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Benzoylecgonine (BZE) is the major toxic metabolite of cocaine and is responsible for the long-term cocaine-induced toxicity owing to its long residence time in humans. BZE is also the main contaminant following cocaine consumption. Here, we identified the bacterial cocaine esterase (CocE) as a BZE-metabolizing enzyme (BZEase), which can degrade BZE into biological inactive metabolites (ecgonine and benzoic acid). CocE was redesigned by a reactant-state-based enzyme design theory. An encouraging mutant denoted as BZEase2, presented a >400-fold improved catalytic efficiency against BZE compared with wild-type (WT) CocE. In vivo, a single dose of BZEase2 (1 mg kg-1 , IV) could eliminate nearly all BZE within only two minutes, suggesting the enzyme has the potential for cocaine overdose treatment and BZE elimination in the environment by accelerating BZE clearance. The crystal structure of a designed BZEase was also determined.

Many false positive and false negative results have been detected in immunoassay analyses of drugs of abuse in urine samples. A method of direct injection of diluted urine into LC/MS/MS was developed and validated for detection and quantitation of Amphetamine, Methamphetamine, MDMA, MDA, Benzoylecgonine, Ecgonine, Norpseudoephedrine, Ephedrine, Tapentadol, Tramadol, O-desmethyltramadol, Tapentadol, Pregabline, Gabapentine and Methadone to avoid the false positive and false negative results in urine samples. Linearity of Amphetamine, Methamphetamine MDMA, MDA, Benzoylecgonine, Ecgonine, Norpseudoephedrine and Ephedrine was (60-2400ng/mL), for Tapentadol, Tramadol, O-desmethyltramadol, and Methadone was (50-1600 ng/mL), and for Pregabline and Gabapentine was (100-4000ng/mL) and r2 ˃ 0.992 for all analysts. A 440 urine samples have been analyzed using both immunoassay technique and LC/MS/MS by direct injection method giving a good comparison to illustrate how this method was specific, accurate, precise, and applicable for forensic urine samples

Homeopathic products are still a controversial issue in modern medicine, understood as complementary or alternative medicine (CAM). In this particular case, homeopathic products prepared from Atropa belladonna extracts may present specific problems due to the effects derived from its components. This article applies a simple, rapid, reliable method to the analysis of different homeopathic products obtained from Atropa belladonna; drugs containing high concentration of plant extracts; and Atropa belladonna seeds. The method was based on a simple solid-phase preconcentration method followed by ultra-high pressure liquid chromatography (UHPLC) coupled to high resolution mass spectrometry using Exactive-Orbitrap as an analyser. An in-house database was set and atropine and scopolamine were the compounds detected at highest concentrations in homeopathic products from Atropa belladonna extracts (4.57 and 2.56 μg/kg, respectively), in Belladonna ointment (4007 and 1139 μg/kg, respectively) and Belladonna seeds (338 and 32.1 mg/kg, respectively). Other tropane alkaloids such as tropine, apoatropine, aposcopolamine, tropinone, homatropine, and anisodamine were detected at lower concentrations (0.04-1.36 μg/kg). When untargeted analysis was performed, other tropane alkaloids were identified in the tested samples, such as ecgonine (0.003 μg/kg), benzoylecgonine (0.56 μg/kg), calystegines A (19.6 μg/kg), B (33.1 μg/kg), and C (1.01 μg/kg). Finally other compounds present in the homeopathic products, such as sugars (fructose, glucose, and lactose) or amino acids (valine, ornithine, leucine, and phenylalanine), were identified.

In this work, we establish that cocaine binding to the Fab fragment of a recombinant humanized anti-cocaine mAb (h2E2) can be directly and easily quantitated using simple and inexpensive absorption and fluorescence measurements, employing dyes typically used for differential scanning fluorimetry, DASPMI and SYPRO Orange. For concentrated samples of the Fab fragment, absorbance spectroscopy employing these dyes reveals the number of cocaine sites present, using either DASPMI (by measuring the increase in dye absorbance) or SYPRO Orange (by measuring the change in dye maximal absorbance wavelength). Interestingly, we observed that cocaine binding to the Fab fragment had a much different effect on the SYPRO Orange dye absorbance than previously reported for the intact h2E2 mAb, resulting in a large decrease in the total dye absorbance for the Fab fragment, in contrast to previous results with the intact h2E2 mAb. For dilute samples of Fab fragment, a dye fluorescence emission spectroscopy assay was developed to quantitate the number of cocaine (and other high affinity cocaine metabolites) binding sites via the ligand-induced decrease in fluorescence emission of both of these extrinsic dyes. The difference in the cocaine titrations for the high affinity (Kd < 30 nM) ligands, cocaine, cocaethylene and benzoyl ecgonine and the low affinity (Kd > 30 μM) ligands, norcocaine, ecgonine methyl ester, and ecgonine were obvious using this assay. These simple, direct, and inexpensive techniques should prove useful for evaluation of other small molecule antigen binding Fab fragments, enabling quantitation and rapid biochemical assessments necessary for determining Fab fragment suitability for in vivo uses and other assays and experiments.

We developed and validated a new rapid and sensitive gas chromatography-tandem mass spectrometry method for the determination of cocaine and its metabolites benzoylecgonine, norcocaine, ecgonine methyl esther and cocaethylene in hair of consumers. Hair samples were firstly decontaminated with three subsequent dichloromethane washes, then incubated for one hour with M3® buffer to promote analytes solubilization and stabilization and finally solid phase extracted. All extracts were derivatized and injected into GC–MS/MS with electron impact ionization. Multiple Reaction Monitoring was used for the acquisition of characteristic analytes ion transitions reaching a high sensitivity 0.01 ng/mg COC and metabolites limit of quantification. The method was linear in the COC and metabolites calibration ranges (LLOQ-10 ng/mg and LLOQ-1 ng/mg, respectively). Intra-assay and inter-assay precision were always lower than 15 %, accuracy never exceeded ± 6.6 %. The main advantages of the presented method are the fast, simple and innovative pretreatment procedure together with the instrumental sensitivity that allowed to measure also less concentrated metabolites.

For the first time in China, the chemical profiling of cocaine specimens was performed at the National Narcotics Laboratory. An ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-QTOF-MS) method was developed and validated for simultaneous analysis of 14 cocaine alkaloids and 5 main adulterants. Among them, ecgonine methyl ester, ecgonine, benzoylecgonine, and norcocaine were identified by comparing with the standard materials; tropacocaine, 3,4,5-trimethoxycocaine, cis-/trans-cinnamoylcocaine were tentatively identified based on the exact masses of protonated molecules and product ions; six unidentified alkaloids of 182/1.47, 316/9.54, 659/9.85, 316/9.87, 420/10.34, and 420/10.85 were marked with 'extracted mass/retention time' for convenience. Minimum sample preparation and analysis time were required, which was suitable for routine analysis. Based on the semi-quantitative data set of 14 alkaloid impurities in 131 linked/unlinked cocaine samples, 50 combinations of pretreatment methods and distance/correlation measurements were tested for their potential discrimination power for cocaine profiling, and Logarithm/Pearson exhibited the best result. After hierarchical cluster analysis (HCA), 183 cocaine samples collected from 2011 to 2015 were classified into 7 major groups. Moreover, 37 groups of linked samples were found within and between provinces, which provide intelligence for the case connection and revealing of the distribution networks. Our results highlighted the practical utilities of drug profiling, especially to support the investigation through operational intelligence and to improve the knowledge related to the drug trafficking through strategic intelligence. Copyright © 2016 John Wiley & Sons, Ltd.

