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.     

Multidimensional gas and liquid chromatographic approaches to trace-level environmental analysis

Summary Multidimensional approaches are becoming increasingly popular for the trace-level determination of microcontaminants in environmental samples. Hyphenated procedures with which sample treatment-plus-separation is combined on-line with a spectroscopic detector such as a (tandem) mass spectrometer, are especially useful because of their high identification power. Socalled comprehensive techniques, which possess an extremely high separation efficiency, are a promising new addition to the list. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

HPLC with Diode-Array Detection for Determination of Leflunomide in Tablets

We have developed and validated a simple HPLC method for analysis of leflunomide in tablets. Method conditions were determined by assay of a photodegraded sample of leflunomide. Optimum chromatographic performance was obtained with a C₁₈ column and acetonitrile-water as mobile phase. Comparison of spectra recorded with a diode-array detector during elution of the leflunomide peak enabled determination of method specificity. The method is highly sensitive (detection limit 10 ng mL⁻¹) and robust to deliberate variation of the conditions (RSD of peak area < 2.0%). Precision and accuracy were adequate over the concentration range 10 to 100 μg mL⁻¹. These results show the proposed method is suitable for its intended use.     

High-performance liquid chromatographic analysis of dinitrobenzene isomers

Summary An efficient, reproducible and rapid high-performance liquid chromatographic method, in normal phase mode, for the analysis of the three dinitrobenzene isomers is described. The method affords good linearity for each isomer in the range 10–160 g ml^–1. The total analysis time is only 10 minutes, and the method shows an accuracy of ±1.25% with a coefficient of variation from 0.30% to 2.85% for different levels of the dinitrobenzene isomers.     

Determination of vitamin A in pharmaceutical preparations by high-performance liquid chromatography with diode-array detection

Summary A very simple high-performance liquid chromatographic method for determination of Vitamin A in pharmaceutical preparations without the need for saponification was developed. A reversed-phase (Nova-Pack C₁₈, 4 m) column was used with a mobile phase of acetonitrile-tetrahydrofuran-water (55378) and a flowrate of 1.5 ml/min. Sample treatment only consisted of the extraction of retinol acetate content from capsules or tablets with methanol. Total extraction was achieved by shaking vigorously with the aid of magnetic stirring for three hours at room temperature. No change of solvent is necessary to introduce the sample in the chromatographic system. This method is suitable for routine quantification of Vitamin A.     

High-performance liquid chromatographic analysis of azlocillin in serum

Summary A rapid and sensitive high performance liquid chromatographic method is described for the determination of azlocillin in serum. This method involves a short manual protein precipitation of the sample followed by an injection into a PR 18 column for separation and quantitation. The mobile phase was a 22% (V/V) solution acetonitrile in phosphate buffer pH 4.8 at a flow rate of 2,5 ml/min. The spectrophotometer detector was set at 220 nm with a sensitivity of 0.08 AUFS.     

Trace enrichment of polar pollutants from water and the influence of humic substances

Summary A practical liquid chromatographic early-warning system for polar pollutants in water ought to have detection limits at a level below a microgram per litre. Using ultraviolet absorbance detection this normally necessitates trace enrichment of the samples. In this study ten different sorbents have been tested with respect to their enrichment capacity. The hydrophobic polymeric PLRP-S material, in combination with a C-18 modified analytical column, proved to be the best choice. The influence of humic substances, present in surface water, was also studied. Lowering of the sample pH, resulted in a significant increase in the interferences due to the humic substances.     

On-column catalysis of hydratization equilibria during liquid chromatographic analysis of acetaldehyde and formaldehyde

Summary The aldehydes were separated by reversed phase chromatography on a polymer-based support. Acid catalysis on the column was needed to obtain sufficiently fast equilibrium between acetaldehyde and its hydrate. Deuterium labeled acetaldehyde (D₃) yielded complete separation from the nonlabeled compound, probably due to a difference in hydratization. Fluorescent products were obtained by reaction between the aldehydes and a mixture of ammonia and dimedone (5.5-dimethyl-1.3-cyclohexanedione). The fluorescence was detected at 460 nm after excitation at 390 nm. A reaction time of 42 seconds at 90°C in a knitted teflon capillary produced detection limits of 0.5 and 1 ng for acetaldehyde and formeldehyde, respectively. A method for preparation of a stable reagent with low background fluorescence is described.     

