Simple and Sensitive Molecularly Imprinted Polymer - Mn-Doped ZnS Quantum Dots Based Fluorescence Probe for Cocaine and Metabolites Determination in Urine.

A new molecularly imprinted polymer (MIP)-based fluorescent artificial receptor has been prepared by anchoring a selective MIP for cocaine (COC) on the surface of polyethylene glycol (PEG) modified Mn-doped ZnS quantum dots (QDs). The prepared material combines the high selectivity attributed to MIPs and the sensitive fluorescent property of the Mn-doped ZnS QDs. Simple and low cost methods have therefore been optimized for assessing cocaine abuse in urine by monitoring the fluorescence quenching when the template (COC) and also metabolites from COC [benzoylecgonine (BZE) and ecgonine methyl ester (EME)] are present. Fluorescence quenching was not observed when performing experiments with other drugs of abuse (and their metabolites) or when using nonimprinted polymer (NIP)-coated QDs. Under optimized operating conditions (1.5 mL of 200 mg L(-1) MIP-coated QDs solution, pH 5.5, and 15 min before fluorescence scanning) two analytical methods were developed/validated. One of the procedures (direct method) consisted of urine sample 1:20 dilution before fluorescence measurements. The method has been found to be fast, precise, and accurate, but the standard addition technique for performing the analysis was required because of the existence of matrix effect. The second procedure performed a solid phase extraction (SPE) first, avoiding matrix effect and allowing external calibration. The limits of detection of the methods were 0.076 mg L(-1) (direct method) and 0.0042 mg L(-1) (SPE based method), which are lower than the cutoff values for confirmative conclusions regarding cocaine abuse.

Solid phase microextraction and gas chromatography-mass spectrometry methods for residual solvent assessment in seized cocaine and heroin.

A simple sample pre-treatment method based on solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) has been optimized and validated for the assessment of 15 residual solvents (2-propanol, 2-methylpentane, 3-methylpentane, acetone, ethyl acetate, benzene, hexane, methylcyclohexane, methylcyclopentane, m-xylene, propyl acetate, toluene, 1,2,4-trimethylbenzene, dichloromethane, and ethylbenzene) in seized illicit cocaine and heroin. DMSO and DMF as sample diluents were found to offer the best residual solvent transference to the head space for further adsorption onto the SPME fiber, and the developed method therefore showed high sensitivity and analytical recovery. Variables affecting SPME were fully evaluated by applying an experimental design approach. Best conditions were found when using an equilibration time of 5 min at 70 °C and headspace sampling of residual solvents at the same temperature for 15 min. Method validation, performed within the requirements of international guidelines, showed excellent sensitivity, as well as intra- and inter-day precision and accuracy. The proposed methodology was applied to 96 cocaine samples and 14 heroin samples seized in Galicia (northwestern Spain) within 2013 and 2014.

Determination of direct alcohol markers: a review.

Alcohol is the most popular legal drug used in our society today, and its consumption by pregnant women remains an important public health problem. Gestational alcohol consumption can result in a continuum of adverse fetal outcomes known as fetal alcohol spectrum disorder (FASD). Effective strategies are needed to prevent the increasing adoption of risky drinking behaviors. Because ethanol itself is only measurable for a few hours after ethanol intake in conventional matrices including blood, urine, and sweat, these matrices are only useful to detect recent ethanol exposure. Since approximately early 2000, the non-oxidative ethanol metabolites have received increasing attention because of their specificity and, in some cases, wide time window of detection in non-conventional matrices including hair and meconium. In the attempt to update analytical methods for the determination of non-oxidative markers of alcohol, the objective of this study is to review published studies that measure fatty-acid ethyl esters (FAEE), ethyl glucuronide (EtG), and phosphatidylethanol (PEth) in alternative biological matrices, focusing on the extraction and detection methods and full analytical conditions used.

Rapid determination of quetiapine in blood by gas chromatography-mass spectrometry. Application to post-mortem cases.

A simple, fast and sensitive method for the determination of quetiapine in human blood has been developed and validated. The method involved a basic liquid-liquid extraction procedure and subsequent analysis by gas chromatography-mass spectrometry, previous derivatization with bis(trimethylsilyl)-trifluoro-acetamide and chorotrimethylsilane (99 : 1). The methods of validation included linearity with a correlation coefficient > 0.99 over the range 0.02-1 µg ml(-1), intra- and interday precision (always < 12%) and accuracy (mean relative error always < 12%) to meet the bioanalytical acceptance criteria. The limit of detection was 0.005 µg ml(-1). The procedure was further applied to post mortems from the Institute of Legal Medicine, University of Santiago de Compostela.

