Measurement of benzoylecgonine and cocaine in urine, separation of various cocaine metabolites using reversed-phase high-performance liquid chromatography

The application of reversed-phase high-performance liquid chromatography to the measurement of benzoylecgonine and cocaine in urine is described. Following a simple extraction and clean-up procedure, chromatography is performed using a column containing an octadecylsilica coated packing, elution with 17% acetonitrile in pH 2.7 phosphate buffer and ultraviolet detection at 200 or 235 nm. The detection limit is ca.0.1 microgram of drug per ml urine, and using the ethyl ester of benzoylecgonine as an internal standard, benzoylecgonine and cocaine are quantified with coefficients of variation of 7.0 and 2.8%, respectively. The procedure has been applied to urines from subjects receiving intranasal cocaine, and compared to the enzyme multiplied immunoassay technique. The chromatography procedure also permits the separation of norcocaine and benzoylnorecgonine.     

Synthesis, properties, and reactivity of cocaine benzoylthio ester possessing the cocaine absolute configuration

One aspect of immunopharmacotherapy for cocaine abuse involves the use of a catalytic monoclonal antibody (mAb) to degrade cocaine via hydrolysis of the benzoate ester. A cocaine benzoylthio ester analogue provides a means to implement high-throughput selection strategies to potentially isolate mAbs with high activity. The required analogue was synthesized starting from (-)-cocaine hydrochloride and possessed the cocaine absolute configuration. Key points in the preparation were the introduction of the sulfur atom at C-3 via a bromomagnesium thiolate addition to the exo face of anhydroecgonine, separation of C-2 diastereomers, recycling of a C-2 thio ester byproduct, and formation of the necessary C-2 methyl and C-3 benzoylthio esters. Effects resulting from the lower electronegativity and greater hydrophobicity of sulfur compared to oxygen were observed. These characteristics could result in interesting drug properties. Furthermore, the analogue was found to be a substrate for catalytic mAbs that hydrolyze cocaine as monitored by HPLC and also spectrophotometry by coupling cleavage of the benzoylthio ester to the disulfide exchange with Ellman's reagent. Screening antibody libraries with the new cocaine analogue using the spectroscopic assay provides an avenue for the high-throughput identification of catalysts that efficiently breakdown cocaine.     

Quantification of cocaine and metabolites in exhaled breath by liquid chromatography-high-resolution mass spectrometry following controlled administration of intravenous cocaine

Breath has been investigated as an alternative matrix for detecting recent cocaine intake; however, there are no controlled cocaine administration studies that investigated the drug’s disposition into breath. Breath was collected from 10 healthy adult cocaine users by asking them to breathe into a SensAbues device for 3 min before and up to 22 h following 25 mg intravenous (IV) cocaine dosing on days 1, 5, and 10, and assayed with a validated liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method to quantify breath cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), and norcocaine. The assay was linear from 25 to 1,000 pg/filter, extraction efficiencies were 83.6–126 %, intra- and inter-assay imprecision was <10.6 %, and bias was between ?8.5 and 16.8 %. No endogenous or exogenous interferences were observed for more than 75 tested. Analytes were generally stable under short-term storage conditions. Ion suppression was less than 46 %. Of breath specimens collected after controlled cocaine administration, 2.6 % were positive for cocaine (26.1–66 pg/filter, 1–9.5 h), 0.72 % BE (83.3–151 pg/filter, 6.5–12.5 h), and 0.72 % EME (50–69.1 pg/filter, 6.5–12.5 h); norcocaine was not detected. Methanolic extraction of the devices themselves, after filters were removed, yielded 19.2 % positive cocaine tests (25.2–36.4 pg/device, 10 min–22 h) and 4.3 % positive BE tests (26.4–93.7 pg/device, 10 min–22 h), explaining differences between the two extraction techniques. These results suggest that the device reflects the drug in oral fluid as well as lung microparticles, while the filter reflects only drug-laden microparticles. A sensitive and specific method for cocaine, BE, EME, and norcocaine quantification in breath was developed and validated. Cocaine in breath identifies recent cocaine ingestion, but its absence does not preclude recent use.

