Determination of cocaine and its metabolites in brain tissue using high-flow solid-phase extraction columns and high-performance liquid chromatography.

A new solid-phase extraction procedure for the determination of cocaine and some of its metabolites in brain tissue, using high-flow co-polymeric sorbents is reported as a substantial improvement on our recently reported procedure. The recovery of cocaine, norcocaine and cocaethylene was excellent as was the reproducibility of the extraction. The use of high-flow sorbents allowed the easy extraction of tissue without the need for a time-consuming lipase digestion, regardless of sample viscosity or physical nature. The use of these solid-phase columns provided many advantages over the more commonly used solvent extraction, including an increase in extraction speed and efficiency, reduced operator time, reduced solvent use and disposal volumes and exceptional extract quality. The procedure was successfully applied to rabbit brains spiked with cocaine, benzoylecgonine, norcocaine and cocaethylene.     

A rapid method for the determination of cocaine in brain tissue.

A rapid procedure is described for the extraction and analysis of brain samples for cocaine and benzoylecgonine. Human brain tissue was sectioned at autopsy, and samples were subjected to a lipase digestion, subsequent to solid-phase extraction. The distribution of cocaine and benzoylecgonine throughout different regions of the brain was determined by high-performance liquid chromatography.     

The determination of cocaine and its major metabolite, benzoylecgonine, in postmortem fluids and tissues by computerized gas chromatography/mass spectrometry

An analytical procedure for the simultaneous determination of both cocaine and benzoylecgonine in postmortem fluid and tissue samples has been developed by using computerized gas chromatography/mass spectrometry and gas chromatography using a nitrogen/phosphorus (N/P) detector. Both methods are accurate and sensitive and allow the determination of tissue concentrations of cocaine and benzoylecgonine as low as 0.015 microgram/mL.     

Segmental analysis for cocaine and metabolites by HPLC in hair of suspected drug overdose cases

Hair samples of eight postmortem cases were analyzed in segments of 1 to 3 cm for cocaine, benzoylecgonine and cocaethylene. Samples were prepared for analysis by digestion in 0.1 M HCl and subsequent extraction with mixed-mode solid-phase extraction columns. Measurement was made by reversed-phase, narrow-bore HPLC and fluorescence detection using two laboratory-made internal standards. The concentrations were in the region of 0.29-316 ng/mg of hair for cocaine, 0.43-141 ng/mg of hair for benzoylecgonine and 0.93-1.83 ng/mg of hair for cocaethylene. All eight investigated cases had cocaine-positive segments. In six of the cases, all segments were positive, suggesting regular cocaine use and two showed in-between negative segments indicating an interruption or a change of the abuse intensity. The results showed a second, remarkable observation, i.e. enormous concentration differences (factor >150) for both cocaine and benzoylecgonine between the different subjects. Furthermore, interindividual cocaine/benzoylecgonine ratios ranged from 0.02 to 8.43. We believe these observations could in part be attributed to both some of the still existing limitations in the analytical approach(es), especially the mandatory hair washing steps, and in our still too limited knowledge of the hair incorporation processes. Nevertheless, in some cases, segmental analysis proved to be an important tool to distinguish, together with postmortem examination, deadly chronic abuse from single acute drug overdosage.     

Segmental analysis for cocaine and metabolites by HPLC in hair of suspected drug overdose cases

Hair samples of eight postmortem cases were analyzed in segments of 1 to 3 cm for cocaine, benzoylecgonine and cocaethylene. Samples were prepared for analysis by digestion in 0.1 M HCl and subsequent extraction with mixed-mode solid-phase extraction columns. Measurement was made by reversed-phase, narrow-bore HPLC and fluorescence detection using two laboratory-made internal standards. The concentrations were in the region of 0.29–316 ng/mg of hair for cocaine, 0.43–141 ng/mg of hair for benzoylecgonine and 0.93–1.83 ng/mg of hair for cocaethylene. All eight investigated cases had cocaine-positive segments. In six of the cases, all segments were positive, suggesting regular cocaine use and two showed in-between negative segments indicating an interruption or a change of the abuse intensity. The results showed a second, remarkable observation, i.e. enormous concentration differences (factor >150) for both cocaine and benzoylecgonine between the different subjects. Furthermore, interindividual cocaine/benzoylecgonine ratios ranged from 0.02 to 8.43. We believe these observations could in part be attributed to both some of the still existing limitations in the analytical approach(es), especially the mandatory hair washing steps, and in our still too limited knowledge of the hair incorporation processes. Nevertheless, in some cases, segmental analysis proved to be an important tool to distinguish, together with postmortem examination, deadly chronic abuse from single acute drug overdosage.     