Analysis of ethyl glucuronide in hair samples by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Many different biomarkers can be used to evaluate ethanol intake. Ethyl glucuronide (EtG) is a direct phase II and minor metabolite of ethanol formed through the UDP-glucuronosyl transferase-catalyzed conjugation of ethanol with glucuronic acid. Its investigation is of interest in both clinical and forensic contexts because of the wide window of detection. A sensitive LC-MS/MS procedure has been developed and fully validated according to the guidelines of forensic toxicology for the analysis of EtG in hair. Sample preparation and chromatographic separation were thoroughly optimized. The analysis was performed in the multiple reaction monitoring mode using the transitions m/z 221 → 203 (for the quantification) and 221 → 85 or 75 (for the qualification) for EtG, and m/z 226 → 208 (for quantification) and 226 → 75 or 85 (for qualification) for EtG-D5, used as the internal standard. Analyses were carried out using an Inertsil ODS-3 column (100 × 3 mm i.d., 3 µm particle size) and a mobile phase composed of formic acid and acetonitrile. Various SPE cartridges and solvents were tested in order to obtain the highest recoveries and cleanest extracts. The assay linearity of EtG was confirmed over the range from 20 to 2500 pg mg(-1), with a coefficient of determination (R(2) ) above 0.99. The lower limit of quantitation (LLOQ) was 20 pg mg(-1) and the limit of detection was 10 pg mg(-1). Intra- and inter-day assays were less than 15% except at the LLOQ (20%). The analytical method was applied to 72 post-mortem hair samples. EtG concentration in the hair ranged from 0 to 653 pg mg(-1) hair.

Chromatographic determination of drugs of abuse in vitreous humor using solid-phase extraction.

A simple method is presented for the simultaneous determination of morphine, 6-acetylmorphine, codeine, cocaine, benzoylecgonine, cocaethylene, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in vitreous humor by high-performance liquid chromatography with photodiode array detector after solid-phase extraction with Oasis® HLB cartridges and dichloromethane as eluent. The chromatographic process was carried out using an XTerra® RP8 column (250 × 4.6 mm i.d., 5 µm particle size) and a mobile phase composed of acetonitrile and pH 6.5 phosphate buffer in gradient mode. A linear response from the detector was obtained within the concentration range of 0.1-4 µg ml(-1) , with correlation coefficients higher than 0.99. The limits of detection were lower than 30 ng ml(-1) for all the drugs studied, the coefficients of variation fluctuated between 0.1 and 12.4%, and the average recoveries were higher than 78% for all the drugs except for EDDP, with a value of 66.4%. Finally, the proposed method was applied to 15 vitreous humor samples coming from individuals who had died from opiate and/or cocaine overdose, showing consumption of cocaine in 14 cases, methadone in five cases and heroin in three cases. Average concentrations of 0.30 µg ml(-1) for morphine, 0.24 µg ml(-1) for 6-acetylmorphine, 0.10 µg ml(-1) for codeine, 0.81 µg ml(-1) for cocaine, 1.26 µg ml(-1) for benzoylecgonine, 0.15 µg ml(-1) for cocaethylene, 0.11 µg ml(-1) for methadone and 0.68 µg ml(-1) for EDDP were obtained.

A new method for quantifying prenatal exposure to ethanol by microwave-assisted extraction (MAE) of meconium followed by gas chromatography-mass spectrometry (GC-MS).

Ethanol is a legal and widely available substance. There are health and social consequences associated with its abuse. One of the most important problems is related to alcohol consumption during pregnancy. In fact, prenatal ethanol exposure can be associated with fetal alcohol spectrum disorder (FASD), a term used to describe a wide range of potentially lifelong effects that include physical, mental, behavioral, and learning disabilities. Fatty acid ethyl esters (FAEEs), which are non-oxidative metabolites of ethanol, are currently used as biomarkers of direct ethanol consumption in different matrices, including hair, blood, skin surface, and meconium. Analysis of these compounds in meconium reveals exposure to alcohol during the second and third trimesters of pregnancy. An important finding for evaluation of gestational ethanol exposure is the fact that FAEEs do not cross the placenta. Because they accumulate in the fetal gut from approximately the 20th week of gestation until birth, this provides a wide window of detection of chronic exposure to alcohol. The sum of the concentrations of all the FAEEs, with a cutoff of 2 nmol g(-1) or 600 ng g(-1) meconium, has been recommended as evidence of maternal alcohol use. We introduce a novel technique to quantify ethyl myristate, ethyl palmitate, ethyl stearate, and their deuterated analogues (as internal standards, IS) in meconium using microwave-assisted extraction (MAE) coupled with gas chromatography-mass spectrometry (GC-MS). Limits of detection and quantification were 50 and 100 ng g(-1) for all analytes except ethyl stearate (LOD 100 ng g(-1) and LOQ 500 ng g(-1)). Calibration curves were linear from the LOQ to 5000 ng g(-1). The validated method was applied to the analysis of 81 meconium samples.