A method was developed using hydrophilic interaction liquid chromatography (HILIC), a versatile mode of liquid chromatography, to determine cocaine, its main degradation products, benzoylecgonine and ecgonine, main pyrolytic products, anhydroecgonine and anhydroecgonine methyl ester, to be used as standards, and a frequent contaminant, levamisole. Detection of all compounds was achieved using two detectors connected in series: charged aerosol detector (CAD) and ultraviolet detector (UV). No method was found in the literature using HILIC coupled to CAD and UV to determine these compounds. The method was carried out with a Phenomenex Kinetex HILIC (150 mm × 4.6 mm ID; 2,6 µm) column with mobile-phase acetonitrile: 10 mM ammonium acetate pH 6.3 (75:25 v/v) with flow rate 0.8 mL min−1 and oven temperature set at 30 °C. CAD detection was performed using a nitrogen pressure of 35 psi, range of 100 pA and the UV detection at 200 nm. Validation parameters such as selectivity, linearity, precision and accuracy were evaluated. Methods were successfully validated to detect and quantify all related substances.

The presence of ecgonine in urine has been proposed as an appropriate marker of cocaine use. Only a few methods have been published for their determination along with cocaine and the rest of its metabolites. Due to their high polarity and consequent solubility in water, these have low recoveries, which is why it is necessary to increase the sensitivity, by the formation of hydrochloric salts or multiderivatization of the analytes or by performing two solid-phase extractions (SPEs), considerably increasing the time and cost of the analysis. This work describes a fast and fully validated procedure for the simultaneous detection and quantification of ecgonine, ecgonine-methyl-ester, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, cocaethylene, cocaine, norcocaine, and norcocaethylene in human urine (500 μL) using one SPE and simple derivatization. Separation and quantification were achieved by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) in selected-ion monitoring mode. Quantification was performed by the addition of deuterated analogs as internal standards. Calibration curves were linear in the adopted ranges, with determination coefficients higher than 0.99. The lower limits of quantification ranged from 2.5 to 10 ng/mL. The intra- and inter-day precision, calculated in terms of relative standard deviation, were 1.2%-14.9% and 1.8%-17.9%, respectively. The accuracy, in terms of relative error, was within a ± 16.4% interval. Extraction efficiency ranged from 84% to 103%. Compared with existing methods, the procedure described herein is fast, since only one SPE is required, and cost-effective. In addition, this method provides a high recovery for ecgonine, resulting in a better alternative to the previously published methods.

Cocaine-related deaths have increased since the early 1990s in Europe, including the UK. Being multi-factorial, they are difficult to define, detect and record. The European Monitoring Centre for Drugs and Drug Addiction commissioned research to: describe trends reported to Special Mortality Registries and General Mortality Registers; provide demographic and drug-use characteristic information of cases; and establish how deaths are identified and classified. A questionnaire was developed and piloted amongst all European Monitoring Centre for Drugs and Drug Addiction Focal Point experts/Special Mortality Registries: 19 (63%) responded; nine countries provided aggregated data. UK General Mortality Registers use cause of death and toxicology to identify cocaine-related deaths. Categorisation is based on International Classification of Diseases codes. Special Mortality Registries use toxicology, autopsy, evidence and cause of death. The cocaine metabolites commonly screened for are: benzoylecgonine, ecgonine methyl ester, cocaethylene and ecgonine. The 2000s saw a generally accelerating upward trend in cases, followed by a decline in 2009. The UK recorded 2700–2900 deaths during 1998–2012. UK Special Mortality Registry data (2005–2009) indicate: 25–44 year-olds account for 74% of deaths; mean age=34 (range 15–81) years; 84% male. Cocaine overdoses account for two-thirds of cases; cocaine alone being mentioned/implicated in 23% in the UK. Opioids are involved in most (58%) cocaine overdose cases.

Background Drug-Drug Interactions (DDIs) represent a common problem in clinical practice during drug treatments. DDIs can both induce the development of adverse drug reactions or reduce the clinical efficacy of each drug. Objectives The main objective of this review was to analyze the pharmacokinetic and pharmacodynamic DDIs in cocaine consumers, focusing the interest on their clinical implications. Methods The PubMed, Embase and Cochrane library databases were searched for articles published until January 10, 2017. Secondary search included articles cited in reference lists identified by the primary search. Papers were deemed eligible if they included any form of words: "adverse drug reaction", "drug interactions", "poly-therapy", "cocaine", "systemic diseases". Results In this review, the nodal points treated concern: i) cocaine biochemical metabolism described for both, inactive benzoylecgonine and ecgonine methyl esters and norcocaine active metabolites. We provided evidences of concepts deriving from rat/mice experimental studies speculating a translation approach to human in order to treat cocaine overdose. ii) Drug-drug interactions, which come out from clinical evidences as the case of CYP450 family enzyme inhibitors or inductors modulating cocaine toxicity. Particularly, we highlighted the lack of knowledge concerning cocaine and CYP3A4 inhibitors (such as ketoconazole, nefazodone, erythromycin, and clarithromycin). We recorded the worst association of cocaine and beta-blockers by direct and indirect action, particularly at postsynaptic levels on dopamine and norepinephrine reuptake, sympathetic activation and increase of heart rate, blood pressure and cardiovascular toxicity. Cocaine also induces increase in serotonin synaptic activity leading to the development of a serotoninergic syndrome when used with drugs that affect serotonin pathway. Genetic (i.e. glutathione peroxidase-1 deficiency) and epigenetic factors (i.e. microRNAs) may be involved in drug-drug interactions in cocaine-users are also being introduced. Conclusion DDIs represent an important potential complication in cocaine users in clinical setting. The knowledge of DDIs can also be used to select treatments for patients, thus optimizing clinical response and minimizing toxicity.

Today, forensic hair analysis is considered to be a standard method for identifying chronic drug users since information about drug use stored and located in hair can cover several months to even years. When interpreting these results, one should be aware of all kind of pitfalls. External factors such as bleaching might influence the analytical result. Although the effect of hydrogen peroxide on cocaine in a solution was described before, it was never investigated whether the described reaction products (ecgonine methylester, benzoylecgonine, hydroxynorcocaine and dihydroxycocaine) are indeed found on contaminated or user hair. Since it is of great importance in forensic hair analysis to know whether cocaine and/or reaction products are detectable in hair after bleaching, matrix-assisted laser desorption/ ionization mass spectrometric imaging (MALDI-MSI) was used to study the effect of hydrogen peroxide treatment on incorporated cocaine in hairs. Cocaine oxidation products were identified in a solution based on MS/MS spectra and spatial distribution of these products in hair was explored using MALDI TOF-MS. All images were accomplished by spraying alpha-Cyano-4-hydroxycinnamic acid (CHCA) as a MALDI-matrix. Images revealed a loss of detectability of cocaine and its reaction products in hairs already after a short bleaching period. Since all compounds of interest are found in the hydrogen peroxide and wash solution, these findings indicate that all evidence of cocaine use might be lost after a hair bleaching treatment. Therefore, forensic toxicologists should take into consideration whether hair samples were bleached before making any conclusions from hair analysis results.

In this study, the procedure for analyzing cocainic drugs and metabolites (anhydroecgnonine, anhydroecgnonine methyl ester, benzoylecgonine, cocaine, cocaethylene, ecgnonine ethyl ester, and ecgnonine methyl ester) in whole blood samples obtained from drugs and driving cases using a mixed mode solid phase extraction (SPE) C8/SCX cartridge (Clean Screen DAU206), is described. This extraction and analysis procedure allows forensic analysts to differentiate between drivers who have used “crack cocaine” against those using regular cocaine. Samples of whole blood (containing deuterated internal standards) were diluted with an aqueous phosphate buffer (pH 6). Each sample was applied to a conditioned SPE column. The filtrate was collected and adjusted to pH 2. The sorbent was rinsed with deionized (DI) water, aqueous hydrochloric acid and methanol. After drying, cocaine, cocathylene, benzoylecgnonine, and anhydroecgnonine methyl ester were eluted from the SPE column with 3 mL of an elution solvent consisting of methylene chloride–isopropanol–ammonium hydroxide. The original filtrate was applied to a second conditioned SPE column and washed with DI water, aqueous hydrochloric acid and methanol. After drying the SPE sorbent, anhydroecgnonine was eluted with methanol containing 4% ammonium hydroxide (3 mL). The eluates were combined and evaporated to dryness, and the residue was dissolved in the mobile phase for analysis by LC-MS/MS in positive multiple reaction monitoring mode (MRM). Chromatography was performed in gradient mode employing a C18 column and a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. The total run time for each analysis was within five minutes. The limits of quantitation/detection for this method were determined to be 0.5 ng mL−1 and 1.0 ng mL−1 respectively. The method was found to be linear from 1.0 ng mL−1 to 100.0 ng mL−1 (r2 > 0.995). The recoveries of the noted cocaine type drugs were found to be greater than 90%. This method was applied to twenty positive cocaine completed drugs and driving cases, it was found that anhydroecgnonine concentrations ranged from 0 ng mL−1 to 23 ng mL−1, anhydroecgonine methyl ester concentrations ranged from 0 ng mL−1 to 66 ng mL−1, while cocaine was found to range from 25 ng mL−1 to 250 ng mL−1, cocaethylene ranged from 0 ng mL−1 to 80 ng mL−1, benzoylecgnonine concentrations were found to be in the range 90 ng mL−1 to 790 ng mL−1, ecgonine methyl ester concentrations ranged from 40 ng mL−1 to 500 ng mL−1 and ecgonine ethyl ester concentrations were found to be in the range 0 ng mL−1 to 180 ng mL−1.

A method to assay cocaine (COC), its metabolites benzoylecgonine (BZE), ecgonine (ECG), ecgonine methyl esther (EME), and benzoylnorecgonine (BNE), pyrolytic products anhydroecgonine (AEC) and anhydroecgonine methyl ester (AEME) and adulterant levamisole (LEV) was developed and validated by liquid chromatography-mass spectrometry (LC-MS) using a chemometric approach including a two-level factorial design in the screening step and face-centered central composite design (FCCCD) to achieve the optimization. The method was carried out on positive electrospray ionization (ESI+) with a flow of 1 mL min−1 in isocratic mode consisting of 53% methanol and 47% ammonium acetate 10 mmol L−1 pH 6.3. The chromatographic separation was obtained with a Phenomenex Luna C18(2) column (250 mm × 4.6 mm, particle size 5 μm), with the temperature set at 31 °C. Validation parameters such as specificity, linearity, precision and accuracy were evaluated. The method was linear over the concentration range of 1–100 ng mL−1 for COC, AEME, EME, LEV, BZE and ECG and 5–100 ng mL−1 for AEC and BNE. The method was successfully applied to identify and quantify the analytes.

Transformations of cocaine and eleven of its metabolites were investigated in untreated municipal sewage at pH ≈ 7 and 9, 23, and 31 °C. Results indicated that hydrolysis-possibly bacterially mediated-was the principal transformation pathway. Residues possessing alkyl esters were particularly susceptible to hydrolysis, with pseudo-first-order rate constants varying from 0.54 to 1.7 day(-1) at 23 °C. Metabolites lacking esters or possessing only a benzoyl ester appeared stable. Residues lacking alkyl esters did accumulate through hydrolysis of precursors, however. As noted previously, this may positively bias cocaine utilization estimates based on benzoylecgonine alone. Reported variability in metabolic excretion was used in conjunction with transformation data to evaluate different approaches for estimating cocaine loading. Results indicate that estimates derived from measurands that capture all major cocaine metabolites, such as COCtot (the sum of all measurable metabolites) and EChyd (the sum of all metabolites that can be hydrolyzed to ecgonine), may reduce uncertainty arising from variability in metabolite transformation and excretion, possibly to ≈ 10 % RSD. This is more than a two-fold reduction relative to estimates derived from benzoylecgonine (26 % RSD), and roughly equivalent to reported uncertainties from sources that are not metabolite-specific (e.g., sampling frequency, flow variability). They and other composite measurands merit consideration from the sewage epidemiology community, beginning with efforts to evaluate the stability of the total cocaine load under realistic sewer conditions.

This study is part of a larger project designed to investigate the prevalence of psychoactive drug (PAD) use among Brazilian drivers. In this paper we describe the development and validation of an analytical method to analyze 32 prescription and illicit PADs (amphetamines, benzodiazepines, cocaine, cannabis, opioids, ketamine and m-CPP) and metabolites in oral fluid samples collected with a Quantisal™ device. Samples were extracted with ethyl acetate:hexane and analyzed by LC-MS/MS. Instrumental LOD ranged from 0.26 to 0.65 ng/mL. Mean procedural recoveries at 1.3 ng/mL (LLOQ) ranged from 50% to 120% for 24 compounds. Recoveries were concentration independent, with the exception of femproporex, heroin and ecgonine methyl-ester (EME) for which the recovery decreased significantly at higher levels (13 and 52 ng/mL). RSD was20% for all compounds at all spiking levels. Ion suppression due to the matrix was20% for most compounds, and higher than 60% for EME and diethylpropion. Analysis was performed against a in-matrix standard curve. About 10% of the 2235 oral fluid samples collected from drivers on Brazilian Federal highways were positive (≥LOD) for at least one analyte investigated. Alone or in combination with other drugs, cocaine/metabolites were the analytes most detected in the samples (129; 5.8%), followed by amphetamines/metabolite (69; 3.1%), benzodiazepines (28; 1.2%), cannabinoids (23; 1.1%) and opioids (8; 0.4%). Detection of at least two PADs from different classes accounted for 9.3% of the 236 positive samples. Cocaine was found at higher levels in the samples (up to 1165 ng/mL). Preventive measures aimed at reducing the use of PADs by drivers in Brazil will certainly contribute to decrease the country's highway death rates.

A novel approach has been developed for the illicit drugs quantitative determination using dried blood spots (DBS) on filter paper. The illicit drugs tested were opiates (morphine and its 3- and 6-glucuronide metabolites, codeine, 6-monoacetylmorphine), cocainics (ecgonine methylester, benzoylecgonine, cocaine, cocaethylene) and amphetamines (amphetamine, methamphetamine, MDA, MDMA, MDEA). The described method, requiring a small blood volume, is based on high performance liquid chromatography coupled to tandem mass spectrometry using on-line extraction. A Whatman card 903 was spotted with 30μL of whole blood and left overnight to dry at room temperature. A 3-mm diameter disk was removed using a manual punch, suspended in 150μL of water for 10min with ultrasonication, and then 100μL was injected in the on-line LC-MS/MS system. An Oasis HLB was used as an extraction column and a C18 Atlantis as an analytical column. The chromatographic cycle was performed with 20mM ammonium formate buffer (pH 2.8) (solvent A) and acetonitrile/solvent A (90:10, v/v) gradient in 16min. Detection was performed in positive electrospray ionization mode (ESI+) with a Quattro Micro (Waters). Recoveries of all analytes were up to 80%. DBS were stored in duplicate at 4°C and -20°C for up to 6 months. Illicit drugs seemed to be much more stabled at -20°C. Furthermore, it was tested whether analysis of DBS may be as reliable as that of whole blood investigating authentic samples; significant correlations were obtained. This DBS assay has potential as rapid, sensitive and inexpensive option for the illicit drugs determination in small blood volumes, which seems of great interest in suspected cases of driving under the influence of drugs.

