Evaluation of an improved general unknown screening procedure using liquid chromatography-electrospray-mass spectrometry by comparison with gas chromatography and high-performance liquid-chromatography--diode array detection

This paper presents an improved, comprehensive liquid chromatography-electrospray-mass spectrometry (LC-ES-MS) general unknown screening (GUS) procedure for drugs and toxic compounds and its comparison with conventional techniques in routine laboratory conditions. Chromatographic separation involved an X-TERRA MS C18, 3.5 microm (100 mm x 1 mm i.d.) column together with a 25-min long gradient of acetonitrile in pH 3, 2 mM ammonium formate delivered at a 50 microl/min flow rate. Two different in-source collision-induced dissociation voltages were alternated, both in the positive and in the negative ion modes. Reconstructed spectra were then obtained in both polarities by adding up spectra obtained with low and high energy, resulting in spectra presenting a sufficient number of specific fragment ions for unambiguous and fast identification of compounds. Two large mass spectral libraries of drugs and toxic compounds were built and an efficient automated signal processing, library searching and report editing algorithm developed. Using a common, efficient solid-phase extraction procedure, this LC-ES-MS technique was compared to GC-MS and HPLC-DAD GUS procedures for the identification of a priori unknown compounds in 51 serum samples consecutively sent to the laboratory for GUS. The present LC-MS method identified 75% of the compounds contained in these samples (versus 66% for GC-MS and 71% for HPLC-DAD), including 8% that the other two techniques failed to identify (versus 8% for GC-MS and 9.5% for HPLC-DAD). Therefore, it is complementary to GC-MS and/or HPLC-DAD and helps enlarge the range of drugs detected in clinical toxicology. It could be useful as well in forensic toxicology to confirm a positive result, as 38% of all the compounds were detected by the three techniques and 36% by two of them.     

Automated screening procedure using gas chromatography-mass spectrometry for identification of drugs after their extraction from biological samples

A novel analytical screening procedure has been developed, using computer-controlled gas chromatography-mass spectrometry (GC-MS), to detect 120 drugs of interest to road safety. This paper describes GC-MS methodology suitable for use on extracts of biological origin, while extraction procedures will be the subject of a future communication. The method was devised to identify drugs in extracts of blood samples, as part of an investigation into the involvement of drugs, other than alcohol, in road accidents. The method could be adapted to screen for other substances. The method depends on a "macro" program which was written to automate the search of GC-MS data for target drugs. The strategy used was to initially search for each drug in the database by monitoring for a single characteristic ion at the expected retention time. If a peak is found in this first mass chromatogram, a peak for a second characteristic ion is sought within 0.02 min of the first and, if found, the ratio of peak areas calculated. Probable drug identification is based on the simultaneous appearance of peaks for both characteristic ions at the expected retention time and in the correct ratio. If the ratio is outside acceptable limits, a suspected drug (requiring further investigation) is reported. The search macro can use either full mass spectra or, for enhanced sensitivity, data from selected ion monitoring (which requires switching between groups of ions during data acquisition). Quantitative data can be obtained in the usual way by the addition of internal standards.     

Systematic toxicological analysis of drugs and their metabolites by gas chromatography-mass spectrometry.

Gas chromatographic-mass spectrometric (GC-MS) procedures for the systematic toxicological analysis of several categories of drugs relevant to clinical toxicology, forensic toxicology and doping control are reviewed. Papers from 1981 to 1991 are taken into consideration. They describe the detection of acute or chronic intoxication and the detection of drug abuse. Screening procedures are included for the following categories: barbiturates and other sedative-hypnotics, anticonvulsants, benzodiazepines, antidepressants, phenothiazine and butyrophenone neuroleptics, central stimulants (amphetamines, cocaine), hallucinogens (LSD, phencyclidine, tetrahydrocannabinol), opioid (narcotic) and other potent analgesics, non-opioid analgesics, antihistamines (histamine H1-receptor blockers), antiparkinsonian drugs, beta-blockers (beta-adrenoceptor blockers), antiarrhythmics (class I and IV), diuretics, laxatives and their metabolites. Methods for confirmation of results obtained by screening procedures using immunoassay or chromatographic techniques are also included. GC-MS procedures for the simultaneous detection of several categories of drugs, the so-called "general unknown analysis", are reviewed. The toxicological question to be answered and the consequence for the choice of an adequate method, the sample preparation and the chromatography itself are discussed. The basic information about the biosample assayed, work-up, GC column, mass spectral detection mode, reference data and sensitivity of each procedure are summarized in tables, arranged according to the category of drug. Examples of typical GC-MS applications are presented. Fragment ions that are suitable for mass spectral screening for particular categories of drugs and for general unknown are tabulated.     