Determination of fentanyl, metabolite and analogs in urine by GC/MS.

A rapid and sensitive method for the simultaneous determination of alfentanyl, sufentanyl and fentanyl (and its major metabolite norfentanyl) in urine was developed and validated. The method involved a liquid-liquid extraction in alkaline conditions, derivatization with pentafluoropropionic anhydride to improve the sensitivity for norfentanyl and subsequent analysis in GC/MS. The LODs are 0.08 ng ml(-1) for all substances (0.04 ng ml(-1) for alfentanyl). Intra- and inter-day precision coefficient of variation was always below 15%; mean relative error (accuracy) was always below 15%. The method was linear for all analytes, with quadratic regression of calibration curves always higher than 0.99. The method was applied to real samples of subjects who had received therapeutic doses of fentanyl, showing its suitability for the determination of low levels of these substances. The method was also applied to a subject whose death was attributed to fentanyl overdose.

Microwave-assisted extraction: a simpler and faster method for the determination of ethyl glucuronide in hair by gas chromatography-mass spectrometry.

Alcohol is the most frequently abused "addictive substance" that causes serious social problems throughout the world; thus, alcoholism is of particular interest in clinical and forensic medicine. Alcohol biomarkers are physiological indicators of alcohol exposure or ingestion and may reflect the presence of an alcohol use disorder. The glucuronide conjugation is a minor pathway of ethanol metabolism. Ethyl glucuronide (EtG) is a marker of recent alcohol consumption that detects alcohol use reliably over a definite time period. The present paper describes a new method for the determination of EtG in hair. It is based both in the microwave-assisted extraction (MAE), to extract the analyte from hair samples, and gas chromatography-mass spectrometry (GC-MS), to identify and quantify the EtG in selected ion monitoring (SIM) mode. The method was applied to 15 hair samples from occasional alcohol users, obtaining positive results in all cases. It was fully validated, including a linear range (0.3-10 ng/mg) and the main precision parameters. In summary, the use of microwave-assisted extraction turned out to be a substantially simpler, faster, and a more sensitive procedure than any other conventional sample preparations.

Determination of ketamine and amphetamines in hair by LC/MS/MS.

A liquid chromatography-tandem mass spectrometry method was developed for the determination of ketamine (with its metabolite norketamine) and some amphetamines (amphetamine, methamphetamine, methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine). This method was developed to determine these compounds in hair and is able to simultaneously quantify all of them in human hair. Hair samples (20 mg) were washed and pulverized, and an extraction with formic acid (0.01%) and ultrasonication for 4 h was used. Deuterated analogs of the analytes were used as internal standards for quantification. Linearity from 0.5 to 25 ng/mg was obtained for both ketamine (and norketamine) and amphetamines with correlation coefficients exceeding 0.99. The limit of detection and the limit of quantification obtained were 0.1 and 0.5 ng/mg, respectively, for ketamine and amphetamines. A total of 25 hair samples from known drug abusers (relating to designer drug consumption or consumption of amphetamines) were examined by this validated method. The results show that the proposed method is suitable for testing these drugs in a single sample of hair. In addition, it is simpler and faster than analysis by conventional methods such as gas chromatography-mass spectrometry, which usually require a more laborious extraction procedure and, in most of cases, an additional derivatization process.

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

The present paper describes a method for the simultaneous determination of cocaine and cocaethylene in plasma. It was based in the extraction of the analytes by solid-phase microextraction (SPME), and gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify the analytes in selected ion monitoring (SIM) mode. The method showed to be very simple, rapid and sensitive. The method was validated for the two compounds, including linearity (range 25-1000 ng/mL) and the main precision parameters. It was applied to ten plasma samples from cocaine and alcohol users, obtaining positive results in all cases.

Microwave-assisted extraction and HPLC-DAD determination of drugs of abuse in human plasma.

