Low dose exposure to HBCD, CB-153 or TCDD induces histopathological and hormonal effects and changes in brain protein and gene expression in juvenile female BALB/c mice.

Developmental health risks of chronical exposure to low doses of foodborne persistent organic pollutants (POP) are recognized but still largely uncharacterized. Juvenile female BALB/c mice exposed to either HBCD, CB-153 or TCDD at doses relevant to human dietary exposures (49.5 µg, 1.35 µg and 0.90 ng kg-1 bw-1 day-1, respectively) for 28 days displayed histopathological changes in liver (HBCD, CB-153, TCDD), thymus (HBCD, CB-153) and uterus (HBCD), reduced serum oestradiol 17ß (E2) levels (HBCD), increased serum testosterone (T) levels (CB-153) and an increased T/E2 ratio (HBCD). Proteomics analysis of brain provided molecular support for the HBCD-induced reduction in E2. Neural gene expression analysis, confirmed effects on 18 out of 30 genes previously found to be affected after exposure to higher doses to the same pollutants. Our findings indicate that exposure to POP at low doses is associated with subtle, but toxicological relevant effects on post-natal development in female mice.

European official control of food: Determination of histamine in fish products by a HPLC-UV-DAD method.

The evaluation of histamine content in fish and fishery products, responsible for scombroid poisoning, is essential to guarantee the safety of food. EU regulation requires validated analytical methods to ensure the verification of compliance with food law in official control activity. To this aim a previous gradient RP-HPLC method with DAD detection was modified and validated, according to international guidelines. The reliability of results was tested by analysing fish reference materials within the participation in European proficiency tests. The method has been used for the analysis of real samples consisting of several fish-based products with considerable differences in matrix composition. This characteristic is of great relevance to be able of apply the method in the field of official control.

New High-performance Liquid Chromatography-DAD Method for Analytical Determination of Arbutin and Hydroquinone in Rat Plasma.

Natural substances present in herbal preparations should be carefully used because they can give toxic or therapeutic effects despite of their amount or the way of administration. The safety of products of vegetable origin must be assessed before commercialisation by monitoring the active ingredients and their metabolites. This study was therefore designed to identify and quantify arbutin and its metabolite hydroquinone, naturally present in Arctostaphylos uva-ursi (L.) Spreng plant in rat plasma, after an acute and subacute administration of aqueous arbutin solution in Wistar rats. For this purpose a reversed-phase high-performance liquid chromatography coupled with photodiode array detection was developed to assess the pharmacokinetic of arbutin and hydroquinone in plasma of female rats treated with aqueous arbutin solutions. The detection (arbutin: 0.0617 µg/ml and hydroquinone 0.0120 µg/ml) and quantification (arbutin: 0.2060 µg/ml and hydroquinone: 0.0400 µg/ml) limits were determined. At the arbutin concentration level of 10.7 µg/ml repeatability was 13.33% and its recovery 93.4±6.93%, while at the hydroquinone concentration level of 10.6 µg/ml repeatability was 11.66% and its recovery 92.9±7.75%. Furthermore the method was fully validated and the obtained data indicate that the new method provides good performances.

Dietary exposure of juvenile female mice to polyhalogenated seafood contaminants (HBCD, BDE-47, PCB-153, TCDD): comparative assessment of effects in potential target tissues.

Fish represents source of nutrients and major dietary vehicle of lipophilic persistent contaminants. The study compared the effects of two legacy and two emerging fish pollutants (Hexabromocyclododecane HBCD; 2,2',4,4'-Tetrabromodiphenyl ether BDE-47; 2,2',4,4',5,5'-Hexachlorobiphenyl PCB-153; 2,3,7,8-Tetrachlorodibenzo-p-doxin TCDD) in juvenile female mice exposed through a salmon based rodent diet for 28 days (dietary doses: HBCD 199 mg/kg bw/day; BDE-47 450 µg/kg bw/day; PCB-153 195 µg/kg bw/day; TCDD 90 ng/kg bw/day). Dose levels were comparable to previously reported developmental Lowest Observed Adverse Effect Levels. None of the treatments elicited signs of overt toxicity, but HBCD increased relative liver weight. All compounds caused changes in liver, thymus and thyroid; spleen was affected by BDE-47 and PCB-153; no effects were seen in uterus and adrenals. Strongest effects in thyroid follicles were elicited by PCB-153, in thymus and liver by BDE-47. HBCD and BDE-47 induced liver fatty changes, but appeared to be less potent in the other tissues. HBCD, BDE-47 and TCDD increased serum testosterone levels and the testosterone/estradiol ratio, suggesting a potential involvement of pathways related to sex steroid biosynthesis and/or metabolism. The results support the role of toxicological studies on juvenile rodents in the hazard characterization of chemicals, due to endocrine and/or immune effects.

