Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

Low-volume,high-sensitivity assay for cadmium in blood and urine using conventional atomic absorption spectrophotometry

An assay for cadmium in whole blood and urine using deuterium background-correction electrothermal atomic absorption spectroscopy (D(2)-ETAAS) was developed. Cadmium (in a 1- to 2-ml sample) was bound to 15 mg anion-exchange resin, interfering ions were removed in a 2-ml Bio-Spin column, and cadmium was extracted into 100 microl 1M nitric acid for analysis. Cadmium in the sample extract was concentrated 7-fold for blood and 10-fold for urine over the starting material. These steps produced cadmium atomic absorption traces with high signal to background ratios and allowed analysis against aqueous standards. At approximately 0.1 ng Cd/ml, mean intra- and interassay coefficients of variation were 11-12%. Cadmium recovery for 0.1 to 0.6 ng added cadmium was 107+/-4% for blood and 94+/-4% for urine (mean+/-SE, n=3). The mean detection limit (mean + 3 x SD of blank) was 0.008 ng/ml for blood and 0.003 ng/ml for urine. Samples from "unexposed" animals including humans ranged from 0.051+/-0.000 to 0.229+/-0.035 ng/ml. Values were approximately 10-fold lower than those obtained by the method of Stoeppler and Brandt using Zeeman background-correction ETAAS. This new high-sensitivity, low-volume assay will be useful for epidemiological studies, even those involving children, and will provide a means to help determine the contribution of cadmium to disease incidence in the general population.     

Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry

The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 microl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 microm I.D.x 30 mm 10 microm Kromasil C(18) pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 microm I.D.x 100 mm 3.5 microm Kromasil C(18) analytical column. Loading flow rates up to 100 microl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M-H](-) at m/z 217.08. The method was validated over the concentration range 0.2-40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4-7.3 and 7.0-8.1%, respectively, and the recoveries were in the range 96.2-97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.     

Determination of trenbolone and its metabolite in bovine fluids by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed for the determination of trenbolone in bovine urine and serum. The aim was a control of the misuse of trenbolone in food-producing animals. The procedure involved, in both cases, a preliminary solid-phase clean-up followed by a liquid-liquid extraction for urine samples after a preliminary enzymatic hydrolysis. The extracts have been directly analysed by reversed-phase LC-MS-MS in selected reaction monitoring (SRM), acquiring two diagnostic product ions from the chosen precursor [M+H](+). The procedures were validated across the concentration range of 1-1500 ng/ml. The linearity, the inter- and intra-day accuracy and precision have been determined. The procedure was specific and the accuracy values were better than 20% at the limit of quantitation of spiked samples. The limit of quantification (LOQ) and the limit of detection (LOD) were, respectively, 1 ng/ml and 350 pg/ml for urine and serum. According to the draft, SANCO/1805/2000, we determined the decision limit CCalpha and the detection capability CCbeta. The recovery values for urine ranged from 87 to 128%, and for plasma the recovery was 70+/-4%. The procedure proved to be simple and suitable for routine and confirmatory purposes such as those developed for residue studies.     

Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3 microm, 150 x 4.6 mm I.D.), while the mobile phase consisted of 75 mM sodium acetate-acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9+/-4.0% for non-deproteinized samples and 93.2+/-4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean+/-SD concentrations in the darkness period were 23.18+/-7.44 pg/ml for free melatonin and 82.5+/-36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23+/-2.22 and 7.40+/-5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01+/-2.31 and 28.55+/-6.95 pg/ml for the free and total fraction, respectively.     

