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.     

Simultaneous determination of oxalate, thiosulfate, and thiocyanate in urine by ion-exclusion chromatography with electrochemical detection

Summary A sensitive ion-exclusion chromatographic method has been developed for determination of oxalate, thiosulfate, and thiocyanate. The method is based on separation of these anions on a polymethacrylate-based, weakly acidic cation-exchange resin (TSKgel OApak-A) and detection by means of a glassy carbon (GC) electrode electrochemically modified with polyvinylpyridine (PVP), palladium, and iridium oxide (PVP/Pd/IrO₂). The electrochemical behavior of oxalate, thiosulfate, and thiocyanate at this chemically modified electrode (CME) have been investigated by cyclic voltammetry. The results indicated that electrocatalytic oxidation of these anions by the electrode was efficient and that the sensitivity, stability, and lifetime of the electrode were relatively high. Combined with ion-exclusion chromatography the PVP/Pd/IrO₂ electrode was used as the working electrode for amperometric detection of these anions. All linear ranges were over two orders of magnitude and detection limits, defined asS/N=3, were 9.0×10⁻⁷ mol L⁻¹ for oxalate, 6.7×10⁻⁷ mol L⁻¹ for thiosulfate, and 5.6×10⁻⁷ mol L⁻¹ for thiocyanate. Correlation coefficients were all>0.998. Coupled with microdialysis sampling the method has been successfully applied to the determination of oxalate, thiosulfate, and thiocyanate in urine.     

Sensitive determination of probenecid in urine samples by reversed-phase liquid chromatography and UV-visible detection using solid-phase extraction techniques for sample clean-up

Summary This study describes a rapid method for the determination of probenecid in human urine by liquid chromatography with UV detection at 254 nm, after clean-up through a C8 solid-phase extraction column. Liquid chromatography was carried out on a C18-bonded phase using an acetonitrile-acetate buffer (pH=4) gradient elution. Ethacrynic acid was used as internal standard. The system has been applied to the determination of probenecid in the 0.10–100.0 g/ml concentration range; the limit of detection was 5 ng/mL.     

Determination of chloride in biological fluids as pentafluorobenzylchloride by reversed-phase high-performance liquid chromatography and UV detection

Summary A reversed-phase high-performance liquid chromatographic method for the determination of chloride in plasma, urine, saliva, sweat and aqueous solution is described. Chloride, in solution in aqueous acetone, is converted by means of pentafluorobenzyl bromide into pentafluorobenzyl chloride. This derivative is separated on a ODS-5 m reversed-phase column using isocratic elution with acctonitrile/water, 50/50, v/v, at a flow rate of 2.0 ml/min, and detected by a UV detector at 264 nm. The method is rapid, accurate and sufficiently sensitive for the determination of chloride in less than 10 l sample volume of a biological fluid.     

Determination of ambroxol in dog plasma by high-performance liquid chromatography and UV detection

Summary A new sensitive HPLC-UV method has been developed and validated for the determination of amboroxol in dog plasma enabling the investigation of a newly developed 75 mg ambroxol-containing retard capsule of EGIS Pharmaceuticals Ltd., Budapest, Hungary. A gradient method was used for removing the longer retained plasma components of no interest. The separation was performed on a BDS Hypersil C18 (5 μm, 250×2.1 mm) analytical column, supplied with a 10 mm guard column containing the same packing material. The detection was performed at 210 nm. The calibration curve was linear in the range 25–2000 ng·mL⁻¹. Nerisopam (EGIS-6775) was used as internal standard. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

The analysis of ethinyloestradiol in tablets by liquid chromatography with electrochemical detection

Summary The analysis of ethinyloestradiol in tablets has been carried out by reversed phase high performance liquid chromatography (HPLC) using electrochemical detection. The drug was eluted with aqueous acetonitrile containing sodium acetate as background electrolyte. The mobile phase was used as extracting solvent, and 4-tertpentylphenol was used as internal standard.     

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.     