Open image in new window

Graphical abstract ?

     

Development of a screening method for cocaine and cocaine metabolites in urine using solvent microextraction in conjunction with gas chromatography

A simple, quick and inexpensive screening method for cocaine and cocaine metabolites has been developed. Drug extraction was achieved using the relatively new technique of solvent microextraction (SME). Complete analysis is achieved in 13 min, using, a 6-min extraction with a 2-microl drop followed by separation on a gas chromatograph. The developed procedure was tested as a screening method for cocaine and cocaine metabolites in spiked urine samples. Using SME, concentrations as low as 0.125 microg ml(-1) of cocaine, ecgonine methyl ester, cocaethylene and anhydroecgonine methyl ester were measurable with relative standard deviation values averaging 9.0%.     

A hydrolysis procedure for the analysis of total cocaine residues in wastewater

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.     

Determination of cocaine in human urine, plasma and red blood cells by gas-liquid chromatography

This paper describes a sensitive and reliable method for the determination of cocaine in human urine, plasma and red blood cells. Cocaine is extracted into cyclohexane from the biological materials at slightly alkaline pH, reduced with lithium aluminium hydride, acylated with pentafluoropropionic anhydride and detected by an electron capture detector. When compared with a gas chromatography-mass spectrometry method the results of cocaine determination correlated highly (r = 0.986). When cocaine was given intravenously to volunteer subjects only 0.2-1.4% of the administered dose was excreted as unmetabolized cocaine in the first 9 h after administration. Plasma and red blood cell levels of cocaine were also determined by this method after intravenous administration.     

Simultaneous quantification of nicotine,opioids, cocaine,and metabolites in human fetal postmortem brain by liquid chromatography tandem mass spectrometry

A validated method for simultaneous LCMSMS quantification of nicotine, cocaine, 6-acetylmorphine (6AM), codeine, and metabolites in 100 mg fetal human brain was developed and validated. After homogenization and solid-phase extraction, analytes were resolved on a Hydro-RP analytical column with gradient elution. Empirically determined linearity was from 5–5,000 pg/mg for cocaine and benzoylecgonine (BE), 25–5,000 pg/mg for cotinine, ecgonine methyl ester (EME) and 6AM, 50-5000 pg/mg for trans-3-hydroxycotinine (OH-cotinine) and codeine, and 250–5,000 pg/mg for nicotine. Potential endogenous and exogenous interferences were resolved. Intra- and inter-assay analytical recoveries were ≥92%, intra- and inter-day and total assay imprecision were ≤14% RSD and extraction efficiencies were ≥67.2% with ≤83% matrix effect. Method applicability was demonstrated with a postmortem fetal brain containing 40 pg/mg cotinine, 65 pg/mg OH-cotinine, 13 pg/mg cocaine, 34 pg/mg EME, and 525 pg/mg BE. This validated method is useful for determination of nicotine, opioid, and cocaine biomarkers in brain.     

Simultaneous determination of cocaethylene and cocaine in blood by gas chromatography with surface ionization detection

Summary Cocaethylene together with cocaine spiked in human whole blood has been found measurable at high sensitivities by capillary gas chromatography with surface ionization detection. The drugs could be rapidly extracted by Sep-Pak C₁₈ cartridges with recovery of more than 60%. The calibration curves for both cocaethylene and cocaine using cocapropylene as internal standard were linear in the range 50–300 pmol mL⁻¹ of whole blood. The detection limits of cocaethylene and cocaine were 5–10 pmol mL⁻¹ (0.1–0.2 pmol on column if recovery is 100%). Cocaethylene could be determined for whole blood obtained from rats (ca. 200 g body wt.), which had received subcutaneous injection of 10 mg cocaine hydrochloride and 2.0 mL of 30% (v/v) ethanol 3 h before sampling; the mean levels of cocaethylene and cocaine were 101 and 1230 pmol mL⁻¹, respectively.     