Nickel absorption and kinetics in human volunteers

Mathematical modeling of the kinetics of nickel absorption, distribution, and elimination was performed in healthy human volunteers who ingested NiSO4 drinking water (Experiment 1) or added to food (Experiment 2). Nickel was analyzed by electrothermal atomic absorption spectrophotometry in serum, urine, and feces collected during 2 days before and 4 days after a specified NiSO4 dose (12 micrograms of nickel/kg, n = 4; 18 micrograms of nickel/kg, n = 4; or 50 micrograms of nickel/kg, n = 1). In Experiment 1, each of the subjects fasted 12 hr before and 3 hr after drinking one of the specified NiSO4 doses dissolved in water; in Experiment 2, the respective subjects fasted 12 hr before consuming a standard American breakfast that contained the identical dose of NiSO4 added to scrambled eggs. Kinetic analyses, using a compartmental model, provided excellent goodness-of-fit for paired data sets from all subjects. Absorbed nickel averaged 27 +/- 17% (mean +/- SD) of the dose ingested in water vs 0.7 +/- 0.4% of the same dose ingested in food (a 40-fold difference); rate constants for nickel absorption, transfer, and elimination were not significantly influenced by the oral vehicle. The elimination half-time for absorbed nickel averaged 28 +/- 9 hr. Renal clearance of nickel averaged 8.3 +/- 2.0 ml/min/1.73 m2 in Experiment 1 and 5.8 +/- 4.3 ml/min/1.73 m2 in Experiment 2. This study confirms that dietary constituents profoundly reduce the bioavailability of Ni2+ for alimentary absorption; approximately one-quarter of nickel ingested in drinking water after an over-night fast is absorbed from the human intestine and excreted in urine, compared with only 1% of nickel ingested in food. The compartmental model and kinetic parameters provided by this study will reduce the uncertainty of toxicologic risk assessments of human exposures to nickel in drinking water and food.     

Serum selenium levels in healthy slovak children and adolescents

Blood serum selenium levels were measured in 891 healthy children and adolescents (aged 11–18 yr, 450 girls and 441 boys) residing in both rural and urban areas from eight regions of Slovakia. Subjects were divided into four age groups (11–12 y, 13–14 y, 15–16 y, and 17–18 y). Serum selenium concentration was determined by the electrothermal atomic absorption spectrometric method. The mean (±SD) serum selenium concentrations were 0.750 ±0.255 μmol/L in girls and 0.773 ±0.235 μmol/L in boys. A large proportion of the individuals (25.7% in girls, 18.1% in boys) exhibited serum selenium levels under 0.57 μmol/L (45 μg/L). An increasing trend of the serum selenium values with age has been observed in both boys (p < 0.01) and girls (p < 0.05). Boys had higher serum selenium levels in the all age groups but the differences were not statistically significant.     

Selenium levels in related biological samples: human placenta, maternal and umbilical cord blood, hair and nails.

A study on selenium levels has been carried out in human placenta, maternal and umbilical cord blood, hair and nails of a group of 50 mothers and in the hair of the newborns. The determinations were perfomed by electrothermal atomic absorption spectrometry. The selenium concentration obtained for each sample type was as follows: For the human placenta the values obtained were between 0.56 and 1.06 microg/g (mean +/- standard deviation: 0.81 +/- 0.02 microg/g). The levels for the umbilical cord blood were 51.1-104.2 microg/l (76.3 +/- 6.5 microg/l). For the maternal blood the values measured were between 57.3 and 117.9 microg/l (90.0 +/- 15.2 microg/l), and for hair and nails were 0.22-1.5 microg/g (0.60 +/- 0.37 microg/g) and 0.46-1.57 microg/g (0.90 +/- 0.27 microg/g), respectively. For the hair of the newborns the values obtained were between 0.40 and 2.53 microg/g (1.04 +/- 0.48 microg/g). The effect of different variables as age, habitat, nutritional index or gestation age of the mothers on the selenium concentration in the samples was studied. The influence of the habitat is significant with a confidence level of 95% for the selenium concentration in maternal blood and umbilical cord blood samples. The influence of the mothers' age is significant with a confidence level of 95% for the selenium concentration in the umbilical cord blood samples. For the placenta samples, the effect of the nutritional index is significant with a confidence level of 95%. There is a positive correlation between samples of umbilical cord blood and the newborns' hair, between placenta and umbilical cord, and between cord blood and maternal blood.     