Determination of hydrazine and 1,1-dimethylhydrazine as salicyldehyde derivates by liquid chromatography with electrochemical detection

Summary Determination of hydrazine and 1,1-dimethylhydrazine after derivatization with salicylaldehyde was done using high-performance liquid chromatography with electrochemical detection. The oxidation of the phenolic group of salicylaldazine (S-HY) and salicylaldehyde-1,1-dimethylhydrazone (S-UDMH) was optimized with respect to ionic strength, pH, and applied potential. Less than 5 ng of S-HY and S-UDMH could be detected. The detection limits for hydrazine and 1,1-dimethylhydrazine solutions were estimated to be 0.025 and 0.20 ppm, respectively.     

High-performance liquid chromatography of biphenols and bis (hydroxyphenyl) propanes (dianes) with voltammetric and UV photometric detection

Summary The retention behaviour of mono- and dihydroxy derivatives of biphenol and bis(hydroxyphenyl)propanes was studied on chemically bonded C-18, CN-, phenyl, phenoxypropyl- and NH₂ stationary phases and on silica gel. The effects of the mobile phase, i.e., of the methanol content, the ionic strength, pH and the contents of cationic and anionic ion-pairing agents on the retention data were investigated. In the reversed-phase systems, all the substances are satisfactorily separated except for 3- and 4-hydroxybiphenyl; these isomers can be separated on silica gel, using a mixture of heptane and propanol as the mobile phase. Sodium dodecylsulphate, present at concentrations higher than the critical micelle concentration (about 10⁻² M, depending on the methanol content), causes a decrease in the retention times and an improvement in the separation. UV absorption spectra, calibration curves and the detection limit values were obtained at two wavelengths and at various salt concentrations in the mobile phase. Hydrodynamic voltammograms of the solutes were measured in a carbon fibre detector and the calibration curves and detection limit values were measured at the optimal voltage (+1.2 V/Ag/AgCl) as a function of the mobile phase composition. For most test substances voltammetric detection is two to three times more sensitive than UV detection. The combination of a UV and a voltammetric detector in series has been utilized to identify the products of microbial degradation of biphenyl.     

Fluorometric determination of isoniazid and its metabolites in urine by high performance liquid chromatography

Summary A rapid, simple, and accurate method was developed for the determination of isoniazid and its metabolites (isonicotinic acid, acetylisoniazid and isonicotinylglycine) in urine by high-performance liquid chromatography. Urine is diluted with the mobile phase. After centrifugation, an aliquot of the supernatant is injected into the chromatograph. Isoniazid and its metabolites are separated by reversed-phase ionpairing chromatography with a mobile phase containing propanesulfonate and detected by fluorometry using postcolumn derivatization at high temperature (150°C) with hydrogen peroxide. The method was applied to the analysis of urine from patients receiving isoniazid therapy.     

Determination of urinary 5-S-cysteinyldopamine by high-performance liquid chromatography with electrochemical detection

Summary 5-S-Cysteinyldopamine, a new metabolite of dopamine, was determined in urine by high-performance liquid chromatography with electrochemical detection. The catechol was detected in 14 of 21 melanoma patients and 7 of 21 normal subjects; the highest values were 657 μg/day for melanoma patients and 44 μg/day for normal subjects. These results suggest that the cysteine conjugate may arise from autoxidation of dopamine but tyrosinase may also participate in the oxidation.     

Structure of hydroxycinnamic acid derivatives established by high-performance liquid chromatography with photodiode-array detection

Summary The present paper describes the development of a method for the differentiation of hydroxycinnamic acid derivatives by HPLC with photodiode-array detection. Furthermore spectral data of the compounds under investigation are given. Whereas p-coumaric acid derivatives are distinguishable from caffeic and ferulic acid derivatives by the positions of their spectrum maxima and their convexity interval value, it is not possible to distinguish between caffeic and ferulic acid derivatives because of overlapping spectrum maxima and convexity interval values.     

