution (pH 12.5) and of a heartcut consisting of four precolumn bed volumes of eluent directed to waste followed by ten precolumn bed volumes of eluent directed to the analytical column. Analytical separation is performed with acetonitrile-water (30:70) containing 0.005 mol/l of tetrabutylammonium hydrogensulphate (pH 8.3) (which is also the desorption eluent during heartcutting) on a polymeric analytical column (PLRP-S). With 25 ml of surface water, spiked at 0.25 and 1 microgram/l, applied to the precolumn, recoveries for all components were over 85% with a relative standard deviation (n = 5) of ca. 9% at 0.25 microgram/l and ca. 2% at 1 microgram/l. Detection limits in surface water samples are 0.05-0.1 microgram/l. Owing to automation, the total analysis time is ca. 30 min.     

Trace-Level Determination of Phenol by Liquid Chromatography with On-Line Precolumn Technology and Fluorescence Detection

Abstract

A strongly basic anion-exchange resin is used for the trace enrichment and automated sample handling of phenol, with subsequent determination by reversed-phase liquid chromatography with fluorescence detection. Because of the presence of high concentrations of ionic compounds in the water samples tested, phenol is first trapped on a relatively long precolumn filled with a highly hydrophobic packing material; during this step, (in) organic anions which are not retained, are flushed to waste. In the next step, phenol is desorbed from this column at high pH and sorbed in a small zone (“peak compression”) on a short precolumn containing the anion exchanger.

In the analysis of tap and river water samples, the detection limit was found to be 10ppt (1:10¹¹).     

Trace-level determination of polar phenolic compounds in aqueous samples by high-performance liquid chromatography and on-line preconcentration on porous graphitic carbon

The use of porous graphitic carbon (PGC) was investigated for the trace enrichment and the on-line liquid chromatographic separation of polar phenolic compounds (phenol, di- and trihydroxybenzenes, aminophenols, etc.) from aqueous samples. Comparison between retentions obtained with PGC and with the copolymer-based sorbent PRP-1 showed similar variations of the capacity factors with the mobile phase composition, but an inverse retention order. The capacity factor of a very polar analyte, such as 1,3,5-trihydroxybenzene (phloroglucinol), is 1000 in pure water, whereas this analyte is not retained by C₁₈-silica and is poorly retained by PRP-1 (k′ = 3 in water). A precolumn packed with PGC can be coupled to a PGC analytical column for simple separation in the reversed-phase mode. This methodology has been applied to the direct determination of pyrocatechol, resorcinol and phloroglucinol below the 0.1 μg/1 level in a 50-ml sample.     

Determination of quinolones and fluoroquinolones in hospital sewage water by off-line and on-line solid-phase extraction procedures coupled to HPLC-UV

In this work, the development of two solid-phase extraction procedures (off-line and on-line formats) for the identification and quantification of several (fluoro)quinolones in hospital sewage water by HPLC-UV is described. Both procedures are based on the use of C18 and anion exchange (SAX) sorbents for the preconcentration and clean-up steps, respectively, and all variables influencing both steps were optimised. In the off-line format, after its pH was adjusted to 2.5, sample was preconcentrated on a C18 cartridge and eluted with 4 mL of methanol/ammonia (94/6). The methanolic extract must be diluted up to 10 mL with water to allow quantitative retention of the analytes on the SAX cartridge. In the on-line format, the addition of 2.5% of NH4Cl to the sewage water sample (pH = 2.5) was necessary to increase the breakthrough volumes of the analytes in the C18 precolumn. Quantitative transfer of the (fluoro)quinolones from the C18 precolumn to the SAX precolumn was accomplished by pumping 2 mL of a mixture methanol/water (40/60, pH = 9.2) at 2 mL min(-1). Elution of the analytes from the SAX precolumn by means of the chromatographic mobile phase required the inclusion of an additional isocratic step at the beginning of the gradient program. Both off-line and on-line solid phase extraction procedures coupled to HPLC-UV were applied to the analysis of a sewage water sample collected in the sewer system at the output of the St Dimphna Hospital (Geel, Belgium). The fluoroquinolone ciprofloxacin was found in this sample and quantified at 5.8 +/- 0.4 microg L(-1) (off-line method) and 5.6 +/- 0.5 microg L(-1) (on-line method). The analysis of spiked samples containing the seven (fluoro)quinolones studied provided quantitative recoveries in all cases with low RSD values (from 6 to 12%), and all the analytes could be identified by means of their UV spectra with match factors varying from 950 to 985 depending on the (fluoro)quinolone.     