A sensitive liquid chromatographic method for the spectrophotometric determination of urinary trans,trans-muconic acid

Benzene is a human carcinogen and its metabolite, urinary trans,trans-muconic acid (ttMA), is a biomarker for risk assessment. However, most of the existing methods were not sensitive enough for monitoring of low level exposure. This paper describes a HPLC-UV method for ttMA determination with enhanced selectivity and sensitivity. A 30 mg OasisMAX cartridge was used to clean-up 50 microl of urine sample and gradient elution was performed on a Zorbax SB-C(18) column (30 degrees C). ttMA was detected at wavelength 263 nm using a UV diode array detector (DAD). The two mobile phases used were (A) 150 mM ortho-phosphoric acid containing of 9% (v/v) methanol; and (B) 125 mM ortho-phosphoric acid containing 30% (v/v) acetonitrile. The method was validated with 61 urine samples collected from non-occupationally benzene exposed individuals and 14 quality control specimens from an international quality assessment scheme. The urinary ttMA concentrations (mean+/-S.D.microg/g creatinine) were 90+/-34 for smokers (n=26), 49+/-39 for non-smokers (n=21) and 23+/-18 for non-smoking hospital staff (n=14). A correlation coefficient, r=0.99 was found with 14 external quality specimens for ttMA ranged from 0.4 to 6.8 mg/l. The recovery and reproducibility were generally over 90% and the detection limit was 5 microg/l.     

Liquid chromatography-tandem mass spectrometry method for determination of phencynonate in rat blood and urine

A sensitive and specific high-performance liquid chromatographic assay with electrospray ionization mass spectrometry detection (LC-ESI-MS) has been developed and validated for the identification and quantification of the novel anticholinergic drug phencynonate in rat blood and urine. The sample pretreatment involves basification and iterative liquid-liquid extraction with ethyl ether-dichloromethane (2:1, v/v) solution, followed by LC separation and positive electrospray ionization mass spectrometry detection. The chromatography was on BetaBasic-18 column (150 mm x 2.1mm i.d., 3 microm). The mobile phase was composed of methanol-water (85:15, v/v), containing 0.5 per thousand formic acid, which was pumped at a flow-rate of 0.2 ml/min. Thiencynonate was selected as the internal standard (IS). Simultaneous MS detection of phencynonate and IS was performed at m/z 358.4 (phencynonate), m/z 364 (thiencynonate), and the selected reaction ion monitoring (SRM) of the two compounds was at 156. Phencynonate eluted at approximately 5.25 min, thiencynonate eluted at approximately 5.10 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1-100 ng/ml in rat blood and 1-500 ng/ml in rat urine. The lower limit of quantification (LLOQ) was reproducible at 1 ng/ml in both of rat blood and urine. The precision measured was obtained from 2.92 to 9.76% in rat blood and 4.17 to 9.76% in rat urine. Extraction recoveries were in the range of 69.57-79.49% in blood and 56.85-64.86% in urine. This method was successfully applied to the identification and quantification of phencynonate in pharmacokinetic studies.     

Serum and urine levels of interleukin-8 in patients with non-Hodgkin's lymphoma

Angiogenesis plays an important role in many types of cancer. Interleukin-8 (IL-8) is known to be a pro-inflammatory and pro-angiogenic cytokine, and IL-8 has been reported to be associated with tumor progression, prognosis and survival in several types of cancers. However, the role of IL-8 in non-Hodgkin's lymphoma (NHL) has not been fully determined. Here, we evaluated the usefulness of measuring serum and urine IL-8 levels in patients with NHL. We developed reference intervals for serum and urine IL-8 level in 131 control individuals. We measured serum IL-8 and urine IL-8 levels in patients with NHL, and we compared the concentrations with those of control individuals. The reference intervals for serum IL-8 and urine IL-8 corrected by creatinine (Cr) were 15.9-430.3 pg/mL and 0.0-28.4 pg/mg Cr, respectively. The concentrations of urine IL-8/Cr were significantly higher in patients than in controls (48.9+/-194.4 vs. 5.2+/-13.8 pg/mg Cr, P<0.001). However, there were no significant differences in serum IL-8 concentrations between NHL patients and controls (159.2+/-40.4 vs. 99.6+/-107.1 pg/mL; P=0.099). Receiver operating characteristic (ROC) analysis gave 0.83 and 0.43 ROC area values for urine IL-8/Cr and serum IL-8, respectively. There was no correlation between the serum and urine concentrations of IL-8 and clinical variables, the only exception being the international prognostic index (IPI), which showed a marginal correlation with urine IL-8/Cr levels (P=0.07). This study indicated that urine IL-8/Cr levels might be useful as a diagnostic marker of NHL.     