Optimized conditions for simultaneous determination of opiates, cocaine and benzoylecgonine in hair samples by GC--MS

The present paper describes a qualitative and quantitative method for the simultaneous detection of opiates, cocaine and benzoylecgonine from human hair samples. Every step of the analytical procedure was studied to find the optimized conditions. Nine different incubation systems were examined. The influence of different pH values of samples on the isolation of analytes from the incubation media by Bond Elut cartridges and the stability of the compounds of interest in the different incubation media and conditions were investigated. The extracting power of different incubation media was studied as well. The phosphate buffer 0.1 N at pH 5 was chosen as the extraction medium in an optimized procedure for simultaneous determination of opiates, cocaine and benzoylecgonine in hair samples. The method developed was validated. Recoveries were 90% for morphine (M), 81% for 6-monoacetylmorphine (6-AM), 90% for codeine (CD), 86% for cocaine (C) and 90% for benzoylecgonine (BE). Relative standard deviation for inter-day precision was better than 12%. The limits of detection resulted as 0.05 ng/mg for M and C, as 0.08 for 6-AM and as 0.2 ng/mg for BE. Forty hair samples collected from drug abusers admitted to centers for detoxification treatment were analyzed obtaining 23 positive results for opiates and/or cocaine. Twelve hair specimens longer than 10 cm were analyzed following a sectional approach. In the six positive cases, it was interesting to find that the 6-AM/M ratio generally decreased for each sample from the proximal segment to the distal segments. Moreover, the 6-AM/M ratio was generally lower than 1 in the intermediate and distal segments.     

Validation of a method to detect cocaine and its metabolites in nails by gas chromatography-mass spectrometry

The objective of the present work was to compare previously published methods and provide validation data to detect simultaneously cocaine (COC), benzoylecgonine (BE) and norcocaine (NCOC) in nail. Finger and toenail samples (5mg) were cut in very small pieces and submitted to an initial procedure for external decontamination. Methanol (3 ml) was used to release analytes from the matrix. A cleanup step was performed simultaneously by solid-phase extraction (SPE) and the residue was derivatized with pentafluoropropionic anhydride/pentafluoropropanol (PFPA/PFP). Gas chromatography-mass spectrometry (GC-MS) was used to detect the analytes in selected ion monitoring mode (SIM). Confidence parameters of validation of the method were: recovery, intra- and inter-assay precision, as well as limit of detection (LOD) of the analytes. The limits of detection were: 3.5 ng/mg for NCOC and 3.0 ng/mg for COC and BE. Good intra-assay precision was observed for all detected substances (coefficient of variation (CV)<11%). The inter-assay precision for norcocaine and benzoylecgonine were <4%. For intra- and inter-assay precision deuterated internal standards were used. Toenail and fingernail samples from eight declared cocaine users were submitted to the validated method.     

HPLC-DAD determination of opioids, cocaine and their metabolites in plasma

High performance liquid chromatography with diode array detection (HPLC-DAD) was used to develop a method for the simultaneous determination of morphine, codeine, 6-acetylmorphine (6AM), cocaine, benzoylecgonine (BEG), cocaethylene, methadone and its metabolite, 2-ethylidene-1,5-dimethyldiphenylpyrrolidine (EDDP), in plasma. Following solid-phase extraction with Bond Elut Certify cartridges, chromatography was performed on an X-Terra RP8 column (250 mm x 4.6 mm i.d., 5 microm particle size), using acetonitrile-phosphate buffer pH 6.53 as mobile phase and elution in the gradient mode. The detector response was linear at concentrations over the range 0.1-10 microg/mL in plasma, and the correlation coefficients for the eight drugs studied were all higher than 0.99. The average extraction recoveries from plasma ranged from 60% for BEG to 95% for methadone. The precision was acceptable, with coefficients of variation oscillating between 2.55% and 6.45%. The accuracy was found to be within satisfactory limits (+/- 8.1%). Finally, the method was applied to 21 plasma samples from fatal overdoses, obtaining positive results for two or more drugs.     