A sample preparation procedure using microwave energy is proposed for the determination of morphine, 6-acetylmorphine, codeine, cocaine, cocaethylene, benzoylecgonine, methadone, and 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine in human plasma. A screening asymmetrical factorial design was used to identify the most suitable extraction conditions as regards solvent, temperature, and extraction time. The target drugs were quantified by high-performance liquid chromatography with diode-array detection. The use of microwave energy was found to reduce solvent consumption and extraction time compared with solid-phase extraction. The detector response was linear over the drug concentration range of 0.05-2.0 microg/mL in human plasma. The precision and accuracy were good, with values less than 8% and 7%, respectively. Drug recoveries from spiked samples ranged from 69 to 81%. The proposed method was successfully applied to a number of forensic cases.

Solid-phase microextraction for the determination of cocaine and cocaethylene in human hair by gas chromatography-mass spectrometry.

A method for the simultaneous determination of cocaine (COC) and cocaethylene (CE) in human hair was developed, using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) as analytical technique to identify and quantify the drugs. Selected ion monitoring (SIM) mode was used to obtain higher sensitivity. The deuterated-labeled analogues were used as internal standards. The detector response was linear for the drugs studied over the range 0.4-15 ng/mg, with correlation coefficients higher than 0.995. The coefficients of variation oscillated between 0.65% and 14.18% and the accuracy was in the range from 0.73% to 11.20%. The limits of quantitation and detection were found to be acceptable. Finally, this method was applied to 15 hair samples from cocaine users, obtaining positive results in all cases. The mean concentrations were 5.39 ng/mg (range: 0.43-8.98 ng/mg) for cocaine and 1.11 ng/mg (range: 0.42-2.23 ng/mg) for cocaethylene.

Determination of cocaine and its metabolites in plasma by porous membrane-protected molecularly imprinted polymer micro-solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A selective molecularly imprinted polymer synthesized for the selective retention of cocaine (COC) and its metabolites [benzoylecgonine (BZE), ecgonine methyl ester (EME), and cocaethylene (CE)] was used as a solid adsorbent for assessing cocaine abuse by plasma analysis. The MIP beads (50mg) were loaded inside a cone shaped device made of a polypropylene (PP) membrane for micro-solid-phase extraction (µ-SPE). High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used for quantifying the analytes after MIP-µ-SPE. The best retention capabilities were reached when loading plasma samples (within the 0.1-5.0mL range), previously adjusted to pH 5.5 by orbital-horizontal shaking (150rpm, 50°C) for 10min. Analyte elution was achieved by subjecting the MIP-µ-SPE device to ultrasound (37kHz, 325W) with 10mL of dichloromethane/2-propanol/ammonium hydroxide (76:20:4) for 8min. After eluate evaporation to dryness and re-dissolution in 100µL of mobile phase, the MIP-µ-SPE method yielded a pre-concentration factor of 50. Precision was assessed by intra-day and inter-day assays, and accuracy (intraday and inter-day analytical recovery, as well as the analysis of a BTMF 1/11-B control serum sample) show that the developed method is highly precise and accurate. In addition, the limits of detection, ranging from 0.061ngmL(-1) for COC to 0.87ngmL(-1) for BZE, were low enough for confirmative conclusions regarding cocaine abuse. The method was used for screening/quantifying cocaine and metabolites in plasma samples from poly-drug abusers.

Synthesis and characterization of novel molecularly imprinted polymer - coated Mn-doped ZnS quantum dots for specific fluorescent recognition of cocaine.

Mn-doped ZnS quantum dots (QDs) coated with a molecularly imprinted polymer (MIP) material selective toward cocaine and its metabolites have been prepared and applied to cocaine (COC) and metabolites assessment by spectrofluorimetry. Ultrasound irradiation (37kHz) was novelty used for performing the Mn-doped ZnS QDs synthesis as well as for preparing the QD based MIP-coated composite by precipitation polymerization (imprinting process). This fact allowed the synthesis to be accomplished in four hours. In addition, the use of ultrasound irradiation during MIP-QDs synthesis increased the homogeneity of the QDs size, and reduced nanoparticles agglomeration. MIP was 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. The fluorescence of MIP-coated QDs was quenched by the template (COC) and also by metabolites from COC such as benzoylecgonine (BZE), and ecgonine methyl ester (EME). Quenching was not observed when performing experiments with non-imprinted polymer (NIP)-coated QDs; and also, fluorescence quenching of MIP-coated QDs was not observed by other drugs of abuse and metabolites (heroin and cannabis abuse). This fact indicates that the prepared material recognize only COC (template) and metabolites.

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.

Quantification of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in meconium for detection of alcohol abuse during pregnancy: Correlation study between both biomarkers.