Structure of hydroxycinnamic acid derivatives established by high-perfomance liquid chromatography with photodiode-array detection

Summary The present paper describes the development of a method for the differentiation of hydroxycinnamic acid derivatives by HPLC with photodiode-array detection. Furthermore spectral data of the compounds under investigation are given. Whereas p-coumaric acid derivatives are distinguishable from caffeic and ferulic acid derivatives by the positions of their spectrum maxima and their convexity interval value, it is not possible to distinguish between caffeic and ferulic acid derivatives because of overlapping spectrum maxima and convexity interval values.     

Improved detection of leucovorin in mixed folates and antifolates by reversed-phase liquid chromatography and on-line post-column UV irridiation

Summary An improved, reversed-phase, high-performance liquid chromatography (HPLC) method with dual UV-fluorescence detection for simultaneous determination of the antifolate methotrexate, the folate leucovorin and their two main metabolites 7-hydroxymethotrexate and 5-methyltetrahydrofolate, respectively is presented. The fluorescence intensity of leucovorin could be significantly increased by on-line, post-column irradiation with UV at 254 nm thus lowering the limit of detection for leucovorin to 0.2 ng absolute at a signal-to-noise ratio 31.     

Determination of iodine anion in dried kelp and iodized throat tablets by reversed-phase ion-pair liquid chromatography with direct UV detection

Summary In this paper, the amount of iodine anion in dried kelp and iodized throat tablets was determined by reversed-phase ion-pair chromatography. A 300×4 mm I.D. columns packed with -Bondapak-C18 (5 m) was used and distilled water containing 10 mmol/l trimethylphenyl ammonium bromide was used as the eluent. UV detection at 231 nm was selected to monitor the iodine anion.     

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.     

Solid-phase microextraction coupled with microcolumn liquid chromatography for the analysis of amitriptyline in human urine

Summary Solid-phase microextraction (SPME) is a solvent-free sample-preparation technique that enables isolation and pre-concentration of analytes from a sample on a thin film coating a fused-silica fiber. In this study SPME coupled with microcolumn liquid chromatography (micro LC) has been used for the determination of four tricyclic antidepressants (amitriptyline, imipramine, nortriptyline, and desipramine) in human urine. SPME conditions which affect extraction efficiency were optimized, and under the optimum conditions the system was a few hundred times more sensitive than direct LC analysis without SPME. For amitriptyline the detection limit was 3 ng mL⁻¹ and the calibration curve was linear in the range of 5–500 ng mL⁻¹. The SPME-micro LC method has been applied to the analysis of amitriptyline in patient’s urine.     

Determination of phenolic antioxidants in pharmaceutical formulations by liquid chromatography and migration study on HDPE packagings

Summary The performance of UV diode-array, spectrometric and electrochemical detectors was compared in the chromatographic analysis of trace amounts of six phenolic antioxidants. Quantitative validation was undertaken; the linearity range was wider using UV detection although the limits of detection were lower with electrochemical detection. UV detection was applied both to identification of an antioxidant in a hydrophilic suspension and to a migration study.     

Development of a 6-hydroxychroman-based derivatization reagent: application to the analysis of 5-hydroxytryptamine and catecholamines by using high-performance liquid chromatography with electrochemical detection

We developed a novel derivatization reagent, (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (alpha-CA), which exhibits intense EC response. NPCA successfully yielded alpha-CA derivatives of primary amines by a two-step derivatization procedure. Following pre-column derivatization with NPCA, a simultaneous determination of alpha-CA derivatives of neuroactive monoamines [dopamine (DA), epinephrine, and 5-hydroxytryptamine (5-HT)], their monoamine oxidase metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid) and their catechol-O-methyltransferase metabolites [3-methoxytyramine (3-MT) and normetanephrine (NMN)] was completely achieved using our HPLC-EC method. Using an HPLC equipped with coulometric electrode-array detection system, the resultant alpha-CA derivatives of NMN, 5-HT, DA and 3-MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4-fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16-60 fmol on column (signal-to-noise ratio 3). The proposed HPLC method was applied to determine 5-HIAA, HVA, alpha-CA-5-HT and alpha-CA-DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions.