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Butyrylcholinesterase is a major cocaine-metabolizing enzyme in humans and other primates, catalyzing hydrolysis to ecgonine methylester. Increasing butyrylcholinesterase activity may be a treatment for cocaine addiction. We evaluated the effect of 30-min pretreatment with horse-derived butyrylcholinesterase (5-15,000 U i.v.) or with the selective butyrylcholinesterase inhibitor cymserine (10 mg/kg i.v.) on the metabolism of cocaine (17 mg/kg i.p.) in anesthetized rats. Venous blood samples were collected for two hours after cocaine administration and later assayed for cocaine and metabolites by gas chromatography/mass spectroscopy. Whole brains were collected after the last blood sample and similarly assayed. Butyrylcholinesterase significantly increased plasma and brain ecgonine methylester levels and decreased cocaine plasma half-life from 26.2 min (saline) to 16.4 min (15,000 U). Butyrylcholinesterase had no significant effect on plasma or brain cocaine or benzoylecgonine levels. Cymserine had no effect on any variable. These findings suggest that butyrylcholinesterase treatment may have benefits in enhancing cocaine metabolism and in increasing levels of ecgonine methylester, which may have a protective action against cocaine.

A method was developed for simultaneously analyzing cocaine (COC), benzoylecgonine (BZE), norbenzoylecgonine (BNE), norcocaine (NCOC), ecgonine (ECG), ecgonine methyl ester (EME), m-hydroxybenzoylecgonine (HBZE), anhydroecgonine methyl ester (AEME), cocaethylene (CE), norcocaethylene (NCE), and ecgonine ethyl ester (EEE) in blood, urine, and muscle. Available deuterated analogs of these analytes were used as internal standards. Proteins from blood and muscle homogenate were precipitated with cold acetonitrile. After the removal of acetonitrile by evaporation, the supernatants and urine were subjected to solid-phase extraction. The eluted analytes were converted to their hydrochloride salts and derivatized with pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. The derivatized products were analyzed by a gas chromatograph (GC)/mass spectrometer by selected ion monitoring. The limit of detection (LOD) for COC, BZE, NCOC, EME, CE, NCE, and EEE was 2ng/ml, while the LODs for BNE, ECG, HBZE, and AEME were 25, 640, 50, and 13 ng/ml, respectively. This method was successfully applied in analyzing 13 case samples from aviation accident pilot fatalities and motor vehicle operators. AEME concentrations found in the 13 samples were consistent with those produced solely by the GC inlet pyrolysis of COC controls in blood. Anhydroecgonine cannot be used as a marker for the abuse of COC by smoking because it is also pyrolytically produced from COC metabolites on the GC inlet. The developed method can be effectively adopted for analyzing COC and related compounds in urine, blood, and muscle by a single extraction with increased sensitivity through formation of hydrochloride salts and using a one-step derivatization.

Electrochemical properties of cocaine and its metabolites such as benzoyl ecgonine (BE), ecgonine, and methyl ester ecgonine have been studied by means of square-wave voltammetry at a hanging mercury drop electrode. In a phosphate buffer at pH = 7 cocaine give rise to a single voltammetric peak at about - 1.490 V vs Ag/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. Methyl ester ecgonine, an another hydrolysis product of cocaine, is electrochemically inactive at the mercury electrode, too. BE undergoes reduction owing to its ester group, giving raise to a voltammetric peak within the potential interval from - 1.010 V 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) dimerization and than (ii) protonation leading to final chemical electrochemically inactive products, most probably ecgonine and benzaldehyde. The rate of the latter 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 electrochemicall 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.

Human monoclonal antibodies (mAbs) designed for immunotherapy have a high potential for avoiding the complications that may result from human immune system responses to the introduction of nonhuman mAbs into patients. This study presents a characterization of cocaine/antibody interactions that determine the binding properties of the novel human sequence mAb 2E2 using three-dimensional quantitative structure-activity relationship (3D-QSAR) methodology. We have experimentally determined the binding affinities of mAb 2E2 for cocaine and 38 cocaine analogues. The K(d) of mAb 2E2 for cocaine was 4 nM, indicating a high affinity. Also, mAb 2E2 displayed good cocaine specificity, as reflected in its 10-, 1500-, and 25000-fold lower binding affinities for the three physiologically relevant cocaine metabolites benzoylecgonine, ecgonine methyl ester, and ecgonine, respectively. 3D-QSAR models of cocaine binding were developed by comparative molecular similarity index analysis (CoMSIA). A model of high statistical quality was generated showing that cocaine binds to mAb 2E2 in a sterically restricted binding site that leaves the methyl group attached to the ring nitrogen of cocaine solvent-exposed. The methyl ester group of cocaine appears to engage in attractive van der Waals interactions with mAb 2E2, whereas the phenyl group contributes to the binding primarily via hydrophobic interactions. The model further indicated that an increase in partial positive charge near the nitrogen proton and methyl ester carbonyl group enhances binding affinity and that the ester oxygen likely forms an intermolecular hydrogen bond with mAb 2E2. Overall, the cocaine binding properties of mAb 2E2 support its clinical potential for development as a treatment of cocaine overdose and addiction.

Background: Concurrent use of cocaine and alcohol results in formation of a cocaine homolog and metabolite—cocaethylene. Methods: To characterize cocaethylene pharmacology, ten paid volunteer subjects were given deuterium-labeled (d 5 ) cocaine (0.3, 0.6, and 1.2 mg/kg and cocaine placebo) by a 15-min constant rate intravenous injection 1 h after a single oral dose of ethanol (1 g/kg) or ethanol and cocaine placebo using a double-blind, crossover design. Six of the same volunteers subsequently received a 1.2 mg/kg dose of cocaine alone. A small (7.5 mg) nonpharmacologically active dose of deuterium-labeled cocaethylene-d 3 was concurrently administered with the cocaine to enable calculation of absolute cocaethylene formation and clearance. Plasma and urine cocaine, cocaethylene, and benzoylecgonine concentrations, physiologic and subjective effects were measured. Results: When co-administered with ethanol, 17±6% (mean±S.D.) of the cocaine was converted to cocaethylene. Cocaethylene peak plasma concentrations and AUC increased proportionally to the cocaine dose. Ethanol ingestion prior to cocaine administration decreased urine benzoylecgonine levels by 48% and increased urinary cocaethylene and ecgonine ethyl ester levels. Subjects liked and experienced more total intoxication after the combination of cocaine and ethanol than after either drug alone. Conclusions: In the presence of ethanol, the altered biotransformation of cocaine resulted in 17% of an intravenous cocaine dose being converted to cocaethylene and relatively lower urinary concentrations of benzoylecgonine.