Piezoelectric affinity sensors for cocaine and cholinesterase inhibitors

We report here the development of piezoelectric affinity sensors for cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized cocaine derivative and an anti-cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization, pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface.The detection of cocaine was based on a competitive assay. The change of frequency measured after 300 s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35 Hz (with an imprecision 3%, n = 3) while the presence of 100 pmol l⁻¹ cocaine decreased the binding by 11%. The limit of detection was consequently below 100 pmol l⁻¹ for cocaine. The total time of one analysis was 15 min.This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO - a competitive inhibitor - served as binding element for cholinesterase in a competitive assay.     

Concentration profiles of cocaine, pyrolytic methyl ecgonidine and thirteen metabolites in human blood and urine: determination by gas chromatography-mass spectrometry

When cocaine is smoked, a pyrolytic product, methyl ecgonidine (anhydroecgonine methyl ester), is also consumed with the cocaine. The amount of methyl ecgonidine formed depends on the pyrolytic conditions and composition of the illicit cocaine. This procedure describes detection of cocaine and 10 metabolites--cocaethylene, nor-cocaine, nor-cocaethylene, methyl ecgonine, ethyl ecgonine, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine and ecgonine--in blood and urine. In addition, the detection of pyrolytic methyl ecgonidine and three metabolites--ecgonidine (anhydroecgonine), ethyl ecgonidine (anhydroecgonine ethyl ester) and nor-ecgonidine (nor-anhydroecgonine)--are included. The newly described metabolites, ethyl ecgonidine and nor-ecgonidine, were synthesized and characterized by gas chromatography-mass spectrometry (GC-MS). All 15 compounds were extracted from 3 mL of blood or urine by solid-phase extraction and identified by a GC-MS method. The overall recoveries were 49% for methyl ecgonine, 35% for ethyl ecgonine, 29% for ecgonine and more than 83% for all other drugs. The limits of detection were between 0.5 and 4.0 ng/mL except for ecgonine, which was 16 ng/mL. Linearity for each analyte was established and in all cases correlation coefficients were 0.9985-1.0000. The procedure was applied to examine the concentration profiles of analytes of interest in post-mortem (PM) blood and urine, and in urine collected from living individuals (LV). These specimens previously were shown to be positive for the cocaine metabolite, benzoylecgonine. Ecgonidine, the major metabolite of methyl ecgonidine, was present in 77% of PM and 88% of the LV specimens, indicating smoking as the major route of cocaine administration. The new pyrolytic metabolites, ethyl ecgonidine and nor-ecgonidine, were present in smaller amounts. The urine concentrations of nor-ecgonidine were 0-163 ng/mL in LV and 0-75 ng/mL in PM specimens. Ethyl ecgonidine was found only in PM urine at concentrations 0-39 ng/mL. Ethanol-related cocaine metabolites, ethyl ecgonine or cocaethylene, were present in 69% of PM and 53% of cocaine-positive LV specimens, implying alcohol consumption with cocaine use. The four major metabolites of cocaine--benzoylecgonine, ecgonine, nor-benzoylecgonine and methyl ecgonine--constituted approximately 88 and 97% of all metabolites in PM and LV specimens, respectively. The concentrations of nor-cocaine and nor-cocaethylene were consistently the lowest of all cocaine metabolites. At benzoylecgonine concentrations below 100 ng/mL, ecgonine was present at the highest concentrations. In 20 urine specimens, benzoylecgonine and ecgonine median concentrations (range) were 54 (0-47) and 418 ng/mL (95-684), respectively. Therefore, detection of ecgonine is advantageous when benzoylecgonine concentrations are below 100 ng/mL.     

