Compounds In Aqueous Samples Research Articles

Full automation of derivatization—solid-phase microextraction–gas chromatography–mass spectrometry with a dual-arm system for the determination of organometallic compounds in aqueous samples

The determination of organometallic compounds in aqueous samples by in-vial derivatization and headspace solid-phase microextraction (SPME)–gas chromatography (GC)–mass spectrometry (MS) has been fully automated using a Twin PAL dual-arm robotic system. Linearity, accuracy, sensitivity for a series of n-methyl, n-ethyl, and n-phenyl metal substituted chloride compounds of tin, lead, and mercury were investigated. The automated method was compared to similar manual methods and improved precision, speed and throughput was achieved. By originally programming the Twin PAL dual-arm system with the supplier’s software (Cycle Composer, Version 1.5.0) the arms on the robot were only able to work in sequence. However, in order to have a flexible system and exploit time efficiently the robotic arms must work simultaneously. This was accomplished by programming the robot with the new software package called Cruise Control 4-2 for Twin PALs. Compared to Cycle Composer, Cruise Control 4-2 enhanced the speed and throughput of the automated system further. In addition, with a built-in crash prevention protocol and an improved user interface a more user-friendly system was obtained.

LC-MS analysis in the aquatic environment and in water treatment ? a critical review

LC-MS has become an invaluable technique for trace analysis of polar compounds in aqueous samples of the environment and in water treatment. LC-MS is of particular importance due to the impetus it has provided for research into the occurrence and fate of polar contaminants, and of their even more polar transformation products. Mass spectrometric detection and identification is most widely used in combination with sample preconcentration, chromatographic separation and atmospheric pressure ionization (API). The focus of the first part of this review is directed particularly toward instruments and method development with respect to their applications for detecting emerging contaminants, microorganisms and humic substances (HS). The current status and future perspectives of 1) mass analyzers, 2) ionization techniques to interface liquid chromatography (LC) with mass spectrometry (MS), 3) methods for preconcentration and separation with respect to their application for water analysis are discussed and examples of applications are given. Quadrupole and ion trap mass analyzers with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are already applied in routine analysis. Time-of-flight (TOF) mass spectrometers are of particular interest for accurate mass measurements for identification of unknowns. For non-polar compounds, different ionization approaches have been described, such as atmospheric pressure photoionization (APPI), electrochemistry with ESI, or electron capture ionization with APCI. In sample preconcentration and separation, solid phase extraction (SPE) with different non-selective sorbent materials and HPLC on reversed-phase materials (RP-HPLC) play the dominant role. In addition, various on-line and miniaturized approaches for sample extraction and sample introduction into the MS have been used. Ion chromatography (IC), size-exclusion chromatography (SEC), and capillary electrophoresis (CE) are alternative separation techniques. Furthermore, the issues of compound identification, matrix effects on quantitation, development of mass spectral libraries and the topic of connecting analysis and toxicity bioassays are addressed.

Development of plasticised PVC sensing films for the determination of BTEX compounds in aqueous samples

A novel plasticised PVC polymer membrane as a sensing film for the determination of BTEX compounds using ATR-FTIR spectroscopy is demonstrated. A range of 10 plasticised PVC phases have been investigated using toluene and tetrachloroethylene as test analytes. Both analyte enrichment rates and infrared absorbance values were considered when choosing a suitable polymer for sensing. An enhancement in analyte absorbance at the characteristic IR absorption bands was noted as the plasticiser concentration in the film was increased. 2% PVC with 75% diisooctyl azelate was found to show promising results for simultaneous determination of the BTEX compounds. All BTEX analytes can be measured in less than 8 min. A study of a multicomponent sample demonstrated that analyte enrichment times were influenced by the presence of even one additional analyte component in the sample.