Multi-residue analysis of pharmaceutical compounds in aqueous samples

Pharmaceutical compounds are nowadays an emerging group of organic pollutants in aquatic systems. Several methodologies have already been published to measure these pollutants in the environment, showing the difficulties to take into account the various compounds belonging to numerous therapeutical and chemical groups. In order to develop environmental monitoring, there is a need for a less costly and time-consuming multi-component procedure. The work presented here deals with the development of an extraction procedure which enables the measurement of a wide spectrum of pharmaceuticals at trace levels (ng 1(-1)) with quite simple equipment (i.e. GC-MS with single quadruple as analyzer). The analyzed compounds comprise anti-inflammatories, antidepressants and hypolipidic drugs. The reliability and sensitivity have been tested on 18 different compounds (7 basic compounds and 11 acidic drugs) extracted simultaneously and analyzed by GC-MS. The optimized procedure has been successfully applied to the analysis of wastewaters, surface waters and drinking waters from the following areas: first the Cortiou rocky inlet, in the Mediterranean Sea (South coast of France), highly impacted by the Marseilles wastewater treatment plant effluent and secondly the Hérault watershed by studying drinking water, surface water and wastewater. In both cases, the level of pharmaceuticals was totally unknown. Results obtained have demonstrated the suitability of the method for multi-residue analysis of different types of water matrices.     

Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography–mass spectrometry

In recent years, a new class of designer drugs has appeared on the drugs of abuse market in many countries, namely, the so-called beta-keto (bk) designer drugs such as mephedrone (bk-4-methylmethamphetamine), butylone (bk-MBDB), and methylone (bk-MDMA). The aim of the present study was to identify the metabolites of mephedrone in rat and human urine using GC-MS techniques and to include mephedrone, butylone, and methylone within the authors’ systematic toxicological analysis (STA) procedure. Six phase I metabolites of mephedrone were detected in rat urine and seven in human urine suggesting the following metabolic steps: N-demethylation to the primary amine, reduction of the keto moiety to the respective alcohol, and oxidation of the tolyl moiety to the corresponding alcohols and carboxylic acid. The STA procedure allowed the detection of mephedrone, butylone, methylone, and their metabolites in urine of rats treated with doses corresponding to those reported for abuse of amphetamines. Besides macro-based data evaluation, an automated evaluation using the automated mass spectral deconvolution and identification system was performed. Mephedrone and butylone could be detected also in human urine samples submitted for drug testing. Assuming similar kinetics in humans, the described STA procedure should be suitable for proof of an intake of the bk-designer drugs in human urine.     

Recently developed GC/MS and LC/MS methods for determining NSAIDs in water samples

Pharmaceuticals have become major targets in environmental chemistry due to their presence in aquatic environments (following incomplete removal in wastewater treatment or point-source contaminations), threat to drinking water sources and concern about their possible effects to wildlife and humans. Recently several methods have been developed for the determination of drugs and their metabolites in the lower nanogram per litre range, most of them using solid-phase extraction (SPE) or solid-phase microextraction (SPME), derivatisation and finally gas chromatography mass spectrometry (GC-MS), gas chromatography tandem mass spectrometry (GC-MS/MS) and liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS). Due to the elevated polarity of non-steroidal anti-inflamatory drugs (NSAIDs), analytical techniques based on either liquid chromatography coupled to mass spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS) after a previous derivatisation step are essential. The most advanced aspects of current GC-MS, GC-MS/MS and LC-MS/MS methodologies for NSAID analysis are presented.     