Brain concentrations of cocaine and benzoylecgonine in fatal cases

Since cocaine in blood rapidly hydrolyzes to benzoylecgonine, cocaine concentrations determined in postmortem blood may not reflect the presence or concentration of cocaine in the body at the time of death. The interpretative value of the determination of cocaine and benzoylecgonine in brain tissue was investigated. Cocaine and benzoylecgonine were quantitated by coextraction and formation of the propyl derivative of benzoylecgonine followed by selected ion monitoring gas chromatography/mass spectrometry (GC/MS) using electron ion impact ionization. Cocaine and benzoylecgonine were found to be evenly distributed throughout the brain. Cocaine and benzoylecgonine concentrations were stable in frozen brain tissue (-4 degrees C) on reanalysis after 1 to 3 months of storage, and in refrigerated tissue (10 degrees C) after 30 days of storage. Blood, brain, and liver concentrations of cocaine and benzoylecgonine in 37 cocaine overdose cases and 46 cases in which cocaine was incidental to the cause of death were reviewed. The ratios of cocaine/benzoylecgonine in the toxic cases (brain mean 14.7 and blood mean 0.64) were clearly different from those found in the incidental cases (brain mean 0.87 and blood mean 0.27). The brain/blood ratios of cocaine and benzoylecgonine concentrations generally were characteristic of the time elapsed since cocaine dosing. In cocaine overdose cases, the mean ratio was 9.6 for cocaine and 0.36 for benzoylecgonine. These are within the range found in animal studies for brain/blood ratios of cocaine and benzoylecgonine 0.5 to 2 h after cocaine administration. In incidental cases, the brain/blood ratios were mean 2.5 for cocaine and 1.4 for benzoylecgonine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of cocaine and its polar transformation products and metabolites in human urine

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 μg/ml) and ecgonine methyl ester (0–177 μg/ml) were substantial and averaged about one tenth the concentrations of benzoylecgonine (0–1074 μg/ml) and cocaine (0–1221 μg/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.     

Testing human hair for drugs of abuse. IV. Environmental cocaine contamination and washing effects

Active cocaine use results in sequestration of parent drug in hair. In addition, hair has unique physicochemical properties that permit absorption of cocaine from the environment. When hair is tested for evidence of cocaine, it is important to consider whether the positive test resulted from active drug use or environmental contamination. In a series of laboratory experiments, it was found that exposure of ‘cut’ hair to cocaine vapor (‘crack’ smoke) and to aqueous solutions of cocaine hydrochloride resulted in significant contamination of hair samples. Similar results were obtained with two subjects who were exposed to cocaine vapor in an unventilated room. The amount of contamination adsorbed by hair depended upon both time and extent of exposure. Washing the hair samples with methanol removed >70% of the cocaine contaminant after cocaine vapor exposure, but was less effective (<50%) following contamination with aqueous cocaine. Shampoo treatment cycles (overnight soaking) progressively removed increasing amounts of cocaine from the contaminated hair, but residual cocaine remained after 10 cycles. Studies were also performed to determine the usefulness of benzoylecgonine as a marker of active cocaine administration. Small amounts of benzoylecgonine (ca. 1 ng/mg) were formed in hair as a result of environmental contamination with cocaine. Also, it was found that benzoylecgonine could be adsorbed from illicit cocaine contaminated with benzoylecgonine. It was concluded that positive hair test results should be interpreted cautiously due to the possibility of environmental contamination from cocaine and related constituents.     

可卡因及其代谢物苯甲酰爱康宁胶体金检测试剂盒的研制

目的根据胶体金免疫层析的原理,建立一种可以在现场快速检测可卡因及其代谢物苯甲酰爱康宁的方法。方法将胶体金标记的抗可卡因单克隆抗体均匀浸涂在玻璃纤维膜上,用点膜仪将可卡因的完全抗原以及羊抗鼠多抗在硝酸纤维素膜上划线,分别作为可卡因的检测区（T）和质控区（C）。并且对样品垫用0.2%Tween 20和0.5%PVA封闭处理。样本（尿样、唾液、血样等）中游离的可卡因及其代谢物同包被的完全抗原分别免疫竞争结合胶体金标记的抗可卡因单抗。肉眼观察试剂盒的质控区和检测区上有无紫红色带来判读结果。结果可卡因胶体金检测试剂盒对65种毒品、药物的特异性测试中仅识别可卡因及其代谢物。对人体尿样中的可卡因和苯甲酰爱康宁检测阈值均为300ng/mL。与GC/MS对照试验,其准确性分别为98.9%和98.6%。在常温下能稳定保存2年。结论该可卡因检测试剂盒在5min内可以检测出样本中的可卡因及其代谢物成分。     