Determination of daidzein and genistein in soybean foods by automated on-line in-tube solid-phase microextraction coupled to high-performance liquid chromatography

An automated on-line method for the determination of the isoflavones, daidzein and genistein, was developed using in-tube solid-phase microextraction coupled to high-performance liquid chromatography (in-tube SPME–HPLC). In-tube SPME is a new extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. Daidzein, genistein and their glucosides tested in this study were clearly separated within 8 min by HPLC using an XDB-C 8 column with diode array detection. In order to optimize the extraction of these compounds, several in-tube SPME parameters were examined. The glucosides daidzin and genistin were analyzed as aglycones after hydrolysis because the glucosides were not concentrated by in-tube SPME. The optimum extraction conditions for daidzein and genistein were obtained with 20 draw/eject cycles of 40 μl of sample using a Supel-Q porous layer open tubular capillary column. The extracted compounds were easily desorbed from the capillary by mobile phase flow, and carryover was not observed. Using the in-tube SPME–HPLC method, the calibration curves of these compounds were linear in the range 5–200 ng/ml, with a correlation coefficient above 0.9999 ( n=18), and the detection limits ( S/ N=3) were 0.4–0.5 ng/ml. This method was successfully applied to the analysis of soybean foods without interference peaks. The recoveries of aglycones and glucosides spiked into food samples were above 97%.

Analysis of aromatic amines in water samples by liquid–liquid–liquid microextraction with hollow fibers and high-performance liquid chromatography

Liquid–liquid–liquid microextraction (LLLME) with hollow fibers in high-performance liquid chromatography (HPLC) has been applied as a rapid and sensitive quantitative method for the detection of four aromatic amines (3-nitroaniline, 4-chloroaniline, 4-bromoaniline and 3,4-dichloroaniline) in environmental water samples. The preconcentration procedure was induced by the pH difference inside and outside the hollow fiber. The target compounds were extracted from 4-ml aqueous sample (donor solution, pH∼13) through a microfilm of organic solvent (di- n-hexyl ether), immobilized in the pores of a hollow fiber (1.5 cm length×0.6 mm I.D.), and finally into 4 μl of acid acceptor solution inside the fiber. After a prescribed period of time, the acceptor solution inside the fiber was withdrawn into the microsyringe and directly injected into the HPLC system for analysis. Factors relevant to the extraction procedure were studied. Up to 500-fold enrichment of analytes could be obtained under the optimized conditions (donor solution: 0.1 M sodium hydroxide solution with 20% sodium chloride and 2% acetone; organic phase: di- n-hexyl ether; acceptor solution: 0.5 M hydrochloric acid and 500 m M 18-crown-6 ether; extraction time of 30 min; stirring at 1000 rev./min). The procedure also served as a sample clean-up step. The influence of humic acid on the extraction efficiency was also investigated, and more than 85% relative recoveries of the analytes at two different concentrations (20 and 100 μg/l) were achieved at various concentration of humic acid. This technique is a low cost, simple and fast approach to the analysis of polar compounds in aqueous samples.

Development of an infrared hollow waveguide sampler for the detection of organic compounds in aqueous solutions with limited sample volumes.

In this study, an infrared (IR) hollow waveguide sampler was developed to detect organic compounds in aqueous samples with sample volumes less than 50 microL. This sampler was prepared by coating a thin hydrophobic film inside the IR hollow waveguide. After injecting a certain amount of aqueous solution, organic compounds could be absorbed into the hydrophobic film by partitions. By removing the residual water in the hollow waveguide sampler with a nitrogen purging gas, the absorbed organic compounds could be sensed using IR radiation. To investigate the applicability of this hollow waveguide sampler in the detection of small amounts of aqueous samples, an analytical working function was developed following an examination of the parameters which influence the analytical signals. Such factors as the volume of the aqueous solution, the sample concentration, the length of the hollow waveguide, and the sensitivity of this method were investigated. Excellent agreement between the analytical and theoretical predicted values was observed. Upon examining the linear relationship between the analyte signals and the concentration, the regression coefficients were generally higher than 0.998 in the examined concentration range of 1 to 100 ppm. Under the condition that the sample volume was 300 microL and based on three-times the spectra noise level, the calculated detection limits for this method were found at around 1 ppm for the examined analytes.