Determination of benzoylecgonine and cocaine in biologic fluids by automated gas chromatography

A method is described for extraction of the cocaine metabolite benzoylecgonine, conversion to the butyl ester derivative and gas chromatographic analysis using packed or capillary columns. Using a capillary column, cocaine and benzoylecgonine may be determined simultaneously. The extraction scheme has been designed to facilitate processing of large numbers of samples generated in pharmacokinetic studies. Structural analogues, m-toluylecgonine and m-toluylecgonine methyl ester, are used as internal standards. Concentrations as low as 10 ng/ml in 1-ml samples of plasma or urine are readily determined. Between-run coefficients of variation were 1.01% for cocaine and 4.18% for benzoylecgonine for concentrations of 75 and 350 ng/ml, respectively.     

Microassay for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine by high-performance liquid chromatography

An improved method for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine using reversed-phase high-performance liquid chromatography with ultraviolet detection is described. Following solid-phase extraction, chromatography was performed using a column containing an octadecylsilica-coated packing, eluted with 6% acetonitrile in phosphate buffer, pH 2.1, and detected at 233 nm. Using 80-microliters samples, the detection limit is 18 ng/ml for benzoylecgonine and benzoylenorecgonine and 35 ng/ml for cocaine and norcocaine. The coefficients of variation range from 3.5% (benzoylecgonine) to 7.0% (norcocaine). The procedure has been applied to samples of guinea pig plasma, urine and amniotic fluid and human urine.     

Analysis of cocaine and two metabolites in dried blood spots by liquid chromatography with fluorescence detection: a novel test for cocaine and alcohol intake

An original HPLC method coupled to spectrofluorimetric detection is presented for the simultaneous analysis in dried blood spots (DBS) of cocaine and two important metabolites, namely benzoylecgonine (its main metabolite) and cocaethylene (the active metabolite formed in the presence of ethanol). The chromatographic analysis was carried out on a C8 column, using a mobile phase containing phosphate buffer (pH 3.0)-acetonitrile (85:15, v/v). Native analyte fluorescence was monitored at 315 nm while exciting at 230 nm. A fast and feasible sample pre-treatment was implemented by solvent extraction, obtaining good extraction yields (>91%) and satisfactory precision values (RSD<4.8%). The method was successfully applied to DBS samples collected from some cocaine users, both with and without concomitant ethanol intake. The results were in good agreement with those obtained from plasma samples subjected to an original solid-phase extraction procedure on C8 cartridges. The method has demonstrated to be suitable for the monitoring of cocaine/ethanol use by means of DBS or plasma testing. Assays are in progress to apply this method on the street, for the control of subjects suspected of driving under the influence of psychotropic substances.     

Magnetic dispersive solid‐phase extraction based on modified magnetic nanoparticles for the detection of cocaine and cocaine metabolites in human urine by high‐performance liquid chromatography–mass spectrometry

An easy‐to‐handle magnetic dispersive solid‐phase extraction procedure was developed for preconcentration and extraction of cocaine and cocaine metabolites in human urine. Divinyl benzene and vinyl pyrrolidone functionalized silanized Fe₃O₄ nanoparticles were synthesized and used as adsorbents in this procedure. Scanning electron microscopy, vibrating sample magnetometry, and infrared spectroscopy were employed to characterize the modified adsorbents. A high‐performance liquid chromatography with mass spectrometry method for determination of cocaine and its metabolites in human urine sample has been developed with pretreatment of the samples by magnetic dispersive solid‐phase extraction. The obtained results demonstrated the higher extraction capacity of the prepared nanoparticles with recoveries between 75.1 to 105.7% and correlation coefficients higher than 0.9971. The limits of detection for the cocaine and cocaine metabolites were 0.09–1.10 ng/mL. The proposed magnetic dispersive solid‐phase extraction method provided a rapid, environmentally friendly and magnetic stuff recyclable approach and it was confirmed that the prepared adsorbents material was a kind of highly effective extraction materials for the trace cocaine and cocaine metabolites analyses in human urine.     

