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.

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.

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.

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.