Although cocaine is typically the second-most identified drug of abuse in drug-testing programs, there is surprisingly little quantitative information on excretion patterns following different routes of administration. This report details the urinary excretion and terminal elimination kinetics for cocaine and eight metabolites [benzoylecgonine (BZE), ecgonine methylester (EME), norcocaine (NCOC), benzoylnorecgonine (BNE), m-hydroxy-BZE (m-HO-BZE), p-hydroxy-BZE (p-HO-BZE), m-hydroxy-COC (m-HO-COC), and p-hydroxy-COC (p-HO-COC)]. Six healthy males were administered approximately equipotent doses of cocaine by the intravenous (IV), smoking (SM), and inhalation (IN) routes of administration. Urine specimens were collected for a minimum of three days after drug administration, screened by immunoassay (EMIT and TDX, 300 ng/mL), and analyzed by GC-MS. Mean Cmax values were generally as follows: BZE > EME > COC > BNE approximately p-HO-BZE > m-HO-BZE > m-HO-COC > NCOC > p-HO-COC. Elimination half-lives for cocaine and metabolites were generally shorter following s.m., intermediate after i.v., and longest following i.n. administration. m-HO-BZE demonstrated the longest half-life (mean range 7.0-8.9 h), and cocaine displayed the shortest (2.4-4.0 h). Mean detection times were extended progressively by lowering cutoff concentrations. The maximum increases were approximately 55% at 50 ng/mL for the TDx assay (e.g., the detection time for the last consecutive positive changed from 32.8 h to 50.6 h for i.v. cocaine) and up to 39% for GC-MS at a cutoff concentration of 40 ng/mL (e.g., the detection time for the last consecutive positive changed from 34.8 h to 48.1 h for i.v. cocaine). Sensitivity, specificity, and predictive values for EMIT and TDx were comparable at the 300-ng/mL cutoff concentration; but at lower cutoff concentrations, predictive values of positive results for TDx were diminished indicating a higher risk of false-positive results, that is, positive results that failed to meet administrative cutoff criteria. Detection of positive results was significantly enhanced through the use of the "Zero Threshold Criteria Method", a method developed by the authors to differentiate false-negatives from true-negatives. The method was based on establishing mean immunoassay response (MIR) baselines and variance (SD) in assays of drug-free specimens. Arbitrary thresholds (MIR + 0.5 SD, MIR + 1 SD, MIR + 2 SD) were utilized to evaluate all negative specimens. Apparent true positives were identified by the presence of BZE at or above 40% GC-MS cutoff concentrations. With these criteria, up to 111 false-negative specimens were confirmed as true-positive specimens; this was in addition to the 208 true positives detected at recommended cutoff concentrations. This represents a 50% increase in positive detection rates through the use of this methodology. Such methodology is recommended for further evaluation by drug-testing programs for enhancement of positive detection rates and as an alternative to creatinine testing for dealing with dilute specimens that test negative by initial tests, but contain quantifiable concentrations of drugs of abuse.

Recent reports have shown that anti-cocaine catalytic monoclonal antibody 15A10 reduces the toxic effect of cocaine by increasing its breakdown to systemically inert products ecgonine methylester and benzoic acid. This study reports the microencapsulation of antibody 15A10 using biodegradable poly (lactic-glycolic) acid (PLGA) by double emulsion technique. Formulation parameters such as protein loading, polymer molecular weight and the presence of zinc carbonate were studied for their effects on in-vitro release of antibody from microspheres. The initial burst release was decreased by the reduction of the protein (as % of total ingredients) in the formulation. Although changing the polymer molecular weight did not cause a reduction in initial burst release, it was effective in improving the release rate. The inclusion of zinc carbonate in microsphere preparation resulted in increase in initial burst release. An in-vivo study in mice revealed the presence of antibody in blood up to ten days following subcutaneous injections. These data demonstrate a potential for a sustained-release formulation of monoclonal antibody 15A10 for treatment of cocaine addiction.

Cocaine metabolism has been studied previously with respect to the formation of predominant hydrolytic or hepatotoxic metabolites via oxidative pathways. In the present study, cocaine and eight of its metabolites (norcocaine, ecgonine methyl ester, benzoylecgonine, benzoylnorecgonine, 3-hydroxy-benzoylecgonine, cocaethylene, norcocaethylene, and ecgonine ethyl ester) were incubated with microsomes from rat liver, kidney, lung, and brain. Qualitative analysis of the metabolites produced was performed using solid phase extraction (SPE), trimethylsilylation, and GC/MS. It was found that the metabolites with a free carboxylic group (e.g., benzoylecgonine) were not further oxidized by microsomal enzymes and their presence in urine or blood may therefore be due to hydrolysis of the respective alkylated entities. Although microsomes from all organs exhibited oxidative metabolism, significant differences were noted. Kidney microsomes produced essentially the same results as liver, but aryl hydroxylated metabolites were not found in incubations with lung and brain microsomes. N-Hydroxy-norcocaine was found only in traces with brain microsomes. It appears that cocaine is converted to N-hydroxy-norcocaine (which is the precursor of toxic metabolites) not only in the liver but also in other organs of rat. This might be relevant in the development of lung toxicity observed in smokers of cocaine ("crack").

Tobacco smoking and cocaine use often co-occurs and the frequency of smoking has been positively correlated with the likelihood of cocaine use. In addition, nicotine pretreatment has been shown to increase the rate of cocaine self-administration in rats and to enhance cue-induced cocaine craving in humans. The present study was conducted to investigate whether nicotine pretreatment via a transdermal patch alters the behavioral, physiological, and pharmacokinetic effects of an acute dose of cocaine in nondependent human volunteers. Seven male tobacco smokers who used cocaine occasionally provided informed consent and participated in this placebo-controlled, four-visit study. Following pretreatment with a transdermal nicotine patch (placebo, 14 mg), subjects were challenged with an acute dose of intranasal cocaine (placebo, 0.9 mg/kg). Nicotine pretreatment attenuated cocaine-induced increases in reports of "high" and "stimulated" and increased the latency to detect cocaine effects and cocaine-induced euphoria. Nicotine did not alter cocaine's effects on heart rate, skin temperature, and blood pressure or plasma cocaine, benzoylecgonine (BE), or ecgonine methylester (EME) concentrations. Our findings indicate that nicotine pretreatment alters some of the positive subjective effects of cocaine in humans without affecting cocaine's effects on physiologic responses or pharmacokinetic profiles.

The in vitro stability of cocaine (COC) was monitored in fresh whole blood and plasma stabilized with potassium fluoride (0.25%) for as long as 15 days. The samples were stored at 4 degreesC, 20 degreesC and 40 degreesC. Additionally, fresh plasma samples containing either benzoylecgonine (BZE), ecgonine methyl ester (EME) or ecgonine (ECG) were stored at 4 degreesC and 20 degreesC. Data were established using subsequent solid-phase extraction procedures and high-performance liquid chromatography coupled to atmospheric pressure ionization mass spectrometry for isolation and quantitation of COC, BZE, EME, and ECG. COC, BZE, and EME concentrations decreased with increasing storage temperature and time after an apparent first-order reaction kinetic. Only ECG appeared to be stable at storage temperatures as high as 20 degreesC for the entire observation period. At 40 degreesC, the amount of ECG produced from hydrolysis of COC still totalled 80% of the initial COC concentration. Hydrolysis of COC to EME occurred more rapidly in plasma than in blood. The dynamic degradation profiles obtained were dependent on the storage temperature. The conversion of COC to BZE, EME, and ECG appeared to be stoichiometric at all time intervals at storage temperatures of 4 degreesC and 20 degreesC. The presence of any hydrolysis product of COC in blood or plasma constitutes confirmatory evidence of COC incorporation, and determination of ECG seems most promising even in samples stored under unfavorable conditions.

