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 fast screening MALDI method for the detection of cocaine and its metabolites in hair

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry was used for the rapid detection of cocaine, benzoylecgonine and cocaethylene in hair. Different MALDI sample preparation procedures have been tested and the employment of a multi-layer 'graphite-sample-electrosprayed alpha-cyano-4-hydroxycinnamic acid (HCCA)' yielded the best results for standard solutions of the target analytes. The same approach was subsequently applied to hair samples that were known to contain cocaine, benzoylecgonine and cocaethylene, as determined by a classical GC-MS method. It was however necessary to extract hair samples by incubating them in methanol/trifluoroacetic acid for a short time (15 min) at 45 degrees C; 1 microl of the obtained supernatant was deposed on a metal surface treated with graphite, and HCCA was electrosprayed on it. This procedure successfully suppressed matrix peaks and was effective in detecting all the target analytes as their protonated species. The results obtained give further confirmation of the effectiveness of the MALDI for detecting drugs and their metabolites in complex biological matrices. The method can be useful as a fast screening procedure to detect the presence of cocaine and metabolites in hair samples.     

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.     

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.     

Cocaethylene,the in vivo product of cocaine and ethanol,is a narcotic more potent than its precursors

The molecular conformation and supramolecular architecture of cocaethylene [systematic name: ethyl (1R ,2R ,3S ,5S )‐3‐benzoyloxy‐8‐methyl‐8‐azabicyclo[3.2.1]octane‐2‐carboxylate], C₁₈H₂₃NO₄, have been determined for the first time. Cocaethylene is a narcotic produced in vivo when cocaine and ethanol are administered concomitantly. The intra‐ and intermolecular features of cocaethylene and its less potent narcotic precursor cocaine are very similar. The only molecular difference is in the conformation of the methyl group of the ethoxycarbonyl group. Similar to cocaine, the carboxylate atoms and the α‐C atom are coplanar in cocaethylene, but the methyl C atom of the ethyl group is bent by ca 90° away from this plane in the narcotic reported here. The main supramolecular motif is a one‐dimensional chain stabilized by weak C—H…O contacts.     

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.     

A validated positive chemical ionization GC/MS method for the identification and quantification of amphetamine, opiates, cocaine, and metabolites in human postmortem brain

A sensitive and specific method for the simultaneous detection and quantification of amphetamine, opiates, and cocaine and metabolites in human postmortem brain was developed and validated. Analytes of interest included amphetamine, morphine, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, ecgonine ethyl ester, cocaethylene, and anhydroecgonine methyl ester. The method employed ultrasonic homogenization of brain tissue in pH 4.0 sodium acetate buffer and solid phase extraction. Extracts were derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide and N,O-bis(trimethylsilyl) trifluoroacetamide. Separation and quantification were accomplished on a bench-top positive chemical ionization capillary gas chromatograph/mass spectrometer with selected ion monitoring. Eight deuterated analogs were used as internal standards. Limits of quantification were 50 ng/g of brain. Calibration curves were linear to 1000 ng/g for anhydroecgonine methyl ester and 6-acetylmorphine, and to 2000 ng/g for all other analytes. Accuracy across the linear range of the assay ranged from 90.2 to 112.2%, and precision, as percent relative standard deviation, was less than 16.6%. Quantification of drug concentrations in brain is a useful research tool in neurobiology and in forensic and postmortem toxicology, identifying the type, relative magnitude, and recency of abused drug exposure. This method will be employed to quantify drug concentrations in human postmortem brain in support of basic and clinical research on the physiologic, biochemical, and behavioral effects of drugs in humans.     

Determination of cocaine and cocaethylene in urine by solid-phase microextraction and gas chromatography-mass spectrometry

In order to evaluate recent cocaine exposure or its coingestion with ethanol, a simple and sensitive solid-phase microextraction (SPME) procedure for determination of cocaine and cocaethylene in urine was developed and validated. A polydimethylsiloxane fibre (100 microm) was submersed in the urine sample for 20 min under magnetic stirring after alkalinization with solid buffer (NaHCO(3):K(2)CO(3), 2:1). Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify the analytes in selected ion monitoring mode (SIM). The limits of quantification were 5.0 ng/mL for both analytes. Good inter- and intra-assay precision was also observed (coefficient of variation <9%).     

UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in oral fluid for DUID assessment

An ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry method for the direct analysis in oral fluid (OF) of several abused drugs and metabolites in a single chromatographic run was set up and validated. Amphetamine, methamphetamine, morphine, O-6-monoacetylmorphine, cocaine, codeine, methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine, methylenedioxyamphetamine, methadone, benzoylecgonine (BEG), Δ9-tetrahydrocannabinol (THC), ketamine, and cocaethylene were determined in a single chromatographic run with no sample pretreatment, after addition of the respective deuterated internal standards. The method was designed to perform a confirmation analysis on the residual OF samples after the preliminary on-site screening test, and it was applied on preservative buffers from different devices (Mavand Rapidstat, Concateno DDS, and Greiner Bio-One) or on neat OF samples. The method was suitable to be applied to the small amounts of sample available for the confirmatory analysis after the preliminary on-site screening or on undiluted OF samples. Limits of detection varied from 5 (morphine) to 0.2 ng/mL (methamphetamine, MDMA, BEG, and cocaethylene). The method was linear for all the substances involved, giving quadratic correlation coefficients of >0.99 in all the different preservative buffers checked. In addition, repeatability and accuracy were satisfactory for the majority of the substances, except for a few cases. The developed method was subsequently applied to 466 residual samples from on-site screening performed by police officers. Of these samples, 74 showed the presence of cocaine and metabolites; THC was detected in 49 samples. Two samples showed codeine and morphine while MDMA was detected in 11 samples and ketamine in four samples.     

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.     

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.     

Screening of cocaine and its metabolites in human urine samples by direct analysis in real-time source coupled to time-of-flight mass spectrometry after online preconcentration utilizing microextraction by packed sorbent

Microextraction by packed sorbent (MEPS) has been evaluated for fast screening of drugs of abuse with mass spectrometric detection. In this study, C8 (octyl-silica, useful for nonpolar to moderately polar compounds), ENV⁺ (hydroxylated polystyrene-divinylbenzene copolymer, for extraction of aliphatic and aromatic polar compounds), Oasis MCX (sulfonic-poly(divinylbenzene-co-N-polyvinyl-pyrrolidone) copolymer), and Clean Screen DAU (mixed mode, ion exchanger for acidic and basic compounds) were used as sorbents for the MEPS. The focus was on fast extraction and preconcentration of the drugs with rapid analysis using a time-of-flight (TOF) mass spectrometer as the detector with direct analysis in a real-time (DART) source. The combination of an analysis time of less than 1 min and accurate mass of the first monoisotopic peak of the analyte and the relative abundances of the peaks in the isotopic clusters provided reliable information for identification. Furthermore, the study sought to demonstrate that it is possible to quantify the analyte of interest using a DART source when an internal standard is used. Of all the sorbents used in the study, Clean Screen DAU performed best for extraction of the analytes from urine. Using Clean Screen DAU to extract spiked samples containing the drugs, linearity was demonstrated for ecgonine methyl ester, benzoylecgonine, cocaine, and cocaethylene with average ranges of: 65–910, 75–1100, 95–1200, and 75–1100 ng/mL (n = 5), respectively. The limits of detection (LOD) for ecgonine methyl ester, benzoylecgonine, cocaine, and cocaethylene were 22. 9 ng/mL, 23. 7 ng/mL, 4. 0 ng/mL, and 9.8 ng/mL respectively, using a signal-to-noise ratio of 3:1.     

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 norcocaine in plasma and cell cultures using high-performance liquid chromatography

A new simple high-performance liquid chromatographic (HPLC) method was developed for the determination of cocaine and norcocaine. Cocaine and norcocaine in biological samples were buffered to pH 9.0, extracted with diethyl ether and reextracted in a 0.1% aqueous solution of tetramethylammonium hydrogen sulfate (TMAHS) with a theoretical yield of extraction of 100%. The HPLC elution of cocaine and norcocaine was performed using a Spherisorb RP-18, 100 mm x 4.6 mm I.D., 5 microns particle size column with a mobile phase containing acetonitrile-0.1% TMAHS aqueous solution (60:40). The compounds were entirely separated, and a reliable limit of quantitation was set at 20 ng/ml when extracted from 0.5 ml of plasma. No interference with 26 other drugs was found. Cocaine and norcocaine stability studies showed that their half-lives in human plasma incubated at 37 degrees C were 50.8 and 43.2 min, respectively. In contrast, plasma from dogs or rats exhibited only weak or no enzymatic esterase activity towards cocaine and norcocaine resulting in less rapid degradation. Hydrolysis could be efficiently inhibited with sodium fluoride and prevented by storage of the sample at -20 degrees C. The highly sensitive assay also allowed the assessment of the oxidative metabolism pathway of cocaine to norcocaine in primary rat hepatocyte cultures.     

