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.     

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.     

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.     

Surface-activated chemical ionization ion trap mass spectrometry for the analysis of cocaine and benzoylecgonine in hair after extraction and sample dilution

Surface-activated chemical ionization (SACI) was employed for the analysis of cocaine and its metabolite, benzoylecgonine, extracted from hair. Following decontamination and acid hydrolysis procedures on the hair sample, the sample solution was diluted (1:10) and directly analyzed by liquid chromatography/surface-activated chemical ionization multiple collisional stage single reaction monitoring mass spectrometry (LC/SACI-MS(3)-SRM) without solid-phase extraction (SPE) pre-purification and concentration procedures. To increase the selectivity of the method, MS(3) was chosen instead of the less selective MS/MS. This data was compared with that achieved using gas chromatography/mass spectrometry (GC/MS), the reference method used by the Italian Government Institute of Health protocol. The limits of detection (LODs) were 0.003 ng/(mg hair) for cocaine and 0.02 ng/(mg hair) for benzoylecgonine and the limits of quantitation (LOQs) were 0.01 ng/(mg hair) for cocaine and 0.04 ng/(mg hair) for benzoylecgonine. The squared correlation coefficient (R(2)) of the calibration curve was 0.9887-0.9980 for cocaine and 0.9987-0.9997 for benzoylecgonine. The percent accuracy error was 2-5% for both cocaine and benzoylecgonine using the LC/SACI-MS(3)-SRM approach, whereas it was higher for benzoylecgonine (20-25%) using the LC/SACI-MS/MS-SRM approach compared with the GC/MS data due to hair matrix contamination. In both cases, high precision was achieved (1-3% precision error), which confirmed the stability of the developed methods.     

Development and validation of an analytical method for the simultaneous determination of cocaine and its main metabolite, benzoylecgonine, in human hair by gas chromatography/mass spectrometry

A new, simple and rapid procedure has been developed and validated for the determination of cocaine and its main metabolite, benzoylecgonine, in human hair samples. After extraction from within the hair matrix by a mixture of methanol/hydrochloric acid (2:1) at 65 degrees C for 3 h, and sample cleanup by mixed-mode solid-phase extraction (SPE), the extracts were analyzed by gas chromatography/mass spectrometry (GC/MS), after derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide with 5% chlorotrimethylsilane. Using a sample size of only 20 mg of hair, limits of detection (LODs) and quantitation (LOQs) were, respectively, 20 and 50 pg/mg for cocaine, and 15 and 50 pg/mg for benzoylecgonine, achieving the cut-off values proposed by the Society of Hair Testing for the analysis of these compounds in hair. The method was found to be linear (weighing factor of 1/x) between the LOQ and 20 ng/mg for both compounds, with correlation coefficients ranging from 0.9974 to 0.9996 for cocaine; and from 0.9981 to 0.9994 for benzoylecgonine. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The sample cleanup step presented a mean absolute recovery greater than 90% for both compounds. The developed method may be useful in forensic toxicology laboratories for the analysis of cocaine and benzoylecgonine in hair samples, taking into account its speed (only 3 h are required for the extraction of the analytes from within the matrix, whereas 5 h or even overnight extractions have been reported) and the low limits achieved (using a single quadrupole mass spectrometer, which is available in most laboratories).     

Simultaneous analysis of cocaine and its metabolites in urine by capillary electrophoresis-electrospray mass spectrometry using a pressurized liquid junction nanoflow interface

A new method for the simultaneous separation of cocaine and four metabolites in urine by CE-ESI-MS via a pressurized nanoliquid junction interface was developed. The resolution of cocaine, cocaethylene, benzoylecgonine, norcocaine, and ecgonine methyl ester was achieved in a polyvinyl-alcohol-coated capillary with 75 μm id × 50 cm total length, using a 15 mM ammonium formate electrolyte solution (pH 9.5) in less than 15 min. In addition, to enhance sensitivity, a field-amplified sample injection (FASI) was evaluated in terms of injection time and sample solvent composition. The limits of detection achieved with the FASI method ranged from 1.5 to 10 ng/mL for all the compounds. The detection of the studied compounds was performed using an ion-trap mass spectrometer in a positive ionization mode. A mixture of methanol:water (80:20 v/v) containing 0.1% v/v of formic acid was employed as spray liquid and delivered at ~200 nL/min. Under optimal CE-MS conditions, linearity was assessed in the concentration range of interest for all analytes with correlation coefficients r2 ≥ 0.9913. Intra- and inter-day precision provided a relative standard deviation lower than 1.54% for migration times and lower than 12.15% for peak areas. Finally, urine samples, spiked with the standard mixture, were extracted using a solid-phase extraction procedure and injected under FASI conditions, providing recoveries from 80% to 94% for all analytes.     