Rationale: The importance of genetic factors in the development of alcoholism has been demonstrated repeatedly. However, the impact of a family history of alcoholism on the development of other drug use has been less thoroughly studied. Objective: The present study was conducted to investigate whether individuals with a positive family history of alcoholism (FHP) differ from individuals without a history (FHN) in their pharmacokinetic profile, subjective and physiological response to an acute intranasal dose of cocaine (0.9 mg/kg). Methods: Nine FHP and nine FHN male occasional cocaine users provided informed consent and participated in this double-blind, placebo-controlled, two-visit study. Responses to cocaine were assessed via a joystick device, the Addiction Research Center Inventory, visual analog scales and heart rate. Plasma concentrations of cocaine and its metabolites, benzoylecgonine and ecgonine methylester also were measured. Results: There were no significant differences between FHP and FHN subjects in subjective reports of intoxication, physiologic responses or plasma cocaine and benzoylecgonine concentrations following cocaine administration. Plasma levels of the cocaine metabolite ecgonine methylester were significantly higher in FHP subjects from 50 to 120 min post-cocaine administration compared to FHN subjects. Conclusions: Our findings indicate that family history of alcoholism does not appear to influence the behavioral and physiological responses to acute cocaine administration, but that some aspects of cocaine metabolism may be different between the two groups.

The aim of this study was to determine the effect of chronic cocaine infusion on urine cocaine, ecgonine methylester and benzoylecgonine concentrations to establish if they varied with dose and duration of cocaine administration. Male rats were continuously infused with cocaine at either 6 or 18 mg kg−1 daily for 13 days. Three urine samples taken over the course of the infusion period showed that cocaine, ecgonine methylester and benzoylecgonine concentrations varied with the dose administered and the duration of administration. Cocaine, ecgonine methylester and benzoylecgonine concentrations were 2–3 times greater in the high-dose group than the low-dose group at each sampling time point. These decreased, respectively, from 7.0 ± 1.1, 26.7 ± 4.5 and 29.5 ± 5.4 μg mL−1 to 2.5 ± 0.5, 10.5 ± 1.8 and 11.8 ± 1.5 μg mL−1 in the high-dose group and from 1.0 ± 0.2, 7.8 ± 1.5 and 6.3 ± 0.1 μg mL−1 to 0.5 ± 0.1, 4.0 ± 0.6 and 3.1 ± 0.4 μg mL−1 in the low-dose group (P < 0.05) over the infusion period. We also studied the pharmacokinetic and metabolic profile of an intravenous bolus dose of 2.5 mg kg−1 cocaine hydrochloride after a similar cocaine infusion in rats. Cocaine pharmacokinetics and the profile of ecgonine methylester, benzoylecgonine and norcocaine were no different from rats chronically infused with saline for the same period. Altered cocaine metabolism could not explain the effect of the duration of cocaine infusion on altered metabolite concentrations in urine. Ecgonine methylester/benzoylecgonine urine concentration ratios did not alter with duration of infusion (1.2 ± 0.2 and 1.1 ± 0.2 in the high-dose group at the first and last time point) and were not affected by the dose of cocaine (1.3 ± 0.6 and 1.2 ± 0.1 at corresponding times in the low-dose group (P > 0.05)). We conclude that chronic cocaine infusion does not alter cocaine metabolism. This was not reflected by absolute cocaine metabolite urine concentrations, which varied with time, but was represented by urine ecgonine methyl ester/benzoylecgonine concentration ratios.

Analyte 2000, a four-channel fiber optic biosensor (FOB), was used for analysis of cocaine and its metabolites (COC) in human urine using a competitive fluorescence immunoassay. Binding of antibenzoylecgonine monoclonal antibody (mAb) to the casein-benzoylecgonine Ag-coated, tapered optical fibers was inhibited by COC. Bound mAb, which inversely correlated with COC concentration, was quantitated by fluorescence produced by evanescent excitation of bound cyanine dye-tagged antimouse antibody (CY5-Ab). The effective concentration range of benzoylecgonine (BE) for inhibiting the fluorescent signals was 0.75-50 ng/mL, with IC50 of 9.0 ng/mL. This FOB had similar affinities for BE, cocaine, and cocaethylene, but very low affinities for ecgonine and ecgonine methyl ester. A sensitivity of 100% and a specificity of 96% were achieved when 54 human urine specimens were analyzed by FOB (cutoff, 300 ng/mL COC) and GC-MS (cutoff, 150 ng/mL BE). All results were in agreement except for one positive FOB result with a GC-MS BE concentration of 148 ng/mL. In addition, regeneration and reuse of the fiber for multiple analyses were performed successfully with no carryover from specimens containing high COC concentrations to specimens containing low COC concentrations.

A gas chromatographic-mass spectrometric (GC-MS) method was used for the simultaneous analysis of cocaine and its metabolites (norcocaine, benzoylecgonine, norbenzoylecgonine, ecgonine methylester, cocaethylene, and the o-, m, and p-hydroxy derivatives of cocaine and benzoylecgonine) in meconium specimens collected from newborns prenatally exposed to the drug in an effort to determine which of these metabolites are more relevant in the confirmation of immunoassay-positive specimens. Significant metabolites included benzoylecgonine and its m- and p-hydroxy derivatives. Reanalysis of immunoassay-positive meconium specimens that previously failed to confirm by GC-MS for benzoylecgonine revealed that the GC-MS confirmation rate could be substantially enhanced by inclusion of m- and p-hydroxybenzoylecgonine in the analysis along with benzoylecgonine. Several specimens were found to be positive for hydroxylated derivative(s) in the absence of benzoylecgonine itself.

The etiology of seizures associated with cocaine use is unclear. Because cocaine seizures are relatively uncommon, they should be diagnosed by exclusion and a neurological workup to rule out central nervous system (CNS) catastrophe should be made. This report describes the clinical findings, treatment, and blood cocaine and metabolite concentrations in a patient who, on two separate occasions, had seizures associated with crack cocaine ingestion. Approximately 1 hour after the ingestion incidents, the patient had multiple, generalized seizures that abated spontaneously. His workup for CNS bleeding, infection, and trauma was negative. Cocaine concentrations on the first incident peaked at 2.48 mg/L and on the second incident peaked at 3.9 mg/L. Other clinical findings included tachycardia, hypertension, diaphoresis, and disorientation. Blood cocaine and metabolite analysis revealed extremely high concentrations. Other than the incident of seizures and transient cardiovascular aberrations, these high concentrations were tolerated by the patient without further sequelae. A review of cocaine-induced seizures and treatment is included.