Automated headspace solid-phase dynamic extraction for the determination of amphetamines and synthetic designer drugs in hair samples

The technique of automated headspace solid-phase dynamic extraction (SPDE) coupled with gas chromatography–mass spectrometry was evaluated for the determination of amphetamines and synthetic designer drugs in hair samples. Headspace SPDE is a novel method for the solventless extraction of organic compounds in aqueous samples. In a so-called inside needle capillary absorption trap a hollow needle with an internal coating of polydimethylsiloxane is used as extraction and preconcentration medium. Sampling is performed on the solution headspace by passing the gas through the device actively by a syringe. Analytes present in the sample are sorbed onto the deposited stationary phase. The syringe needle is placed into the injection port of a GC and rapid heating of the metal needle induces the desorption of analytes. For the determination of amphetamine, methamphetamine, 3,4-methylendioxyamphetamine (MDA), 3,4-methylendioxymethamphetamine, 3,4-methylendioxyethylamphetamine (MDEA), 3,4-methylendioxyphenyl-2-butanamine and N-methyl-1-(3,4-methylendioxyphenyl)-2-butanamine in human hair samples, 10 mg of hair were hydrolysed with sodium hydroxide. After absorption of analytes for an on-coating derivatization procedure the SPDE needle was directly placed into the headspace of a second vial containing N-methyl-bis(trifluoroacetamide). A validation procedure revealed absolute analyte recoveries between 10.2 and 16.7%. Linearity was obtained from 0.1 to 20 ng/mg with coefficients of correlation between 0.992 and 0.999. Intra- and inter-day precision were determined at two different concentrations and resulted in ranges between 1.4 and 4.1% (intra-day) and 4.2–14.6% (inter-day). Limits of detection between 0.03 ng/mg (MDA) and 0.19 ng/mg (MDEA) were achieved. Results indicated that SPDE is a rapid and sensitive method for the analysis of biological samples. Compared to solid-phase microextraction we found a higher extraction rate coupled with a faster automated operation.

Automated on-line in-tube solid-phase microextraction coupled with high performance liquid chromatography for the analysis of bisphenol A, alkylphenols, and phthalate esters in foods contacted with plastics

An automated on-line method for the analysis of endocrine disruptors related to plastics was developed using in-tube solid-phase microextraction coupled with high performance liquid chromatography (in-tube SPME/HPLC). In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. Suspected endocrine disrupting compounds, bisphenol A, two alkylphenols, and nine phthalate esters, tested in this study were well separated with a Hypersil ODS column with diode array detection using acetonitrile/water as a mobile phase. In order to optimize the extraction of these compounds, several in-tube SPME parameters were examined. The optimum extraction conditions were 20 draw/eject cycles of 40 μL of sample using a Supel-Q PLOT capillary column. The extracted compounds were easily desorbed from the capillary by mobile phase flow, and carryover was not observed. Using in-tube SPME/HPLC method, the calibration curves of these compounds were linear in the range from 1 to 500 ng/mL, with correlation coefficient above 0.991 (n = 18), and the detection limits (S/N = 3) were 0.1–4.0 ng/mL. This method was successfully applied to the migration test from plastics and the analysis of food samples.

Comprehensive-trace level determination of methyltin compounds in aqueous samples by cryogenic purge-and-trap gas chromatography with flame photometric detection.

A comprehensive method was developed for the sensitive and fast determination of trace levels of methyltin compounds in aqueous samples. Tin compounds in aqueous solution at pH 5 were converted to the corresponding volatile hydrides: CH3SnH3, (CH3)2SnH2, and (CH3)3SnH, by reaction with potassium borohydride. A CP-4010 purge and trap injector (PTI) was used to purge analyte species from water directly. The volatile derivatives were base-line separated on a capillary column in an Angilent-6890 gas chromatograph by a suitable temperature program and were detected by a flame photometric detector (FPD). The detection limits were 18 ng L-1 for monomethyltin, 12 ng L-1 for dimethyltin, and 3 ng L-1 for trimethyltin, respectively. This method was successfully applied to the determination of methyltin compounds in different aqueous samples.

Determination of phenols in water using liquid phase microextraction with back extraction combined with high-performance liquid chromatography

Liquid phase microextraction with back extraction (LPME/BE) combined with high-performance liquid chromatography (HPLC) was studied for the determination of a variety of phenols in water samples. The target compounds were extracted from 2-ml aqueous sample adjusted to pH 1 (donor solution) through a microliter-size organic solvent phase (400-μl n-hexane), confined inside a small PTFE ring, and finally into a 1-μl basic aqueous acceptor microdrop suspended in the aforementioned solvent phase from the tip of a microsyringe needle. After extracting for a prescribed time, the microdrop was taken back into the syringe and directly injected into an HPLC for detection. Factors relevant to the extraction procedure were studied. At the optimized extraction conditions, a large enrichment factor (more than 100-fold) can be achieved for most of the phenols within 35 min. The detection limit range was 0.5–2.5 μg/l for different analytes in aqueous samples. The results demonstrate the suitability of the LPME/BE approach to the analysis of polar compounds in aqueous samples.