Determination of cocaine and its metabolites using cation-selective exhaustive injection and sweeping-MEKC

We have employed a high-sensitivity on-line preconcentration method, cation-selective exhaustive injection (CSEI) and sweeping MEKC, for the analysis of cocaine, benzoylecgonine, norcocaine, and cocaethylene. We monitored the effects of several of the CSEI-sweeping-MEKC parameters - including the pH, the concentrations of SDS and organic modifier, the injection length of the high-conductivity buffer, and the injection time of the sample - to optimize the separation process. The optimal BGE was 100 mM phosphoric acid (pH 1.8) containing 75 mM SDS with 10% 2-propanol and 10% tetrahydrofuran as the organic modifier. In addition, electrokinetic injection of the sample at 15 kV for 900 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancements for cocaine, norcocaine, and cocaethylene ranged from 2.06 x 10(4) to 3.96 x 10(4); for benzoylecgonine it was 1.75 x 10(3); the coefficients of determination exceeded 0.9958. The LODs, based on an S/N ratio of 3:1, of sweeping-MEKC ranged from 33.5 to 52.8 ng/mL; in contrast, when using CSEI-sweeping-MEKC the sensitivity increased to range from 29.7 to 236 pg/mL. Under the optimal conditions, we analyzed cocaine in a human urine sample prepared using off-line SPE to minimize the influence of the matrix. The recovery of the SPE efficiency was satisfactory (ca. 74.9-87.6%). Our experimental results suggest that, under the optimal conditions, the CSEI-sweeping-MEKC method can be used to determine cocaine and its metabolites with high sensitivity in human urine.     

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.     

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.     

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.     

Profiling of cocaine by micellar electrokinetic chromatography

The potential of micellar electrokinetic chromatography (MEKC) for the profiling of cocaine samples is described. An MEKC system containing sodium dodecyl sulfate (SDS) and methanol was optimized using a test mixture of cocaine, its common impurities (benzoylecgonine, norcocaine, tropacocaine, and trans-cinnamoylcocaine), and several degradation products. The effect of pH, percentage modifier, and concentration surfactant on the separation has been investigated. The optimal separation buffer for cocaine samples consisted of 75 mM SDS, 17.5% methanol, and 25 mM borate (pH 8.3) and was well suited to separate components of diverse polarity in one run. Various cocaine seizures have been analyzed with the MEKC system and their signatures were compared. The electrokinetic chromatograms obtained were characteristic, and differences and similarities among the samples could easily be observed. Several impurities were identified in the samples by means of migration times and comparison of recorded and library UV spectra. The composition of the samples was determined semiquantitatively using relative corrected peak areas.     

Direct tandem mass spectrometry for the simultaneous assay of opioids,cocaine and metabolites in dried urine spots

A micro-analytical method based on spotting urine samples (20 μL) onto blood/urine spot collection cards followed by air-drying and extraction (dried urine spot, DUS) was developed and validated for the screening/confirmation assay of morphine, 6-methylacetylmorphine (6-MAM), codeine, cocaine and benzoylecgonine (BZE). Acetonitrile (3 mL) was found to be a useful solvent for target extraction from DUSs under an orbital-horizontal stirring at 180 rpm for 10 min. Determinations were performed by direct electrospray ionization tandem mass spectrometry (ESI-MS/MS) under positive electrospray ionization conditions, and by using multiple reaction monitoring (MRM) with one precursor ion/product ion transition for the identification and quantification (deuterated analogs of each target as internal standards) of each analyte. The limits of detection of the method were 0.26, 0.94, 1.5, 1.1, and 2.0 ng mL⁻¹, for cocaine, BZE, codeine, morphine and 6-MAM, respectively; whereas, relative standard deviations of intra- and inter-day precision were lower than 8 and 11%, respectively, and intra- and inter-day analytical recoveries ranged from 94 ± 4 to 105 ± 3%. The small volume of urine required (20 μL), combined with the simplicity of the analytical technique makes it a useful procedure for screening/quantifying drugs of abuse. The method was successfully applied to the analysis of urine from polydrug abusers.     