Determination of seven selected antipsychotic drugs in human plasma using microextraction in packed sorbent and gas chromatography–tandem mass spectrometry

A method using microextraction by packed sorbent (MEPS) and gas chromatography–tandem mass spectrometry (GC-MS/MS) is described for the determination of seven antipsychotic drugs in human plasma. The studied compounds were chlorpromazine (CPZ), haloperidol (HAL), cyamemazine, quetiapine, clozapine, olanzapine (OLZ), and levomepromazine; promazine, protriptyline, and deuterated CPZ were used as internal standards. The validation parameters included selectivity, linearity and limits of detection and quantitation, intra- and interday precision and trueness, recovery, and stability and were studied according to internationally accepted guidelines. The method was found to be linear between the lower limit of quantitation and 1000 ng/mL, except for OLZ and HAL (200 ng/mL), with determination coefficients higher than 0.99 for all analytes, and extraction efficiencies ranged from 62 to 92 %. Intra- and interday precision ranged from 0.24 to 10.67 %, while trueness was within a ±15 % interval from the nominal concentration for all analytes at all studied levels. MEPS has shown to be a rapid procedure for the determination of the selected antipsychotic drugs in human plasma, allowing reducing the handling time and the costs of analysis. Furthermore, GC-MS/MS has demonstrated to be a powerful tool for the simultaneous quantitation of the studied compounds, enabling obtaining adequate selectivity and sensitivity using a sample volume of as low as 0.25 mL.     

Simultaneous analysis of flunixin, naproxen, ethacrynic acid, indomethacin, phenylbutazone, mefenamic acid and thiosalicylic acid in plasma and urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

Simple and reproducible high-performance liquid chromatographic (HPLC) and gas chromatographic-mass spectrometric (GC-MS) methods have been developed for the simultaneous analysis of several acidic drugs in horse plasma and urine. Although the capillary GC-MS column provided better separation of the drugs than the reversed-phase C8 (3 microns, 75 mm) HPLC column, the total analysis time with HPLC was shorter than the total analysis time with GC-MS. The HPLC system equipped with a diode-array detector provided simultaneous screening (limit of detection 100-500 ng/ml) and confirmation (limit 1.0 micrograms/ml) of the drugs. The HPLC system equipped with fixed-wavelength ultraviolet and fluorescence detectors provided a relatively sensitive screening [limit of detection 50-150 ng/ml for ultraviolet and 10 ng/ml for fluorescence (naproxen only) detectors] of the drugs. However, the positive samples had to be confirmed by using either the diode-array detector or the GC-MS system. The GC-MS system provided simultaneous screening and confirmation of the drugs at very low concentrations (20-50 ng/ml).     

Comparison of immunoassay and gas chromatography-mass spectrometry for measurement of polycyclic aromatic hydrocarbons in contaminated soil

Polycyclic aromatic hydrocarbons (PAHs) are frequently encountered in the environment and may pose health concerns due to their carcinogenicity. A commercial enzyme-linked immunosorbent assay (ELISA), was evaluated as a screening method for monitoring PAHs at contaminated sites. The ELISA was a carcinogenic PAH (C-PAH) RaPID assay testing kit that cross-reacts with several PAHs and utilizes benzo[a]pyrene (BaP) as a calibrator. Soil samples were extracted with 50% acetone in dichloromethane (DCM) for analysis by ELISA and gas chromatography-mass spectrometry (GC-MS). The overall method precision was within ±30% for ELISA and within ±20% for GC-MS. Recovery data for spiked soils ranged from 46 to 140% for BaP as determined by ELISA. Recoveries data of the GC-MS surrogate standards, 2-fluorobiphenyl and chrysene, were greater than 70%. The GC-MS procedure detected a total of 19 priority PAHs (2-6-ring PAHs) including seven probable human carcinogens (4-6-ring B2-PAHs). The ELISA results were compared to GC-MS summation results for the total 19 target PAHs as well as for the subset of the seven B2-PAH compounds. For all soil samples, the PAH concentrations derived from ELISA were greater than the sum of B2-PAH concentrations obtained by GC-MS. ELISA determinations were also frequently greater than the results obtained by GC-MS for the total 19 PAH compounds. This discrepancy can be expected, since the ELISA is a screening assay for the detection of several related PAHs while the GC-MS procedure detects priority PAH compounds. Thus, only a subset of PAHs (e.g. 19 PAHs) in the soil samples were measured by GC-MS while additional PAHs, including alkylated PAHs, and PAH derivatives have been demonstrated to be cross-reactive in the C-PAH ELISA. Results of paired tests show that the PAH data from ELISA and GC-MS methods are significantly different (P<0.001), but highly correlated. The ELISA data had a strong positive relationship with the GC-MS summation data for the B2-PAHs as well as for the 19 PAHs targeted by the GC-MS method. Results indicate that the ELISA may be useful as a broad screen for monitoring PAHs in environmental samples.     