Construction and validation of automated purge-and-trap–gas chromatography for the determination of volatile organic compounds

An automated purge-and-trap chromatographic system for the determination of dissolved volatile organic compounds in aqueous samples was built in the laboratory with minimum cost both in the construction and routine operation. This system was built upon a commercial gas chromatograph with full automation capability using self-developed hardware and software. The use of a multi-sorbent bed quantitatively trapped a wide range of volatile organic compounds at ambient temperature, including the extremely volatile ones such as dichlorofluoromethane (CFC-12). Flash heating for rapid desorption and adequate plumbing for minimizing dead volume resulted in excellent chromatographic separation at above-ambient temperatures, which eliminated the need for cryogen for cooling at the head of the column, a second refocusing stage, or entire GC oven for refocusing. This cryogen-free system was tested with standard solutions and environmental samples for determining hydrocarbons with flame ionization detection, and halogenated compounds with electron-capture detection. An innovative method was also developed for validating the system’s linearity for extremely volatile compounds. By introducing ambient air, which usually contains constant levels of anthropogenic halocarbons, e.g., CFC-12 and CFC-11 (CCl 3F), the need to prepare aqueous standards containing extremely volatile compounds is avoided, hence providing a convenient method for evaluating a purge-and-trap system.

Determination of Sn- and Pb-organic compounds by solid-phase microextraction-gas chromatography-atomic emission detection (SPME-GC-AED) after in situ propylation with sodium tetrapropylborate

A method is presented for the simultaneous derivatisation and solid-phase microextraction (SPME) of organotin and organolead compounds in aqueous samples or extracts, based on gas chromatographic separation and atomic emission detection (GC-AED). Derivatisation is carried out in situ in aqueous solution by sodium tetrapropylborate, which has only recently become commercially available. The use of the tetrapropylborate reagent allows the simultaneous derivatisation and determination of all environmentally relevant organotin (butyl- and phenyltin) and organolead (methyl- and ethyllead) compounds. Optimum reaction and extraction conditions are established. The effect of temperature on the combined derivatisation/extraction step is studied, showing that the derivatisation yield has an optimum at elevated temperature, while the extraction efficiency generally decreases with increasing temperature. For practical reasons, it was decided to carry out the determination at room temperature with a reaction/extraction time of 20 min. Under these conditions, detection limits in the range of 0.2 ng l−1 (as metal) could be achieved with headspace sampling and a 20 ml sample volume. The repeatability of measurements in the low ng l−1 range is between 2 and 9% and the relative standard deviation of the method is generally lower than 5% within the calibrated range.

Automated on-line in-tube solid-phase microextraction followed by liquid chromatography/electrospray ionization-mass spectrometry for the determination of chlorinated phenoxy acid herbicides in environmental waters.

A method for the determination of six chlorinated phenoxy acid herbicides in river water was developed using in-tube solid-phase microextraction (SPME) followed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from a sample directly into an open tubular capillary by repeated draw/eject cycles of the sample solution. Simple mass spectra with strong signals corresponding to [M-H]- and [M-RCOOH]- were observed for all herbicides tested in this study. The best separation of these compounds was obtained with a C18 column using linear gradient elution with a mobile phase of acetonitrile-water containing 5 mmol l-1 dibutylamine acetate (DBA). To optimize the extraction of herbicides, several in-tube SPME parameters were examined. The optimum extraction conditions were 25 draw/eject cycles of 30 microliters of sample in 0.2% formic acid (pH 2) at a flow rate of 200 microliters min-1 using a DB-WAX capillary. The herbicides extracted by the capillary were easily desorbed by 10 microliters acetonitrile. Using in-tube SPME-LC/ESI-MS with time-scheduled selected ion monitoring, the calibration curves of herbicides were linear in the range 0.05-50 ng ml-1 with correlation coefficients above 0.999. This method was successfully applied to the analysis of river water samples without interference peaks. The limit of quantification was in the range 0.02-0.06 ng ml-1 and the limit of detection (S/N = 3) was in the range 0.005-0.03 ng ml-1. The repeatability and reproducibility were in the range 2.5-4.1% and 6.2-9.1%, respectively.