This article presents results from 47 meconium samples, which were analyzed for fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) for detection of gestational alcohol consumption. A validated microwave assisted extraction (MAE) method in combination with GC-MS developed in the Institute of Forensic Science (Santiago de Compostela) was used for FAEE and the cumulative concentration of ethyl myristate, ethyl palmitate and ethyl stearate with a cut-off of 600ng/g was applied for interpretation. A simple method for identification and quantification of EtG has been evaluated by ultrasonication followed solid phase extraction (SPE). Successful validation parameters were obtained for both biochemical markers of alcohol intake. FAEE and EtG concentrations in meconium ranged between values lower than LOD and 32,892ng/g or 218ng/g respectively. We have analyzed FAEE and EtG in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. Certain agreement between the two biomarkers was found as they are both a very specific alcohol markers, making it a useful analysis for confirmation.

Matrix solid phase dispersion assisted enzymatic hydrolysis as a novel approach for cocaine and opiates isolation from human hair.

The possibility of assisting enzymatic hydrolysis (EH) procedures by sample disruption mechanisms inherent to matrix solid phase dispersion (MSPD) has been explored in the current study. EH of hair specimens from poly-drug abusers was assisted by dispersing/blending the sample (0.05 g) with alumina (2.25 g) before loading the dissolved enzyme (6 mL of 1 mg mL(-1) Pronase E in 1.4 M/1.4 M Tris/HCl, pH 7.3) through the hair-alumina solid phase packaged inside a disposable MSPD syringe. The MSPD-EH method was developed, and it proved to offer quantitative results when isolating cocaine, benzoylecgonine (BZE), codeine, morphine and 6-monoacethylmorphine (6-MAM) from human hair samples. The procedure allows an on column clean-up/pre-concentration procedure of the isolated targets by attaching a previously conditioned Oasis HLB cartridge to the end of the MSPD syringe. The EH procedure of human hair with Pronase E can therefore be shortened to approximately 30 min. Within this time, sample blending/dispersion, MSPD syringe package, elution (EH when dissolved Pronase E is passing through the sample-dispersant bed), and extract clean-up and target pre-concentration stages are achieved. Gas chromatography-mass spectrometry (GC-MS) was used for determining each target after elution from the Oasis HLB cartridges with 2 mL of 2% (v/v) acetic acid in methanol, concentration by N2 stream evaporation, and dried extract derivatization with N-methyl-tert-butylsilyltrifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS). The method was validated according to the guidance for bioanalytical method validation of the US Department of Health and Human Services, Food and Drug Administration. The simplicity of the proposed approach makes it a useful procedure for screening/quantifying drugs of abuse in hair specimens from poly-drug abusers.

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 (20µL) 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.

Simultaneous determination of cocaine and opiates in dried blood spots by electrospray ionization tandem mass spectrometry.

A sample pre-treatment method based on blood spot collection filter cards was optimized as a means of using small volume samples for the screening and confirmation of cocaine and opiates abuse. Dried blood spots (DBSs) were prepared by dispersing 20 µL of whole blood specimens previously mixed with the internal standards (deuterated analogs of each target), and subjecting the whole DBS to extraction with 5 mL of methanol under orbital-horizontal shaking (180 rpm) for 10 min. Determinations were based on direct electrospray ionization tandem mass spectrometry (ESI-MS/MS) by injecting the re-dissolved methanol extract with the delivery solution (acetonitrile-water-formic acid, 80:19.875:0.125) at a flow rate of 60 µL min(-1), and using multiple reaction monitoring (MRM) mode with the m/z (precursor ion)→m/z (product ion) transitions for acquisition. Matrix effect has been found to be statistically significant (Multiple Range Test) when assessing cocaine, BZE, codeine and morphine, and the use of the standard addition method (dispersion of whole blood previously mixed with standards onto the filter papers) was needed for accurate determinations. The developed DBS-ESI-MS/MS procedure offered good intra-day and inter-day precisions (lower than 10% and 12%, respectively), as well as good intra-day and inter-day accuracies (inter-day absolute recoveries, expressed as the mean analytical recovery over three target concentration levels, of 103%, 100%, 101%, 98% and 100% for cocaine, BZE, codeine, morphine and 6-MAM, respectively). The high sensitivity inherent to MS/MS determinations combined with the minimal dilution of sample allowed low limits of quantification for all targets, and the developed method results therefore adequate for cocaine and opiates screening and confirmation purposes. The procedure was finally applied to DBSs prepared from whole blood from polydrug abusers, and results were compared with those obtained after a conventional sample pretreatment method based on solid phase extraction for plasma specimens and gas chromatography-mass spectrometry.