Photo- and thermal-stability studies on benzimidazole anthelmintics by HPLC and GC-MS

Photo- and thermal-stability of the anthelmintics Albendazole, Mebendazole and Fenbendazole as in solid as in solution form has been investigated, by using a Xenon arc lamp as a radiation source, according to the ICH guideline for the drug stability tests. The degradation process was monitored by a HPLC method. All drugs showed high photosensitivity in solution but a reliable stability in solid form and when exposed to a temperature up to 50 degrees C. Two main degradation products from hydrolysis of the carbamic groups were identified by GC-MS. Validation studies demonstrated high accuracy (recovery 94 to 106%) and precision (RSD under 4.6%) of the HPLC method. The analytical procedure was successfully applied to the control of the drugs in the respective pharmaceutical formulations.     

Optimization of the derivatization reaction and the solid-phase microextraction conditions using a D-optimal design and three-way calibration in the determination of non-steroidal anti-inflammatory drugs in bovine milk by gas chromatography-mass spectrometry

An experimental design optimization is reported of an analytical procedure used in the simultaneous determination of seven non-steroidal anti-inflammatory drugs (NSAIDs) in bovine milk by gas chromatography with mass spectrometry detection (GC-MS). This analytical procedure involves a solid-phase microextraction (SPME) step and an aqueous derivatization procedure of the NSAIDs to ethyl esters in bovine milk. The following NSAIDs are studied: ibuprofen (IBP), naproxen (NPX), ketoprofen (KPF), diclofenac (DCF), flufenamic acid (FLF), tolfenamic acid (TLF) and meclofenamic acid (MCL). Three kinds of SPME fibers - polyacrylate (PA), polydimethylsiloxane/divinylbenzene (PDMS/DVB) and polydimethylsiloxane (PDMS) - are compared to identify the most suitable one for the extraction process, on the basis of two steps: to determine the equilibrium time of each fiber and to select the fiber that provides the best figures-of-merit values calculated with three-way PARAFAC-based calibration models at the equilibrium time. The best results were obtained with the PDMS fiber. Subsequently, 8 experimental factors (related to the derivatization reaction and the SPME) were optimized by means of a D-optimal design that involves only 14 rather than 512 experiments in the complete factorial design. The responses used in the design are the sample mode loadings of the PARAFAC decomposition which are related to the quantity of each NSAID that is extracted in the experiment. Owing to the fact that each analyte is unequivocally identified in the PARAFAC decomposition, a calibration model is not needed for each experimental condition. The procedure fulfils the performance requirements for a confirmatory method established in European Commission Decision 2002/657/EC.     

气相色谱-质谱法同时筛检人血液中26种常见毒药物

建立了气相色谱-质谱法(GC-MS)同时快速筛检人血液中26种常见毒药物的新方法.通过对样品前处理方法的摸索及GC-MS分析条件的优化,采用磷酸盐缓冲液(pH 3.5)稀释血样后,乙醚萃取,分段选择离子监测(SIM)法鉴定,并用提取离子进一步验证,可以同时检测甲胺磷、毒鼠强、地西泮、尼可刹米、利多卡因、苯巴比妥、阿托品等7大类共26种常见毒药物,回收率大部分达80%～90%,检测限为0.01 mg/L.本法用色谱保留时间、质谱特征离子同时定性,消除了血液中复杂基体的干扰,适用于中毒患者血液的应急检测.     