The determination of six taste and odour compounds in water using Ambersorb 572 and high resolution mass spectrometry.

A method for the analysis of six taste and odour causing compounds in aqueous samples using the granular adsorbent, Ambersorb 572, and gas chromatography-high resolution mass spectrometry (GC-HRMS) has been developed. This method for the determination of geosmin, 2-methylisoborneol (2-MIB), 2-isopropyl-3-methoxypyrazine (IPMP), 2-isobutyl-3-methoxypyrazine (IBMP), 2,3,6-trichloroanisole (236-TCA) and 2,4,6-trichloroanisole (246-TCA) is highly productive [up to 40 samples per day + 23 quality control (QC) samples] and provides rapid (24-48 h) turnaround times. The analytes are extracted from water by the addition of Ambersorb 572 to the sample bottle and rolling for 1 h. The adsorbent is isolated by filtration and allowed to air dry for 1 h. The Ambersorb 572 is transferred to an autosampler vial and the analytes are desorbed into dichloromethane. The extract is analysed by GC-HRMS at 7000 resolving power (RP). Quantification is performed by isotope dilution and internal standard techniques utilizing d3-geosmin, d3-2-MIB, d5-246-TCA and 2-sec-butyl-3-methoxypyrazine (s-BMP). Method precisions of 3.5-5.8% and accuracies of +/- 5.7-8.9% were obtained. Reporting detection limits (RDLs) of 1.0 ng L-1 were obtained for 2-MIB, geosmin, IPMP and IBMP, while RDLs of 2.0 ng L-1 were obtained for 236-TCA and 246-TCA.

Polypyrrole-coated capillary coupled to HPLC for in-tube solid-phase microextraction and analysis of aromatic compounds in aqueous samples.

In-tube solid-phase microextraction (SPME) based on a polypyrrole (PPY)-coated capillary was investigated for the extraction of aromatic compounds from aqueous solutions. The PPY-coated capillary was coupled on-line to HPLC that was programmed with an autosampler to achieve automated in-tube SPME and HPLC analysis. Three groups of aromatics, including both polar and nonpolar compounds, were examined. The results demonstrated that the PPY coating had a higher extraction efficiency than the currently used commercial capillary coatings, especially for polycyclic aromatic compounds and polar aromatics due to the increasing pi-pi interactions, interactions by polar functional groups, and hydrophobic interactions between the polymer and the analytes. In addition to the functional groups in the PPY coating, which contributed to the higher extraction efficiency and selectivity toward analytes, the coating's porous surface structure,which was revealed by electron microscopy experiments, provided a high surface area that allowed for high extraction efficiency. It was found that the extraction efficiency and selectivity could be tuned by changing the coating thickness. The preliminary study of the extraction mechanism indicated that analytes were extracted onto the PPY coating mainly by an adsorption mechanism. The method was used for the extraction and analysis of both polar and nonpolar aromatics in aqueous samples.

Speciation of organoarsenic compounds by polypyrrole-coated capillary in-tube solid phase microextraction coupled with liquid chromatography/electrospray ionization mass spectrometry

In-tube solid phase microextraction (SPME) is an on-line extraction technique for compounds in aqueous samples, in which analytes are extracted and concentrated from the sample directly into a coated capillary by repeated aspirate/dispense steps. In this paper, a polypyrrole (PPY) coated capillary and several commercially available capillaries (used for GC separation) were used to examine their extraction efficiencies to the organoarsenic compounds studied. Compared with commercial capillaries that were currently used for in-tube SPME, the PPY coated capillary has shown better extraction efficiency to most of the compounds studied, especially to the anionic species, due to the inherent multi-functionality of pyrrole polymer. For the first time, this PPY coated capillary has been used for automated in-tube SPME and speciation of organoarsenic species in aqueous samples when coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Organoarsenic compounds in water samples and arsenobetaine in a certified reference material (DORM-2) were analyzed by this method.

