Constantinos Maravelias

Zinc: a multipurpose trace element

Zinc (Zn) is one of the most important trace elements in the body and it is essential as a catalytic, structural and regulatory ion. It is involved in homeostasis, in immune responses, in oxidative stress, in apoptosis and in ageing. Zinc-binding proteins (metallothioneins, MTs), are protective in situations of stress and in situations of exposure to toxic metals, infections and low Zn nutrition. Metallothioneins play a key role in Zn-related cell homeostasis due to their high affinity for Zn, which is in turn relevant against oxidative stress and immune responses, including natural killer (NK) cell activity and ageing, since NK activity and Zn ion bioavailability decrease in ageing. Physiological supplementation of Zn in ageing and in age-related degenerative diseases corrects immune defects, reduces infection relapse and prevents ageing. Zinc is not stored in the body and excess intakes result in reduced absorption and increased excretion. Nevertheless, there are cases of acute and chronic Zn poisoning.

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.

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 μg/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.

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 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.

Phagocytosis of the protozoon Tetrahymena pyriformis as an endpoint in the estimation of cocaine salt and cocaine freebase toxicity.

Cells of the ciliated protozoon Tetrahymena pyriformis strain W, grown in a peptone‐yeast medium, usually contain many phagocytic vacuoles. The phagocytic activity of this protozoon was studied in vivo using heat‐inactivated yeast stained with carmine after exposing the cultures for 1 hour to different doses of cocaine hydrochloride or cocaine freebase (crack) (0.5, 1 or 2 mg/100 ml of protozoan culture).The number of vacuoles formed indicated the phagocytic activity. Cocaine hydrochloride and crack caused a decrease of the phagocytic activity of the protozoon (p < 0.05) when compared to the control cultures. Furthermore, the two chemical forms of cocaine, salt and free‐base respectively, caused quantitatively different effects on the phagocytic activity. Crack produced an extensive decrease in phagocytosis, compared to equal concentrations of cocaine hydrochloride. These results suggest a possible relationship between cocaine abuse and the suppression of phagocytosis that may contribute to the impairment of immunity in drug misusers.

The effects of morphine, cocaine, amphetamine and hashish on the phagocytosis of the protozoon Tetrahymena pyriformis strain W.

Cells of the ciliated protozoon Tetrahymena pyriformis, strain W, grown in a peptone-yeast medium usually contain many phagocytic vacuoles. The phagocytic activity of this protozoon was studied in vivo using heat-killed yeast stained with carmine dye and after exposing the cultures for 2 hr to morphine (20 mug/ml), cocaine (20 mug/ml), amphetamine (0.5 mug/ml) or hashish (0.1 mug/ml). The number of vacuoles formed indicated the phagocytic activity after treatment with the drugs of abuse. Amphetamine caused a slight increase (P < 0.05) in the phagocytic activity of the protozoon, whereas morphine, cocaine and hashish each caused a significant (P < 0.01) decrease in this activity.

Development and Validation of a Simple GC-MS Method for the Simultaneous Determination of 11 Anticholinesterase Pesticides in Blood—Clinical and Forensic Toxicology Applications

Abstract:  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 (R2 ≥ 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 %Er 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.