The role of liquid chromatography-mass spectrometry in the determination of heroin and related opioids in biological fluids.

The opioid most commonly sold in the illicit market is heroin. This substance, classified as an analgesic narcotic drug, has an extremely short half-life, and it is rapidly metabolized to 6-monoacetyl-morphine and further to morphine. Morphine is principally metabolized by conjugation to morphine-3 and morphine-6 glucuronides. Morphine itself is a potent analgesic that is frequently used in the pharmacological intervention of cancer pain. The toxicological and clinical evaluation of heroin and morphine have stimulated pharmacokinetic studies in human and animal models. Although a number of methods exist to determine opiates and their metabolites, liquid chromatography (LC) appears to be the technique that can separate without any pretreatment the lipophilic and the hydrophilic analytes of the complete metabolic profile of heroin and/or morphine. Moreover, mass spectrometry (MS) used as a detector for liquid chromatography is unique, because it offers universality and selectivity. Furthermore, efforts have been made to develop LC/MS interfaces that could overcome the previous problem of poor sensitivity. For this reason, in recent years LC combined with MS has been applied to the analysis of opiates--parent drugs and metabolites--in biological fluids. This article reviews the existing literature on the determination, using liquid chromatography coupled to mass spectrometry, of opiate metabolites found in different biological matrices after the administration of the parent compounds.

Determination of lorazepam in plasma and urine as trimethylsilyl derivative using gas chromatography-tandem mass spectrometry.

A procedure based on gas chromatography-tandem mass spectrometry for identification and quantitation of lorazepam in plasma and urine is presented. The analyte was extracted from biological fluids under alkaline conditions using solid-phase extraction with an Extrelut-1 column in the presence of oxazepam-d5 as the internal standard. Both compounds were then converted to their trimethylsilyl derivatives and the reaction products were identified and quantitated by gas chromatography-tandem mass spectrometry using the product ions of the two compounds (m/z 341, 306 and 267 for lorazepam derivative and m/z 346, 309 and 271 for oxazepam-d5 derivative) formed from the parent ions by collision-induced dissociation in the ion trap spectrometer. Limit of quantitation was 0.1 ng/ml. This method was validated for urine and plasma samples of individuals in treatment with the drug.

Determination of opiates and cocaine in hair as trimethylsilyl derivatives using gas chromatography-tandem mass spectrometry.

An analytical method for the determination of heroin, 6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine, and cocaethylene in human hair using gas chromatography-tandem mass spectrometry is presented. The analytes were extracted from finely cut hair with methanol at 56 degrees C for 18 h in the presence of nalorphine as the internal standard. After the incubation, methanol was evaporated to dryness, and all the analytes, except heroin, cocaine, and cocaethylene, were converted to their trimethylsilyl derivatives. The reaction products were identified and quantitated using product ions formed from the parent ions by collision-induced dissociation in the ion-trap mass spectrometer. This method provided excellent sensitivity and specificity for analytes at the concentrations usually found in the keratin matrix.

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 microl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C18 reversed-phase column with a mobile phase consisting of water-acetonitrile (71:29, v/v) containing 0.05 M Na2HPO4 and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1-500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.

Issues in methodology and applications for therapeutic drug monitoring of fluoxetine and norfluoxetine enantiomers.

