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A mixture of sodium [1- 14 C]acetate and DL -[4- 3 H]phenylalanine ( 3 H/ 14 C  1.0) was administered to Erythroxylon coca plants by painting an aqueous solution of these compounds on the leaves. The leaves were removed after two weeks and yielded labeled cocaine ( 3 H/ 14 C  11.4)and cuscohygrine. A systematic degradation of the cocaine indicated that 96% of the 3 H was located in the para -position of its benzoyl moiety, and the 14 C was located in the ecgonine residue at positions in accord with the accepted biosynthesis of this alkaloid. The cuscohygrine, containing a negligible amount of 3 H, was found to have 67% of its 14 C located on the carbonyl carbon. Cocaine, isolated from a second crop of leaves, harvested 10 weeks after the initial feeding, was still labeled at specific positions.

A method was developed whereby the major metabolites of cocaine (ecgonine and benzoylecgonine) were extracted from human urine, butylated and subsequently isolated and identified on Polygram silica gel sheets. A comparison of the butylated ecgonine thin-layer chromatographic technique (BETT) with the enzyme multiplied immunoassay technique (EMIT) for benzoylecgonine indicated complete agreement on the presence or absence of cocaine metabolites in 88% of the human urine samples analyzed. Disagreement on the positive urine samples was ascribed to the lower level of sensitivity of BETT (3–5 μg/ml of ecgonine or benzoylecgonine) in comparison to EMIT (1 μg/ml benzoylecgonine). Of the urine samples positive for the cocaine metabolites 18% were negative for other drugs of abuse and 71% were positive for methadone. Cross-reactivity studies with EMIT indicated a high degree of specificity for benzoylecgonine. The BETT and EMIT assays were found to be highly reliable, valid and sensitive tests for the presence of cocaine metabolites in human urine.

Structure-activity relationships were determined for cocaine congeners in counteracting the depolarization induced by the action of veratridine on voltage-dependent sodium channels of synaptoneurosomes from mouse brain cortex, and their potency was compared to those determined previously on Na+ uptake and batrachotoxinin binding. Cocaine, norcocaine, (+)-pseudococaine, (-)-pseudococaine, (+)-neopseudococaine, benzoyltropine, benzoylpseudotropine, ecgonine methylester, atropine, WIN-35,428, WIN-35,140, WIN-35,065-3, WIN-35,004, and procaine were tested for their potency in inhibiting depolarization as measured by the distribution of the lypophilic cation [3H]triphenylmethylphosphonium across the membrane. All of the tested compounds inhibited the veratridine-induced depolarization in a competitive manner. The structure-activity relationships were similar to those for inhibition of 22Na+ uptake in mouse brain homogenates, and the potency of these local anesthetics in inhibiting veratridine-induced uptake of [3H]triphenylmethylphosphonium correlated well with their potency in inhibiting [3H]batrachotoxinin A 20-α-benzoate binding in mouse brain synaptosomes.

A sensitive and reliable method was developed to determine quantitatively cocaine and its metabolites benzoylecgonine and ecgonine. The method involved the formation of fluoro derivatives which were separated on 3% and 5% OV-1 columns and detected in picomole qunatities using an electron capture detector. Ecgonine and benzoylecgonine were derivatized with a mixture of hexafluoroisopropanol- heptafluorobutyric anhydride (1:2). Cocaine was first reduced by LiAlH 4 and them acylated by pentafluoropropionic anhydride. Benzoylecgonine, but not ecgonine, could also be determined by reduction and subsequent acylation. This provided the basis for the determination of cocaine, benzoylecgonine and ecgonine from the same sample. Cocaine could be determined in urine and plasma by this method.

