Simultaneous determination of cocaine, ecgonine methyl ester, benzoylecgonine, cocaethylene and norcocaine in dried blood spots by ultra-performance liquid chromatography coupled to tandem mass spectrometry.

Cocaine (COC) is one of the most widely abused drugs in the world and its sensitive and its reliable measurement in blood is of great importance in the field of forensic and clinical toxicology. Additionally, the determination of COC metabolites such as benzoylecgonine (BZE), cocaethylene (CE), ecgonine methyl ester (EME), and norcocaine (NCOC) are also of complementary diagnostic value. The quantification of COC and metabolites in dried blood spots (DBS) may be an alternative to conventional collection methods with several advantages, including easier, on-site, collection, transportation and storage. In this study, we present a simple and comprehensively validated UPLC-MS/MS assay to measured COC, BZE, EME, NCOC and CE in DBS. The evaluated assay was linear from 5-500 ng mL-1. Precision assays presented CV% of 1.27-6.82, and accuracy in the range of 97-113.78%. Low haematocrit values had a negative impact in the assay accuracy. COC, BE, NCOC and CE measurements can be made reliably in DBS stored for 14 days at room temperature, as well as at -20 °C and 45 °C. All evaluated compounds can be measured in DBS maintained at -20 °C for 14 days. DBS sampling can be used for the clinical evaluation of the exposure to COC, being an alternative for collection, short-term storage and transportation of blood at room and high temperatures.

Simultaneous determination of drugs and pesticides in postmortem blood using dispersive solid-phase extraction and large volume injection-programmed temperature vaporization-gas chromatography-mass spectrometry.

A d-SPE protocol followed by gas chromatography-mass spectrometry (GC-MS) analysis using large volume injection-programmed temperature vaporization (LVI-PTV) was optimized for simultaneous quantification of 14 pesticides, drugs of abuse, prescription drugs and metabolites in human postmortem blood without derivatization. The validated method showed good repeatability, linearity, intermediate precision, and recovery. LOQs were 0.02 or 0.03µg/mL. The method showed to be fast and easy-to-implement in a forensic laboratory and was satisfactorily applied for the analysis of 10 postmortem blood real samples. Six samples contained cocaine (0.04-3.13µg/mL), two 3,4-methylenedioxymethamphetamine hydrochloride (MDMA, 0.04-0.09µg/mL) and two carbamazepine (0.08-0.98µg/mL). Other analytes found were carbofuran (27.3µg/mL), the metabolite 7-aminoflunitrazepam (1.12µg/mL), amitriptyline (0.21µg/mL) and diazepam (0.03µg/mL).

Perinatal undernutrition facilitates morphine sensitization and cross-sensitization to cocaine in adult rats: a behavioral and neurochemical study.

The development of sensitization to the locomotor effects of morphine and cross-sensitization between morphine and cocaine were evaluated in adult rats submitted to a protein malnutrition schedule from the 14th day of gestation up to 30 days of age (D-rats), and compared with well-nourished animals (C-rats). Dose-response curves to morphine-induced locomotor activity (5, 7.5, 10 or 15 mg/kg, i.p., every other day for 5 days) revealed a shift to the left in D-rats compared to C-rats. This implies that D-rats showed behavioral sensitization to the lower dose of morphine used (5 mg/kg), which was ineffective in C-rats. Furthermore, when a cocaine challenge (10 mg/kg, i.p) was given 48 h after the last morphine administration, only D-rats exhibited cross-sensitization in morphine-pretreated animals (7.5 and 10 mg/kg). In order to correlate the differential response observed with the functioning of the mesocorticolimbic dopaminergic system, extracellular dopamine (DA) levels were measured in the nucleus accumbens (core and shell) and the dorsal caudate-putamen. A challenge with cocaine in morphine pre-exposed animals produced an increase in DA release, but only in the nucleus accumbens "core" of D-rats. Similar DA levels were found in the nucleus accumbens "shell" and in the dorsal caudate-putamen of both groups. Finally, these results demonstrate that D-rats had a lower threshold for developing both a progressive behavioral sensitization to morphine and a cross-sensitization to cocaine. In accordance with these behavioral findings, a higher responsiveness of the nucleus accumbens core, expressed by increased DA levels, both basal and after cocaine challenge, was observed in D-rats.

Validation of an ion-trap gas chromatographic-mass spectrometric method for the determination of cocaine and metabolites and cocaethylene in post mortem whole blood.

The coingestion of cocaine (COC) and ethanol is a very frequent occurrence and is known to increase the risk of morbidity and mortality. The formation occurs of a transesterification product, the cocaethylene (CE), which is even more toxic than cocaine. In order to study the role of ethanol as an agent of interaction in lethal cocaine intoxication, and to establish its influence in post mortem cocaine concentrations, an ion-trap gas chromatographic-mass spectrometric method (GC-MS) was validated to quantify simultaneously the agent and its biotransformation products, benzoylecgonine (BE), ecgoninemethylester (EME) and the 'biomarker' of the interaction, the CE present in whole blood. Deuterated internal standards were added to 2 ml of post mortem whole blood and extracted in Bond Elut Certify columns. The residues were evaporated and derivatized with N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA). Detection was performed by electron impact ionization. The monitored ions were m/z 82/85 for EME-tert-butyldimethylsilyl (TBDMS)/EME-d3-TBDMS; m/z 182/185 for COC/COC-d3; m/z 196/199 for CE/CE-d3 and m/z 282/285 for BE-TBDMS/BE-d3-TBDMS. The limits of detection and quantification were found to be 25 ng and 50 ng ml(-1), respectively, for COC and CE, and 50 and 100 ng ml(-1) for BE and EME. Accuracy was different for each of the compounds, varying from 65 to 98%. The dynamic range of the assay was 50-2000 ng ml(-1).