Porous membrane-protected molecularly imprinted polymer micro-solid-phase extraction for analysis of urinary cocaine and its metabolites using liquid chromatography – Tandem mass spectrometry

Porous membrane-protected micro-solid phase extraction (μ-SPE) using a molecularly imprinted polymer (MIP) as an adsorbent has been proposed as an integrated extraction-cleanup procedure for isolating cocaine (COC) and its metabolites [benzoylecgonine (BZE), ecgonine methyl ester (EME), and cocaethylene (CE)] from human urine. MIP beads have been synthesized using COC as a template molecule, ethylene dimethacrylate (EDMA) as a functional monomer, divinylbenzene (DVB) as a cross-linker, and 2,2′-azobisisobutyronitrile (AIBN) as an initiator. High performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) has been used for quantifying the analytes after MIP-μ-SPE. Variables such as urine pH, adsorption temperature and time, mechanical (orbital-horizontal) stirring; and composition of the eluting solution and eluting time, were evaluated. The proposed method was shown to be precise and accurate [relative standard deviations (RSDs) of intra- and inter-day tests ranging from 3 to 8% and from 2 to 10%, respectively]; and analytical recoveries in the range of 89–100%). In addition, excellent accuracy was also verified after analyzing a FDT +25% control material for BZE. The detection limits were in the range of 0.16–1.7 ng L⁻¹, low enough for confirmative conclusions regarding cocaine abuse. The method was finally applied for screening/quantifying cocaine and metabolites in urine samples from poly-drug abusers.     

Analysis of cocaine and benzoylecgonine in urine by using multisyringe flow injection analysis‐gas chromatography‐mass spectrometry system

In this paper, a method was described to determine cocaine (COC) and benzoylecgonine (BZE) in human urine samples by GC‐MS detection. The extraction of analytes from urine samples was achieved in an Oasis hydrophilic–lipophilic balance column (20 mm×3.9 mm id, dp=25 μm; Waters, USA), incorporated in a multisyringe flow injection system, used for the sample treatment. Finally, to improve the volatility of the BZE, an in‐line derivatization reaction with N,O‐bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane was made microwave‐assisted in order to reduce the reaction time. The results showed that the proposed method is a good alternative for the analysis of COC and BZE in urine samples because it offers advantages compared with those described in the literature, which include simplicity in the sample treatment, the sensitivity and selectivity necessary to determine the analytes of interest at low levels in the urine and high sample throughput.     

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%.     

Screening of Benzoylecgonine in Urine by Cyclobond Solid Phase Extraction and High Performance TLC

Abstract

A method is described for screening of the cocaine metabolite benzoylecgonine in urine at 0.2 μ/ml using a Cyclobond cyclodex^?trin solid phase extraction cartridge and high performance thin layer chromatography. Results are compared with those obtained using liquid-liquid and diatomaceous earth column extraction and C-18 solid phase extraction.     

Potentiometric detection in UPLC as an easy alternative to determine cocaine in biological samples

The analytical methods which are often used for the determination of cocaine in complex biological matrices are a prescreening immunoassay and confirmation by chromatography combined with mass spectrometry. We suggest an ultra‐high‐pressure liquid chromatography combined with a potentiometric detector, as a fast and practical method to detect and quantify cocaine in biological samples. An adsorption/desorption model was used to investigate the usefulness of the potentiometric detector to determine cocaine in complex matrices. Detection limits of 6.3 ng mL^?1 were obtained in plasma and urine, which is below the maximum residue limit (MRL) of 25 ng mL^?1. A set of seven plasma samples and 10 urine samples were classified identically by both methods as exceeding the MRL or being inferior to it. The results obtained with the UPLC/potentiometric detection method were compared with the results obtained with the UPLC/MS method for samples spiked with varying cocaine concentrations. The intraclass correlation coefficient was 0.997 for serum (n =7) and 0.977 for urine (n =8). As liquid chromatography is an established technique, and as potentiometry is very simple and cost‐effective in terms of equipment, we believe that this method is potentially easy, inexpensive, fast and reliable. Copyright © 2014 John Wiley & Sons, Ltd.     

Analysis of anabolic steroids in hair by GC/MS/MS

A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) method is described for the detection of anabolic steroids, usually found in keratin matrix at very low concentrations. Hair samples from seven athletes who spontaneously reported their abuse of anabolic steroids, and in a single case cocaine, were analyzed for methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl derivative and finally analyzed by GC/MS/MS. For detection of cocaine and benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at 56 degrees C then overnight in a thermostatic bath at the same temperature. After the incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with the athletes' reports, confirming that hair is a valid biological matrix to establish long-term intake of drugs.     

Transformation of cocaine during water chlorination

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