A fast analytical approach for the simultaneous quantitative screening for illicit drugs in serum and urine without tedious chromatographic separation steps was developed by combining solid-phase extraction (SPE) followed by flow-injection analysis (FIA) with ionspray-ionization and tandem mass spectrometry (MS-MS) detection using a PE Sciex API 300 triple-quadrupole MS. MS-MS analysis was performed by sequentially isolating the precursor ions of the analytes and their deuterated standards with subsequent fragmentation and monitoring of one fragment ion for each substance. A multiple-reaction monitoring experiment was set up for morphine (MO), codeine (COD), amphetamine (AMP), benzoylecgonine (BZE), and their deuterated analogues. For method evaluation, serum samples spiked with 2-1000 ng of each drug and deuterated standards were extracted by mixed-mode SPE, redissolved in CH3CN-NH4OAc-buffer, and directly injected by flow injection into the ionspray source. The specificity of this new method was demonstrated by testing compounds with similar chemical structure for interferences from the analytes of interest (e.g., dihydromorphone, morphine glucuronide, and 6-monoacetylmorphine with MO; dihydrocodeine and hydrocodone with COD; cocaine [COC] and ecgonine methylester with BZE; methamphetamine with AMP). The possibility of interferences of such compounds with the FIA-ionspray-MS-MS screening method is discussed. Spiked serum samples and serum and urine samples from drug addicts and victims of drug abuse were analyzed with FIA-MS-MS and, after derivatization, with gas chromatography-mass spectrometry (GC-MS). Comparable quantitative results were obtained with both methods; no interferences with metabolites or other compounds were found. The FIA-ionspray-MS-MS method is a fast, quantitative, sensitive, and highly specific alternative method to drug-screening by immunoassays, high-performance liquid chromatography, and GC-MS. It can be used for the simultaneous detection of different drugs and metabolites such as opiates, COC, AMP derivatives, and many other drugs.

Ten 3 beta-ecgonine analogues were synthesized and characterized by 1H and 13C NMR, MS, and elemental analysis. The compounds were synthesized as (-)-stereoisomers from (-)-cocaine. These compounds were assessed for their ability to inhibit [3H]cocaine binding to rat striatal tissue and to inhibit [3H]DA uptake into rat striatal synaptosomes. In this series of compounds, the length of the spacer between the aryl group and the tropane skeleton ranged from 1 to 4 bond distances, and conformational flexibility of the linkage and orientation of the aryl ring system were controlled by various types of linkages. The most potent of the analogues was methyl-(1R-2-exo-3-exo)-8-methyl-3-(beta-styrenyl)-8-azabicyclo[3. 2.1] octane-2-carboxylate. One of the less potent compounds was found to inhibit [3H]cocaine binding and [3H]DA uptake with significantly different IC50 values, in contrast to 14 other 3 beta-substituted analogues. Molecular modeling and CoMFA analysis were used to obtain a rigorous structure-function relationship for the studied compounds. The results showed that the potencies of these 3 beta-substituted ecgonine methyl esters were dominated by steric effects and were acutely sensitive to the distance between the aryl ring and the tropane skeleton and to the orientation of the aryl ring system relative to the tropane skeleton. The current study provides a clearer picture of the shape and size of the putative hydrophobic binding pocket for the 3 beta substituent at the cocaine receptor as well as emphasizing the importance of a drug's free energy of solvation in obtaining structure-activity relationships.

Ecgonine is formed by the hydrolysis of both ester linkages in cocaine and is of interest as both a potential metabolite and a postmortem and postcollection artifact in biological specimens. Its extreme polarity and water solubility make its isolation very difficult, particularly from complex matrices such as postmortem whole blood. This procedure describes an initial protein precipitation followed by two derivatization steps, the first of which is to propylate organic acids and primary and secondary amines and the second of which is to form the p-nitrobenzoyl ester of organic alcohols. The resultant derivatized products are then subjected to cleanup using a standard extraction with n-butyl chloride as the solvent. Analysis of the extracts is performed by gas chromatography-mass spectrometry using deuterated internal standards, although the adducts would also be suitable for analysis by high-performance liquid chromatography. Significant quantities of ecgonine have been identified in postmortem whole blood samples using this method.

The use of cocaine use has been associated with adverse developmental effects in humans, and cocaine administration produces developmental toxicity in animal models. However, whether the adverse effects produced during organogenesis are due directly to the effects of cocaine or its metabolites remains to be established. This study was therefore undertaken to compare the morphological effects of cocaine and its metabolites, ecgonine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in whole embryo culture (WEC) using early somite stage ICR mice. Cocaine produced a concentration-dependent induction of defects including effects on craniofacial development such as neural tube closure defects (NTDs). Concentrations of cocaine of 51.4 μ m or more produced dysmorphogenesis and 100% of the embryos exhibited NTDs at 441 μ m . EME also induced defects at concentrations of 400 μ m or above. Neither ecgonine nor BE altered embryogenesis at concentrations of 2000 μ m or less. The incidence of cocaine-induced NTDs was dependent on the length of exposure to cocaine. At 294 μ m , exposures of 3 hr or more were required to alter development when evaluated at the end of a 24-hr culture period. Lower cocaine concentrations required longer exposure periods (6 or 12 hr) to produce dysmorphogenesis. The incidence of NTDs appears to follow the area under the concentration time curve and is not solely dependent on the peak cocaine concentration in the medium. Exposure of conceptuses to a combination of cocaine and EME produced a high incidence of NTDs. These results suggest that the concentration of cocaine or EME required to induce NTDs in vitro is higher than the teratogenic concentration in vivo . Additionally, the time required for high concentrations of cocaine to induce NTDs is longer than the serum half-life of cocaine reported in vivo following a single administration. Thus, NTDs produced by cocaine administration appear not to be due solely to the effect of cocaine or its metabolites on the conceptus but may involve effects on extraembryonic and/or maternal tissues as well.

Although gas chromatography/mass spectrometry (GC/MS) is the most powerful technique for analysing cocaine and its hydrolysis products (benzoylecgonine, ecgonine methyl ester and ecgonine) in urine, the sample pretreatment required is rather laborious and involves a large number of steps. This paper reports a continuous method for the separation (based on the retention properties of cocaine and its major metabolites on a reversed phase C18 column), preconcentration (evaporation under nitrogen) and derivatization (collection of the residue in a few microlitres of derivatizing reagent) of cocaine and its metabolites in urine samples for subsequent analysis by GC/MS. Calibration graphs are linear from 5 to 3000 ng/mL; the detection limits are 1 ng/mL (cocaine and ecgonine methyl ester), 20 ng/mL (benzoylecgonine) and 200 ng/mL (ecgonine). The method was applied to illicit cocaine samples and to urine from a drug addict.

