Development and validation of a reversed-phase HPLC method for CYP1A2 phenotyping by use of a caffeine metabolite ratio in saliva.

CYP1A2 is important for metabolizing various clinically used drugs. Phenotyping of CYP1A2 may prove helpful for drug individualization therapy. Several HPLC methods have been developed for quantification of caffeine metabolites in plasma and urine. Aim of the present study was to develop a valid and simple HPLC method for evaluating CYP1A2 activity during exposure in xenobiotics by the use of human saliva. Caffeine and paraxanthine were isolated from saliva by liquid-liquid extraction (chlorophorm/isopropanol 85/15v/v). Extracts were analyzed by reversed-phase HPLC on a C18 column with mobile phase 0.1% acetic acid/methanol/acetonitrile (80/20/2 v/v) and detected at 273nm. Caffeine and paraxanthine elution times were <13min with no interferences from impurities or caffeine metabolites. Detector response was linear (0.10-8.00µg/ml, R(2) >0.99), recovery was >93% and bias <4.47%. Intra- and inter-day precision was <5.14% (n=6). The limit of quantitation was 0.10µg/ml and the limit of detection was 0.018±0.002µg/mL for paraxanthine and 0.032±0.002µg/ml for caffeine. Paraxanthine/caffeine ratio of 34 healthy volunteers was significantly higher in smokers (p<0.001). Saliva paraxanthine/caffeine ratios and urine metabolite ratios were highly correlated (r=0.85, p<0.001). The method can be used for the monitoring of CYP1A2 activity in clinical practice and in studies relevant to exposure to environmental and pharmacological xenobiotics.

Development and application of GC-MS method for monitoring of long-term exposure to the pesticide cypermethrin.

Cypermethrin (CPMN) is a synthetic pyrethroid used as an insecticide in large-scale commercial agricultural applications as well as for domestic purposes. In the present study a gas chromatography-mass spectrometry (GC-MS) based method was developed and validated for the quantitation of CPMN metabolites, 3-phenoxybenzoic acid (3-PBA) and cis- and trans- 3-(2,2-dichlorovinyl)-2,2-dimethyl-1-cyclopropane (cis- and trans- Cl2 CA). The developed method was applied for the monitoring of CPMN metabolites in hair of laboratory animals (rabbits) intentionally exposed per os to CPMN at 40 (low dose) and 80 (high dose) mg/kg weight/day for 16 weeks. The analytical method comprises three main steps: isolation of analytes from hair, analytes derivatization, and subsequent instrumental analysis by GC-MS. The limits of detection ensured by the method are 4.0, 3.9 and 1.0 pg/mg hair for cis-Cl2 CA, trans-Cl2 CA and 3-PBA, respectively. The instrument responce is linear (r(2) > 0.99) in the investigated concentrations range from 25 to 1000 pg/mg. With and between-run precision as well as accuracy were estimated and found satisfactory. Analytes were efficiently isolated by solid-liquid extraction from hair with recoveries greater than 84.8% for cis-Cl2 CA, 87.2% for trans-Cl2 CA and 96.4% for 3-PBA. Rabbit's hair showed increasing levels for all metabolites (metabolites accumulation in a time and dose dependent manner) over time and in a dose-dependent manner. The developed experimental procedure could be used for biomonitoring of population exposure to CPMN.

Hypospadias in offspring is associated with chronic exposure of parents to organophosphate and organochlorine pesticides.

We have currently evaluated the possible association between hypospadias and exposure to organophosphorus (OP) and organochlorine (OC) pesticides. For this purpose, we measured the dialkyl phosphate metabolites of organophosphate pesticides (DAPs) in the hair and blood, as well as OC pesticides (DDTs, HCHs) in the hair collected from children with hypospadias and their parents. The concentration of HCHs in the hair samples obtained from mothers was higher than that previously reported for people working in open cultivations, while the concentration of DDTs in the hair samples obtained from mothers, fathers and their children with hypospadias was much higher than that previously reported for occupationally exposed individuals. The DMP concentration in hair samples obtained from mothers was much higher not only from that reported for the general population, but even higher than that reported for occupationally exposed individuals. Furthermore, SUMDEPs and SUMDAPs in the hair samples obtained both from the hypospadiac boys, as well as from their parents were higher than the corresponding values previously reported for the general population. Our study supports the hypothesis that organophosphate and organochlorine pesticide exposure may be a potential risk factor for hypospadias.

Development and application of LC­APCI­MS method for biomonitoring of animal and human exposure to imidacloprid.

Imidacloprid (IMI) is a relatively new neuro-active neonicotinoid insecticide and nowadays one of the largest selling insecticides worldwide. In the present study a LC­APCI­MS based method was developed and validated for the quantification of imidacloprid and its main metabolite 6-chloronicotinic acid (6- CINA) in urine and hair specimens. The method was tested in biomonitoring of intentionally exposed animals and subsequently applied for biomonitoring of Cretan urban and rural population. The developed analytical method comprises two main steps of analytes isolation from specimen (solid­ liquid extraction with methanol for hair, liquid­liquid extraction with methanol for urine) and subsequent instrumental analysis by LC­APCI­MS. The developed method was applied for the monitoring of IMI and 6-ClNA in hair and urine of laboratory animals (rabbits) intentionally fed with insecticide at low or high doses (40 and 80 mg kg(-1) weight d(-1) respectively) for 24 weeks. The analytes were detected in the regularly acquired hair and urine specimens and their found levels were proportional to the feeding dose and time of exposure with the exception of slight decline of IMI levels in high dose fed rabbits after 24 weeks of feeding. This decline can be explained by the induction of IMI metabolizing enzymes by the substrate. After testing on animal models the method was applied for pilot biomonitoring of Crete urban (n = 26) and rural (n = 32) population. Rural but not urban population is exposed to IMI with 21 positive samples (65.6%) and found median concentration 0.03 ng mg(-1). Maximum concentration detected was 27 ng mg(-1)

Investigation of in vivo toxicity of hydroxylamine sulfate and the efficiency of intoxication treatment by α-tocopherol acetate and methylene blue.