We report a sample pretreatment approach for the analysis of total cocaine residues in wastewater that eliminates the need for two key assumptions often made in estimating cocaine utilization from measurement of its benzoylecgonine metabolite: that benzoylecgonine is neither degraded nor generated during transport in a sewer system, and that it is excreted as a constant fraction of cocaine ingested. By adding NaOH and incubating samples at 55 °C, cocaine and its principal metabolites are efficiently hydrolyzed into ecgonine, anhydroecgonine, and norecgonine. Ecgonine, estimated to represent between 37% and 90% (on a molar basis) of cocaine residues, can be directly determined (without preconcentration via solid-phase extraction (SPE)) by reversed-phase (RP) or hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC/MS/MS). If samples are subjected to SPE, anhydroecgonine can also be determined; this metabolite (and its precursors) represents ≈7% of urinary cocaine residues (based on spot collections from living individuals). Although a reference standard for norecgonine is not commercially available, such nortropanes are also a minor fraction (up to 2%) of urinary cocaine residues. The stability of two human markers (cotinine and creatinine) to the hydrolysis procedure was also investigated. Results obtained by applying the hydrolysis approach for the analysis of total cocaine in an untreated municipal wastewater sample (obtained from Baltimore, MD) were generally in excellent agreement with those obtained from split samples analyzed using a more comprehensive solid-phase extraction RPLC/MS/MS method as described in our previous work. In particular, total tropane-based cocaine residues were found to be hydrolyzed to ecgonine with 98-99% efficiency.

The question posed by this research involves how frequently one can expect to contact cocaine in day-to-day living experiences where drug use may not normally be suspected. Issues concerning contamination are germane to medico-legal investigators who evaluate the significance of drug test results in (1) questioned deaths, (2) public health concerns, (3) drugs crimes, and (4) drug use toxicological tests potentially caused by external contamination, such as hair, sweat, and skin swabs. Previous surface studies focus mainly on currency drug contamination; few have addressed other common surfaces. Public surfaces handled by a large number of people, such as building entrance door handles, bank currency dispensing machines, food store shopping carts, and service station fuel pumps within the New Haven, Connecticut metropolitan area were tested. Socio-economically, the distribution of items tested ranged from working-class to upper-middle class areas. Results were obtained using the Securetec Drugwipe II® which is an immunological, on-site test device. Precautions were taken to avoid carry-over and other potential contamination from handling including single-use latex gloves which were pre-tested as a negative control immediately before swabbing. Analysis was performed according to the manufacturer’s instructions and in the laboratory to standardize conditions. Drugwipe II® limit of detection (LOD) is ∼50 ng. Test results revealed 78% positive for cocaine substances as follows: fuel pump buttons for credit card authorization, 100% positive (n = 10 individual locations); ATM machines for currency withdrawal, 100% positive (n = 10 individual locations); grocery store shopping carts, 70% positive; academic building entrance doors, 30% positive (n = 10); and shopping mall entrance/exit doors, 100% positive (n = 5). Forensic scientists and medico-legal investigators responsible for interpreting surface test results are cautioned to consider contamination before ascribing drug activity to a specific individual. For legal proceedings these results are indicative but not conclusive because immunological tests are considered presumptive and may react with the parent substance (cocaine), related compounds (benzoylecgonine, ecgonine methyl ester, ecgonine, nor-ecgonine, and cocaethylene) and immunologically similar materials.

The contradiction noted previously, that the quantity of the cocaines in the leaves on a branch remains almost constant for 8 successive leaves with the exception of that of the youngest leaf, while the composition of the cocaines in the leaves alters during their growth, - young leaves contain more cinnamylcocaines than cocaine and old leaves more cocaine than cinnamylcocaines, - is explained as being caused by the continuous formation of fresh cocaines in the leaves, and the continuous decomposition of an almost equal quantity of cocaines containing less cocaine and more cinnamylcocaines than the percentages in the fresh formed cocaines. Reasons for the quicker decomposition of the cinnamylcocaines in the leaves are indicated. In agreement with this explanation it is noted, that the acids of the esters of l-nor-ecgonine consist for the main part of formic acid, benzoic acid being not present. The way in which l-nor-formylecgonine can be formed in the leaves from the cinnamylcocaines is indicated.

The amino acid formerly1) separated from Java coca leaves is l-nor-ecgonine, which Einhorn has obtained by oxidation of l-ecgonine with potassium permanganate in alkaline solution. It is present in the leaves as acid esters. The calcium salis of these esters and also that of l-nor-ecgonine itself are soluble in the mixture of benzene and methyl alcohol boiling at 58° and also in the mixture of benzene and ethyl alcohol boiling at 68°.

The specific rotation of l-ecgonine in different solutions is discussed. The decomposition of 0.5g of cocaine in 20 cm3 of 2 n.hydrochloric acid is complete in 5 hours, as is also that of 0.25 g in 20 cm3 of n/2 acid. A method is indicated for the determination of the quantity of l-ecgonine in a mixture containing a compound with leavo rotation, which is not altered by boiling with 20% caustic potash solution. The following principle results were obtained with this method. The quantity of ecgonidine formed by boiling l-ecgonine, l-benzoylecgonine and l-cocaine with hydrochloric acid is greater the stronger the acid and the longer the time of boiling. The esters are split up first into ecgonine and this is then partly transformed into ecgonidine. d-Φ-Ecgonine is not converted by boiling for 12 hours in a strong solution of hydrochloric acid. The l-ecgonine of the esters is partly converted into ecgonidine by boiling with 20% potash. At ordinary temperatures the esters of l-ecgonine are partially converted into d-Φ-ecgonine by alkalis in alcohol or acetone. This is a result of their easy conversion into their d-isomers while they are still undecomposed and in contact with an alkali. By boiling with water benzoylecgonine and also cocaine form only small quantities of ecgonidine. l-Cocaine heated at 115°–120° is converted first to the extent of 50% into ecgonidine methylester and benzoic acid; this acid then forms a benzoate with the remaining 50% of l-cocaine, which is transformed on heating into l-ecgonine methyl ester and benzoic acid anhydride, while the latter ester is changed on longer heating into its d-isomer. d-Cocaine is also transformed by heating at 115–120° into ecgonidine but with much more difficulty. The ecgonines and the cocaines are all converted on heating with excess benzoic acid at 115–120° into ecgonidine. l-Ecgonine forms a compound with barium chloride (2 mol. of ecgonine + 1 mol. of BaCl2) which is slightly soluble in boiling absolute alcohol, but insoluble in boiling benzene.

Methods are indicated by which the absence of l-benzoylecgonine and the presence of an ester of l-ecgonine in Java coca leaves can be established.

By heating l-ecgonine in weakly acidified aqueous solution and also in strong hydrochloric acid solution, l-ecgonine is converted to the extent of 10.5-12.5% into d-φ-ecgonine, an equilibrium mixture of ecgonines being formed. Both l- and d-φ-ecgonine give black crystals like l-ecgonidine with hydriodic acid solution containing iodine; these crystals, when boiled with water, give solutions which show respectively leavo- and dextro-gyratory rotations. On boiling l-ecgonine as an alkali-salt in saturated alkali chloride solution, another equilibrium is reached between the ecgonines; the sodium salt gave a 86.9% conversion into d-φ-ecgonine, the potassium salt one of 69% and the calcium salt one of 78.5%.

The leaves, 20 g. are mixed with finely pulverised quick lime, containing a quantity of calcium oxide corresponding exactly with the moisture content of the leaves. After remaining for 24 hours in a closed flask they are transferred to an extractor containing a mixture of 100 cm3 of anhydrous benzene and 5 cm3 of absolute methyl alcohol. A quantity of ammonia solution, containing exactly 1.938 g of water is added and the leaves mixed with the solvents. After 24 hours, finely pulverised quick lime possessing a quantity of calcium oxide corresponding with the quantity of water used in the ammonia solution is added and after mixing, the extractor closed until the following day. Percolation is then started in a slow rate. As soon as the solvent mixture is run down a quantity of the mixture of anhydrous benzene and absolute methyl alcohol boiling at 58° is added to the leaves. Percolation with this solvent mixture is finished when the percolate mesures 800 cm3. The mixture boiling at 58° is distilled from this percolate and the remaining benzene solution is filtered from the calcium salts insoluble in benzene. The residue from the benzene solution is shaken with 25 cm3 of n/5 HCl solution and 100 cm3 of ether. The quantity of bases, the cocaines and the methyl ester of ecgonine can be determined in the hydrochloric acid solution so obtained. The quantity of l-nor-ecgonine can be determined in the mixture of calcium salts.