Liquid chromatographic analysis with electrochemical detection for metoclopramide in human plasma

Summary A simple, reproducible liquid-chromatographic method for determination of metoclopramide anti-emetic and anti-spasmodic agent is described. Metoclopramide is extracted from alkalinized plasma into diethylether, then separated with a sperisorb CN column and measured through amperometric detection at +1,4 volt vs Ag/ AgCl. The average extraction recovery was 92%. Standard curves were linear and reproducible from 0.05 to 1.5 mg/ L. Within-day CV was better than 6%.     

On-line trace enrichment — column liquid chromatography of polar pollutants in surface water using bifunctional membrane-based extraction-disk cartridges

Summary An on-line trace-enrichment system, using a bifunctional membrane extraction-disk cartridge, has been combined with a column liquid chromatography separation for the simultaneous determination of basic, neutral and acidic pollutants in surface water. The enrichment device consisted of a specially constructed holder containing both C-18 and cation-exchange disks. The holder can contain up to 25 disks of 0.5 mm thickness and 4.6 mm diameter. Before trace enrichment of 20 ml of surface water (pH 3.0), calcium ions were removed from the sample by means of an oxalic acid precipitation. Desorption of the cartridge was at elevated temperature using reversed-phase gradient elution; detection was performed with a diode-array UV absorbance detector. The detection limits for the test compounds in surface water are typically 0.5–2 g/l; the calibration graphs are linear from the lower limit of determination up to 50 g/l.     

High-performance liquid chromatographic analysis of isoxazolylpenicillins in plasma and urine samples

Summary A simple and fast liquid chromatographic method is described, applicable to the routine analysis of isoxazolylpenicillins (cloxacillin, dicloxacillin, flucloxacillin) in biological fluids (plasma, urine). The method is based on a simple dilution step employed to destroy the protein binding, which is over 95%, and allows the detection of concentrations down to 10μg/ml. In order to analyze concentrations of less than 10μg/ml, a liquid-liquid extraction with dichloromethane must be executed prior to the reversed-phase analysis with absorbance detection at 206nm. The minimum detectable amounts of the isoxazolylpenicillins with this procedure are between 2.5 and 5.1 ng in 100μl plasma samples. The stability of the penicillin samples in aqueous solutions (stock solutions, eluents) was investigated and no significant degradation was observed during the storage and analysis of the samples. Furthermore, the degree of protein binding was established by using a suitable ultrafiltration technique, and the usefulness of the developed procedures in pharmacokinetic studies was demonstrated.     

Improvement of on-line solid-phase extraction for determining phenolic compounds in water

Summary Modifying the most common design for the on-line coupling of a precolumn to reversed phase LC with diode array detection has resulted in reduction of the broadening of the peaks which results when the compounds of interest are strongly retained by a highly hydrophobic sorbent. The modification consists of the desorption of the analytes trapped on the precolumn solely by the organic solvent used to modify the solvent strength of the mobile phase. Results obtained using this design were compared with those obtained with the conventional design, with C₁₈ and PLRP-S precolumns. The performance of the system was also tested with a highly cross-linked styrene-divinylbenzene copolymer (ENVI-chrom P) precolumn for the determination of phenolic compounds in real samples. The advantages and disadvantages are discussed. Ion-pair solid phase extraction is used in order to increase the breakthrough volumes of more polar compounds, mainly phenol. The use of the new design enables phenolic compounds to be determined at the low μg L⁻¹ level with limits of detection ranging between 0.1 and 2 μg L⁻¹ in tap water when a 10 mL sample was analyzed.     

Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

Summary An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min⁻¹, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.     

Determination of eleven priority EPA phenolics at ng L−1 levels by on-line solid-phase extraction and liquid chromatography with UV and electrochemical detection

Summary The eleven priority, EPA phenolic pollutants were determined by liquid chromatography followed by two detectors in series; UV and electrochemical. Three different adsorbents, Envi-Carb (a carbon black) and two functionalized polymeric resins, Bond Elut PPL and another synthesized in our laboratory with an ocarboxybenzoyl moiety, were compared for solid-phase extraction (SPE) to detect lower concentrations of the eleven phenolics in natural waters. Higher recoveries were obtained using the functionalized polymeric adsorbents compared with Envi-Carb. When real samples were analysed, the synthetic adsorbent gave lower interference than Bond Elut PPL and phenol was determined at low levels with no humic and fulvic acid inter-ference when Na₂SO₃ was added. The linearity range for most compounds in tap water was 0.05–20 μg L⁻¹ and the limits of detection were <35 ng L⁻¹. Repeatability and reproducibility between days for real samples spiked at 0.1 μg L⁻¹, expressed as relative standard deviation, were <8% and 10%, respectively.     