The present study investigated the actions of cocaine and some of its metabolites and analogs upon the synaptosomal (P2) synthesis and release of dopamine appearing from [14C]phenylalanine. Also examined was the influence of reserpine upon these actions of cocaine. The P2 preparations from the rat caudate nucleus were incubated with the drugs for [14C]phenylalanine and, after filtration, the particulates and medium fractions were analyzed for [14C]dopamine and [14C]phenylalanine. Labelled dopamine in the medium was taken as a measure of its release by any addition of drug. Cocaine, norcocaine and the synthetic cocaine analogs WIN 35428 and WIN 35-065(2) each stimulated both the total (medium plus particulates) formation and the release of dopamine, while benzoylecgonine and ecgonine were without effect. Reserpine inhibited the synthesis and enhanced the release of dopamine. An addition of reserpine blocked the stimulating effect of cocaine, norcocaine, WIN 35428 and WIN 35-065(2) on synthesis and furthermore, these drugs had, in the same experiments, inhibitory effects on the synthesis, additive to the reserpine-induced inhibition. Benzoylecgonine and ecgonine were only weakly inhibitory in the presence of reserpine. Stimulation, rather than additive inhibition, by cocaine was observed in the presence of exogenous dopamine, and amphetamine increased the synthesis in the presence of either reserpine or added dopamine. Pretreatment of rats with reserpine also blocked the stimulation of synthesis by cocaine and WIN 35428. The uptake of labelled substrate was not affected by addition of drug, or by the pretreatment.

Through the use of an O-(o-iodobenzoyl) ecgonine derivative (3), tritium labelled O-benzoyl ecgonine has been prepared, by catalytic hydrogenolysis, in sufficiently high specific activity for use in radioimmunoassay studies for cocaine metabolites. The ease of converting O-benzoyl ecgonine to cocaine also makes this labelled substrate potentially useful in metabolism studies of the latter.

Working on a small scale the following two methods have given a conversion of 92-95% into cocaine. I. To 1-2 cm3 of n/10 ecgonine, 1 cm3 of methyl alcohol and 20 cm3 of benzene were added, and 10 cm3 of the solvents were distilled off. To the remaining solution 0.1 cm3 of methyl alcohol, 0.05 cm3 of benzoyl chloride and 2 g of sodium carbonate were added and the solution heated in a closed vessel for 20 hours at 35-40°. After the addition of 1 cm3 of methyl alcohol and 10 cm3 of benzene, 10 cm3 of the solvents were distilled off and the remaining solution was boiled for 10 hours with 0.5 cm3 of benzoyl chloride. II. To 1-2 cm3 of n/10 ecgonine, 1 cm3 of methyl alcohol, 0.15 g of benzoic acid and 20 cm3 of benzene were added, and 10 cm3 of the solvents distilled off. To the remaining solution of ecgonine benzoate 0.1 cm3 of methyl alcohol and 0.4 g of benzoic anhydride were added. After 5-10 days at ordinary temperature in a closed vessel 0.1 cm3 of methyl alcohol, 0.05 cm3 of benzoyl chloride and 2 g of sodium carbonate were added and the solution heated for 20 hours at 35-40°. With a hydrochloric acid methyl alcohol solution and sodium carbonate, benzoyl ecgonine is partly decomposed by heating it in benzene solution at 35-40°, giving about 25% of cocaine and about 75% of ecgonine, the benzoic acid split off being converted almost completely into the methyl ester.

The technique of “reaction gas chromatography” involving dehydrogenation of heterocyclic compounds by a platinum-firebrick catalyst was applied to alkaloids possessing the tropane moiety in their structures. The reaction chromatogram of 3-tropanol, from which most of these alkaloids may be considered to be derived, exhibited several peaks, among which were those due to pyrrole, methylpyrrole, pyrrolidine, pyridine, piperidine, and toluene. These products were also produced by atropine, homatropine, scopolamine, ecgonine, and cocaine when these alkaloids were examined under similar experimental conditions. Atropine, homatropine, and scopolamine exhibited some additional peaks which were correlated with their structures.

