Accidental poisoning after ingestion of "aphrodisiac" berries: diagnosis by analytical toxicology.

BACKGROUND: A large number of plants, seeds, and berries have been used for medicinal, psychotropic, or aphrodisiac purposes for a thousand years. Mandragora officinarum belongs to the family of Solanaceae and is traditionally known as an aphrodisiac and is closely associated with witchcraft. OBJECTIVES: In this study we report a case of an accidental poisoning after ingestion of some "aphrodisiac" berries and the contribution of the toxicological analysis in the case investigation. CASE REPORT: A 35-year-old man was admitted to the hospital with clinical signs and symptoms of an anticholinergic syndrome. The diagnosis of the poisoning was made by the toxicological analysis of the patient's urine. The cause of the poisoning was revealed by his girlfriend's disclosure that the patient had intentionally consumed some "aphrodisiac" berries to enhance his sexual performance. Subsequently, berries similar to the ones consumed were sent to the laboratory. The analysis of the urine and the berries revealed the presence of hyoscyamine and scopolamine; the berries were identified as Mandragora officinarum berries. Decontamination and symptomatic treatment were proven effective for the control of this poisoning. The patient recovered completely after hospitalization for 4 days. CONCLUSION: This case report indicates the importance of analytical toxicology in diagnosis of intoxications after the consumption of unknown plants or plant products and presents the clinical aspects of Mandragora intoxication.

Pre-irradiation exposure of peripheral blood lymphocytes to glutaraldehyde induces radiosensitization by increasing the initial yield of radiation-induced chromosomal aberrations.

Glutaraldehyde (GA) is a high production volume chemical that is very reactive with a wide spectrum of medical, scientific and industrial applications. Since human exposure in anthropogenic and occupational environment occurs frequently, GA has been extensively tested for genotoxic activity in vitro and in vivo. However, there are conflicting results in the literature and there is a lack of information concerning the combined effects of exposure to both GA and ionizing radiation in human cells. In the present study, the results obtained using conventional cytogenetic analysis do not suggest a statistically significant clastogenic or genotoxic activity of GA when concentrations in the range of 10(-6) to 10(-2) mM were applied. However, a 24-h pre-irradiation exposure of human peripheral blood lymphocytes (PBLs) to non-genotoxic doses of GA showed a statistically significant (P > 0.05) increase in chromosomal radiosensitivity. The observed increase may be an effect of GA-induced alterations in the cell-cycle and feedback control mechanisms during the cell-cycle transition points or it may be a consequence of an effect of GA either on the DNA repair capacity of the cells after irradiation or on the initial induction of radiation-induced chromosomal damage. To elucidate the mechanism underlying the obtained radiosensitization, conventional cytogenetics, the G2 chromosomal radiosensitivity assay and premature chromosome condensation methodologies were applied. The results support the hypothesis that pre-irradiation exposure of PBLs to GA induces radiosensitization by increasing the initial yield of chromosomal aberrations following irradiation.

Validated method for the simultaneous determination of methadone and its main metabolites (EDDP and EMDP) in plasma of umbilical cord blood by gas chromatography-mass spectrometry.

A sensitive and specific GC/MS method for the determination of methadone (MDN) and its two main metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), in plasma samples obtained from venous and arterial umbilical cord blood and maternal blood has been developed, optimized and validated. Specimen preparation includes protein precipitation with acetonitrile and simultaneous solid-phase extraction of the three analytes. Methadone-d9 was used as internal standard for the determination of MDN and EMDP, while EDDP-d3 for EDDP. Limits of detection were 0.6 microg/L for MDN and 0.3 microg/L for EDDP and EMDP, while limits of quantification were 2.0 microg/L for MDN and 1.0 microg/L for EDDP and EMDP. The calibration curves were linear up to 2000 microg/L for MDN and up to 1000 microg/L for EDDP and EMDP. Absolute recovery ranged from 94.8 to 99.7% for all three analytes. Intra- and interday accuracy was less than 5.3 and 5.5%, respectively, while intra- and interday precision was less than 3.5 and 5.0%, correspondingly, for all analytes. The method proved suitable for the determination of MDN and its two main metabolites in plasma samples obtained from umbilical cord and maternal blood of a woman participating in a MDN maintenance program, during the prenatal and postpartum period.

Development and validation of an EI-GC-MS method for the determination of methadone and its major metabolites (EDDP and EMDP) in human breast milk.