OBJECTIVES: Investigation of hydroxylamine sulfate toxicity mechanism in vivo and estimation of α-tocopherol acetate and methylene blue efficiency in poisoning treatments. METHODS: In vivo experiments were conducted on 102 Wistar Han rats. The experiments investigated the hematotoxic and oxidative stress effects of hydroxylamine sulfate in acute and subacute toxicity treatment of animals. Electron Spin Resonance was used for quantitative determination of blood and liver tissue parameters alterations after intoxication. The osmotic fragility of erythrocytes, lipid peroxidation intensity and level of SH-groups in liver of rats were determined by established biochemical assays. RESULTS: Hydroxylamine sulfate cause an acute hematotoxicity and oxidative stress in vivo as demonstrated by the appearance of free oxidized iron in blood, reduced glutathione content and increased lipid peroxidation in liver. The experimental studies showed the formation of Hb-NO, MetHb in erythrocytes and as well of stable complex of reduced iron (Fe(2+)) with hydroxylamine sulfate. Methylene blue treatment does not reduce the Hb-NO or MetHb levels in intoxicated animals while administration of α-tocopherol acetate reduces substantially lipid peroxidation. CONCLUSIONS: Oxidative stress is a key mechanism of acute hematotoxicity caused by hydroxylamine sulfate. Methylene blue is not suitable antidote in case of hydroxylamine intoxication.

Analytical methods of biological monitoring for exposure to pesticides: recent update.

Extensive use of synthetic pesticides for agricultural and nonagricultural purposes began in the past 50 years. As a result of their wide and extensive application, exposure to hazardous pesticides is a concern to the general population and occupationally exposed persons. Robust methods are therefore needed for measuring markers of pesticide exposure. This article presents a review of the most recently published analytical methodologies and instrumentations developed for and applied to biological monitoring of exposure to pesticides of various classes. Most of the methods reviewed here are based on chromatography combined with mass spectrometry detection. This work clearly demonstrates that although gas chromatography still appears to be the most widely employed technique for pesticide analysis in various biological samples, recently a trend has been observed toward the use of liquid chromatography coupled with tandem mass spectrometry.

Pitfalls in drying oils identification in art objects by gas chromatography.

Drying oils identification in art objects is an important step in the scientific investigation of the artifact which provides conservators and art historians with valuable information concerning materials used and painting techniques applied. The present communication is devoted to pitfalls and troubleshooting in drying oils identification by means of GC-MS analysis of fatty acids composition in a microsample of an art object. We demonstrate that in the case of nonlinear instrument response the ratios of palmitic to stearic (P/S), distinctive for each oil type and used for drying oil identification, depend on sample dilution so that different dilutions of the same sample can give different P/S ratios. This phenomenon can hinder drying oil identification and lead to erroneous interpretations. This is an important observation as nowadays very often the P/S ratio is calculated from the corresponding peak area ratios or by the use of one-point calibration method. In these approaches, the linearity of the instrument response is not controlled and ensured. In the case analyzed, the nonlinear instrument response was attributed to incomplete sample evaporation in the injector. Packing of the glass liner with deactivated glass wool improved the sample evaporation and ensured the linearity of the instrument response and independence of the P/S ratio from sample dilution.

Impact of biological factors on binding media identification in art objects: identification of animal glue in the presence of Aspergillus niger.

The materials and especially organic materials used for creation of art objects can be utilized by various microorganisms for their growth and facilitate the microbial colonization of the object. An understanding of the chemical alterations in artefacts caused by the presence of microorganisms can be crucial for correct identification of the materials initially used for the artefact creation--nowadays an important step in restoration and/or art-historical investigation of the art object. The present article describes a model experiment in which we investigated the possible chemical alterations in animal glue films used as substrate for growth of the fungus Aspergillus niger. The sterilized animal glue solution was poured into Petri dishes, inoculated with Aspergillus niger, and subsequently incubated at 15 degrees C for 0, 7, 9, 14, and 28 days. After interruption of incubation, the content of the Petri dish was analyzed for amino acid composition by the GC-MS based method. It was found that the growth of Aspergillus niger on animal glue films did not cause significant changes in the amino acid composition of the film and had no impact on animal glue identification.

Hair analysis used to assess chronic exposure to the organophosphate diazinon: a model study with rabbits.

The main purpose of the present study was to determine whether hair analysis would be a suitable method to assess chronic exposure of rabbits to the pesticide diazinon. A controlled study was designed, in which white rabbits of the New Zealand variety were systemically exposed to two dosage levels (15 mg/kg per day and 8 mg/kg per day) of the pesticide, through their drinking water, for a period of 4 months. Hair samples from the back of the rabbits were removed before commencing the experiment and at the end of the dosing period. Parallel experiments with spiked hair were carried out in order to design a simple and efficient method of extraction of diazinon from hair. The hair was pulverized in a ball mill homogenizer, incubated in methanol at 37 degrees C overnight, liquid-liquid extracted with ethyl acetate and measured by chromatography techniques (GC-NPD and GC-MS) for confirmation. The concentration of the diazinon in the hair of the exposed animals ranged from 0.11 to 0.26 ng/mg hair. It was concluded that there is a relationship between the administered dose and the detected pesticide concentration in hair. Finally, it seems that hair analysis may be used to investigate chronic exposure to the pesticide.