The calcium salt of l-ecgonine is soluble in methyl and ethyl alcohols and also in anhydrous benzene. It crystallises in fine needles. Ecgonine forms a compound with calcium chloride. The lead salt also crystallises in needles and is readily soluble in alcohol and benzene. An almost complete conversion of l-ecgonine into l-benzoylecgonine is obtained in 5 days at ordinary temperature in aqueous acetone with the addition of 2–3 times the theoretical quantity of benzoic anhydride. By the action of benzoic anhydride and methyl alcohol on benzoylecgonine at ordinary temperature the anhydride of benzoylecgonine is formed as the main product, which can then give cocaine and benzoylecgonine with methyl alcohol. The conversion into cocaine obtained, however, by using 7 times the theoretical quantity of benzoic anhydride was only 80%. Benzoylecgonine when treated with methyl alcohol and 3.5 times the theoretical quantity of benzoyl chloride at ordinary temperature gives an almost complete conversion into cocaine.

The configurations of (±)-allococaine and (±)-allopseudococaine have been determined, for the first time in an unambiguous way, by means of NMR spectroscopy. The known configurations of (±)-cocaine and (±)-pseudococaine receive independent confirmation. The preferential conformation of the piperidine ring of the tropane nucleus is found to be the chair form in all four isomers, and also in the methyl esters of the four corresponding isomers of ecgonine. The preparation of (±)-allococaine and (±)-allopseudococaine has been improved.

Opiates, cocaine, and metabolites were quantified by liquid chromatography-mass spectrometry (LC-MS) in 284 urine specimens, collected thrice weekly, to monitor possible drug relapse in 15 pregnant heroin-dependent women. Opiates were detected in 149 urine specimens (52%) with limits of quantification (LOQ) of 10-50 microg/L. Morphine, morphine-3-glucuronide, and/or morphine-6-glucuronide were positive in 121 specimens; 6-acetylmorphine, a biomarker of heroin ingestion, was quantifiable in only 7. No heroin, 6-acetylcodeine, papaverine, or noscapine were detected. One hundred and sixty-five urine specimens (58%) from all 15 participants were positive for one or more cocaine analytes (LOQ 10-100 microg/L). Ecgonine methylester (EME) and/or benzoylecgonine were the major cocaine biomarkers in 142. Anhydroecgonine methylester, a biomarker of smoked cocaine, was positive in six; cocaethylene and/or ecgonine ethylester, biomarkers of cocaine and ethanol co-ingestion, were found in 25. At the current Substance Abuse Mental Health Services Administration cutoffs for total morphine (2000 microg/L), codeine (2000 microg/L), 6-acetylmorphine (10 microg/L), and benzoylecgonine (100 microg/L), 16 opiate- and 29 cocaine-positive specimens were identified. Considering 100 microg/L EME as an additional urinary cocaine biomarker would identify 51 more positive cocaine specimens. Of interest is the differential pattern of opiate and cocaine biomarkers observed after LC-MS as compared to gas chromatography-mass spectrometry analysis.

The structure of the hydrated gold(III) tetrachloride salt of L-ecgonine {hydronium tetrakis[(1R,2R,3S,5S,8S)-3-hydroxy-8-methyl-8-azoniabicyclo[3.2.1]octane-2-carboxylate pentakis[tetrachloridoaurate(III)] hexahydrate}, (C(9)H(16)NO(3))(4)(H(3)O)[AuCl(4)](5).6H(2)O, demonstrates an unprecedented stoichiometric relationship between the cations and anions in the unit cell. The previous tropane alkaloid structures, including the related hydrochloride salts, all have a cation-anion ratio of 1:1, as does the anhydrous salt described here, namely (1R,2R,3S,5S,8S)-3-hydroxy-8-methyl-8-azoniabicyclo[3.2.1]octane-2-carboxylate tetrachloridoaurate(III), (C(9)H(16)NO(3))[AuCl(4)]. The hydrated salt, however, consists of four monopositive N-protonated units of the alkaloid and five [AuCl(4)](-) counter-ions, plus seven solvent water molecules. The H atom required for change balance has been assigned to a water molecule. In addition, the hydrate has a novel arrangement, with all seven of the water molecules and all of the O atoms in the cations participating in an alternating arrangement of interleaved sheets of the anionic species. Both the hydrate and the anhydrous salt of the same toxicologically important marker for cocaine show that the cation and anion are in close proximity to each other, as was found in the gold(III) tetrachloride salt of L-cocaine.

As laboratories are called upon to develop novel, fast, and sensitive methods, here we present a completely automated method for the analysis of cocaine and its metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and cocaethylene) from whole blood. This method utilizes an online solid-phase extraction (SPE) with high performance liquid chromatographic separation and tandem mass spectrometric detection. Pretreatment of samples involve only protein precipitation and ultracentrifugation. An efficient online solid-phase extraction (SPE) procedure was developed using Hysphere MM anion sorbent. A gradient chromatography method with a Gemini C6-Phenyl (50mmx3.00mm i.d., 5microm) column was used for the complete separation of all components. Analysis was by positive ion mode electrospray ionization tandem mass spectrometry, using multiple reaction monitoring (MRM) to enhance the selectivity and sensitivity of the method. For the analysis, two MRM transitions are monitored for each analyte and one transition is monitored for each internal standard. With a 30-microL sample injection, linearity was analyte dependent but generally fell between 8 and 500ng/mL. The limits of detection (LODs) for the method ranged from 3 to 16ng/mL and the limits of quantitation (LOQs) ranged from 8 to 47ng/mL. The bias and precision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as7%, and %precision as9% for all components at each QC level.

A new approach for fast and sensitive electrochemiluminescence (ECL) detection of narcotic drugs on a microchip after separation by micellar electrokinetic chromatography (MEKC) is presented, taking the cocaine and its hydrolysate ecgonine as the test analytes. The mixture of hydrophilic BMIMBF4 ionic liquid (IL) and sodium dodecyl sulfate (SDS) was used directly as the buffer of MEKC with less noisy baselines, lower electrophoretic current and satisfactory separation performance.

Understanding cocaine and metabolites urinary excretion following smoking is important for interpretation of urine test results in judicial, workplace and treatment settings. In National Institute on Drug Abuse approved studies on a secure research unit, six subjects smoked placebo, 10, 20, and 40 mg cocaine with a precise dose delivery device and six different subjects smoked 42 mg cocaine in a glass pipe. Urine specimens (n = 700) were collected for up to seven days and analyzed for cocaine (COC), benzoylecgonine (BE), ecgonine methylester (EME), m-hydroxybenzoylecgonine (mOHBE), p-hydroxybenzoylecgonine (pOHBE), norbenzoylecgonine (NBE), and ecgonine (EC) by gas chromatography–mass spectrometry. Results (mean ± SE) for the 40-mg precise delivery doses are as follows: COCBEEMEmOHBEpOHBENBEECCutoff(ng/mL)10201025252550Cmax(ng/mL)4085±23039196±15694638±1548222±69540±227614±347852±211Tmax(h)2.2±0.36.6±0.95.6±1.47.8±0.54.4±1.56.0±2.09.3 ±1.3Lastpositive(h)5510616455553280 Mean Cmax for all analytes linearly increased with increasing dose. Tmax was not dose-dependent. All metabolites were detected in some subjects within 2 h. EC concentrations were significantly higher after smoked cocaine in a precise delivery coil compared to a glass “crack” pipe.