Rapid confirmation of enzyme multiplied immunoassay technique (EMIT) cocaine positive urine samples by capillary gas-liquid chromatography/nitrogen phosphorus detection (GLC/NPD)

A rapid gas-liquid chromatographic (GLC) method was developed for the confirmation of benzoylecgonine (BE) positive urine samples screened by the enzyme multiplied immunoassay technique (EMIT) assay. The procedure is performed by solvent extraction of BE from 0.1 or 0.2 mL of urine, followed by an aqueous wash of the solvent and evaporation. The dried residue was derivatized with 50 microL of pentafluoropropionic anhydride and 25 microL of pentafluoropropropanol at 90 degrees C for 15 min. The derivatizing reagents were evaporated to dryness, and the derivatized BE, and cocaine if present, were reconstituted and injected into the gas chromatograph. The column was a 15-m by 0.2-mm fused silica capillary column, coated with 0.25 micron of DB-1, terminating in a nitrogen phosphorus detector (NPD). Cocaine and the pentafluoro BE derivatives retention times were 3.2 and 2.6 min, respectively. Nalorphine was used as reference or internal standard with a retention time of 4.78 min. The complete procedure can be performed in approximately 1.5 h. The EMIT cutoff between positive and negative urine samples is 300 ng/mL of BE. The lower limit of sensitivity of this method is 25 ng of BE extracted from urine. Validation studies resulted in confirmation of 101 out of 121 EMIT cocaine positive urine samples that could not be confirmed by thin-layer chromatography (TLC). This represents 84% confirmation efficiency.     

High-performance liquid chromatography with column switching for the determination of cocaine and benzoylecgonine concentrations in vitreous humor

A sample concentration technique was adapted for the determination of cocaine and benzoylecgonine (BE) concentrations in vitreous humor. Vitreous humor (0.5 mL) was diluted 1:1 with water and applied through a filter onto a 3-cm preconcentration column. Following a simple wash step, the analytes were flushed directly onto a reversed-phase analytical high-performance liquid chromatography (HPLC) system. Absolute recoveries were high (above 90%) and the chromatograms were free from interference. Analysis for the drug and its breakdown product was performed using ultraviolet (UV) visible photodiode array detection, which allowed confirmation of peak identity. Recognizable UV spectra could be measured with as little as 20 ng on column. Comparison of the drug levels in 27 blood and vitreous humor samples showed that, while there was only a low correlation between the blood and vitreous concentrations (R = 0.70), vitreous cocaine and BE determinations were good indicators of antemortem cocaine use. In almost all cases, the vitreous BE concentrations were higher than the cocaine concentrations. The technique was easy to perform and the vitreous samples were especially compatible with this low-labor analytical procedure.     

Levels of cocaine and its metabolites in washed hair of demonstrated cocaine users and workplace subjects

In a study of subjects in drug rehabilitation programs, cocaine and cocaine metabolite levels were determined in the hair of 75 subjects who had produced cocaine-positive urine results. The hair was analyzed after being washed with the 3.75 h wash procedure developed by this laboratory. In addition, results of testing 73 non-users are presented, as well as levels of cocaine, benzoylecgonine (BE), cocaethylene, and norcocaine from workplace population samples. The data support a recommendation of reporting as positive a sample with cocaine of 500 pg/mg hair and either a 5% ratio of benzoylecgonine (BE) to cocaine in samples, or the presence of cocaethylene at 50 pg/mg hair, or norcocaine at 50 pg/mg hair for samples < or =2000 pg cocaine/mg hair. For samples with cocaine present at >2000 pg/mg hair, the data indicate that a ratio of 5% BE may be an overly conservative approach. In appropriately washed hair samples, cocaine users can produce hair levels of <5% BE and thus a minimum BE cutoff in lieu of a ratio could be considered.     

