Panagiota Nikolaou

A fully validated method for the simultaneous determination of 11 antidepressant drugs in whole blood by gas chromatography–mass spectrometry

Antidepressant drugs are widely used for the treatment of depression and other psychiatric disorders and as a result they are involved in numerous clinical and forensic cases. The aim of this study was the development, optimization and validation of a simple, specific and sensitive GC/MS method for the simultaneous determination of 11 antidepressant drugs and 4 of their metabolites (amitriptyline, citalopram, clomipramine, fluoxetine, fluvoxamine, maprotiline, desmethyl-maprotiline, mirtazapine, desmethyl-mirtazapine, nortriptyline, paroxetine, sertraline, desmethyl-sertraline, venlafaxine and desmethyl-venlafaxine) in whole blood. The combination of solid-phase extraction with derivatization using heptafluorobutyric anhydride efficiently reduced matrix effect and improved sensitivity of the method. In this assay, protriptyline was used as internal standard. Absolute recovery values for all analytes were ranged from 79.2 to 102.6%. LODs and LOQs were found to be between 0.30-1.50 μg/L and 1.00-5.00 μg/L, respectively. The calibration curves were linear (R(2)≥0.990) within the range of 5.00-1000 μg/L for all analytes. Accuracy expressed as the % E(r) was found to be between -12.3 and 12.2%. Precision expressed as the % RSD was found to be less than 11.7% for all antidepressants. The developed method proved to be suitable for routine work and it was used to successfully analyze more than 2500 clinical and forensic blood samples.

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.

AH-7921: the list of new psychoactive opioids is expanded

AH-7921 is a structurally unique synthetic opioid analgesic that has recently entered the drug arena in Europe, the USA, and Japan. Although it was synthesized and patented in the mid-1970s, it was first identified in a seized sample purchased via the Internet in July 2012 and formally brought to the attention of the European Union early warning system in August 2012 by the United Kingdom. Several in vitro experiments and animal model studies established the morphine-like analgesic action of AH-7921 as a μ-opioid receptor agonist that has been found to be several times more potent than codeine and at least as potent as morphine. This novel psychoactive substance has already led to eight non-fatal intoxications and 16 deaths in Sweden, the United Kingdom, Norway, and the USA. Thus, AH-7921 is a current public health risk, and better international collaboration, effective legislation and continuous community alertness are needed to tackle this current growing problem. The aim of this review is to summarize the current knowledge about this drug concerning its chemistry, pharmacology, and toxicology, as well as its international legal status. The limited existing analytical methodologies for the determination of AH-7921 in biological samples are also presented. Published or reported AH-7921-related cases, fatalities, or intoxications, and self reports from drug users are reviewed.

2C-I-NBOMe, an "N-bomb" that kills with "Smiles". Toxicological and legislative aspects.

Abstract Substituted phenethylamines are a class of designer drugs that have recently emerged in the drug abuse market. Such substances remain legal to use, possess, and supply until these compounds become classified as scheduled. 2C-I-NBOMe or 25I-NBOMe is the N-benzyl-derivative of the iodo-substituted dimethoxy-phenethylamine (2C-I) that appeared recently in the drug market under the street name “N-Bomb”. Due to its high potency, intoxications and fatal cases related to 2C-I-NBOMe use are increased worldwide. The use and trafficking of this substituted phenethylamine is banned only in some countries. A comprehensive review was performed using PubMed and Medline databases, together with additional non-peer reviewed information sources, including books and publications of state authorities in different countries, regarding chemistry, availability, pharmacology, and toxicology of 2C-I-NBOMe. Intoxications or lethal cases, published or reported, as well as the current legislation on this newly introduced drug are also reviewed.

Development and validation of a GC/MS method for the determination of tadalafil in whole blood

Tadalafil is a phosphodiesterase type 5 (PDE-5) inhibitor and it is used in the treatment of pulmonary arterial hypertension and erectile dysfunction. A sensitive and specific method is described for the determination of tadalafil in whole blood. Tadalafil and its internal standard (protriptyline) were isolated from the matrix by solid phase extraction, and were analyzed by gas chromatography/mass spectrometry (GC/MS) after derivatization by N,O-bis(trimethylsilyl)-trifluoracetamide (BSTFA) with 1% trimethylchlorsilane (TMCS). Limits of detection and quantification for tadalafil were 0.70 and 2.00 μg/L, respectively. The calibration curve was linear between 2.00 and 500.0 μg/L, with a correlation coefficient higher than 0.991. The values obtained for intra- and inter-day accuracy was found to be between -10.5 to 8.5% and -4.2 to 4.5%, respectively, while intra- and inter-day precision were less than 8.4 and 11.2%, correspondingly. Absolute recovery was determined at three concentration levels and ranged from 92.1 to 98.9%. The proposed method is the first fully validated GC/MS method for the determination of tadalafil in whole blood and it can be routinely applied by toxicological laboratories, for pharmacokinetic studies, for therapeutic drug level monitoring or for the investigation of related forensic cases.