ESCV meeting on quality control

A scheme addressing analytical quality in marine chemistry has been established within the UK. The National Marine Analytical Quality Control (NMAQC) scheme is primarily intended to underpin the co-ordinated National Monitoring Programme established in 1993/1994. In this paper we review the progress of the scheme during its first 4 years of operation with respect to the determination of concentrations of organochlorine compounds in aqueous samples, and relate performance in the exercises undertaken to date with the requirements of the NMP The further development of the NMAQC scheme in respect of these determinands is also discussed.

In-membrane preconcentration/membrane inlet mass spectrometry of volatile and semivolatile organic compounds.

The on-line determination of volatile and semivolatile organic compounds (SVOCs) is reported using membrane inlet mass spectrometry with in-membrane preconcentration (IMP-MIMS). Semivolatile organic compounds in aqueous samples are preconcentrated in a flow-through silicone hollow-fiber membrane inlet held in a GC oven. The sample stream is replaced with air, and the SVOCs are thermally desorbed into the mass spectrometer by rapid heating of the membrane. The method is evaluated for the on-line determination of 4-fluorobenzoic acid, 3,5-difluorobenzoic acid, 2-chlorophenol, p-tert-butylphenol, and dimethyl sulfoxide (DMSO) in water. The selectivity of the IMP-MIMS technique for SVOCs in the presence of VOCs is demonstrated. Cryotrapping and a rapid gas chromatographic separation step were added between the membrane and the mass spectrometer ion source for the determination of SVOCs in complex mixtures. The procedure is demonstrated for the determination of dimethyl sulfoxide (DMSO) in equine urine, using internal standardization with DMSO-d6. Full-scan electron ionization (EI) mass spectrometric detection showed good linearity (R = 0.998) and RSDs, relative to the internal standard, of 2.2% for desorption only and 4.6% for desorption and cryotrapping.

Determination of Butyltin Compounds in Aqueous Samples by Gas Chromatography with Flame Photometric Detector and Headspace Solid-Phase Microextraction after in-situ Hydride Derivatization

A method for the extraction and determination of butyltin species in aqueous samples by solid-phase microextraction (SPME) combined with a capillary gas chromatography—flame photometric detector (GC-FPD) is described. The butyltin species was converted to its hydride form by sodium tetrahydroborate (NaBH4) in a closed headspace vial prior to extraction. A laboratory-assembled SPME device including a fused-silica fiber and a modified microsyringe protection part was used throughout the experiment. The extraction was an equilibrium process that depended on the butyltin species partitioning between the liquid phase and the fiber. When the equilibrium was reached, the fiber was directly transferred to a GC column under the protection of a microsyringe, where the analyte was thermally desorbed inside the heated injector and subsequently, separated in a HP-1 capillary column and detected by a laboratory-made flame photometric detector using quartz surface-induced tin emission. The detection limits based on the signal equal to 3 times of the base-line noise were 0.2 ¼g/L, 0.2 ¼g/L, 0.1 ¼g/L and 0.02 ¼g/L for monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT) and tetrabutyltin (TeBT), respectively. The recovery ranged from 85% to 117%. The proposed method has been applied to determination of butyltin species in various aqueous samples.

Simple and rapid determination of amphetamine, methamphetamine, and their methylenedioxy derivatives in urine by automated in-tube solid-phase microextraction coupled with liquid chromatography-electrospray ionization mass spectrometry.

A simple and rapid method for the determination of amphetamine, methamphetamine, and their 3,4-methylenedioxy derivatives in urine samples was developed using automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). In-tube SPME is an extraction technique for organic compounds in aqueous samples in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC-MS analyses of stimulants were initially performed by liquid injection onto an LC column to determine spectra. Five stimulants tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M+H]+ were observed for all stimulants. The stimulants were well separated with a Supelcosil LC-CN column using acetonitrile/50mM ammonium acetate (15:85) as a mobile phase. In order to optimize the extraction of stimulants, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 35 microL of sample in 50mM Tris-HCI (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary column. The stimulants extracted by the capillary were easily desorbed by mobile phase flow, and carryover of stimulants was not observed. Using in-tube SPME-LC-ESI-MS with selected ion monitoring, the calibration curves of stimulants were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9985 (n = 18) and detection limits (S/N = 3) of 0.38-0.82 ng/mL. This method was successfully applied to the analysis of human urine samples without interference peaks. The recoveries of stimulants spiked into urine samples were above 81%.