Improved method for selenium determination in biological samples by gas chromatography

A gas chromatographic assay employing electron-capture detection for the determination of selenium in biological samples is reported. A calibration curve of 4-nitro-o-phenylenediamine derivative of selenium as a function of peak area was linear from 5–1000 pg. The limit of detection for the electron-capture detector was approximately 0.5 pg. Recoveries of selenium added to various biological materials ranged from 95–105%. This procedure reduces the number of transfers thereby reducing errors associated with losses or contamination. One advantage of the present method is that interfering compounds occurring in previously employed chromatographic methods are eliminated. This procedure can be used for routine microanalysis of selenium. Samples containing less than 2 ng selenium in 200 μl of biological fluid can be routinely analyzed using this method.     

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.     

Determination of selenium in serum by FI-HG-AAS and calculation of dietary intake

A method was developed for the determination of selenium concentration in serum by flow injection-hydride generation-atomic absorption spectrometry (FI-HG-AAS) following microwave digestion of serum samples and reduction of selenate to selenite. The detection limit of the method was 0.3 μg Se/L and the characteristic concentration, corresponding to the 0.0044 absorbance signal, was 0.12 μg Se/L. The results from the analysis of two Seronorm standard reference materials showed good agreement with the certified values. The method was then used to analyze selenium in sera of Austrian and Slovenian people for the calculation of dietary intakes. The selenium concentrations in sera of mothers at delivery, their neonates, and the male and female adults were 71 ± 14, 42 ± 6, 75 ± 21, and 65 ± 16 μg/L for the Austrians and 62 ± 15, 34 ± 7, 70 ± 12, and 66 ± 15 μg/L for the Slovenians. The dietary intakes of selenium of the mothers and the male and the female adults were calculated as 52, 37, and 46 μg/d for the Austrians and 45, 38, and 32 μg/d for the Slovenians.     

Stability studies of selected doping agents in urine: caffeine

The stability of caffeine in urine samples has been studied. A high-performance liquid chromatography (HPLC) method for the quantification of caffeine in urine samples was validated for that purpose. The method consists of a liquid-liquid extraction at alkaline pH with chloroform-2-propanol (9:1, v/v) with a salting out effect. 7-Ethyltheophylline was used as internal standard (ISTD). Analyses were performed with an Ultrasphere ODS C18 column using water/acetonitrile (90:10, v/v) as a mobile phase at a flow rate of 1 ml/min. Ultraviolet absorption at 280 nm was monitored. Extraction recoveries for caffeine and 7-ethyltheophylline were 81.4+/-6.0 and 87.3+/-5.7%, respectively. The calibration curves were demonstrated to be linear in the working range of 6-30 microg/ml (r2>0.990). The limit of detection and the limit of quantitation were estimated as 0.7 and 2.0 microg/ml, respectively. Precisions in the range of 1.5-9.2 and 4.1-5.8% were obtained in intra- and inter-assay studies, respectively, using control samples containing 10, 14 and 26 microg/ml of caffeine. Accuracies ranging from 2.9 to 7.4% for intra-assay experiments, and from 3.9 to 5.4% in inter-assay studies were obtained. Stability of caffeine in urine samples was evaluated after long- and short-term storage at different temperature conditions. The batches of spiked urine were submitted to sterilization by filtration. No adsorption of the analyte on filters was observed. Before starting stability studies, batches of reference materials were tested for homogeneity. For long-term stability testing, caffeine concentration in freeze-dried urine stored at 4 degrees C and in liquid urine samples stored at 4, -20, -40 and -80 degrees C was determined at several time intervals for 18 months. For short-term stability testing, caffeine concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing was also studied for up to three cycles. The stability of caffeine was also evaluated in non-sterile samples stored at -20 degrees C for 18 months. No significant loss of the compound was observed at any of the investigated conditions.     