Summary Competition feeding experiments with sodium acetate-(3H) and sodium acetoacetate-(3-14C) in isolated root cultures of Datura metel yielded preferential incorporation of radiocarbon into the C3-bridge of tropane alkaloids, showing that acetoacetate is the more direct precursor. Sodium acetate-(1-14C) gave rise to labelling of the carboxyl group of the ecgonine part in cocaine. Radioactive labelled hygrine showed lower incorporation figures into tropane alkaloids and cuskohygrine respectively than does ornithine-(2-14C). Hygrine-a-carboxylic acid is postulated to be a key metabolite in the biosynthesis as well as of pyrrolidine bases as tropane and coca alkaloids. Ecgonine and pseudoecgonine didn't decarboxylate in different species of datura.

Analyzing the fators of “Cocaineinactivation” by rabbit's liver the following results were obtained. (1) By the biological estimation of cocaine it was demonstrated that the liver extract of rabbits could inactivate cocaine and its important mechanism was suggested to be destruction. (2) Its destructive products, methyl alcohol, bezoic acid and ecgonine, were chemically demonstrated. (3) The quantitative micro-determination of benzoic acid was testified to be albe to measure benzoic acid derived from cocaine by rabbitt's liver extract. (4) Using this method, it could be proved that the production of benzoic acid was enzymatically performed. (5) The destructive ability was compared among the tissues of rabbits and the livers of several animals, and the liver of rabbits showed the most active destruction.

Without any sample pretreatment, effervescent beverage fluids were manually sprayed into the primary ion plume created by using a nanoelectrospray ionization source for direct ionization, and the analyte ions of interest were guided into an ion trap mass spectrometer for tandem mass analysis. Functional ingredients (e.g., vitamins, taurine, and caffeine, etc.) and spiked impurity (e.g., cocaine) in various beverages, such as Red Bull energy drink, Coco-cola, and Pepsi samples were rapidly identified within 1.5 s. The limit of detection was found to be 7 ∼ 15 fg (S/N = 3) for cocaine in different samples using the characteristic fragment (m/z 150) observed in the MS3 experiments. Typical relative standard deviation and recovery of this method were 6.9% ∼ 8.6% and 104% ∼ 108% for direct analysis of three actual samples, showing that nanoextractive electrospray ionization tandem mass spectrometry is a useful technique for fast screening cocaine presence in beverages.

The effect of ecgonine, tropine, tropinone and some of their derivatives, tropane, N-methylpyrrolidine, and N-methylpiperidine on impulse summation in the CNS, the conditioned avoidance reflex, antagonism with hexobarbital, synergism with cocaine, and also their toxicity (LD50) were investigated experimentally. Benzoylecgonine, the methyl ester of ecgonine, ecgonine itself, tropine, pseudotropine, carbomethoxytropinone, tropinone, tropane, N-methylpyrrolidine, and N-methylpiperidine were shown to have a definite stimulating effect on the activity of the CNS and, in this respect, are similar to cocaine. It is concluded that compounds whose molecules contain the structure of tropane or one of its fragments have a central stimulant effect and that the stimulant action of cocaine on the CNS may be associated with the tropane moiety of its molecule.