Determination of drugs of abuse in blood is of great importance in clinical and forensic toxicology. This review describes procedures for detection of the following drugs of abuse and their metabolites in whole blood, plasma or serum: Delta9-tetrahydrocannabinol, 11-hydroxy-Delta9-tetrahydrocannabinol, 11-nor-9-carboxy-Delta9-tetrahydrocannabinol, 11-nor-9-carboxy-Delta9-tetrahydrocannabinol glucuronide, heroin, 6-monoacetylmorphine, morphine, morphine-6-glucuronide, morphine-3-glucuronide, codeine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, N-ethyl-3,4-methylenedioxyamphetamine, 3,4-methylenedioxyamphetamine, cocaine, benzoylecgonine, ecgonine methyl ester, cocaethylene, other cocaine metabolites or pyrolysis products (norcocaine, norcocaethylene, norbenzoylecgonine, m-hydroxycocaine, p-hydroxycocaine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine, ethyl ecgonine, ecgonine, anhydroecgonine methyl ester, anhydroecgonine ethyl ester, anhydroecgonine, noranhydroecgonine, N-hydroxynorcocaine, cocaine N-oxide, anhydroecgonine methyl ester N-oxide). Metabolites and degradation products which are recommended to be monitored for assessment in clinical or forensic toxicology are mentioned. Papers written in English between 2002 and the beginning of 2007 are reviewed. Analytical methods are assessed for their suitability in forensic toxicology, where special requirements have to be met. For many of the analytes sensitive immunological methods for screening are available. Screening and confirmation is mostly done by gas chromatography (GC)-mass spectrometry (MS) or liquid chromatography (LC)-MS(/MS) procedures. Basic information about the biosample assayed, internal standard, workup, GC or LC column and mobile phase, detection mode, and validation data for each procedure is summarized in two tables to facilitate the selection of a method suitable for a specific analytic problem.

Optimisation and harmonisation of analytical and statistical methodology have been carried out between two forensic laboratories (Lausanne, CH and Lyon, F) in order to provide drug intelligence for cross-border cocaine seizures. The aim was to improve the gas chromatographic analysis of cocaine samples for profiling. Some important validation parameters were tested to verify the developed method and demonstrate its profiling capacity: the selectivity of the method with retention time reproducibility, the choice of a derivatisation agent improving the chromatography (MSTFA, BSA, TMSI and BSTFA + TMCS 1%), the cutting agents influence (matrix effect), the influence of the sample storage conditions and the sample quantity to weigh for analyses. Eight main alkaloids, which represent the sample signature, have been selected: ecgonine methyl ester, ecgonine, tropacocaine, benzoylecgonine, norcocaine, cis- and trans-cinnamoylcocaine and 3,4,5-trimethoxycocaine. Their stability in the solvent used (CHCl3/pyridine) was demonstrated. In order to reach the final objective, which is the comparison of samples seized and analyzed in two different laboratories, the harmonisation of the profiling method between the two laboratories had to be ensured and is the subject of ongoing research.

An analytical method for determining traditional and emerging drugs of abuse in particulate matter, sewage sludge and sediment has been developed and validated. A total of 41 drugs of abuse and metabolites including cocainics, tryptamines, amphetamines, arylcyclohexylamines, cathinones, morphine derivatives, pyrrolidifenones derivatives, entactogens, piperazines and other psychostimulants were selected. Samples were ultrasound extracted with McIlvaine buffer and methanol, and the extracts were cleaned up by solid phase extraction (SPE) using Strata-X cartridges. Drugs were eluted using methanol and methanol-dichloromethane and determined by liquid chromatography tandem mass spectrometry. The optimum solid-liquid extraction (SLE) conditions were: weight 1g of sample and ultrasound assisted extraction (UAE) with 10mL of methanol-McIlvain buffer (1:1, v/v, pH 4.5) for 10min. Recoveries for all compounds were ≥50% in the three matrices with the exception of ephedrine (EPHE), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), ecgonine methyester (ECME), heroin (HER), 3,4-methylendioxyamphetamine (MDA) and 4-acetoxy N,N'-dimethyltryptamine (4-AcO-DIPT) and methadone (MET). Data acquisition was done by selective reaction monitoring (SRM), and the two most abundant product ions were used for confirmation. Limits of detection were lower than 1.32ngg-1 dry weight (d.w.) and limits of quantification were between 0.12 and 3.96ngg-1 (d.w.). The method was applied to the analysis of particulate matter, where cocaine (COC), benzoylecgonine (BECG), ecgoninemethylester (ECME), cocaethylene (COCET), methadone (MET) and codeine (COD) were mostly detected. In the case of dehydrated sludge, opioids are at higher concentration than cocainics and some emerging drugs such as 4-methoxyamphetamine (PMA), ketamine (KET) and bufotenine (BUF) were detected. In sediment COC, 4-methoxyphencyclidine (4-MeO-PCP), MET and BECG were most relevant compounds. © 2015 Elsevier B.V., This work has been supported by the Spanish Ministry of Economy and Competitiveness through the projects (GCL2011-29703-C02-02). M. Jesús Andrés-Costa also thanks the Spanish Ministry for the FPI grant.

Objectives Cocaine intoxication leads to over 500,000 emergency department visits annually in the United States and ethanol cointoxication occurs in 34% of those cases. Cardiotoxicity is an ominous complication of cocaine and cocaethylene overdose for which no specific antidote exists. Because infusion of lipid emulsion (Intralipid) can treat lipophilic local anesthetic toxicity and cocaine is an amphipathic local anesthetic, the authors tested whether lipid emulsion could attenuate cocaine cardiotoxicity in vivo. The effects of lipid emulsion were compared with the metabolically inert sulfobutylether-β-cyclodextrin (SBE-β-CD; Captisol) in an isolated heart model of cocaine and cocaethylene toxicity to determine if capture alone could exert similar benefit as lipid emulsion, which exhibits multimodal effects. The authors then tested if cocaine and cocaethylene, like bupivacaine, inhibit lipid-based metabolism in isolated cardiac mitochondria. Methods For whole animal experiments, Sprague-Dawley rats were anesthetized, instrumented, and pretreated with lipid emulsion followed by a continuous infusion of cocaine to assess time of onset of cocaine toxicity. For ex vivo experiments, rat hearts were placed onto a nonrecirculating Langendorff system perfused with Krebs-Henseleit solution. Heart rate, left ventricle maximum developed pressure (LVdevP), left ventricle diastolic pressure, maximum rate of contraction (+dP/dtmax), maximum rate of relaxation (–dP/dtmax), rate-pressure product (RPP = heart rate × LVdevP), and line pressure were monitored continuously during the experiment. A dose response to cocaine (10, 30, 50, and 100 μmol/L) and cocaethylene (10, 30, and 50 μmol/L) was generated in the absence or presence of either 0.25% lipid emulsion or SBE-β-CD. Substrate-specific rates of oxygen consumption were measured in interfibrillar cardiac mitochondria in the presence of cocaine, cocaethylene, ecgonine, and benzoylecgonine. Results Treatment with lipid emulsion delayed onset of hypotension (140 seconds vs. 279 seconds; p = 0.008) and asystole (369 seconds vs. 607 seconds; p = 0.02) in whole animals. Cocaine and cocaethylene induced dose-dependent decreases in RPP, +dP/dtmax, and –dP/dtmaxabs (p

This case discusses the pharmacodynamic interaction between cocaine and propranolol, resulting in hypertension and exaggerated cocaine toxicity.