Hot plate testing of rats was performed to determine the optimal analgesic doses of intracerebroventricularly (ICV) administered cocaine, significant cocaine metabolites, and selected structurally similar molecules. Optimal, (subseizure) analgesic doses for cocaine and selected cocaine analogues were (in microM): cocaine, 0.37; cocaethylene, 0.09; benzoylecgonine, 0.35; norcocaine, 0.43; and ecgonine, 2.1. Ecgonine methyl ester was not analgesic at < or = 3.7 microM. These results, in conjunction with findings on other structurally similar molecules suggest that, of the molecules tested, (a) a hydrophobic group at the C-3 attached carbon is critical for analgesia; (b) a hydrophobic C-2 ester group can enhance analgesic activity (e.g., cocaethylene); (c) the N-methyl position is minimally important for analgesia; and (d) isomeric configurational changes can influence analgesia.

In humans, the plasma enzyme, butyrylcholinesterase (E.C. 3.1.1.8), metabolizes cocaine to the water-soluble, pharmacologically inactive compounds, ecgonine methylester and benzoic acid. Homogeneous enzyme was purified from human plasma and used to determine the enzyme kinetic parameters of Km and Vmax with cocaine as the substrate. The KM (11.9 microM) indicates that cocaine is tightly bound to the four active sites of the native tetramer. The Vmax (1.17 microM/min) is 50-fold greater than cocaine catalytic antibodies. Administration of purified human butyrylcholinesterase to a cocaine-intoxicated patient would be expected to shift the metabolism to the inactive metabolites and reduce the toxicity.

This paper describes a method for the analysis of thermal degradation compounds generated from cocaine during the smoking process, together with chemical and enzymatic biotransformation products which, by virtue of their polarity, are not recovered by existing analytical procedures. The method employs cation exchange solid phase extraction coupled with gas chromatography/mass spectrometry. Compounds identified in urine from subjects of cocaine-related death included cocaine, benzoylecgonine, ecgonine methyl ester and ecgonine, which were measured quantitatively, and ecgonidine, ecgonidine methyl ester, ecgonine ethyl ester, ethyl benzoylecgonine, norcocaine, benzoylnorecgonine, cinnamoylcocaine, and cinnamoylecgonine. The concentrations of ecgonine (0-104 micrograms/ml) and ecgonine methyl ester (0-177 micrograms/ml) were substantial and averaged about one tenth the concentrations of benzoylecgonine (0-1074 micrograms/ml) and cocaine (0-1221 micrograms/ml). These and several of the other compounds identified will be valuable markers for cocaine use, in degraded samples and for indicating the route of administration.

A procedure is presented for the simultaneous identification and quantification of morphine (MOR), codeine (COD), ethylmorphine (EM), 6-monoacetylmorphine (6-MAM), cocaine (COC), benzoylecgonine (BZE), ecgonine methylester (EME) and cocaethylene (CE), contained in the hair of opiates and cocaine addicts. The method involves decontamination in dichloromethane, pulverization in a ball mill, heat-acid hydrolysis, addition of deuterated internal standards, liquid-liquid extraction and gas chromatography/mass spectrometry (GC/MS) after silylation. The limit of detection (LOD) was approximately 0.1-0.8 ng/mg for each drug, using a 30-mg hair sample. The method is reproductible, with a coefficient of variation (CV) of approximately 8-17%. Cocaine and 6-monoacetylmorphine were the major compounds detected in cases of cocaine (14 cases) and heroin (68 cases) intake. Concentrations were in the range 0.4-78.4 ng/mg (COC), 0.0-36.3 ng/mg (BZE), 0.0-1.6 ng/mg (EME), 0.0-2.1 ng/mg (CE), 0.0-84.3 ng/mg (6-MAM), 0.2-27.1 ng/mg (MOR) and 0.1-19.6 ng/mg (COD). An application in forensic sciences, involving multi-sectional analysis, is given.

The hydrolytic metabolism of cocaine into benzoylecgonine, ecgonine methyl ester, and ecgonine was studied in the human hepatoma cell line Hep-G2 and in the nontumorigenic fetal hepatic cell line WRL-68. Also, the toxicological response of these cells to cocaine was compared to previously published results obtained with perfused liver cells and in vivo systems. Our experiments indicated that Hep-G2 appear to have similar metabolic and toxicological patterns to in vivo and perfused cell systems. The WRL-68 tissue culture system was found to be less similar. These results suggest Hep-G2 cells can be utilized to study cocaine metabolism and toxicology, and possibly in studies involving other xenobiotic compounds.

The method for simultaneous determination of cocaine and its four metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and norcocaine) in urine by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) was studied. The mass spectra showed the quasi-molecular ions, [M+H]+ as the base peak. LC/APCI-MS analysis was performed by focusing the characteristic ions at m/ = 186, 290, 200, 304 and 290 for ecgonine, benzoylecgonine, ecgonine methyl ester, cocaine and norcocaine, respectively. Cocaine and its four metabolites were well separated by high performance liquid chromatography (HPLC). The recoveries of cocaine and its metabolites from the spiked urine were 40.3-94.7% by solid-phase extraction with two type cartridges (Bond Elut Certify and Bond Elut SCX).

Cocaine, a naturally occurring alkaloid, is known to affect the immune system. The present authors have demonstrated that peritoneal macrophages, isolated from mice injected with cocaine, have an increased capacity for the production of reactive oxygen intermediates (ROI) and a decreased capacity for the production of reactive nitrogen intermediates (RNI). The present studies were done to determine the effects of certain cocaine metabolites on the induction of ROI and RNI by peritoneal macrophages. C57BL/6 mice were injected i.p. with either saline or 5 mg/kg of one of the following: cocaine, norcocaine, benzoylecgonine, ecgonine methyl ester HCl or ecgonine HCl. The ROI were measured using a chemiluminescence assay and the RNI were measured as nitrite secretion following exposure of isolated M phi to interferon gamma and LPS. Isolated peritoneal M phi from mice injected i.p. with cocaine, norcocaine and benzoylecgonine exhibited an increase in the production of ROI and a concomitant decrease in the production of RNI. However, injections of either ecgonine methylester HCl or ecgonine HCl had no effect on the induction of either ROI or RNI by murine peritoneal M phi. The cocaine metabolites, norcocaine and benzoylecgonine, have been reported to cause hepatic and/or cerebral toxicity. The present study also demonstrated that injection of these metabolites in vivo, also altered M phi functions which were measured in vitro.

We studied the incorporation rates of cocaine and its major metabolites, benzoylecgonine (BE) and ecgonine methylester (EME), into hair from blood. It was demonstrated in our previous report (Nakahara et al. 1992a) that the incorporation rate of cocaine from blood into hair was much higher than those of BE and EME following administration of cocaine. In this study, the incorporation rates of these drugs into rat hair were investigated following independent administration of BE and EME at 10 mg/kg per day for 5 days, and co-administration of cocaine, BE-d3 and EME-d3 at 3, 0.5 and 0.5 mg/kg per day for 10 days. When BE and EME were administered to rats independently, levels of both these drugs were hardly detectable or quite low in hair, though their AUCs in plasma were very high. On co-administration of cocaine, BE-d3 and EME-d3, the deuterium labeled metabolites in hair were negligibly lower in comparison with the unlabeled ones and in particular the concentration of BE in rat hair was significantly higher than that of BE-d3 although the AUC of BE-d3 was higher than that of BE. It was concluded that the incorporation rates of BE and EME into hair were very low in comparison with that of cocaine and most of the BE detected in hair would be a hydrolytic product derived from cocaine in hair matrix after incorporation.