Methadone is used extensively for the maintenance of opioid-addicted pregnant women. Because methadone and the two major metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), are excreted into breast milk, a sensitive and specific gas chromatographic-mass spectrometric method has been developed, optimized, and validated for their quantitative determination in human breast milk. The procedure combined protein precipitation with acetonitrile and solid-phase extraction, using Isolute Confirm HCX mixed-mode SPE columns, with minimal matrix effect. The optimum extraction conditions for all three analytes were evaluated using spiked human breast milk, and the recovery exceeded 93.0%. This assay uses methadone-d(9) as internal standard for the determination of methadone and EMDP, and EDDP-d(3) for the determination of EDDP. Calibration curves were linear within the range of 2.00-1000 microg/L for methadone (R(2) > 0.995) and 1.00-500 microg/L for EDDP (R(2) >0.997) and EMDP (R(2) > 0.991). Intra- and interday accuracy and precision were within the range of 0.8-5.7% and 1.3-5.2%, respectively, for all analytes. The stability study was assessed by fortifying human breast milk with methadone and its metabolites at two different concentrations and keeping the samples at different temperature conditions. The analytes were found to be stable in breast milk at room temperature for at least 4 h and at -20 degrees C for at least one month. The method was used for the determination of methadone and its major metabolites in human breast milk samples obtained from women in the postpartum period participating in a methadone maintenance program.

Development and validation of a simple GC-MS method for the simultaneous determination of 11 anticholinesterase pesticides in blood--clinical and forensic toxicology applications.

Anticholinesterase pesticides are widely used, and as a result they are involved in numerous acute and even fatal poisonings. The aim of this study was the development, optimization, and validation of a simple, rapid, specific, and sensitive gas chromatography-mass spectrometry method for the determination of 11 anticholinesterase pesticides (aldicarb, azinphos methyl, carbofuran, chlorpyrifos, dialifos, diazinon, malathion, methamidophos, methidathion, methomyl, and terbufos) in blood. Only 500 µL of blood was used, and the recoveries after liquid-liquid extraction (toluene/chloroform, 4:1, v/v) were more than 65.6%. The calibration curves were linear (R(2) ≥ 0.996). Limit of detections and limit of quantifications were found to be between 1.00-10.0 and 3.00-30.0 µg/L, respectively. Accuracy expressed as the %E(r) was found to be between -11.0 and 7.8%. Precision expressed as the percent relative standard deviation was found to be <9.4%. The developed method can be applied for the investigation of both forensic and clinical cases of accidental or suicidal poisoning with these pesticides.

A fully validated method for the determination of vardenafil in blood using gas chromatography/mass spectrometry.

Vardenafil (VDN) is one of the three commercially available phosphodiesterase type 5 inhibitors and it is mainly used in the treatment of erectile dysfunction. A sensitive and specific gas chromatography/mass spectrometry (GC/MS) method for the determination of VDN in blood has been developed and validated. Sample preparation included solid-phase extraction and derivatization with N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide (MTBSTFA) and 1% tert-butyldimethylsilylchloride (TBDMSCl). Protriptyline was used as the internal standard for this assay. Limits of detection and quantification for VDN were 0.70 and 2.00 µg/l, respectively. The calibration curves were linear within the dynamic range 2.00-200.0 µg/l with a correlation coefficient higher than 0.991. Absolute recovery ranged from 88.6% to 95.7% for the analyte of interest at three quality control levels. Intra- and inter-day accuracy was found to be between - 6.1% to 10.8% and - 9.3% to 11.6%, respectively, whereas intra- and inter-day precision was < 7.8% and 9.7%, correspondingly. The proposed method is the first fully validated GC/MS method for the determination of VDN in blood samples and it can be used in routine every day analysis by clinical and forensic laboratories for pharmacokinetic studies, for therapeutic drug level monitoring or for the investigation of related forensic cases. A few blood samples analyzed using the developed method is reported herein to demonstrate the suitability of the method.

Development and validation of a highly sensitive GC/MS method for the determination of buprenorphine and nor-buprenorphine in blood.

A sensitive and specific GC/MS method for the determination of buprenorphine (BPN) and its main metabolite nor-buprenorphine (nor-BPN) in blood has been developed, optimized and validated. Sample preparation includes solid-phase extraction of both analytes and their derivatization with acetic anhydride in pyridine. BPN-d4 was used as internal standard for the determination of both analytes. Limits of detection and quantification for BPN and nor-BPN were 0.02 and 0.05 µg/L, respectively. The calibration curves were linear within the dynamic range of each analyte (0.05-30.0 µg/L) with a correlation coefficient higher than 0.996. Absolute recovery ranged from 90.2 to 97.6% for both analytes and their internal standard. Intra- and inter-day accuracy was found to be between -5.40 to 1.73% and -2.45 to 2.80%, respectively, while intra- and inter-day precision were less than 5.8 and 4.7%, for both analytes. The method was applied to real blood samples obtained from patients that follow BPN maintenance program. The developed method can be used in routine every day analysis by clinical and forensic laboratories, for pharmacokinetic studies, for therapeutic drug level monitoring in order to adjust BPN dosage of BPN maintained patients or for the investigation of forensic cases.

Effect of cocaine and crack on the ploidy status of Tetrahymena pyriformis: a study using DNA image analysis.