A standardization of the analytical procedures for monitoring of fluoxetine and norfluoxetine enantiomers is described. Simultaneous determination of fluoxetine and norfluoxetine enantiomers in plasma and serum was performed by high-performance liquid chromatography with a chiral stationary phase, using ultraviolet absorbance detection. The analytes were extracted from the biologic matrix by alkalinization with NaOH and solid-phase extraction. Stability studies were conducted in EDTA, lithium-heparinized plasma and in serum spiked with the analytes stored at +4 degrees C for 1 week and at -20 degrees C for 1 month. Furthermore, stability studies in NaOH and in the extraction solvents were executed. Using this methodology, EDTA plasma is the most suitable matrix for drug monitoring, even if the storage should not exceed 3 weeks at -20 degrees C. Furthermore, the biologic sample should be left in NaOH for a short time before solid-phase extraction to prevent a degradation of matrix, which would interfere with the chromatographic analysis.

Simultaneous determination of heroin 6-monoacetylmorphine, morphine, and its glucuronides by liquid chromatography--atmospheric pressure ionspray-mass spectrometry.

A new analytical technique has been developed for the simultaneous determination of heroin, 6-monoacetylmorphine, morphine, morphine-6- and 3-glucuronides, and codeine in serum using liquid chromatography coupled with ionspray mass spectrometry. The analytes and the internal standard, nalorphine, were subjected to solid-phase extraction (SPE) using ethyl SPE columns before chromatography. The chromatographic separation of the analytes was achieved using a normal phase column and a water-methanol-acetonitrile-formic acid mobile phase at a flow rate of 230 microL/min. The mass spectrometer was operated in selected-ion monitoring mode. Under these conditions, the limit of quantitation was 0.5 ng/ml for heroin, 4 ng/ml for 6-monoacetylmorphine, 4 ng/ml for morphine, 1 ng/ml for morphine-3-glucuronide, 4 ng/ml for morphine-6-glucuronide, and 4 ng/mL for codeine. Serum levels of heroin metabolites were determined in C57BL/6 inbred mice after a dose of 20 mg/kg heroin administered subcutaneously. 6-monoacetylmorphine showed a peak concentration of 0.93 micrograms/mL serum at 3 min, whereas morphine and morphine-3-glucuronide achieved their peak concentrations of 9.6 and 2.9 micrograms/mL serum at 10 and 20 min, respectively. Finally, the absence of morphine-6-glucuronide and codeine excluded the possibility of their formation from morphine in this animal model.

Hair analysis for nicotine and cotinine: evaluation of extraction procedures, hair treatments, and development of reference material.

Analysis of nicotine and cotinine in human hair can provide information on nicotine intake and exposure to environmental tobacco smoke over a long period of time. Nonetheless, to better assess the usefulness of hair analysis to determine smoking habits or exposures, all procedures have to be standardized. Various solvents were tested as washing solvents to eliminate external contamination from nicotine. Dichloromethane was found effective when used for two washes prior to the extraction. Basic and acid digestion of hair followed by solid phase extraction with Extrelut-3 glass column using dichloromethane:isopropyl alcohol (9:1) as eluting mixture both gave good recoveries of nicotine and cotinine, when compared with extractions reported in the literature. The extraction method was free from substances, which could interfere in the chromatographic analysis. Furthermore, the addition of methanolic HCl to the eluting mixture prevented the loss of nicotine during the evaporation step before chromatography. Chromatography was performed using a reversed-phase column and a U.V. detection at 254 nm. Furthermore, hair treatments (dyes, permanent wave, hydrogen peroxide) caused a major decrease in the nicotine content in hair, and a smaller effect on cotinine levels. However, the effect of various treatments was not reproducible. Several attempts to produce reference materials were carried out. Nicotine and cotinine standard solutions at different concentrations were added to blank hair soaked in dimethylsulfoxide, methanol and water.

The analysis of nicotine in infants' hair for measuring exposure to environmental tobacco smoke.