Determination of kanamycin in serum by solid-phase extraction,pre-capillary derivatization and capillary electrophoresis

An effective method based on solid-phase extraction (SPE) and capillary electrophoresis (CE) for the determination of kanamycin in human serum was developed and validated. Off-line SPE was employed for the isolation of kanamycin from serum on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge. A mixture of 0.2 M borate (pH 10.5)-methanol (50:50, v/v) was used as analyte eluting solvent. After pre-capillary derivatization with o-phthalaldehyde/mercaptoacetic acid reagent, the sample was analyzed by CE with a separation buffer of 30 mM borax, pH 10.0, containing 16% (v/v) methanol. A linear response over the concentration range 5-40 microgram/ml was obtained with a detection limit of 2 microgram/ml. Intra-day and inter-day precision were 6.2 and 10.3% RSD, respectively. Recoveries of approximately 90% were found. For the determination of lower levels of kanamycin (<5 microgram/ml), NH(4)OH (25%, w/v)-methanol (30:70, v/v) was used for analyte elution. After evaporation, reconstitution and derivatization, the sample was analyzed by on-line field-amplified sample stacking (FASS) CE. Good linearity in the concentration range 0.4-5 microgram/ml was obtained with a detection limit of 0.1 microgram/ml. Intra-day and inter-day RSD were 3.4 and 11.2%, respectively. Recoveries of approximately 60% were found. The method was successfully applied to the analysis of kanamycin in sera of tuberculosis patients at peak level and trough level concentrations.     

A specific radioimmunoassay for PGE2 using an antibody with high specificity and a sephadex LH-20 microcolumn for the separation of prostaglandins.

Antiserum against PGE2 was raised in rabbits following immunization with prostaglandin-hen-gamma-globulin conjugate. The antiserum exhibited 14% cross reactivity with PGE1 and far less cross-reaction with heterologous prostaglandins. A microcolumn of Sephadex LH-20 was used for a partial, but sufficient separation of PGE2 from PGE1 and a complete separation from heterologous prostaglandins to ensure a specific RIA for PGE2. The precision of the method in the rage 10-500 picograms showed a coefficient of variation varying between 4 and 13%. The detection limit was 10 picograms corresponding to 15 pg/ml of PGE2 in serum. In order to demonstrate the validity of the method values obtained for non-diuretic rat renal venous serum were compared with those obtained using the isotope derivative method of Bojesen & Buckhave (1972) on the same samples. The concentrations of PGE2 obtained were 239 +/- 25 pg/ml and 250 +/- 58 pg/ml, respectively.     

Determination of plasma melatonin levels by enzyme-linked immunosorbent assay (EIA) in turbot (Scophtalmus maximus L.) and tench (Tinca tinca L.)

An enzymoimmunoassay (EIA) kit for plasma melatonin (MLT) measurements was employed in tench (Tinca tinca) and in turbot (Scophtalmus maximus). Tench and turbot plasma samples were purified with a C18 reversed phase extraction columns because this kit is designed for human serum measurements. The lowest detection limit of the technique was 11.48 pg/well with a sensitivity at 50% binding of 100 pg/well. Intra-assay and inter-assay CV (%) were always less than 5% (n=8), and 9% (n=6) in tench plasma samples, and less than 5% (n=8) and 13% (n=5) in turbot plasma samples, respectively. Correlation coefficients between EIA and RIA measurements in tench and turbot plasma samples were 0.93 and 0.89 (p<0.001) respectively. Diurnal and nocturnal plasma melatonin mean levels were 14.7+/-2.1 pg/ml and 87.4+/-11 pg/ml in tench (n=15), and 3.5+/-0.4 pg/ml and 28.1+/-2.1 pg/ml in turbot (n=15). These species showed a melatonin circadian rhythm as in other animals studied. The results suggest that the commercial kit used in this experiment could be a suitable and alternative method to RIA for plasma MLT determinations in tench and turbot although it is necessary to increase volumes (1ml) and concentrate daytime samples.     

High-performance liquid chromatographic determination of sotalol in biological fluids

A sensitive, selective and reproducible reversed-phase high-performance liquid chromatographic method is described for the quantification of sotalol in human serum and urine. Sotalol and the internal standard, atenolol, were extracted from alkalinized serum and urine (pH 9.0) into 1-butanol—chloroform (20:60, v/v). The organic phase was evaporated, and to the residue was added 0.1 M sulphuric acid (serum analysis) or mobile phase (urie analysis). The mobile phase consisted of 0.01 M phosphate buffer (pH 3.2) and acetonitrile (20:80, v/v) containing 3 mM n-octylsodium sulphate. The flow-rate was 1.5 ml/min. The retention times of atenolol and sotalol were 7 and 10 min, respectively. Ultraviolet detection at 226 nm made it possible to achieve a detection limit of 0.03 μmol/l.