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 GC/MS method for the simultaneous determination of levetiracetam and lamotrigine in whole blood.

A sensitive and accurate gas chromatography-mass spectrometric method was developed and validated for the simultaneous determination of levetiracetam and lamotrigine in whole blood. A solid-phase extraction (SPE) procedure using HF Bond Elut C18 columns followed by derivatization using N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide (MTBSTFA) with 1% tert-butyldimethylsilyl chloride (TBDMSCl) was used. In this assay, levetiracetam-d6 was used as internal standard. Limits of detection and quantification were 0.15 and 0.50 μg/mL, respectively, for both analytes. The method was proved to be linear within the concentration range of 0.50-50.0 μg/mL (R(2) ≥ 0.992) for both analytes. Absolute recovery was found to be at least 90.0 and 97.2% for levetiracetam and lamotrigine, respectively. Intra-day and inter-day accuracy values for both analytes were ranged from -6.5 to 4.2 and -6.6 to 3.0%, respectively, whereas their respective precision values were less than 11.4 and 8.3%. The developed method was successfully used in our laboratory for quantification of levetiracetam and lamotrigine blood concentrations during the investigation of forensic cases where these antiepileptic drugs were involved. This method could also be used for therapeutic drug monitoring purposes.

Stability of Morphine, Codeine, and 6‐Acetylmorphine in Blood at Different Sampling and Storage Conditions

The stability of drugs in biological specimens is a major concern during the evaluation of the toxicological results. The stability of morphine, codeine, and 6‐acetyl‐morphine in blood was studied after different sampling conditions: (i) in glass, polypropylene or polystyrene tubes, (ii) with addition of dipotassium ethylene diamine tetraacetic acid (K2EDTA) or sodium oxalate (Na2C2O4), and (iii) with or without the addition of sodium fluoride (NaF). Spiked blood samples were stored at two different temperatures (4 and −20°C), analyzed after different storage times and after three freeze–thaw cycles. Opiate concentrations were decreased in all conditions, but the most unstable was 6‐acetyl‐morphine. The addition of NaF as preservative improved the stability of opiates at all conditions studied, whereas the type of anticoagulant did not affect the stability of opiates. It was concluded that blood samples should be stored at −20°C in glass tubes containing oxalate and NaF for maximum stability.

Development and validation of an EI-GC/MS method for the determination of sertraline and its major metabolite desmethyl-sertraline in blood.

A sensitive and specific GC/MS method for the determination of sertraline and its main metabolite desmethyl-sertraline in whole blood has been developed, optimized and validated. Sample preparation included solid-phase extraction of both analytes and their derivatization with heptafluorobutyric anhydride (HFBA). Protriptyline was used as internal standard for the determination of both analytes. Limits of detection and quantification for both sertraline and desmethyl-sertraline were 0.30 and 1.00 μg/L, respectively. The calibration curves were linear within the dynamic range of each analyte (1.00-500.0 μg/L) with a correlation coefficient (R(2)) exceeding 0.991. Extraction efficiency ranged from 90.1(± 5.8)% to 95.4(± 3.0)% for sertraline, and from 84.9(± 8.2)% to 107.7(± 4.4)% for desmethyl-sertraline. The precision for sertraline and desmethyl-sertraline was between 3.6-5.5% and 4.7-7.2%, respectively, while the accuracy was in the range of -6.67% to 2.20% and -6.33% to 2.88% for sertraline and desmethyl-sertraline, respectively. The method was applied to real blood samples obtained from patients that follow sertraline treatment and also in cases of forensic interest. The developed method can be used in routine every day analysis by clinical and forensic laboratories, for pharmacokinetic studies, for therapeutic sertraline monitoring or for the investigation of forensic cases where sertraline is involved.