Sequential Electrochemical Polymerization of Aniline and Their Derivatives Showing Electrochemical Activity at Neutral pH

Sequential electropolymerization of aniline followed by an aniline derivative bearing an ion moiety is presented. The studied derivatives contain sulfonic, carboxylate or amino groups. Its electrochemical behavior at acid and neutral pH is studied by cyclic voltammetry combined with quartz crystal microbalance or probe beam deflection in order to assess the mass transfer process involved in these new modified electrodes. All of them show a stable and quasi‐reversible electrochemical behavior at neutral pH that can be attributed to a self‐doping process. These new modified electrodes can be further modified due to the presence of functional groups.     

Determination of tacrolimus (FK 506) in whole blood using liquid chromatography and fluorescence detection

Summary A sensitive and selective liquid chromatographic method, using precolumn derivatization with dansylhydrazine followed by fluorescence detection, has been developed for the determination of tacrolimus (FK 506) in whole blood. After haemolysis, whole blood samples are extracted with diethyl ether and derivatized. After on-line removal of excess dansylhydrazine on a C₁₈ precolumn, the derivative is loaded on to a C₁₈ clean-up column, and a heart cut is subsequently transferred to a graphitized carbon column, where the final separation takes place. The method requires 1 ml of sample and has a limit of quantitation of 3 ng/ml. At the 15 ng/ml level the precision isca 10%, and the response is linear from the limit of quantitation toca 200 ng/ml of FK 506 in whole blood. The capacity of the method is 50 samples/day and about 1000 1-ml samples can be analyzed without changing either clean-up or separation column. Finally, the applicability of the method for therapeutic drug monitoring is shown.     

Optimized determination of thiochrome derivatives of thiamine and thiamine phosphates in whole blood by reversed-phase liquid chromatography with precolumn derivatization

A liquid chromatographic method involving precolumn derivatization for determining thiamine and its phosphate esters in human blood has been optimized. Blood sample stored at - 20 degrees C were haemolysed and deproteinized by perchloric acid. The supernatants of the samples were oxidized by addition of potassium ferricyanide-sodium hydroxide, and phosphoric acid was added to obtain a neutral pH in order to extend the column life. The samples were stable after derivatization for at least 24 h, if protected from light and kept at room temperature. Gradient separation with 140 mmol phosphate buffer (pH 7.0), and methanol, tert-butylammonium hydroxide and dimethylformamide as modifiers, on a 3-microns Chromsphere octadecylsilica column gave an analysis time of 15 min. The method was found to be very suitable for the determination of thiamine components in whole blood. The minimal detectable amount is 0.5 nmol/l and the method is linear to at least 1000 nmol/l. The recovery (98 +/- 3%) and precision are very good.     

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.     

Trace-level determination of 3'-azido-3'-deoxythymidine in human plasma by preconcentration on a silver (I)-thiol stationary phase with on-line reversed-phase high-performance liquid chromatography

A method was developed for the determination of 3'-azido-3'-deoxythymidine (AZT) in plasma. The method is based on the trace enrichment of AZT on a pre-column packed with a silver-loaded thiol stationary phase at pH 11.6. On-line desorption to the reversed-phase liquid chromatographic system is performed by injecting a plug of 50 microliters of 1 M perchloric acid on the silver (I)-thiol pre-column. Two different sample pretreatment methods - protein precipitation with perchloric acid and on-line clean-up via a polymeric PRP-1 pre-column - were applied for the determination of AZT in human plasma. The latter method allows the direct injection of plasma samples into the analytical system and can therefore easily be automated. With both methods detection limits in the order of 10(-8) M AZT were obtained after preconcentration of 1.0 ml of plasma, using UV detection at 267 nm.     

