Degradation Of Cocaine Research Articles

Postmortem redistribution of cocaine and its metabolites, benzoylecgonine and ecgonine methyl ester in humans: Important variables that might be influencing the central blood / peripheral blood ratio

IntroductionA big challenge in forensic toxicology is the correct interpretation of the results of quantitative analyses in postmortem cases. Postmortem drug concentrations not necessarily reflect the drug concentrations at the time of death, due to postmortem changes in drug concentrations caused by postmortem redistribution (PMR). Cardiac blood is more prone to PMR related concentration changes than peripheral blood. Because of this difference in susceptibility to PMR related concentration changes, the ratio of cardiac blood concentration/peripheral blood concentration (C/P) of a drug is an often-used marker of PMR. In this study, we investigated the relationship between different potentially significant variables and the C/P ratios of cocaine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in humans. The aim was to elucidate the mechanisms involved in PMR of these substances and potentially provide guidelines aiding forensic toxicologists in the interpretation of postmortem quantitative results of cocaine and its metabolites. To differentiate between postmortem concentration changes due to redistribution versus degradation of cocaine, the relationships between these variables and metabolite/cocaine ratios were investigated as well. MethodToxicological results of all postmortem cases that were positive for cocaine, BE and/or EME investigated by the Netherlands Forensic Institute between January 1st 2010 and July 31st 2020 were reviewed. The C/P ratios, BE/cocaine ratios and EME/cocaine ratios were determined for all selected cases. Cocaine, BE and/or EME were quantified in both femoral blood and cardiac blood in a total of 148 cases. Ratios were compared between subgroups by performing either a Mann-Whitney U test or a Kruskal-Wallis test followed by post-hoc Mann-Whitney U test. ResultsA statistically significant difference in C/P ratio of EME was observed between trauma and non-trauma cases with median C/P ratios of 2.03 and 1.57, respectively (p value=0.001). A statistically significant difference in EME/cocaine ratio was observed between the BMI subgroups 18.5 – 25.0 kg/m2 and> 25 kg/m2 with median EME/cocaine ratios of 3.79 and 1.58, respectively (p-value<0.001). ConclusionPostmortem cocaine concentrations should be interpreted with caution, considering the occurrence of both PMR and postmortem degradation. When interpreting postmortem toxicological results in cocaine-related fatalities, it might prove useful to take the above-mentioned variables into account.

Cocaine esterase occurrence in global wastewater microbiomes and potential for biotransformation of novel psychoactive substances.

The analysis of drugs in wastewater for forensic purposes has been constantly increasing and the investigation of the potential interaction between drugs or metabolites and sewage microbiota is important. The results demonstrated that cocaine esterase genes were widely distributed in 1142 global wastewater samples collected from 64 countries and linked to several bacterial species. In addition, in silico predictions indicated that carfentanil, 4F-MDMB-BINACA, 5F-MDMB-PICA, MDMB-4en-PINACA and mitragynine might also undergo microbial hydrolysis, in a similar fashion of cocaine degradation by cocaine esterase. In conclusion, it was demonstrated the microbial potential to hydrolyze drugs of abuse in wastewater environments, contributing to the critical evaluation of potential metabolites as biomarkers for microbial and human transformation of drugs in wastewater.

Alternatives for cocaine disposal: An experimental, techno-economic, and environmental comparison between incineration and biological degradation

Worldwide trends in the area under coca crops cultivation have been primarily motivated by illegal advances in Colombia around cocaine production. Thus, alternative ways for the final disposal of cocaine are needed and the evaluation of their possible economic and environmental advantages. This work aimed to compare incineration and biological degradation of cocaine by considering techno-economic and environmental aspects. There was a cocaine removal efficiency of 99.7% when bacterial C1T consortium was grown using cocaine as the sole carbon source. The biological degradation process exhibited a specific energy consumption and total potential environmental impact, 5 and 10 fold lower than the incineration process, respectively. The biological degradation process can also reach similar disposition unit prices as the incineration process (i.e., 1.00 USD/kg).