Trace determination of tamoxifen and 5-fluorouracil in hospital and urban wastewaters

Tamoxifen and 5-fluorouracil are widely used in cancer therapy. They are highly toxic (teratogenic, mutagenic, etc.), as are most of the anticancer drugs. Two methods were set up to analyse these drugs in wastewaters to evaluate the potential for environmental contamination by cytostatic agents. Liquid–liquid extraction followed by purification on OASIS® MCX cartridge and gas chromatography with mass spectrometry detection (GC-MS) was used for the analysis of tamoxifen. 5-Fluorouracil was extracted with an ENV+?(Isolute) cartridge (solid-phase extraction), derivatized with pentafluorobenzyl bromide (PFBBr) and detected by GC-MS. Both methods showed good recoveries (>70%), repeatability (RSD<10%) and limits of detection (LOD 6–15?ng/L). Wastewaters from a residential area, a hospital, and sewage-treatment plants (STPs) were analysed using the analytical methods developed in this study. Tamoxifen was detected in wastewaters of the hospital, residential area, and influent of STPs, but not in treated wastewaters. 5-Fluorouracil in all wastewaters was below the LOD of the analytical method.     

Chemometric approach to the optimisation of LC-FL and GC-MS methods for the determination of nitrite and nitrate in some biological,food and environmental samples

Gas chromatography–mass spectrometry (GC-MS) method and a liquid chromatography–fluorescence (LC-FL) detection method using experimental design and optimisation approach were improved for the quantitative determination of nitrite and nitrate in biological, food and environmental samples. The obtained recoveries of nitrite and nitrate ions from samples based on both GC-MS and LC-FL results ranged from 98.5% to 98.9% for nitrite and 97.9% to 98.4% for nitrate. The precision of these methods, as indicated by the relative standard deviations (RSDs), was within the range from 2.4% to 3.6% for nitrite and 2.5% to 3.8% for nitrate, respectively. The limits of detection of nitrite and nitrate ions from samples based on GC-MS and LC-FL results ranged from 0.01 to 0.14 ng L^?1 for nitrite and 0.02 to 0.71 ng L^?1 for nitrate, respectively. The optimised isolation procedure by central composite design was successfully applied to real samples. The results revealed that the proposed procedure combined with GC-MS and LC-FL techniques is more sensitive, reliable and selective compared to the other methods available for the precise determination of trace levels of nitrite and nitrate in biological, food and environmental samples.     

A downscaled multi-residue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC-MS3)

Within the scope of the European Community member states' residue monitoring plan, illicit administration of anabolic steroids is monitored at slaughterhouse level as well as on living animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a procedure for which high performance liquid chromatographic (HPLC) fractionation prior to GC-MS(n) analysis was no longer required. Analytical results could be obtained within 1 day and only 5 mL urine was needed to carry out the screening procedure. Using the downscaled methodology, all validation criteria described in the European Commission document 2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs) established for anabolic steroids in urine, could be achieved. A higher GC-MS technique's specificity was achieved by detecting the steroids using GC-MS3. Nevertheless, it was decided to screen routinely sampled urine with GC-MS2 whereas GC-MS3 was applied to confirm the presence of anabolic steroid residues in suspected sample extracts.     

Pressure-adjusted continual flow heart-cutting for the high throughput determination of amphetamine-type stimulant drugs in whole blood by fast multidimensional gas chromatography-mass spectrometry

Innovative features and technical improvements in modern bench-top quadrupole gas chromatograph-mass spectrometer (GC-MS) have prepared the way for faster and more cost-effective applications while still maintaining sufficient chromatographic resolution, speed of MS data acquisition and reliability of analytical methodology. In this paper, a short wide-bore capillary column with low film thickness (5 m x 0.32 mm i.d., 0.1 microm) was used a pre-fractionating column and only chosen heart-cuts were transferred to the second chromatographic dimension (15 m x 0.25 mm i.d., 0.25 microm) by means of a pressure-adjusted continual flow type switching device for quantification of five common amphetamine-type stimulant drugs. The instrumental setting used, in combination with carefully optimized operational fast GC and MS parameters, markedly decreased the retention times of the targeted analytes, e.g., amphetamine 0.891 min and methamphetamine 1.037 min, and the total chromatographic runtime (1.700 min), as well as reducing the need for continuous cleaning of the MS ion source and increasing column life compared with conventional GC-MS approaches. The performance of the instrumental configuration and analytical method was evaluated in validation experiments and the method was also applied to authentic samples. The method demonstrates the potential of fast GC-MS in combination with a gas-phase microfluidic Deans switch device for analysing of (semi)volatile compounds, such as amphetamine-type stimulant (ATS) drugs. This should be particularly useful in modern laboratories aiming at cost-efficient analysis as well as the optimum use of available laboratory capacity and instrumentation.     