Cocaine and its metabolites, benzoylecgonine (BZE) and ecgonine methylester (EME), were found in hair samples from ancient Peruvian coca-leaf chewers dating back to ad 1000. Hair was analyzed by gas chromatography/mass spectrometry ( GC MS ) to quantitate the concentrations. The two metabolites were found in higher concentration than the parent drug. The metabolite levels appear to be below that of modern cocaine abusers. Gender does not appear to be a factor in the incorporation of drug into hair.

To the Editor: We have previously reported a method for the analysis of ecgonine together with other polar cocaine metabolites in postmortem blood (1). The findings in that manuscript and our subsequent experience (2,3) have shown that ecgonine is consistently present in postmortem blood specimens; its origins, however, are usually open to debate and discussion (4-6). Application of the method in carefully preserved whole blood specimens from cocaine addicts reporting to an emergency room have also shown ecgonine to be consistently present (3). This suggests that at least in high doses, often chronic cocaine administration, ecgonine will be present during life also. Our intent in the work that led to our original publication was to investigate the utility of ecgonine as a potentially longerlived marker than benzoylecgonine of past cocaine use. All our findings to date however have indicated that the detection windows for benzoylecgonine and ecgonine are approximately the same, at least at our limit of detection (10 ng/mL) (1-3). Other workers have documented the instability of both cocaine and its monoester metabolites, benzoylecgonine and ecgonine methyl ester, during in vitro storage of blood (4,5). It is likely that at least some of the loss of these primary metabolites is via a secondary de-esterification to ecgonine. Consequently, the true value of an assay for ecgonine may be its ability to detect what is likely the final in vitro degradation product following cocaine use. Ecgonine would thus be the analyte of choice in cases in which a specimen has been collected without an enzyme-inhibiting preservative or at temperatures known to be inadequate for the preservation of cocaine or its primary monoester metabolites (4,5). We report a case in which a defendant in a homicide case relied in part on his recent cocaine use as a component of his defense. The specimen was collected in a tube containing EDTA, a chelating agent with no significant enzyme-inhibiting properties, and was stored for approximately 18 months, some of which time included at least some period of room-temperature storage. During that time it was subjected to an analysis for a variety of drugs including cocaine, benzoylecgonine, cocaethylene, and ecgonine methyl ester, all of which proved negative. Following this lengthy storage, the specimen was submitted to us for analysis for the presence of ecgonine, according to the method described in our earlier work (1,2). The testing disclosed the presecnce of ecgoinine in the blood at a concentration of 557 ng/mL. The specimen was negative for other cocaine-related compounds and metabolites including cocaine, benzoylecgonine, ecgonine methyl ester, ecgonine ethyl ester, cocaethylene, and norcocaine. The assay used was a selected ion-monitoring assay; however, the presence of ecgonine was confirmed by full scan mass spectrometry of its derivative. To our satisfaction, this demonstrated that the subject, sometime prior to the collection of the blood specimen, had in fact ingested cocaine, which may have been corroborative of his defense. We did not attempt to estimate the dose, the time since ingestion, or the likely effects of the ingestion on the subject proximate to the time of the specimen collection. The complete absence of other cocaine metabolites suggests that the ecognine was at least partly an artifact of the inadequate preservation of the specimen. Based on this experience, we strongly suggest that ecognine analysis may be an appropriate test to demonstrate cocaine use when the provenance of the specimen is unknown, when preservation has been inadequate, or when other cocaine metabolites have been shown to be undetectable.

To determine whether changes in pituitary responsiveness might account for the lack of corticotropin (ACTH) stimulation following 6 consecutive days of continuous cocaine administration (5 or 25 mg/kg/day, via osmotic minipumps), the hormonal response of vehicle- or cocaine-pretreated male rats was compared. Intravenous injections of synthetic corticotropin-releasing factor (CRF) (0.2, 1, or 5 μg/Kg) elicited dose-dependent increases in ACTH secretion irrespective of whether rats had been previously exposed to cocaine or not. Similarly, in both vehicle- and cocaine-pretreated rats ACTH response to acute injections of the drug was identical, indicating that pituitary corticotrophs remained responsive following continuous administration of cocaine. To determine and compare plasma concentrations of cocaine and its metabolites after continuous or acute administration of the drug, pharmacokinetics analysis of concentration vs. time was ascertained. Circulating concentrations of cocaine from rats continuously exposed to the drug were relatively low throughout the 6 days of exposure. In contrast, intravenous injections of cocaine produced peak concentrations of the drug that were significantly higher than those measured during continuous cocaine infusion. Such peak concentrations in cocaine correlated with marked increases in plasma ACTH levels. Plasma concentrations of the metabolites benzoylecgonine and methyl ester ecgonine followed a pharmacokinetic clearance similar to that of the parent compound, with low concentrations detected during continuous exposure whereas high concentrations were observed following intravenous injections of the drug. Our results suggest two nonmutually exclusive conclusions. First, there may be a critical threshold of cocaine plasma concentrations (as indicated by our results as being over 800 ng/ml) that are necessary for activation of the hypothalamic-pituitary-adrenal axis to occur. Second, stimulation of ACTH secretion may only occur during rapid systemic increases in drug concentrations.

A solid-phase extraction method was developed for the extraction of first-day meconium samples from premature infants of cocaine-dependent mothers. Extracts were analysed by high-performance liquid chromatography and gas chromatography—mass spectrometry for cocaine and its metabolites. Control stools showed no drug. Meconium from cocaine-dependent mothers showed cocaine in the range 0.1–0.78 μg/g. Benzoylecgonine, ecgonine and ecgonine methyl ester were not present in the samples, which suggests that the metabolism of cocaine in the premature neonate is limited.

A high-performance liquid chromatographic method has been developed for the quantitation of the enantiomers of cocaine. Mixtures of the naturally occurring (−)-cocaine and synthetically produced (+)-cocaine were hydrolysed in water to (+) and (−)-benzoyl ecgonine. Esterification with an optically pure 2-octanol resulted in diastereoisomers that could be separated on bare silica gel using an acetonitrile-aqueous ammonium phosphate mobile phase.

Cocaine is a local anesthetic containing a tertiary amine structure derived from the amino alcohol base, ecgonine. Ecgonine is methylated and esterified with benzoic acid to form cocaine or benzoylmethylecgonine (1). Cocaine was first isolated in 1857 and its medical uses have included application as a topical anesthetic in nasal surgery. The drug appears to be metabolized in humans and other mammals by means of plasma and tissue esterases (2). The major urinary metabolites appear to be the water soluble substances, benzoylecgonine and ecgoninemethy-lester (3). Cocaine can also be N-demethylated in the body and the resulting metabolite, norcocaine, has been reported to have good biological activity in a variety of systems (4,5,6).