Cocaine degradation using a rotating biological disc reactor: Techno-economic and environmental analysis using experimental data

A bacterial mixed culture that utilizes cocaine as the sole carbon and energy sources was isolated and used in a Rotary Disc Reactor as an alternative method for the final disposal of seized cocaine. This study aimed to compare the performances of cocaine incineration (oven) and biodegradation (Rotary Disc Reactor), considering economic and environmental aspects. There was a 99.4% cocaine removal efficiency when bacterial C1T consortium was grown in a Rotary Disc Reactor for 42 h. The economic analysis allowed determining the high potential of the biotechnological cocaine degradation to be evaluated at higher scales. Indeed, the unit disposition price of the biotechnological degradation pathway was 58% higher than the calculated value for the incineration process considering an initial cocaine concentration of 30 g/L. Moreover, the economic sensitivity analysis demonstrated a price reduction of 20% in the unit disposition price of the biotechnological degradation using a rotary disc reactor. Further, cocaine degradation using a rotary disc reactor system presented a better environmental performance than the incineration process considering atmospheric and toxicological impact categories because of the low release of hazardous materials to the atmosphere.

Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells.

Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.

Degradation of Cocaine and Benzoylecgonine in Sewage Sludge Batch Tests

The degradation studies of cocaine and its principal metabolites, benzoylecgonine, as emerging pollutants were carried out using various sludge types collected from the RAF Molesworth Sewage Treatment Work (STW) to determine their distribution and persistence in aquatic environment. The methodology, initially characterized the various sludge types before the laboratory batch tests were carried out at different temperatures and times. The results, after 3 hours equilibration of batch samples, showed the removal rates for cocaine and benzoylecgonine to be 91.0% and 90.6%, respectively. The partition coefficients (Kd) for the two substances ranged from 2.7 – 31.9 Kg L-1. Degradation of compounds was found to be both biotic and abiotic and the two processes produced many short chain compounds and several metabolites that include cocaethylene and ecgonine methylester. Also at 4 ± 0.5o C, the degradation of substances occurred slowly, at 5 and 10% but was relatively greater at 19 ± 0.5o C. Compounds degradation was also influenced by both extracellular and intracellular enzyme activities. The batch study is an innovative method in calculating degradation rates of compounds from sewage treatment works. The capabilities of generating removal rates of drugs have also been clearly demonstrated in this work and can be applied to any organic compounds in real life (STWs) situations

(Un)targeted Scanning of Locks of Hair for Drugs of Abuse by Direct Analysis in Real Time-High-Resolution Mass Spectrometry.

Forensic hair evidence can be used to obtain retrospective timelines of drug use by analysis of hair segments. However, this is a laborious and time-consuming process, and mass spectrometric (MS) imaging techniques, which show great potential for single-hair targeted analysis, are less useful due to differences in hair growth rate between individual hairs. As an alternative, a fast untargeted analysis method was developed that uses direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) to longitudinally scan intact locks of hair without extensive sample preparation or segmentation. The hair scan method was validated for cocaine against an accredited liquid chromatography/tandem mass spectrometry (LC/MS/MS) method. The detection limit for cocaine in hair was found to comply with the cutoff value of 0.5 ng/mg recommended by the Society of Hair Testing; that is, the DART hair scan method is amenable to forensic cases. Under DART conditions, no significant thermal degradation of cocaine occurred. The standard DART spot size of 5.1 ± 1.1 mm could be improved to 3.3 ± 1.0 mm, corresponding to approximately 10 days of hair growth, by using a high spatial resolution exit cone. By use of data-dependent product ion scans, multiple drugs of abuse could be detected in a single drug user hair scan with confirmation of identity by both exact mass and MS/HRMS fragmentation patterns. Furthermore, full-scan high-resolution data were retrospectively interrogated versus a list of more than 100 compounds and revealed additional hits and temporal profiles in good correlation with reported drug use.