Headspace solid-phase microextraction of cannabinoids in human head hair samples

A fast method was optimized and validated with the aim to detect cannabinoids (cannabidiol, cannabinol, and delta-9-tetrahydrocannabinol) in human head hair samples. The method was based on an initial procedure of external decontamination of hair samples (10 mg) with petroleum ether, followed by alkaline digestion and further extraction of cannabinoids by means of a headspace solid-phase microextraction technique (HS-SPME). GC-MS was used to identify and quantify the analytes in SIM mode. The LOQs and LODs obtained were 0.07 and 0.12 ng/mg, respectively, for all the studied cannabinoids. The method proved to be simple, rapid, and precise. By using the weighted least squares linear regression (weighting factor 1/x2), the accuracy of the analytical method was improved at the lower end of the calibration curve (from 0.12 to 12 ng/mg; r >0.98). Hair samples collected from eight volunteers (in-patients of a drug abuse rehabilitation clinic) were submitted to the proposed method. Detection of the drugs was observed in samples of the volunteers who reported frequent marijuana use (at least ten times a week).     

Application of liquid-phase microextraction and gas chromatography-mass spectrometry for the determination of polychlorinated biphenyls in blood plasma

This study investigated the feasibility of applying liquid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS) to determine polychlorinated biphenyls (PCBs) in blood plasma. An efficient and simple extraction technique has been developed for the enrichment of PCBs from human blood plasma samples using single-step liquid-phase microextraction (LPME) in conjunction with a hollow fibre membrane (HFM). An eight PCB congener mixture was spiked into 2.5 ml of blood plasma, and the solution was then adjusted to pH 10.5 with a salinity of 20% (w/v) prior to making the total volume to 5 ml with ultrapure water. The porous HFM, filled with 3 microl of organic solvent, was then immersed into the solution, which was continuously agitated at 700 rpm for 30 min. Extract (1 microl) containing the pre-concentrated analytes was then injected into a GC-MS without further pre-treatment. Using an optimised extraction procedure, a large enrichment factor of the analytes, i.e. up to 241-fold was achieved in 30 min. The procedure resulted in a relative standard deviation of < 11% (n = 6), and a linear calibration range from 2.5 to 150 microg/l (r > 0.999), and detection limits between 0.07 and 0.94 microg/l, respectively. To demonstrate the feasibility of the procedure, PCB concentrations were determined in actual blood samples collected from the local population in Singapore using the optimised LPME technique.     

Rapid discrimination of fatty acid composition in fats and oils by electrospray ionization mass spectrometry.

Fatty acids in 42 types of saponified vegetable and animal oils were analyzed by electrospray ionization mass spectrometry (ESI-MS) for the development of their rapid discrimination. The compositions were compared with those analyzed by gas chromatography-mass spectrometry (GC-MS), a more conventional method used in the discrimination of fats and oils. Fatty acids extracted with 2-propanol were-detected as deprotonated molecular ions ([M-H]-) in the ESI-MS spectra of the negative-ion mode. The composition obtained by ESI-MS corresponded to the data of the total ion chromatograms by GC-MS. The ESI-MS analysis discriminated the fats and oils within only one minute after starting the measurement. The detection limit for the analysis was approximately 10(-10) g as a sample amount analyzed for one minute. This result showed that the ESI-MS analysis discriminated the fats and oils much more rapidly and sensitively than the GC-MS analysis, which requires several tens of minutes and approximately 10(-9) g. Accordingly, the ESI-MS analysis was found to be suitable for a screening procedure for the discrimination of fats and oils.     

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.