Gas chromatography-mass spectrometry (GCMS) is the preferred method for the analysis of drugs/metabolites in biological specimens with use of isotopically labeled analogs of the analytes as internal standards (ISs). An important aspect of the chemical derivatization (CD) for GC-MS analysis is that the CD products derived from the analyte and the selected IS must generate ions suitable for designating the analyte and the IS. These ions should not have significant cross contribution (CC), i.e., IS contribution to the intensities of the ions designated for the analyte, and vice versa. With this in mind, the authors have conducted a search of isotopically labeled analogs of commonly abused cocaine and related compounds (cocaine, norcocaine, benzoylecgonine, cocaethylene, ecgonine, ecgonine methyl ester, anhydroecgonine methyl ester) that are commercially available. These ISs and analytes were derivatized with various groups of reagents, and the CD products were analyzed by GC-MS. MS data are presented in two forms: (1) systematic presentation of fullscan spectra; and (2) tabulation of CC data for ions with potential for designating the ISs and analytes. Many (if not most) of these full-scan spectra are not yet available in the open literature and should be of routine reference value to forensic and clinical laboratories that are engaged in the analysis of these drugs/metabolites. Fullscan MS data were further used to select ion pairs with potential for designating the analytes and ISs in quantitative analysis protocols. The CC data of these ion pairs were evaluated using data collected under the selected ion-monitoring mode and summarized in table format. The data exhibited similar CC characteristics in each alkyl, acetyl, or TMS series. Among the potentially usable ion pairs derived from a specific CD group, there was a trend that the ion pairs with higher mass showed better CC data. The CC data derived from the use of ISs labeled with more deuterium atoms were generally more favorable. These data should save enormous amounts of time and effort for practicing laboratories in their search for optimal analytical parameters.

Erythroxylon coca, the shrub from which co-caine is naturally derived, grows indigenously inSouth America. Cocaine was first identified asthe active alkaloid in the coca leaf in 1857. Asfar back as the twelfth century, Incas used co-caine-filled saliva as local anesthesia for ritualtrephinations [1]. In 1884, it was recognized med-ically as a local anesthetic [2]. In the early twenti-eth century, cocaine was used briefly as aningredient in Coca-Cola. In 1906, the UnitedStates began to control cocaine use, and in 1914the Harrison Narcotic Act labeled cocaine asa narcotic. It became a schedule II drug in the1970s. During the last several decades, recreation-al cocaine use has increased, and reports of sideeffects have grown exponentially. As of 2003,34.9 million citizens of the United States(14.7%) have used cocaine at least once, with2.3 million citizens using cocaine within the pastmonth [3].PharmacologyCocaine is absorbed through application to themucosa, ingestion, inhalation, and direct intrave-nous injection. Effects from nasal insufflationbegin rapidly with peak concentrations typicallyreached within 30 to 60 minutes. Intravenousand inhalational routes of cocaine use producenear-immediate distribution throughout the cir-culation. ‘‘Crack’’ is the direct precipitate offree-base cocaine that results from alkalinizationof aqueous cocaine hydrochloride.The relative contributions of cocaine and itsmetabolites to the clinical effects remain some-what unclear. Cocaine is hydrolyzed rapidly byliver and plasma esterases to ecgonine methylester(EME), which accounts for 30% to 50% of theparent product. Nonenzymatic hydrolysis resultsin the formation of the other major metabolite,benzoylecgonine (approximately 40% of the par-ent product). The biologic half-life of cocaine is0.5 to 1.5 hours; Benzoylecgonine and EME, themajor metabolites of cocaine, have half-lives of 5to 8 hours and 3.5 to 6 hours, respectively [4].Minor metabolites, norcocaine and ecgonine,account for the majority of the other degradationproducts. Early studies suggested that cocaine andnorcocaine accounted for majority of the vasculareffects of cocaine [5]. Recent studies, however,demonstrate an active role for many of the metab-olites. Most studies suggest that cocaine and nor-cocaine are the most potent vasoconstrictors;benzoylecgonine and ecgonine have less of an ef-fect. Some studies suggest that EME may resultin mild cerebral vasodilation [6,7]. Sodium-chan-nel antagonist effects occur with cocaine and nor-cocaine [8]. Sodium-channel antagonist effects donot occur with benzoylecgonine or EME [8].Cocaethyleneisauniquemetabolitethatresultsfrom the combined use of alcohol and cocaine [9].In clinical studies, cocaethylene produces hemody-namic effects comparable to those of cocaine. Co-caethylene has a direct myocardial depressanteffect [10] that is independent of any coronary

International audience; A sensitive GC/CI/MS/MS method was developed for the simultaneous determination of cocaine (COC), anhydroecgonine methylester (cocaine pyrolysis product, AEME), ecgonine methylester (cocaine enzymatic hydrolysis product, EME) and cocaethylene (cocaine with ethanol trans-esterification product, COET) in human hair samples. After acid hydrolysis, hair samples were extracted with an automated solid phase extraction (SPE). The analysis of cocaine and its three metabolites was performed using an ion-trap spectrometer in positive chemical ionization with isobutane as gas reagent. The procedure was validated. Weighted linear regression was found appropriate in a concentration range of 0.10-5.00 ng/mg for AEME, 0.05-5.00 ng/mg for COC, EME and COET. The limit of detection was estimated at 0.005 ng/mg for COC and COET, at 0.025 ng/mg for EME, and at 0.050 ng/mg for AEME. Method performance was evaluated in terms of trueness and precision using quality control (QC) samples over the investigated ranges. Method selectivity and robustness were also demonstrated.

Hepatocellular damage is thought to occur as a result of cytochrome P450-mediated oxidation of cocaine to norcocaine (NC), a precursor of the hepatotoxic nitrosonium ion. However, this damage occurs only in male mice, with females exhibiting minimal biochemical and histological signs of hepatocellular stress. The objective of this study was to determine the plasma time course and tissue disposition of cocaine and its metabolites to further investigate the role that metabolism may play in the gender difference observed. Male and female CF-1 mice were orally administered 20 mg/kg cocaine hydrochloride once daily for 7 days. Blood samples were withdrawn at various time points post-injection and analyzed for cocaine and its metabolites benzoylecgonine (BE), norcocaine, ecgonine methyl ester (EME), and ecgonine (E). In addition, tissue concentrations of cocaine and its metabolites were determined in liver, heart, brain, and kidney tissue. The results demonstrated that the plasma elimination half-life of cocaine is nearly three times longer in males versus females. Non-hepatotoxic hydrolysis metabolites BE, EME, and E were higher in female tissues while norcocaine was detected in tissues of male animals only. This study revealed that differences in cocaine pharmacokinetics and the resultant differences in the biodisposition of cocaine and its metabolites in tissues contribute to the mechanism of gender difference seen in cocaine hepatotoxicity.

Although urine analysis remains the standard for detection of drugs of abuse, sweat patches provide a convenient alternative that avoids some of the problems with drug testing such as violations of privacy in observed urination, possibility of disease transmission, and transport of noxious fluids. This study examined minimum length of wear necessary to detect recent or concurrent cocaine use in a convenience sample of active cocaine users and also differences in analyte concentrations with increasing longer-term wear. Twenty-seven subjects (22 active drug users and 5 comparison subjects who did not use drugs) wore short-term ((1/2)h, 1 h, 1(1/2) h, and 2 h), then long-term patches (1, 3, 7, and 14 day). Short- and long-term patches were identical except for duration of wear. The predominant analyte found was cocaine, followed by benzoylecgonine, then ecgonine methylester. The minimum duration that patches must be worn to detect recent or concurrent cocaine use in this sample is more than 2 h and less than or equal to 1 day. Analyte concentrations increase significantly with increasing lengths of wear. However, increases between the one-week and two-week patches were significant for benzoylecgonine only.