The removal of illicit drugs and morphine in two waste water treatment plants (WWTPs) under tropical conditions.

The consumption of drugs of abuse has been recently investigated in Martinique using the back-calculation approach, also called the "sewage epidemiology" method. Results demonstrated a very high consumption considering the international data. Wastewater treatment plants (WWTPs) are located just behind the Martinique island shoreline, and effluents could impact the vulnerable corals and marine seagrass ecosystem. The present article aims to determine a WWTP's efficiency by comparing the influent and effluent of two WWTPs, with different residence times and biological treatments, located either outdoors or indoors. In parallel, a degradation study is conducted using spiked wastewater exposed to tropical and ambient temperatures. Results demonstrate the consistent efficiency of the two processes, especially for the outdoor WWTP which uses the activated sludge process. The positive effect of the tropical temperature is showed by the increase of cocaine degradation at 31°C. Thus, low illicit drug residue concentrations in effluent would indicate that wastewater treatment is efficient and even enhanced under tropical context. This fact should be confirmed with others molecules. Furthermore, our results highlight the need for subsequent studies of sludge contamination because of their local recycling as compost.

Development and validation of an HPLC-DAD method for simultaneous determination of cocaine, benzoic acid, benzoylecgonine and the main adulterants found in products based on cocaine

Here, an HPLC-DAD method was developed and validated for simultaneous determination of cocaine, two cocaine degradation products (benzoylecgonine and benzoic acid), and the main adulterants found in products based on cocaine (caffeine, lidocaine, phenacetin, benzocaine and diltiazem). The new method was developed and validated using an XBridge C18 4.6mm×250mm, 5μm particle size column maintained at 60°C. The mobile phase consisted of a gradient of acetonitrile and ammonium formate 0.05M – pH 3.1, eluted at 1.0mL/min. The volume of injection was 10μL and the DAD detector was set at 274nm. Method validation assays demonstrated suitable sensitivity, selectivity, linearity, precision and accuracy. For selectivity assay, a MS detection system could be directly adapted to the method without the need of any change in the chromatographic conditions. The robustness study indicated that the flow rate, temperature and pH of the mobile phase are critical parameters and should not be changed considering the conditions herein determined. The new method was then successfully applied for determining cocaine, benzoylecgonine, benzoic acid, caffeine, lidocaine, phenacetin, benzocaine and diltiazem in 115 samples, seized in Brazil (2007–2012), which consisted of cocaine paste, cocaine base and salt cocaine samples. This study revealed cocaine contents that ranged from undetectable to 97.2%, with 97 samples presenting at least one of the degradation products or adulterants here evaluated. All of the studied degradation products and adulterants were observed among the seized samples, justifying the application of the method, which can be used as a screening and quantification tool in forensic analysis.

Effects of sewer conditions on the degradation of selected illicit drug residues in wastewater

The stability of five illicit drug markers in wastewater was tested under different sewer conditions using laboratory-scale sewer reactors. Wastewater was spiked with deuterium labelled isotopes of cocaine, benzoyl ecgonine, methamphetamine, MDMA and 6-acetyl morphine to avoid interference from the native isotopes already present in the wastewater matrix. The sewer reactors were operated at 20 °C and pH 7.5, and wastewater was sampled at 0, 0.25, 0.5, 1, 2, 3, 6, 9 and 12 h to measure the transformation/degradation of these marker compounds. The results showed that while methamphetamine, MDMA and benzoyl ecgonine were stable in the sewer reactors, cocaine and 6-acetyl morphine degraded quickly. Their degradation rates are significantly higher than the values reportedly measured in wastewater alone (without biofilms). All the degradation processes followed first order kinetics. Benzoyl ecgonine and morphine were also formed from the degradation of cocaine and 6-acetyl morphine, respectively, with stable formation rates throughout the test. These findings suggest that, in sewage epidemiology, it is essential to have relevant information of the sewer system (i.e. type of sewer, hydraulic retention time) in order to accurately back-estimate the consumption of illicit drugs. More research is required to look into detailed sewer conditions (e.g. temperature, pH and ratio of biofilm area to wastewater volume among others) to identify their effects on the fate of illicit drug markers in sewer systems.