Rapid on-line precolumn high-performance liquid chromatographic method for the determination of benomyl, carbendazim and aldicarb species in drinking water.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of trace concentrations of benomyl, carbendazim, aldicarb, aldicarb sulphoxide and aldicarb sulphone in drinking water. A 10-ml sample of water is passed through a 3-cm precolumn, packed with 5-microns C8 sorbent, at a flow-rate of 5 ml/min. The HPLC system is then switched to an acetonitrile-water gradient elution program. The preconcentrated analytes are eluted from, and separated by, the 3-cm C8 precolumn and determined by UV absorption. The total analytical time is 25 min. The lowest detectable concentrations are in the range of 2.5 x 10(-9)-11.0 x 10(-9) g/ml for the five analytes investigated with 10 ml of sample.     

Direct injection of plasma into column liquid chromatographic systems

Summary Since the late 1970’s several techniques involving the direct injection of biological fluids, like plasma and serum have been published. Most of the systems presented utilize a precolumn for the reception of the biological fluids, trace enrichment and preliminary clean-up before the analyte fraction is transferred to the separation column. The most common solid phase for the precolumn is silanized silica, but many other types have been used. One was especially designed for the purpose of direct injections of biological fluids: the Internal Surface Reversed Phase support. The main problem with direct injections is the development of column instabilities due to the denaturation and precipitation of plasma proteins on the support. It has, however, been possible to design systems which can handle the injection of 25ml total plasma volume without the need to exchange precolumn. The most promising techniques in this respect are the so called “pre-column” venting plug technique and the use of micellar mobile phases. Losses of analytes may be a problem when protein-binding extends 95%. However, remedies are available, such as the use of more hydrophobic solid phases, the addition of competitive binders and pH adjustments of injected solutions.     

Use of cotton as a sorbent for on-line precolumn enrichment of polycyclic aromatic hydrocarbons in waters prior to liquid chromatography determination

Using cotton as a solid-phase extraction sorbent of the precolumn, an on-line coupled precolumn preconcentration-liquid chromatography system with fluorescence detection was developed for the determination of PAHs in aqueous samples. Four PAHs including fluorene, phenanthrene, fluoranthene and benzo[k]fluoranthene were preconcentrated by a precolumn packed with 30 mg of absorbent cotton and then separated by C₁₈ analytical column. When 100 ml of sample was enriched, the proposed procedure provided detection limits in the range of 0.5-57 ng l⁻¹. Several water samples spiked with PAHs were analyzed with recoveries in the range of 92-119% at spiking level of 100 ng l⁻¹ for fluorene, phenanthrene and fluoranthene, and 10 ng l⁻¹ for benzo[k]fluoranthene, respectively.     

Application of an internal surface reversed-phase column for the automated determination of flucycloxuron residues

A liquid chromatographic column-switching system for the automated determination of flucycloxuron, a benzoylphenylurea pesticide, in crop and environmental matrices is described. The system consists of an internal surface reversed-phase (ISRP) column, a phenyl-bonded precolumn and an analytical reversed-phase (RP) C₁₈ column. Sample extracts are evaporated to dryness and dissolved in the mobile phase of the ISRP column. An aliquot of this solution is injected into the column-switching system. Clean-up, with regard to removal of large molecules, is performed on the ISRP column. The flucycloxuron fraction from the ISRP column is concentrated on the phenyl-bonded precolumn. Additional clean-up can be performed by washing the precolumn. Finally, the compound is desorbed from the precolumn and separation and determination of the Z- and E-isomers of flucycloxuron are performed with the analytical RP-C₁₈ column using UV detection at 254 nm. The total analysis time required is 40 min. The reproducibility of the method obtained with the column-switching system, expressed as relative standard deviation, varies between 3.7 and 10% for apple, strawberry, citrus and soil samples for flucycloxuron levels between 0.04 and 0.33 mg/kg. The system showed no loss of analytical performance after more than 300 analyses.     