The effects of cocaine and its primary metabolites, benzoylecgonine and ecgonine, on early embryogenesis were investigated in in vitro cultured chick embryos, and levels of cocaine and benzoylecgonine were measured in the embryo and in the culture medium. Neurulation was the only developmental process shown to be sensitive to cocaine. Closure of neural folds was inhibited within 4 hr of treatment of stage 6-8(-) embryos with 170-340 mug cocaine hydrochloride in 1 ml culture medium. Cocaine levels within embryos treated with 340 mug cocaine hydrochloride decreased from approximately 11 to 5 mug/embryo between 20 min and 4 hr of culture. Cocaine effects were more pronounced in continuously treated embryos.

Cocaine and 11 of its metabolites were identified in a urine specimen from a cocaine user. Four of the metabolites are reported for the first time: ecgonidine, norecgonidine methyl ester, norecgonine methyl ester, and m-hydroxy-benzoylecgonine. The structures of the newly identified metabolites were confirmed by comparison of their gas chromatographic retention times and their electron ionization and chemical ionization mass spectra with the corresponding data obtained on synthesized standards. Other metabolites present were benzoylecgonine, ecgonine methyl ester, ecgonine, ecgonidine methyl ester, norcocaine, p-hydroxycocaine, and m-hydroxycocaine.

Cocaine has pronounced peripheral vasoconstrictor effects. Despite the short half life of cocaine in the body these effects are relatively long-lived. The role of cocaine metabolites in vasoconstriction attributed to cocaine has not been reported. We evaluated the contractile ability of cocaine and its major metabolites in isolated cat cerebral arteries. The primary cocaine metabolite, benzoylecgonine was a potent contractile agent, causing a 50% decrease in cross sectional area at 10(-5) M. This was less than caused by serotonin, but greater than caused by norepinehrine. Ecgonine and cocaine were less active contractile agents than was benzoylecgonine, and ecgonine methyl ester was a mild relaxant.

We measured concentrations of cocaine and its major metabolites (benzoylecgonine, ecgonine methylester, norcocaine, and cocaethylene) in 15 autopsied brain regions of 14 human chronic cocaine users. Only slight differences were observed in concentrations of cocaine and its metabolites amongst the examined brain areas. Although it is likely that some postmortem redistribution of the drug must have occurred, our data are consistent with the possibility that behaviorally relevant doses of cocaine are widely distributed throughout the brain of humans who use the drug on a chronic basis. Consideration should therefore be given to the possible pharmacological and toxicological actions of cocaine in both striatal and extra-striatal brain areas in human users of the drug.

Fingernail and toenail specimens were obtained from 18 suspected cocaine users. The nails were cut, heated under methanolic reflux, and the methanolic extracts were purified by solidphase extraction. Gas chromatography/mass spectrometry was utilized for the qualitative and quantitative analysis of nine cocaine analytes. Comparison of conventional postmortem analysis of blood and urine with nail analysis revealed a marked increase in the detection of cocaine use by nail analysis. Cocaine analytes were present in 14 (82.3%) subjects utilizing nail analysis. Out of those 14 subjects, only 5 (27.7%) were positive by conventional postmortem drug analysis. Cocaine and benzoylecgonine were the predominant analytes in all positive nail specimens. Anhydroecgonine methyl ester, ecgonine methyl ester, ecgonine ethyl ester, cocaethylene, norcocaine, and norbenzoylecgonine were detected in a limited number of specimens. The ratio of cocaine to benzoylecgonine ranged from 2–10:1. These findings suggest that nails may be a useful alternative matrix for the detection of cocaine exposure.

Methodology is presented for the isolation and comparative determination of 16 alkyloyl and 2 heteroaroyl ecgonine methyl esters in illicit cocaine samples. These trace-level alkaloids, five of which are reported for the first time, were partially isolated from cocaine using alumina column chromatography and recrystallization and determined using capillary gas chromatography-mass spectrometry using selected ion monitoring. Acetoxy-, propionoyl-, isobutyroyl-, butyroyl-, 2-methylbutyroyl-, isovaleroyl-, valeroyl-, senecioyl-, trans-4-hexenoyl-, tigloyl-, hexanoyl-, 3′-furanoyl-, trans-2-hexenoyl-, 2′-furanoyl-, trans-3-heptenoyl-, cis-, trans-; trans-, cis-; and trans-, trans-2,4-hexadienoyl- ecgonine methyl esters were detected at levels less than 1 × 10−5 % (w/w relative to cocaine).

Methodology used for the comparative chemical analyses of two illicit cocaine seizures, and its application in a successful criminal prosecution, is described. A description of events leading to the arrest of the defendant and an overview of the jury trial are provided. Illicit cocaine, found in the defendant's suitcase and wallet, was subjected to chemical derivatization and three distinct gas chromatographic methods for the detection and relative quantitation of cocaine manufacturing impurities/by-products. The cocaine impurities included cis- and trans-cinnamoylcocaine, the isomeric truxillines and the hydroxy-cocaines. Among the cocaine manufacturing byproducts detected were benzoylecgonine, ecgonine methyl ester, ecgonine, N-benzoylnorecgonine methyl ester and N-norcocaine. Chemical derivatization of the cocaine samples was accomplished using heptafluorobutyric anhydride and N,O-bis(trimethylsilyl)acetamide. The derivatized impurities/by-products were subjected to capillary gas chromatographic analysis using both flame ionization and electron-capture detectors. The comparative chemical analyses provided a positive correlation between the suitcase and wallet cocaine samples.

To the Editor: In a previous communication, we presented a simple, sensitive, and selective quantitative procedure for ecgonine methyl ester (EME) analysis in urine (1). The between-run precision of this assay was 10%. Using the above procedure we evaluated the long term stability of EME in urine. Ecgonine methyl ester hydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO). Drug free urine was spiked with a known concentration of EME (0.84 mg/mL). The pH was adjusted with sodium hydroxide or hydrochloric acid, and the three urines, pH 3, 5, and 9, were analyzed on the day of sample preparation. Samples were stored in a refrigerator at 4-5~ between analyses. At known time intervals, the samples were taken from the refrigerator and analyzed using our previously described procedure (1). Results, which are reported as percent loss from the day of sample preparation, were calculated using the pH 5 urine as standard. Three years later, the three samples at pH 3, 5, and 9 were analyzed for the presence of EME. For the three-year stability determination, an EME standard was prepared in fresh drug-free urine and was used to calculate the EME concentration in the pH 3 and 5 samples. Table I shows the results of samples that were analyzed for the presence of EME at pH 9 and 3 over a Table I. Ecgonine Methyl Eater Stability In Urine 29-day period, and three years later at pH 3, 5, and 9. EME was unstable under basic conditions, pH 9, with % EME loss* against pH 5.0 standard 100% of the EME being lost in 30 days. Similar results Time pH 5.0

A confirmation procedure for the analysis of cocaine, benzoylecgonine, ecgonine, and phencyclidine (PCP) in human hair using tandem mass spectrometry has been developed. This procedure requires no solvent extraction and thus can examine the metabolites of drugs such as cocaine. Hairs from six cocaine users were examined and the amount of cocaine and its ratio to the metabolites was not correlated to the reported use pattern. Only PCP was detected in the hair from a PCP user, no metabolites were found. In passive exposure experiments, hair was found to tightly absorb PCP from aqueous solutions, which mimics the incorporation of PCP by the body. These results indicate that drugs may be absorbed onto the hair of nonusers and become tightly bound.