Investigation of degradation products of cocaine and benzoylecgonine in the aquatic environment

In this work, ultra-high-performance liquid chromatography (UHPLC) coupled to a hybrid quadrupole time-of-flight mass spectrometer (QTOF MS) has allowed the discovery and elucidation of degradation products of cocaine and its main metabolite benzoylecgonine (BE) in water. Spiked surface water was subjected to hydrolysis, chlorination and photo-degradation (both ultraviolet irradiation and simulated sunlight). After degradation of cocaine, up to sixteen compounds were detected and tentatively identified (1 resulting from hydrolysis; 8 from chlorination; 7 from photo-degradation), three of which are well known cocaine metabolites (BE, norbenzoylecgonine and norcocaine). Regarding BE degradation, up to ten compounds were found (3 from chlorination; 7 from photo-degradation), including one known metabolite (norbenzoylecgonine). Since reference standards were available for the major metabolites, they could be confirmed using information on retention time and fragment ions. The other degradates resulted from chlorination, dealkylation, hydroxylation and nitration, or from a combination of these processes. Several influent and effluent sewage water, and surface water samples were then screened for the identified compounds (known and unknown) using UHPLC–tandem MS with triple quadrupole. BE, norcocaine and norbenzoylecgonine were identified in these samples as major metabolites. Four previously unreported degradates were also found in some of the samples under study, illustrating the usefulness and applicability of the degradation experiments performed in this work.

Transformation of cocaine during water chlorination

The stability of cocaine and its two main human metabolites, benzoylecgonine and ecgonine methyl ester, in chlorine-containing waters has been investigated by direct injection of different reaction time aliquots in a liquid chromatograph (LC) coupled to a quadrupole-time-of-flight mass spectrometer (QTOF-MS). Factors potentially affecting cocaine degradation (the only compound showing a significant decrease in the preliminary study) were evaluated in detail by means of a Box-Behnken experimental design. Sample pH resulted to be the most important variable, increasing both the rate of chlorination-mediated reactions and the ester hydrolysis process. From these reactions, and due to the high mass accuracy measurements obtained with the QTOF system, four by-products could be positively identified: benzoylecgonine, norcocaine, norbenzoylecgonine and N-formylnorcocaine. Finally, their formation and cocaine degradation yields were assessed under chlorination experiments with two real surface water samples. In one of them, showing a low anthropogenic impact, benzoylecgonine and norcocaine were notably generated even after only 1 h of reaction, whereas at higher contact times also norbenzoylecgonine and N-formylnorcocaine could be determined with a lower yield. On the other hand, the second sample, with a higher organic matter content, consumed rapidly the chlorine, so that only benzoylecgonine was produced. These findings point out the convenience of monitoring the described transformation products, in addition to the precursor illicit drug, during drinking water production, taking into account that cocaine traces might be present in water catchments and particularly in areas with high population densities.