Development and Validation of a Method Using Dried Oral Fluid Spot to Determine Drugs of Abuse

A liquid chromatography–mass spectrometry method using dried oral fluid spots was developed and validated for the simultaneous quantification of cocaine, benzoylecgonine, cocaethylene, amphetamine, and 3,4‐methylenedioxymethamphetamine. The oral fluid was applied to a Whatman 903 grade paper and submitted to a drying time of 2.5 h. The extraction procedure was optimized by chemometric approach using simplex centroid design. Spots were extracted with a mixture of acetonitrile, buffer, and methanol. Calibration curves covered a linear concentration range of 40–500 ng/mL. Validation parameters of linearity, precision, accuracy, selectivity, carryover, matrix effects, and stability were evaluated and showed satisfactory results. Spot homogeneity was also satisfactory, with less than 15% of deviation from nominal concentration. Spot volume did not influence accuracy when less than 100 μL of the sample was applied to the spot. The validation of the proposed method suggests a potential application in different scenarios in toxicology.     

Aqueous phase hexylchloroformate derivatization and solid phase microextraction: determination of benzoylecgonine in urine by gas chromatography-quadrupole ion trap mass spectrometry.

A derivatization/solid phase microextraction (SPME) method for the determination of benzoylecgonine in urine was developed. The derivatization is conducted directly in 1 mL of urine while sonicating for 3 min with 12 microL of hexyl chloroformate and 70 microL of a mixture containing acetonitrile:water:hexanol:2-dimethylaminopyridine (5:2:2:1 v/v), yielding benzoylecgonine hexyl ester (BHE) as the product. After the 3 min period, an aliquot of 250 microL is transferred to a vial for SPME. After the desired extraction time the 100 microns polydimethylsiloxane SPME fiber was transferred to the GC-MS for separation and analysis with a quadrupole ion trap mass spectrometer. The hexyl chloroformate derivatization and SPME procedures were optimized for compatibility and sensitivity. The method was found linear for 0.10 to 20.0 micrograms/mL (r2 = 0.999) of benzoylecgonine in urine using benzoylecgonine-d3 as an internal standard (1.5 micrograms/mL). Intra-day precisions were 8.8 and 6.8% RSD for 0.30 microgram/mL and 17 micrograms/mL benzoylecgonine standards in urine (n = 6), respectively. Inter-day precision (n = 3) were < or = 3.3% RSD, indicating good reproducibility. A detection limit of 0.03 microgram/mL (S/N = 3) was achieved, thus making the SPME method a simplified alternative to SPE for GC-MS confirmation after EMIT tests for benzoylecgonine which have a cutoff of 0.30 microgram/mL. Quantitative results by SPME and SPE of two clinical urine specimens known positive for cocaine by EMIT were in excellent agreement. Benzoylecgonine was detected by the derivatization/SPME method in 22 out of 22 other urine specimens known positive for cocaine.     

A rapid method for the extraction of cocaine and benzoylecgonine from body fluids

A method has been developed for the rapid extraction of cocaine and benzoylecgonine from post-mortem blood and urine samples. Solid phase columns containing C8 packing material gave clean, emulsion free extracts from body fluids. The total time for each extraction was approx. 10 min and the system has the capacity for the simultaneous extraction of up to 10 samples. Urine can be extracted directly, blood samples were sonicated before extraction to allow for their easy passage through the columns. The method gave excellent recoveries of cocaine (98-100%) from spiked samples at concentrations of between 50 ng/ml and 10 micrograms/ml. Analysis of the extracts was by high performance liquid chromatography (HPLC).     

Validity testing of commercial urine cocaine metabolite assays: I. Assay detection times, individual excretion patterns, and kinetics after cocaine administration to humans

A validity study of eight commercial urine assays for detection of cocaine metabolite was performed on clinical specimens collected from human subjects who received single 20-mg intravenous doses of cocaine hydrochloride. The specimens were collected under controlled conditions and analyzed in random order under blind conditions. Benzoylecgonine concentration in each specimen also was determined by gas chromatography/mass spectrometry (GC/MS). Mean times of detection of the last positive specimen (greater than or equal to 300 ng/mL of benzoylecgonine equivalents) after cocaine administration varied among seven of the commercial tests from 16.9 to 52.9 h in the following ascending order: Toxi-Lab less than TDx = EMIT dau = EMIT st less than Abuscreen less than Coat-A-Count = Double Antibody. In contrast, a commercial spot test (KDI Quik Test) which was evaluated for detection of cocaine metabolite produced both false positives and false negatives for benzoylecgonine and was not considered to be a valid test for detection of cocaine metabolite. Half-lives of excretion of benzoylecgonine among four subjects varied from 5.9 to 7.9 h, and overall recovery of benzoylecgonine varied from 15.0 to 34.3% of the administered dose of cocaine.