The effect of cocaine and crack on the ploidy status of Feulgen-stained Tetrahymena pyriformis macronuclei using computerized DNA image analysis system was tested. For this purpose, selected doses of 5, 10 and 20 µg (per mL culture) of both drugs were administered for 2, 5 and 20 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the following parameters: Ploidy Balance (PB), Degree of Aneuploidy (DA), skewness and kurtosis. The results have shown a positive effect of both cocaine and crack on PB and on DA of T. pyriformis macronuclei. In particular, our results reveal that the aneugenic effect (which is expressed as a decrease in PB and an increase in DA) of cocaine on T. pyriformis macronuclei follows a dose-dependent manner, while crack induces aneuploidy in a dose-independent manner. Changes in the PB and DA values would induce a disturbance in the cellular density and heterogeneity of chromatin and the increase in skewness and kurtosis values after exposure of T. pyriformis to both drugs, did confirm this hypothesis. These observations were further correlated with alterations in the chromosomal segregation and with damage in mitotic spindle microtubules observed previously. In this study the impact of cocaine and crack on genomic instability and carcinogenesis was further supported and T. pyriformis can be proposed as a model organism to test the nuclear ploidy status after exposure to harmful chemicals and drugs.

Mass intoxication with Datura innoxia--case series and confirmation by analytical toxicology.

BACKGROUND: Anticholinergic plants contain a variety of alkaloids that are toxic if ingested. Datura innoxia belongs to the family of Solanaceae and contains two main toxic alkaloids, atropine and scopolamine. CASE SERIES: In this study we report the case series of seven individuals who were admitted to two different hospitals of Athens with an anticholinergic syndrome. All symptoms manifested after consumption of cooked vegetables (blites). INVESTIGATION: The investigation of the cases revealed that among the vegetables there was also Datura innoxia, which has a similar appearance to blites. Urine and plasma samples of the seven patients, as well as a sample of cooked vegetables, were analyzed with gas chromatography-mass spectrometry. Atropine and scopolamine were confirmed in all urine and vegetable samples, but not in plasma probably because of the delay in sample collection. The urine samples of all patients contained atropine in concentrations between 67.1 and 691.7 ng/mL, while urine concentrations of scopolamine ranged from 32.4 to 186.4 ng/mL. The concentrations of atropine and scopolamine in the cooked vegetables were found to be 0.8 and 1.2 microg/g, respectively. CONCLUSION: All patients recovered completely, although some required mechanical ventilation. The investigation and the presentation of this case series illustrate not only mass intoxication with D. innoxia, but also the utility of analytical toxicology. It also illustrates the dangers of collection of vegetables in the wild.

Development and validation of an EI-GC-MS method for the determination of benzodiazepine drugs and their metabolites in blood: applications in clinical and forensic toxicology.

Benzodiazepines are used widely in daily clinical practice, due to their multiple pharmacological actions. The frequent problems associated with the wide use of benzodiazepines, as well as the multiple incidents of poisonings, led to the necessity for the development of a precise, sensitive and rapid method for the simultaneous determination of the 23 most commonly used benzodiazepines (diazepam, nordiazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam, flunitrazepam, 7-amino-flunitrazepam, triazolam, prazepam, nimetazepam, nitrazepam, temazepam, lormetazepam, clonazepam, camazepam) in blood. A gas chromatographic method combined with mass spectrometric detection was developed, optimized and validated for the determination of the above substances. This method includes liquid-liquid extraction with chloroform at pH 9 and two stages of derivatization using tetramethylammonium hydroxide and propyliodide (propylation), as well as a mixture of triethylamine:propionic anhydride (propionylation). The recoveries were higher than 74% for all the benzodiazepines. The calibration curves were linear within the dynamic range of each benzodiazepine with a correlation coefficient higher than 0.9981. The limits of detection and quantification for each analyte were statistically calculated from the relative calibration curves. Accuracy and precision were also calculated and were found to be less than 8.5% and 11.1%, respectively. The developed method was successfully applied for the investigation of both forensic and clinical toxicological cases of accidental and suicidal poisoning.

Off-line HPLC method combined to LC-MS for the determination of sildenafil and its active metabolite in post-mortem human blood according to confirmation criteria.

A simple HPLC method has been validated for the determination of sildenafil and its active metabolite (N-desmethylsildenafil) in human blood, using an octadecyl silica (ODS) hypersil column. The chromatographic run time is less than 25 min using a mobile phase of 35:65 (v/v) acetonitrile-0.015 M disodium hydrogen phosphate (Na(2)HPO(4)), triethylamine 0.1%, pH 7.4 at 1 mL/min flow rate and UV-vis detection at 230 nm. The method is linear in the concentration range of 10-500 ng/mL (r>0.999, n=5) for each analyte, with relative standard deviation (R.S.D.) less than 5.05%. Interday and intraday errors were found to be < or =11.94%. The limits of detection and quantitation for both analytes were 5.0 ng/mL (s/n>3) and 10.0ng/mL (s/n>10), respectively. The method was applied in two post-mortem human blood samples, concerning two fatal cases from sildenafil citrate use, reported for the first time in Greece, and the results were further confirmed with LC-MS. The method is proposed as supplementary to LC-MS when inadequate mass fragmentation does not provide information appropriate to meet confirmation criteria.