The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle

The in vitro stability of cocaine in horse blood, sheep vitreous humour (VH) and homogenised deer muscle is described. The stability of cocaine in horse blood was of interest because many toxicology laboratories utilise horse blood for the preparation of calibration and check standards and the latter are typically stored during routine use. The storage stability of cocaine in human VH and muscle has not been previously reported. In the absence of blank human VH and muscle, cocaine stability under varying conditions was demonstrated in animal tissues. Blood and VH were stored with and without addition of NaF at room temperature (RT), 4°C and -18°C for 84 days. Muscle homogenates were prepared in water, water/2% NaF, and phosphate buffer (pH 6.0)/2% NaF, and stored for 31 days at RT, 4°C and -18°C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18°C for 13 months. Cocaine and benzoylecgonine (BZE) were extracted using SPE and quantified by GC-MS/MS. Cocaine was stable for 7 days in refrigerated (4°C) horse blood fortified with 1 and 2% NaF. In the absence of NaF, cocaine was not detectable by day 7 in blood stored at RT and 4°C and had declined by 81% following storage at -18°C. At 4°C the rate of cocaine degradation in blood preserved with 2% NaF was significantly slower than with 1% NaF. The stability of cocaine in horse blood appeared to be less than that reported for human blood, probably attributable to the presence of carboxylesterase in horse plasma. Cocaine stored in VH at -18°C was essentially stable for the study period whereas at 4°C concentrations decreased by >50% in preserved and unpreserved VH stored for longer than 14 days. Fluoride did not significantly affect cocaine stability in VH. The stability of cocaine in muscle tissue homogenates significantly exceeded that in blood and VH at every temperature. In preserved and unpreserved samples stored at 4°C and below, cocaine loss did not exceed 2%. The increased stability of cocaine in muscle was attributed to the low initial pH of post-mortem muscle. In tissue from one human case stored for 13 months at -18°C the muscle cocaine concentration declined by only 15% (range: 5-22%). These findings promote the use of human muscle as a toxicological specimen in which cocaine may be detected for longer compared with blood or VH.

Analysis of cocaine and its principal metabolites in waste and surface water using solid-phase extraction and liquid chromatography–ion trap tandem mass spectrometry

A validated method based on solid-phase extraction (SPE) and liquid chromatography-ion trap tandem mass spectrometry (LC-MS/MS) is described for the determination of cocaine (COC) and its principal metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME), in waste and surface water. Several SPE adsorbents were investigated and the highest recoveries (95.7 +/- 5.5, 91.8 +/- 2.2 and 72.5 +/- 5.3% for COC, BE and EME, respectively) were obtained for OASIS HLB(R) cartridges (6 mL/500 mg) using 100 mL of waste water or 500 mL of surface water. Extracts were analysed by reversed-phase (RP) or hydrophilic interaction (HILIC) LC-MS/MS in positive ion mode with multiple reactions monitoring (MRM); the latter is the first reported application of the HILIC technique for drugs of abuse in water samples. Corresponding deuterated internal standards were used for quantification. The method limits of quantification (LOQs) for COC and BE were 4 and 2 ng L(-1), respectively, when RPLC was used and 1, 0.5 and 20 ng L(-1) for COC, BE and EME, respectively, with the HILIC setup. For COC and BE, the LOQs were below the concentrations measured in real water samples. Stability tests were conducted to establish the optimal conditions for sample storage (pH, temperature and time). The degradation of COC was minimal at -20 degrees C and pH = 2, but it was substantial at +20 degrees C and pH = 6. The validated method was applied to a set of waste and surface water samples collected in Belgium.

Rapid and Robust Protection against Cocaine-Induced Lethality in Rats by the Bacterial Cocaine Esterase

There is no approved means to prevent the toxic actions of cocaine. Cocaine esterase (CocE) is found in a rhodococcal strain of bacteria that grows in the rhizosphere soil around the coca plant and has been found to hydrolyze cocaine in vitro. The esteratic activity of CocE (0.1-1.0 mg, i.v.) was characterized and confirmed in vivo by assessing its ability to prevent cocaine-induced convulsions and lethality in the rat. The therapeutic efficiency of the enzyme was demonstrated by the increasing dose of cocaine (100-1000 mg/kg, i.p.) required to produce toxic effects after a single intravenous injection of CocE. The enzyme demonstrated rapid kinetics for cocaine degradation in rat and human serum. Two catalytically inactive mutants of CocE (S117A or Y44F) failed to protect rats from the toxic effects of cocaine, confirming the protective effects are due to hydrolytic activity. However, butyrylcholinesterase, an endogenous cocaine-hydrolyzing enzyme, was inactive (1.3-13 mg, i.v.) in this rat toxicity procedure. Furthermore, CocE did not block the lethality of WIN-35065-2 (560 mg/kg, i.p.), a cocaine analog that lacks the benzoyl ester moiety targeted by CocE. This characterization of CocE provides preliminary evidence that the enzyme could serve as a suitable antidote to cocaine toxicity in humans.