Non-invasive detection of cocaine dissolved in beverages using displaced Raman spectroscopy

We demonstrate the potential of Raman spectroscopy to detect cocaine concealed inside transparent glass bottles containing alcoholic beverages. A clear Raman signature of cocaine with good signal-to-noise was obtained from a ∼300 g solution of adulterated cocaine (purity 75%) in a 0.7 L authentic brown bottle of rum with 1 s acquisition time. The detection limit was estimated to be of the order of 9 g of pure cocaine per 0.7 L (∼0.04 moles L⁻¹) with 1 s acquisition time. The technique holds great promise for the fast, non-invasive, detection of concealed illicit compounds inside beverages using portable Raman instruments, thus permitting drug trafficking to be combated more effectively.     

Thin-layer chromatographic analysis of cocaine and benzoylecgonine in urine.

The sensitivity achieved by the described thin-layer chromatographic (TLC) method greatly exceeds that of previously published TLC methods for the determination of cocaine and its principal metabolite, benzoylecgonine, in urine. Sensitivity for cocaine and benzoylecgonine approaches 0.1 and 0.25 mug/ml, respectively, for a 5.0-ml specimen. A simple extraction with a mixed organic solvent provides the basic mechanism for isolating the drugs from biologic specimens. Cocaine and its metabolites are stable in sulfuric acid solutions but labile in aqueous media containing certain other inorganic and organic acids; therefore, an emphasis on the utilization of sulfuric acid solutions is employed throughout the procedure. An evaluation of sensitivities achieved for cocaine and benzoylecgonine by various detection reagents is presented. The technique is applicable to drug screening programs.     

Sensitive and selective cocaine electrochemical detection using disposable sensors

This paper describes the voltammetric determination of cocaine in presence of three different interferences that could be found in street samples using disposable sensors. The electrochemical analysis of this alkaloid can be affected by the presence of codeine, paracetamol or caffeine, whose oxidation peaks may overlap and lead to false positives. This work describes two different solutions to this problem. On one hand, the modification of disposable carbon sensors with carbon nanotubes allows the voltammetric quantification of cocaine by using ordinary least squares regressions in the concentration range from 10 to 155 μmol L⁻¹, with a reproducibility of 5.6% (RSD, n = 7. On the other hand, partial least squares regressions are used for the resolution of the overlapped voltammetric signals when using screen-printed carbon electrodes without any modification. Both procedures have been successfully applied to the evaluation of the purity of cocaine street samples.     

Micro-HPLC–UV analysis of cocaine and its adulterants in illicit cocaine samples seized by Austrian police from 2012 to 2017

The worldwide consumption of illicit drugs presents a big problem in terms of health care and prosecution. In the recent years, hundreds of novel psychoactive substances came up and were traded through the Internet, but there is still a big demand for classic illicit drugs such as cocaine, heroin, cannabis, and ecstasy. Among these, cocaine particularly is frequently altered not only with excipients but also with other physiologically active substances. The purpose of this work was to estimate the trend of cocaine purity and abundance of its adulterants in samples seized by Austrian police from 2012 to 2017. A micro-HPLC method for quantification of cocaine and its most common adulterants was developed and validated using gradient elution and UV detection at four wavelengths. A total of 110 cocaine samples were analyzed. In all the samples, cocaine was present as hydrochloric salt. Caffeine, procaine, levamisole, phenacetin, lidocaine, and benzocaine were the most abundant adulterants.     

Fast quantitative detection of cocaine in beverages using nanoextractive electrospray ionization tandem mass spectrometry

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.     

Determination of cocaine, benzoylecgonine and ecgonine methyl ester in plasma by reversed-phase high-performance liquid chromatography.

A combined assay is described for cocaine and its major metabolites, benzoylecgonine and ecgonine methyl ester. The method uses electrochemical and ultraviolet detectors in series. A non-silica column is used with high-pH mobile phase. The three compounds are completely separated from other cocaine metabolites. The assay has been suitable for pharmacokinetic studies of cocaine disposition in animal studies.