Cocaine (COC) is one of the most widely abused illicit drugs in America. COC abuse transcends all social, racial, and economic boundaries. Following the introduction in the mid-1980s of a new form of cocaine called crack, cocaine use has been on the rise. Because of its intense high, crack smoking has become very popular. Despite its popularity, crack smoking is a particularly dangerous form of COC use. Additionally, COC and ethanol are frequently used together, resulting in the formation of a biologically active molecule that is nearly as psycoactive as COC but produces a longer lasting and toxic effect. Demonstrating the presence or absence of COC and COC-related molecules in postmortem fluids and/or tissues can have serious legal consequences and may help determine the cause of impairment and/or death. We have developed a simple method for the simultaneous determination of COC and the COC metabolites benzoylecgonine, norbenzoylecgonine, ecgonine methyl ester, ecgonine, and norcocaine, as well as anhydroecgonine methyl ester (a unique byproduct of COC smoking), cocaethylene (a molecule formed by the concurrent use of COC and ethanol) and their related metabolites, anhydroecgonine, norcocaethylene, and ecgonine ethyl ester. This method incorporates a Zymark RapidTrace automated solid-phase extraction system, gas chromatography/mass spectrometry, and PFP/PFPA derivatives. The lower limits of detection ranged from 0.78 - 12.5 ng/mL, and the linear dynamic range for most analytes was 0.78 3200 ng/mL. The extraction efficiencies were from 26 - 84%, with the exception of anhydroecgonine and ecgonine, which were from 1 - 4%. We applied this method to 5 aviation fatalities. This method has proven to be simple, robust, and accurate for the simultaneous determination of COC and 11 COC metabolites in postmortem fluids and tissues.

Electrochemical properties of cocaine and its metabolites such as benzoylecgonine (BE), ecgonine (ECG), and ecgonine methyl ester (EME) have been studied by means of square-wave voltammetry (SWV) at a hanging mercury drop electrode. In a phosphate buffer at pH 7 cocaine gives rise to a single voltammetric peak at about −1.490 V versus AgCl (3 mol/l KCl). At higher pH, BE is spontaneously formed, as a first hydrolysis product of cocaine. BE can further hydrolyse to ecgonine that is electrochemically inactive at the mercury electrode. Ecgonine methyl ester, an another hydrolysis product of cocaine, is electrochemically inactive at the mercury electrode, too. BE undergoes reduction owing to its ester group, giving rise to a voltammetric peak within the potential interval from −1.010 to −1.200 V, depending on the concentration of BE. Thus, at pH 8.5, cocaine solution gives rise to two SW peaks due to the reduction of unhydrolysed cocaine and BE. The reduction of BE undergoes through a complex electrode mechanism coupled by adsorption and two types of following chemical reactions. The reduction electrode product of BE undergoes (i) dimerisation and then (ii) protonation leading to final products, most probably ecgonine and benzaldehyde. The rate of the later chemical reaction is controlled by the proton concentration in the electrolyte solution. Comparing the voltammetric properties of cocaine to that of BE and methyl ester ecgonine, it has been concluded that the electrochemical activity of cocaine originates from the reduction of the same ester group as in the case of BE. The main difference between cocaine and BE is manifested in their tendency for adsorption on the mercury electrode surface. BE adsorbs stronger than cocaine, and hence an adsorptive stripping voltammetric method for determination of cocaine was developed on the basis of BE reduction.

Erythroxylum coca var. coca Lam. And Erythroxylum novogranatense var. novogranatense (Morris) Hieron (E.n. var. novogranatense) were grown in a controlled environment for 52 weeks to monitor the content of hygrine, tropinone, tropacocaine, methyl ecgonine, cocaine and cis- and trans-cinnamoylcocaine in seeds, plant parts, and organs during juvenile growth. Embryos and endosperms of var. coca contained cocaine while only embryos of var. novogranatense contained cocaine. Tropacocaine was present in hypocotyls, stems and roots of var. novogranatense, but not in var. coca. Trans-cinnamoylcocaine was the most abundant alkaloid in cotyledons (12 weeks after seeding) and leaves (24 weeks after seeding) in both var. coca and var. novogranatense. After 52 weeks of seeding growth, cocaine was the preeminent alkaloid in leaves of both taxons with a cocaine content of 0.66 percent in var. coca and 1.04 percent in var. novogranatense.

A series of 2beta-isopropyl ester analogs of cocaine (7-11) was synthesised and evaluated in an in vitro dopamine transporter (DAT) binding assays. Ecgonine HCl (5) was obtained from (-)-cocaine (1) by hydrolysis using 1 N HCl. Acid catalysed esterification of 5 using 2-propanol and HCl gas afforded 2beta-isopropyl ecgonine (6). Compounds 7-9 were obtained via esterification of the 3beta-hydroxyl group of 6 using the appropriate acid chloride. Compound 10 was obtained via selective hydrolysis and re-esterification of 7 using 2-propanol and HCl gas. Compound 11 was obtained by reduction of 9 using H(2)/Pd-C. Compounds 7, 10 and 11 showed high binding affinity to the DAT (as indicated from the inhibition of the binding of [(3)H]WIN 35,428 (3)) with IC(50) values (mean +/- S.E.M.) 208.5 +/- 9.5, 47.43 +/- 1.79 and 11.25 +/- 3.37 nM, respectively). Compound 7 is comparatively as active as cocaine, 10 is ca. fivefold more active than cocaine and 11 is ca. 20-fold more active than cocaine and even twice more active than the radioligand 3. Compound 11, like its methyl ester analog (2' aminococaine), exhibited the highest affinity to the DAT. These results, along with previous results, emphasise the importance of a hydrogen-bond donor group at the 2'-position of cocaine and its isopropyl ester analogs to enhance binding affinity to the DAT.

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.

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.

The calcium salts contain besides the acid esters of l-nor-ecgonine, calcium salts of ordinary acids, one or more esters of ordinary acids and an amide. The quantity of the acid ester of l-nor-ecgonine is determined in the following way. The calcium salts are acidified with a few drops of strong HCl solution and the acids and the esters of ordinary acids extracted by ether. The neutralized HCl solution basified with a known quantity of n/10 NaOH solution and boiled in a Kjehldal apparatus as used for ammonia determinations. The quantity of acid esters is equivalent to the loss of NaOH diminished by the quantity of ammonia obtained.

Our current experiments assess the applicability of on-line sample extraction with coupled rapid chromatography systems to quadrupole orthogonal acceleration time-of-flight (Q-TOF) detection for the quantitative analysis of cocaine (COC), and ecgonine methyl ester (EME) in rat plasma. Experiments were performed on a Q-TOF instrument, operated in the MS/MS mode. Quantitation was achieved utilizing the most prominent parent-daughter transition and internal standard calibration techniques (COC-d3: IS). The calibration curves produced for EME and COC ranged from 5.0 to 10,000 and 0.5 to 10,000 ng/ml, respectively. Equations of regression line and correlation coefficients for the pseudo-multiple reaction monitoring (MRM) ion abundance ratio and the corresponding calibration concentrations (r2) were as follows: y = 0.0003 + 0.0703x (r2 = 0.9921) for EME and y = 0.0032 + 0.0035x (r2 = 0.9997) for COC. The system repeatability, given as percent coefficient of variation (% CV) of mean peak-area ratios, was assessed using 50 injections of a rat plasma sample from the pharmacokinetic study. The analyses were performed over the course of 5 days, rendering % CVs for EME and COC of 0.73 and 0.58, respectively. This method suggests that on-line sample extraction coupled with fast liquid chromatography/quadrupole orthogonal time-of-flight mass spectrometry may be a viable alternative for quantitative analysis of EME and COC in rat plasma.

A pentafluorophenylpropyl (PFPP) bonded silica column has been used for the high-performance liquid chromatography-electrospray ionization-mass spectrometry-mass spectrometry assay (HPLC-ESI-MS-MS) of cocaine (COC) and its metabolite, ecgonine methyl ester (EME) in human urine. COC and EME were used as model basic solutes to demonstrate that a PFPP phase yields excellent results for the assay and validation of drugs in biological fluids. The assay was linear over three orders of magnitude (1.0-1000 ng/ml) and precision and accuracy of the assay was 4 and 15%, respectively. The limit of detection (LOD) for COC and EME was 1.6 and 2.8 pg on column, respectively. In addition, only a simple 1:10 dilution of the urine was necessary for the sample preparation procedure thus saving time on a laborious extraction step. The major advantage of the PFPP phase was the enhancement of the ESI-MS signal by providing good retention and good peak shape of COC and EME with a mobile phase of 90% acetonitrile. The MS signal for COC was a factor of 12 times greater on the PFPP phase than on the C18 phase.