Many cocaine base (crack) exhibits contain traces of sodium bicarbonate. Pseudoecgonine methyl ester and ecgonine methyl ester are formed as artifacts from the effect of sodium bicarbonate and methanol in the injection port of a gas chromatograph. Formation of ecgonine methyl esters was confirmed by comparison to known standards via gas chromatography/mass spectrometry (GC/MS). The mechanisms for artifact formation are hydrolysis of cocaine, followed by esterification and trans-esterification/base catalyzed epimerization.

The effects of acid (NH4Cl, 60 mg/kg) or alkaline (NaHCO3, 400 mg/kg) pretreatment on tissue disposition and urinary excretion of (3H)-cocaine and its metabolites were assessed in rats. Animals were decapitated 4 hr after 1.9 microCi in 40 mg/kg cocaine (Cl-) in experiments on the biological disposition and the plasma, brain, and liver were assayed. In experiments on urinary excretion, 10 microCi were given and urine was assayed after 18, 24, and 48 hr. Pretreatment with NH4Cl produced a decrease of rates of cocaine + norcocaine in the brain as well as of the polar metabolites (benzoylecgonine, ecgonine benzoylnorecgonine). Forty-eight hours after injection of (3H)-cocaine, NH4Cl produced an increase of the rates of cocaine and its metabolites in the urine. Pretreatment with NaHCO3 causes a decrease of total radioactivity (cocaine + metabolites) in the plasma and in the liver, as well as a decrease of the levels of cocaine + norcocaine in the liver, as well as an increase of the rate of polar metabolites that are excreted in the urine. These data suggest that the decrease of pH after acid treatment produces ionization of basic molecules; these do not penetrate as well in the tissues for which they have no affinity. As a consequence, the urinary excretion is more important. It is possible that alkaline treatment accelerates the excretion of cocaine and its metabolites because it increases diuresis.

1. 1. The activity of some cocaine metabolites (norcocaine, benzoylnorecgonine and ecgonine) and of a stereoisomer of cocaine, pseudococaine, was examined on the high affinity uptake of dopamine into isolated striatal synaptosomes. 2. 2. High affinity uptake of dopamine (defined as uptake at 0.5 μM dopamine concentration) into synaptosomal fractions isolated by gradient centrifugation from rat striatum was inhibited competitively by norcocaine and pseudococaine, as well as by cocaine, with KI 50S of 8 x 10 −5 M, 5 x 10 −4 M and 7 x 10 −6 M, respectively. 3. 3. Benzoylnorecgonine and ecgonine methylester inhibited dopamine uptake about 50% at 5 x 10 −3 or higher. Benzoylecgonine and ecgonine showed no inhibition at these concentrations but the benzoyl-derivatives inhibited dopamine uptake slightly at concentrations of 10 −7 or lower.

The present study examines the possible role of sodium channels in the behavioral effects of cocaine. Cocaine congeners are apparent competitive inhibitors of the scorpion toxin-enhanced binding of [3H]batrachotoxinin A 20-alpha-benzoate to sodium channels in mouse cerebrocortical synaptosomes. However, in agreement with the allosteric model for heterotropic cooperative interactions, the compounds produce a concentration-dependent increase in the rate of dissociation of binding. Concentrations that give a 2-fold increase of k-1 are close to Ki values for inhibiting equilibrium binding of [3H]bactrachotoxinin A 20-alpha-benzoate, suggesting that the inhibitory effect on binding results mostly from an increase of the apparent dissociation rate constant. The ester linkage between the tropane and benzoyl ring of cocaine is not essential for the inhibitory potency, and for both the C-2 and C-3 substituents the equatorial position results in a higher potency than the axial position. There is reasonable agreement between the rank order of potencies in blocking the sodium channel and in inhibiting locomotor behavior. The present results do not support a relationship between the capability of cocaine congeners in blocking sodium flux and in inhibiting uptake of dopamine into striatal synaptosomes. However, peak levels of cocaine in the brain of cocaine addicts could be high enough to interfere with sodium channel functioning, possibly contributing to some of cocaine's actions.

Quantitative extraction studies from urine were carried out by addition of cocaine, benzoylecgonine, ecgonine methyl ester and ecgonine to urine samples. After hydrolysis to ecgonine the compounds were analyzed together. Ecgonine was isolated by a cation-exchange resin and purified by an anion-exchange resin. The quantitative determination was performed by GC after silylation with MSTFA. The recovery was 77% at a concentration of 150 μg ecgonine/ml urine. A qualitative determination of ecgonine by GC/MS was possible up to the detection limit of 20 ng/ ml. The method can be applied for the detection of cocaine abuse.

1. The metabolism of [3H]norcocaine, N-hydroxy[3H]norcocaine and cocaine-N-oxide has been investigated in rats after i.v. injection. 2. The biological t 1/2 of norcocaine (dose 2 mg/kg i.v.) in plasma, liver and brain were 0.4, 1.6, 0.5 h, respectively and the compound was not detectable in the central nervous system 6 h after injection. The % dose of norcocaine excreted unchanged in urine and faeces in 96 h were 0.7 and 1.0, respectively. Benzoylnorecgonine, norecgonine, norecgonine methyl ester and an unidentified compound were excreted in urine. 3. The biological t 1/2 of N-hydroxynorcocaine (5 mg/kg i.v.) in brain and plasma were 0.3, 1.6 h respectively and only 1.3 and 1.6% of dose were excreted unchanged in urine and faeces in 96 h. N-Hydroxybenzoylnorecgonine and N-hydroxynorecgonine methyl ester were the major urinary metabolites. N-hydroxynorcocaine was not metabolized to norcocaine in vitro by liver microsomes. Doses of greater than 7.5 mg/kg i.v. resulted in death of rats by cardiorespiratory arrest. 4. Cocaine-N-oxide (50 mg/kg i.v.) yielded ecgonine-N-oxide methyl ester as its major metabolite; other minor metabolites were cocaine (0.5%), norcocaine (1%), benzoylecgonine, ecgonine, ecgonine-N-oxide, along with minor amounts of unmetabolized compound. Lethality of cocaine-N-oxide (100 mg/kg i.v.) was possibly due to metabolism to norcocaine and cocaine.