Capillary-electrochromatographic separations with copolymeric reversed-stationary phase and ion-exchanger-packed columns

A macroporous, spherical, 7 μm, polystyrene–divinylbenzene (PS–DVB), reversed-phase adsorbent (PRP-1) was evaluated as a stationary phase for the capillary electrochromatographic (CEC) separation of neutral, acidic, and basic analytes of pharmaceutical interest. Electroosmotic flow (EOF) for a PRP-1 packed capillary is nearly constant over the pH 2 to 10 range and is higher than for a silica-based C₁₈ packed capillary on the acidic side. EOF increases with an increase in buffer acetonitrile concentration or as applied potential increases. As analyte hydrophobicity increases, analyte retention and migration time increases. Increasing buffer acetonitrile concentration reduces analyte partitioning with the PS–DVB stationary phase and analyte retention and migration time decreases. When exchange sites are present on the PS–DVB copolymer, EOF (EOF is reversed for the anion-exchanger) increases as the exchange capacity increases. An increased exchange capacity also reduces partitioning of the analyte with the PS–DVB matrix and analyte retention and migration time decrease. Because of excellent stability in an acid environment, the PRP-1 packed capillary can be used in strong acid buffer solution and weak acid and base analytes depending on pK_a values can be separated as neutral species and cations, respectively. CEC separations on a PRP-1 capillary of neutral steroids, weak base pharmaceuticals (separation as cations), purines and pyrimidines (as cations), fatty acids (as undissociated species), and sulfa derivatives (as cations) are described. Efficiency for the PRP-1 packed capillary for acetone or thiourea as the analyte is about 6·10⁴ plates m⁻¹.     

Direct HPLC analysis of ketoprofen in horse plasma applying an ADS-restricted access-phase.

Making up part of the unique family of restricted access materials (RAM) the Lichrospher ADS (alkyl-diol silica) sorbents have been developed as special packing materials for precolumns used for LC-integrated sample processing of biofluids. The advantage of such phases consists of direct injection of untreated biological fluids without sample clean-up and elimination of the protein matrix together with an on-column enrichment. The plasma samples, with internal standard phenacetin added (not essential), were brought onto the precolumn (C-18 ADS, 25 micron, 25 x 4 mm i.d.) using a phosphate buffer, 0.1 M, pH 7.0. After washing with the buffer, the ADS column was backflushed with the mobile phase phosphate buffer 0. 05 M pH 7.0: acetonitrile (80:20), thus transporting the analytes onto a reversed-phase column Ecocart 125-3 HPLC cartridge with a LiChrocart 4-4 guard column, both packed with LiChrospher 5 micron 100 RP-18; after separation detection was performed in UV at 260 nm. Essential features of the method include the novel precolumn packing, the absence of sample pretreatment, a quantitave recovery, good precision and accuracy, as well as a considerable reduction of analysis time compared to conventional manual methods applied in bioavailability studies.     

Electrochemical Detection of Phenylurea Herbicides in Liquid Chromatography

Abstract

This paper describes the electrochemical detection of ten phenylurea herbicides after on-line trace enrichment on a small C18 precolumn and liquid chromatography on a C18 analytical column. The method presented shows sub-ppb sensitivity in surface water samples without extensive sample pretreatment. Electrode contamination occurs but does not seriously interfere in the routine analysis of such samples. Selective determination (at 1. 0 V) of metoxuron in the presence of other phenylureas allows the detection of 30 ppt of the herbicide in surface water.