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.     

Simultaneous determination of opiates, methadone, amphetamines, cocaine, and metabolites in human placenta and umbilical cord by LC-MS/MS

LC-MS/MS methods for the quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, hydroxybenzoylecgonine, cocaethylene, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in human placenta and umbilical cord were developed and validated. Specimens (1?±?0.02 g) were homogenized with the Ultra-Turrax T8 disperser and centrifuged, and the supernatant was submitted to solid-phase extraction with Oasis MCX cartridges. Chromatographic separation was performed using an Atlantis T3 analytical column (100?×?2.1 mm, 3 μm) and a gradient of 0.1 % formic acid and acetonitrile. Selectivity was verified in 10 different blank specimens. The method was linear from 1–5 to 100–500 ng/g, depending on the analyte. Limits of detection and quantification ranged from 0.5 to 2.5 ng/g and 1 to 5 ng/g, respectively. Method imprecision was ≤15.3 %, except for MDMA at low quality control (18.1 %); accuracy, 87.1 to 114 %; extraction efficiency, 16.3 to 154.0 % (%CV?=?1.8-39.4 %); matrix effect, ?75.7 to 449.9 % (%CV?=?3.5–50 %); and process efficiency, 8.7 to 316.0 %. The method was applied to authentic placenta and umbilical cord specimens from drug-user pregnant women.     

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.     

Optimization of Sample Preparation and Instrumental Parameters for the Rapid Analysis of Drugs of Abuse in Hair samples by MALDI-MS/MS Imaging

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) has been employed to rapidly screen longitudinally sectioned drug user hair samples for cocaine and its metabolites using continuous raster imaging. Optimization of the spatial resolution and raster speed were performed on intact cocaine contaminated hair samples. The optimized settings (100 × 150 μm at 0.24 mm/s) were subsequently used to examine longitudinally sectioned drug user hair samples. The MALDI-MS/MS images showed the distribution of the most abundant cocaine product ion at m/z 182. Using the optimized settings, multiple hair samples obtained from two users were analyzed in approximately 3 h: six times faster than the standard spot-to-spot acquisition method. Quantitation was achieved using longitudinally sectioned control hair samples sprayed with a cocaine dilution series. A multiple reaction monitoring (MRM) experiment was also performed using the ‘dynamic pixel’ imaging method to screen for cocaine and a range of its metabolites, in order to differentiate between contaminated hairs and drug users. Cocaine, benzoylecgonine, and cocaethylene were detectable, in agreement with analyses carried out using the standard LC-MS/MS method.

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Graphical Abstract ?

     

Determination of different recreational drugs in hair by HS-SPME and GC/MS

A simple procedure combining headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC/MS) to detect and quantify amphetamines, ketamine, methadone, cocaine, cocaethylene and ∆⁹-tetrahydrocannabinol (THC) in hair is described. This procedure allows, in a single sample, even scant, analysis of drugs requiring different analytical conditions. A hair sample (10 mg) is washed and subjected to acidic hydrolysis. Then the HS-SPME is carried out (10 min at 90 °C) for amphetamines, ketamine, methadone, cocaine and cocaethylene. For derivatization of analytes, the fibre is introduced into the headspace of another closed vial containing acetic anhydride. After a chromatographic run, an alkaline hydrolysis for THC analysis is carried out in the same vial containing the hair sample previously used. For adsorption, the solid-phase microextraction needle is inserted into the headspace of the vial and the fibre is exposed for 30 min at 150 °C. For derivatization of analytes, the fibre is introduced into the headspace of another closed vial containing N-methyl-N-(trimethylsilyl)trifluoroacetamide. The GC/MS parameters were the same for both chromatographic runs. The linearity was proved to be between 0.01 and 10.00 ng/mg. The repeatability (intra- and interday precision) was below 10% as the coefficient of variation for all compounds. The accuracy, as the relative recovery, was 96.2–103.5% (spiked samples) and 88.6–101.7% (quality control sample). The limit of detection ranged from 0.01 to 0.12 ng/mg, and the limit of quantification ranged from 0.02 to 0.37 ng/mg. Application of the procedure to real hair samples is described. To the best of our knowledge, the proposed procedure combining HS-SPME and GC/MS is the first one be to successfully applied to the simultaneous determination of most of the common recreational drugs, including THC, in a single hair sample.     

Microfluidic chip based nano liquid chromatography coupled to tandem mass spectrometry for the determination of abused drugs and metabolites in human hair

A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R ² at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.