Monkeys were chronically treated with cocaine, 1 mg/kg subcutaneously twice daily for the first week and four times daily for the subsequent 21 weeks, followed by the same dose of [3H] cocaine injected intravenously. The t12 values of cocaine in the plasma, cerebrospinal fluid, liver, kidney and heart of acutely and chronically treated monkeys were 1.32, 0.85; 0.91, 0.75; 1.60, 1.29; 0.90, 0.91; and 1.02, 0.91 h, respectively. In areas of the central nervous system (CNS) the values for the acute and chronic group ranged between 0.75 – 0.90 and 0.62 – 0.84 h, respectively. With the exception of the temporal cortex, cerebellum and caudate nucleus, the t12 were not significantly different in the two groups. Norcocaine, benzoylnorecgonine, benzoylecgonine, and minor amounts of ecgonine were the metabolites of cocaine in the brain of monkeys. Significant amounts of total radioactivity due to benoylnorecgonine and benzoylecgonine persisted in the CNS of chronically treated monkeys. Norcocaine constituted approximately 3, 21, 8, and 6% of the values of cocaine in the CNS areas in the acutely treated monkeys, and 14, 13, 14, and 16% in the chronically treated ones, 0.25, 0.5, 1 and 2 h after cocaine injection. A biphasic pattern of peak levels in the plasma and bile of chronically treated monkeys indicated enterohepatic circulation of cocaine. Brain to plasma and CSF to plasma ratios of cocaine were higher in the chronic than in the acute group. The amounts of cocaine excreted in urine and feces as a percentage of dose were 0.23 – 5.1 and 0.1 – 0.23 in the acute group, 0.54 – 7.0 and 0.1 – 0.14 in the chronic group. Major excretion of radioactivity occurred in urine within 24 h and the mean values of the total radioactivity in urine and feces (96 h) in the acute and chronic groups were 60.4 and 63.2% of the dose, respectively. Norcocaine, benzoylnorecgonine, benzoylecgonine, ecgonine methyl ester, ecgonine, and unidentified compounds were the urinary metabolites of cocaine in the two groups. The percentage of benzoylecgonine was higher, that of benzoylnorecgonine lower, in the chronic than in the acute group and the values of other metabolites were not markedly different in the two groups. Data support the postulate that chronic treatment of monkeys with cocaine does not produce dispositional tolerance.

1. Beagle dogs were chronically treated with cocaine, 5 mg/kg subcutaneously twice daily for 6 weeks, followed by same dose of [3H]cocaine given intravenously. 2. The t1/2 values of cocaine in plasma, liver, spleen and heart, in acutely and chronically treated dogs, were: 1-2, 1-1; 2-2, 1-8; 1-8, 1-3; 2-0, 1-2 h, respectively. In both groups, cocaine disappeared from all areas of the central nervous system 12-24 h after injection but significant amounts of radioactivity due to benzoylnorecgonine and benzoylecgonine persisted in the CNS even 1 week after administration of cocaine. Brain-to-plasma ratios of cocaine were lower in chronically-treated than in acutely-treated dogs 2 and 4 h after injection. 3. Norcocaine, benzoylnorecgonine, benzoylecgonine and ecgonine were metabolites of cocaine in dog brain in both groups. Norcocaine and benzoylnorecgonine were present in higher amounts in brains of chronically treated dogs. Rate of disappearance of norcocaine was similar to cocaine in both groups. 4. The amounts of cocaine excreted in urine and faeces as percentage of dose were 0-9-5-0, 1-1-6 in the acute and 2-2-3-3 and 0-2-0-3 in the chronically treated dogs. Major excretion of radiactivity occurred in urine within 24 h in both groups. Total radioactivity (65% of dose) in urine plus faeces was similar in both groups. 5. Norcocaine, benzoylnorecgonine, benzoylecgonine, ecgonine, norecgonine, ecgonine methyl ester and unidentified compounds were urinary metabolites of cocaine in both groups. Benzoylnorecgonine and ecgonine were excreted in higher amounts and benzoylecgonine and norecgonine in lower amounts in the acute than in the chronically treated dogs. 6. The possible role of persistence of benzoylnorecgonine and benzoylecgonine (which possessed potent stimulant activity intracisternally) in the CNS is discussed.

To assess the role that action on sodium channels may play in the physiological effects of cocaine and to obtain information on the structure-activity relationships of this action, cocaine, norcocaine, N-allynorcocaine, (+)-pseudococaine, (-)-pseudococaine, (+/-)-allococaine, (+/-)-allopseudococaine, ecgonine, ecgonine methyl ester, O-benzoylecgonine and atropine were tested for their effects on sodium channels. The method employed was a sodium channel specific equilibrium 22Na+ uptake assay with rat brain membrane homogenates. All of the compounds listed with the exception of the ecgonines were found to be single affinity competitive inhibitors of veratridine activation of sodium channels. Ecgonine showed no inhibition at concentrations as high as 10(-3) M while ecgonine methyl ester and O-benzoylecgonine showed inhibition only at very high concentrations. The order of inhibition potencies was found to (+)-pseudococaine congruent to norcocaine congruent to N-allynorcocaine greater than cocaine greater than (-)-pseudococaine congruent to (+/-)-allopseudococaine greater than (+/-)-allococaine greater than atropine greater than O-benzoylecgonine greater than ecgonine methyl ester greater than ecgonine. This ordering of potencies is in good agreement with published reports of the local anesthetic potencies of these agents.