Hair samples were collected from 24 infants (3-36 months) exposed and non-exposed to environmental tobacco smoke. Hair was washed in dichloromethane, digested in NaOH, extracted by solid-phase extraction and analyzed by high-performance liquid chromatography to determine the content of nicotine and cotinine. Nicotine concentration in non-exposed infants (1.3 +/- 1.7 ng/mg hair) was significantly different from that in occasionally exposed infants (6.8 +/- 2.1 ng/mg hair) and that in infants passively exposed to smoke (15.4 +/- 6.7 ng/mg hair). Cotinine could be measured only in passive smokers infants. These findings suggest the possibility of monitoring exposure to environmental tobacco smoke in infants by using nicotine measurement in hair rather than in urine, blood or saliva.

Drug monitoring in nonconventional biological fluids and matrices.

Determination of the concentration of drugs and metabolites in biological fluids or matrices other than blood or urine (most commonly used in laboratory testing) may be of interest in certain areas of drug concentration monitoring. Saliva is the only fluid which can be used successfully as a substitute for blood in therapeutic drug monitoring, while an individual's past history of medication, compliance and drug abuse, can be obtained from drug analysis of the hair or nails. Drug concentrations in the bile and faeces can account for excretion of drugs and metabolites other than by the renal route. Furthermore, it is important that certain matrices (tears, nails, cerebrospinal fluid, bronchial secretions, peritoneal fluid and interstitial fluid) are analysed, as these may reveal the presence of a drug at the site of action; others (fetal blood, amniotic fluid and breast milk) are useful for determining fetal and perinatal exposure to drugs. Finally, drug monitoring in fluids such as cervical mucus and seminal fluid can be associated with morpho-physiological modifications and genotoxic effects. Drug concentration measurement in nonconventional matrices and fluids, although sometimes expensive and difficult to carry out, should therefore be considered for inclusion in studies of the pharmacokinetics and pharmacodynamics of new drugs.

Household and community determinants of exposure to involuntary smoking: a study of urinary cotinine in children and adolescents.

The study examines the role of several potential predictors of urinary cotinine levels in a cross-sectional sample of 1,072 nonsmoking children and adolescents in Latium, Italy, during 1990-1991. As expected, there was a strong relation between passive exposure to smoking and the amount of maternal and paternal self-reported smoking. The urinary cotinine level increased with a decreasing level of paternal education and with an increasing index of household crowding; self-report of recent exposure to smoking outside the home was a strong predictor of the biologic marker. The analysis was then restricted to 346 subjects whose parents claimed that they were nonsmokers and that there were no smokers at home. In this group, however, 57 children reported some active smoking at home by their parents. Those with parents suspected to be "deceivers" had higher level of urinary cotinine than did those truly not exposed. In addition, urinary cotinine in this group was clearly associated with duration of exposure to smoking outside home. The study indicates that both factors related to family circumstances and exposure outside the household setting are strong determinants of urinary cotinine levels. The finding may be considered a direct confirmation that passive smoking among children should be viewed as a specific community responsibility.

Improved clean-up procedure for the high-performance liquid chromatographic assay of clomipramine and its demethylated metabolite in human plasma.

A rapid and selective assay of clomipramine and its metabolite desmethylclomipramine in human plasma, based on high-performance liquid chromatography with UV detection has been developed. The compounds were subjected to solid-phase extraction, using Extrelut 1 cartridges. Recoveries ranged between 88-95% for clomipramine, and 75-80% for desmethylclomipramine. This method has been used for therapeutic monitoring of clomipramine and its metabolite in individuals treated with this drug.

Solid-phase extraction of nicotine and its metabolites for high-performance liquid chromatographic determination in urine.

A solid-phase extraction, using Extrelut-1 glass columns, has been applied to urine samples of both passive and active smokers for high-performance liquid chromatographic determination of nicotine and its metabolites cotinine and trans-3'-hydroxycotinine. Chromatography was performed using a reversed-phase LC8DB column and a mobile phase consisting of water-acetonitrile (80:9, v/v) containing 5 ml triethylamine, 670 mg/l sodium heptanesulphonate, and 0.034 M each of K2HPO4 and citric acid (pH 4.4), at a flow-rate of 1.6 ml/min. The results obtained indicate that solid-phase extraction is a reliable and quick procedure which can be applied also to other nicotine metabolites.