Focused microwave-assisted extraction combined with solid-phase microextraction and gas chromatography–mass spectrometry for the selective analysis of cocaine from coca leaves

An effective combination of focused microwave-assisted extraction (FMAE) with solid-phase microextraction (SPME) prior to gas chromatography (GC) is described for the selective extraction and quantitative analysis of cocaine from coca leaves ( Erythroxylum coca). This approach required switching from an organic extraction solvent to an aqueous medium more compatible with SPME liquid sampling. SPME was performed in the direct immersion mode with a universal 100 μm polydimethylsiloxane (PDMS) coated fibre. Parameters influencing this extraction step, such as solution pH, sampling time and temperature are discussed. Furthermore, the overall extraction process takes into account the stability of cocaine in alkaline aqueous solutions at different temperatures. Cocaine degradation rate was determined by capillary electrophoresis using the short end injection procedure. In the selected extraction conditions, less than 5% of cocaine was degraded after 60 min. From a qualitative point of view, a significant gain in selectivity was obtained with the incorporation of SPME in the extraction procedure. As a consequence of SPME clean-up, shorter columns could be used and analysis time was reduced to 6 min compared to 35 min with conventional GC. Quantitative results led to a cocaine content of 0.70 ± 0.04% in dry leaves (RSD < 5%) which agreed with previous investigations.

The Effect of Lovastatin and Thioridazine on the Degradation of Cocaine in Human Serum In Vitro

The effect of lovastatin and thioridazine on the degradation of cocaine in human serum was studied by incubating therapeutic and toxic concentrations of either drug with cocaine in human serum at 37 degrees C. Without these other drugs, cocaine concentrations decreased by an average of 88% in 120 minutes. Lovastatin at all concentrations studied showed a negligible effect on the degradation of cocaine in human serum and did not alter the pseudocholinesterase activity of the serum. In contrast, in the presence of a therapeutic concentration of thioridazine, cocaine concentrations decreased by only 71% during this period of time, and, with a toxic concentration of thioridazine, cocaine concentrations decreased by only 55%. Thioridazine suppressed the pseudocholinesterase activity of human serum by up to 19% during the 120-minute incubation period, and this effect was more pronounced at higher thioridazine concentrations. These findings suggest that thioridazine may prolong the serum half-life of cocaine by inhibiting the pseudocholinesterase-mediated catabolism of cocaine to ecgonine methyl ester. This may be important in cocaine users who are treated with phenothiazines and other structurally similar drugs. They also may be of interest in cocaine-abusing patients who are treated with phenothiazines for schizophrenic disorders.

Ecgonine methyl ester protects against cocaine lethality in mice.

Plasma cholinesterase (PChE) metabolizes cocaine to ecgonine methyl ester (EME). Limited data demonstrate that EME is a mild vasodilator. Exogenous PChE protects against cocaine-induced seizures and lethality. It is unclear whether this protective effect results from enhanced degradation of cocaine, the loss of active metabolites (benzoylecgonine, norcocaine), or the production of a beneficial metabolite (EME). This study was designed to further investigate the pharmacologic effects of EME. All experiments used female ICR Swiss albino mice weighing 20-30 grams. Mice were acclimated to 12 h alternating light-dark cycles and given food and water ad libitum. Using a randomized, blinded protocol, 80 mice were then pretreated with either IP EME (50 mg/kg) in a 0.9% sodium chloride solution or an equal volume of 0.9% sodium chloride solution as control. Five minutes later, all animals received 126 mg/kg of cocaine IP and were observed for seizures and death. Fatality was compared using a Fisher's exact test, and the time to seizures and death were compared using a Mann-Whitney U statistic. Pretreatment with EME increased survival following cocaine (9/40 vs. 2/40, for EME vs. control, respectively, p<0.05). The median times to seizure and death for both groups were 2.0 vs. 1.5 min (p>0.05), and 4.5 vs. 4.6 min (p>0.05) (EME vs. control for seizures and death, respectively). In this animal model, EME is protective against cocaine lethality. This effect is consistent with the previously described vasodilatory effects of EME. Further studies are indicated to determine whether the increase in EME produced by exogenous PChE administration contributes to the benefits that occur when PChE is given to cocaine-poisoned animals.