A high-performance liquid chromatographic method for determination of flecainide in human plasma using fluorescence detection

Summary A high performance liquid chromatographic method for the determination of flecainide in serum has been developed. The analysis is performed on a microparticulate silica column. The eluate is monitored by fluorescence detection at an excitation wavelength of 300nm and an emission wavelength of 370nm. No sources of interference were identified and a coefficient of variation of less than 8% was observed on repeated flecainide determinations. The method has a good reproducibility, specificity and accuracy, and can be applied in therapeutic drug monitoring of flecainide in patients.     

Automated sample preparation using supported liquid membranes for liquid chromatographic determination of sulfonylurea herbicides

Summary Sample preparation for determination of sulfonylurea herbicides in aqueous samples is investigated. The technique studied utilizes extraction and back extraction in an automated flow system and is coupled on-line to a liquid chromatographic system. The extraction unit consists of an immobilized liquid membrane, separating two aqueous phases. From the acidified donor phase the analytes are extracted into the organic solvent of the membrane. After traversing the membrane they are back extracted into an alkaline/neutral aqueous acceptor phase. They are trapped in the acceptor by dissociation, making them insoluble in the membrane.Studies of the sample preparation system concern factors like channel length of separators, distribution coefficients of analytes and use of a precolumn instead of loop for chromatographic injections. Effects of the internal diameter of the analytical column as well as the detection of the sulfonylurcas are investigated.     

High performance liquid chromatographic analysis of patent blue V in cheese

Summary The HPLC method developed for the analysis of the dye Patent Blue V in extracts from cheese is sufficiently sensitive to detect and measure concentrations above 0.1 ppm with a standard deviation of 3 %. The extraction procedure described gives a recovery from cheese of about 80 %. The method has been applied to commercial samples of cheese and a concentration of the dye of about 0.12 ppm was measured in one case.     

High performance liquid chromatographic analysis of Patent Blue V in cheese

Summary The HPLC method developed for the analysis of the dye Patent Blue V in extracts from cheese is sufficiently sensitive to detect and measure concentrations above 0.1 ppm with a standard deviation of 3%. The extraction procedure described gives a recovery from cheese of about 80%. The method has been applied to commercial samples of cheese and a concentration of the dye of about 0.12 ppm was measured in one case.     

Liquid chromatography of phenolic compounds in natural water using on-line trace enrichment

Summary Two packing materials, C18 and PLRP-S, are studied for on-line trace enrichment of phenolic compounds in water. Various precolumns of different internal diameter are also tested and the addition of an ion-pair reagent to increase retention and thus, breakthrough volumes of phenolic compounds, is studied. Best results are obtained when a PLRP-S precolumn is coupled on-line with a C18 analytical column and DAD detector. Addition of TBA considerably increases breakthrough volumes. In contrast, when a C18 precolumn is used, breakthrough volumes are lower and it is impossible to determine TCP and PCP, under the experimental conditions used, because of interference of other nonpolar compounds in the samples. The performance of the system is evaluated with river and tap water and the preconcentration of 10 ml of sample in a PLRP-S precolumn involves a linear range between 1 g 1^–1 and 20 l^–1 and limits of determination between 0.5 g l^–1 and 1 g l^–1 are obtained.     

High-performance liquid chromatographic assay of bacterial collagenase

Summary The activity of bacterial collagenase Clostridiopeptidase A was estimated using a labelled synthetic peptide, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg, as substrate. The N-protected dipeptide obtained after enzymatic hydrolysis of Leu-Gly peptide bond was quantified by reversed-phase, high-performance liquid chromatography using 4-phenylazobenzyloxycarbonyl-L-Pro-L-Phe as internal standard. The time dependence of the appearance of the hydrolysis product and the dependence of rates of hydrolysis on collagenase concentration were linear. Kinetic parameters for collagenase were determined to test the suitability of the described procedure.