Antisera which react with the major metabolites of cocaine have been prepared in rabbits and a hemagglutination-inhibition (HI) test which detects these metabolites in urine or serum in concentrations of 1 ng/ml is described. A comparison of this test with alternate detection procedures shows excellent agreement at comparable sensitivity levels. HI tests on simian and human biological specimens suggest that the use of cocaine remains detectable for at least 3 days after administration of a minimal pharmacologically active dose. Combination of physical separation of drug metabolites with immunoassay procedures indicates that benzoylecgonine and ecgonine are the immunoreactive cocaine metabolites found in human urine. While it was possible to apply the HI test at maximal sensitivity to human sera and to murine or simian urine specimens, interference was encountered with some human urine specimens. Preliminary data suggest that by reducing the sensitivity of the test to a threshold of 200 ng/ml this interference can be overcome.

A simple and economical radioimmunoassay for benzoylecgonine in blood or urine is described. Haemolysis, decomposition, common anticoagulants and sodium fluoride do not affect the results. A commercially available antiserum is used at a dilution of 1:600. The tracer is radioiodinated p-hydroxybenzoylecgonine. It is prepared by reacting p-acetoxybenzoic anhydride with methyl ecgonine followed by mild hydrolysis and radioiodination by the Iodo-gen method. The product is purified on a disposable silica cartridge and has a specific activity of about 30 TBq mmol−1. Polyethylene glycol is used to separate the bound and free fractions in the assay. The range of the dose-response curve is 0–400 ng ml−1 benzoylecgonine. The assay is largely specific for benzoylecgonine. Cocaine, ecgonine, methylecgonine and cinnamoyl cocaine have slight or negligible cross-reactivities. The inter-assay coefficient of variation is 7·5% and the recovery of benzoylecgonine from ‘spiked’ blood is 103%. The ‘cut-off’ is 20 ng ml−1 benzoylecgonine for both blood and urine. Radioimmunoassay and high-performance liquid chromatography results agree well.

The hydrolysis of cocaine and its N-demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.

Cocaine disposition studies in the rat are described. A mixture of cocaine, cocaine-d3, cocaine-14C, and cocaine-3H was used as a dose. The urine was collected, extracted with organic solvents, and fractions collected using HPLC and TLC. The radioactive cocaine was useful in the separation of metabolites while the deuterium-labeled cocaine, creating an artificial isotopic cluster, provided specific identification of the metabolites by gas chromatography/mass spectrometry. Benzoylecgonine, ecgoninemethylester, ecgonidine, norcocaine, N-hydroxynorcocaine, hydroxycocaine, and hydroxymethoxycocaine were found to be the major metabolites, while ecgonine, benzoylnorecgonine, and cocaine-N-oxide seemed to be present in trace amounts. Dimethoxyhydroxy cocaine was tentatively identified. Several of these newly identified metabolites of cocaine were present in significant concentrations and could contribute to the toxic effects of cocaine.

A high-performance liquid chromatographic procedure for separating and identifying the four isomeric cocaines has been developed. Use of this procedure with an internal standard allows for the determination of the quantity of any isomeric cocaine in an unknown sample. The pitfalls and problems encountered in the use of gas chromatography and mass spectrometry in the analysis of cocaines and ecgonine methyl esters are discussed.

The carbon-13 nmr spectral assignments oi cocaine, O-benzoylecgonine, ecgonine, norcocaine, O-benzylnorecgonine, norecgonine, and N-allylnorcocaine were made. The assignments were primarily based on the correlation of substituent effects within series and selective proton irradiation studies.