Analysis of cocaine, benzoylecgonine, ecogonine methyl ester, and ecgonine by high-pressure liquid chromatography-API mass spectrometry and application to a short-term degradation study of cocaine in plasma.

A method for the determination of cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and ecgonine (ECG) in plasma by liquid chromatography-mass spectrometry (LC-MS-MS) was developed. The analytes were isolated from human plasma by subsequent solid-phase extraction and were separated on a Zorbax Eclipse XDB-C8 narrow-bore column using an ammonium acetate buffer/acetonitrile/methanol gradient. A Turbolonspray source was used for ionization. The analytes were characterized by their particular molecular ion and several fragments. Multiple reaction monitoring (MRM) was used for isolation and quantitation. The assay was rapid, highly sensitive, and reliable. The method was applied to monitor the in vitro degradation of cocaine in plasma. Fresh unpreserved and preserved (0.25% KF) plasma samples were spiked with 1,000 ng cocaine/mL. Aliquots of both series were stored at 4 degrees C and 20 degrees C and were analyzed at selected storage times of up to 15 days. In all samples, degradation of cocaine that was dependent on storage time and temperature and on sample preservation could be observed. The formation of BE did not occur to a significant extent (< 12%, referred to the initial concentration of COC), and its concentration was slightly higher in preserved compared with unpreserved plasma at both storage temperatures chosen. EME was formed in considerably higher amounts compared to BE. As already observed for COC, its formation and degradation were dependent on storage time, temperature, and preservation. EME is suggested to be the major source of ECG, which was detectable in all samples after 1-2 days of storage. Although the degradation of COC was shown to be highly dynamic in nature, the sum of all hydrolysis products of COC accounted for the initial COC concentration at any particular time of storage. Therefore, production of hitherto unknown degradation products of COC seems unlikely. Moreover, the common transformation product of BE and EME appeared to be stable, and ECG is suggested as a promising postcollection artifact.

Importance of vacutainer selection in forensic toxicological analysis of drugs of abuse.

The enzymatic degradation of cocaine in blood samples, even during transport to a forensic laboratory, is a common problem in toxicological analysis. This can be avoided by the use of blood-sampling devices such as gray-top Vacutainers containing the cholinesterase inhibitor sodium fluoride. In the present study, which included 147 authentic cases, blood samples were collected into two different tubes, one containing fluoride/oxalate and one without stabilizing agents. In all cases, both samples were analyzed for drugs of abuse using Abbott FPIA immunoassays after precipitation and gas chromatography-mass spectrometry (GC-MS) for quantitative analysis. The cannabinoid immunoassay showed markedly lower values in the fluoride-containing samples; this was investigated further and could be explained by hemolysis of these samples. In addition, the concentrations of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) were lower in these samples. A stability study with the THCCOOH acyl glucuronide showed that it is unstable in unpreserved serum, which could explain our observation. GC-MS quantitative data for amphetamine and derivatives, opiates, delta9-tetrahydrocannabinol, and 11-hydroxy-delta9-tetrahydrocannabinol were essentially identical; however, they also differed substantially for cocaine, cocaethylene, ecgonine methylester, and benzoylecgonine. Unexpectedly, the concentrations of benzoylecgonine in unpreserved serum were almost half as high as in the fluoride-containing samples.