Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography-mass spectrometry

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI-SPME) with gas chromatography-mass spectrometry (GC-MS) is presented. The main parameters affecting the DI-SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180-0.988 and 0.0156-2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r(2)) were 0.994-0.999. The detection limits for whole blood were 0.05-1 microg x ml(-1), and those for urine 0.01-0.6 microg x ml(-1). Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI-SPME/GC-MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.     

Simultaneous detection of amphetamine-like drugs with headspace solid-phase microextraction and gas chromatography-mass spectrometry

A headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) procedure for the simultaneous detection of methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) in hair has been developed. This method is suitable for the separation of primary and secondary amines, is reproducible, is not time consuming, requires small quantities of sample and does not require any derivatization. It provides sufficient sensitivity and specificity, with limits of detection (LOD) and limits of quantitation (LOQ) for each substance of <0.7 and 1.90 ng/mg, respectively. Intra- and inter-day precision were within 2 and 10%, respectively. This method is suitable for routine clinical, epidemiological and forensic purposes and can be used for the preliminary screening of many other substances (amphetamine, methamphetamine, ketamine, ephedrine, nicotine, phencyclidine, methadone) in hair and other biological matrices such as saliva, urine and blood. We also describe the first application of this HS-SPME-GC-MS procedure to the analysis of hair and saliva samples from young people attending a disco in the Rome area. All positive hair samples were confirmed by the gas chromatography-mass-mass (GC-MS(2)) technique in positive chemical ionization (PCI) mode. Some examples of the use of the method in detecting different drugs are reported.     

Determination of metaldehyde in human serum by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of metaldehyde in human serum samples. Metaldehyde is extensively used as a molluscicide for the control of slugs and snails, and cases of metaldehyde poisoning have been reported. Metaldehyde was headspace-extracted on a polydimethylsiloxane (PDMS) fiber at 70 degrees C for 25 min, desorbed, and analyzed rapidly by GC-MS. The method was validated for limit of detection (LOD), linearity, precision, and recovery. Although the recovery of the sample was very low, the method itself was rapid with a low detection limit of 0.25 microg/ml, R.S.D. value 12.6%, and linearity range 0.5-25.0 microg/ml (r(2)=0.999). The results demonstrated that the SPME-GC-MS method for the analysis of metaldehyde is simple, rapid, solvent-free, and does not require any pre-analysis conversions.     

Sol-gel-based solid-phase microextraction and gas chromatography-mass spectrometry determination of dextromethorphan and dextrorphan in human plasma

A novel solid-phase microextraction (SPME) method was developed for isolation of dextromethorphan (DM) and its main metabolite dextrorphan (DP) from human plasma followed by GC-MS determination. Three different polymers, poly(dimethylsiloxane) (PDMS), poly(ethylenepropyleneglycol) monobutyl ether (Ucon) and polyethylene glycol (PEG) were synthesized as coated fibers using sol-gel methodologies. DP was converted to its acetyl-derivative prior to extraction and subsequent determination. The porosity of coated fibers was examined by SEM technique. Effects of different parameters such as fiber coating type, extraction mode, agitation method, sample volume, extraction time, and desorption condition, were investigated and optimized. The method is rapid, simple, easy and inexpensive and offers high sensitivity and reproducibility. The limits of detection are 0.010 and 0.015 ng/ml for DM and DP, respectively. The precisions for both analytes are below 5% (n=5). The correlation coefficient was satisfactory (r(2)>0.99) for both DM and DP. Linear ranges were obtained from 0.03 ng/ml to 2 microg/ml for DM and from 0.05 ng/ml to 2 microg/ml for DP.     

Rapid determination of acetone in human plasma by gas chromatography-mass spectrometry and solid-phase microextraction with on-fiber derivatization

Acetone is an important volatile disease marker. Due to its nature of activity and volatility, it is a difficult task to measure the concentration of acetone in biological samples with accuracy. In this paper, we developed a novel method for determination of trace amount acetone in human plasma by solid-phase microextraction technique with on-fiber derivatization. In this method, the poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber was used and O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) was first loaded on the fiber. Acetone in plasma sample was agitated into headspace and extracted by solid-phase microextraction (SPME) fiber and subsequently derivatized with PFBHA on the fiber. Acetone oxime was analyzed by gas chromatography-mass spectrometry (GC-MS). Quantitative analysis of acetone in plasma was carried out by using external standard method. The SPME conditions (extraction temperature and time) and the method validation were studied. The present method was tested by determination of acetone in diabetes plasma and normal plasma. Acetone concentration in diabetes plasma was found to be higher than 1.8mM, while in normal plasma was lower than 0.017 mM. The results show that the present method is a potential tool for diagnosis of diabetes.     

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME–GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25–1.05% of methanol and 0.38–0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 μg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1–0.5 μg/0.5 ml for methanol and 0.6 μg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.     

Determination of acrolein by headspace solid-phase microextraction gas chromatography and mass spectrometry

We developed a headspace solid-phase microextraction (headspace SPME) method to measure acrolein in human urine. This new technique resolves some problems with the headspace gas chromatography and mass spectrometry (GC–MS) method which we developed previously. With the original method, a column and a filament were damaged by the injection of air. A 0.5-ml urine (or phosphate-buffered saline) sample in a glass vial containing propionaldehyde as an internal standard was heated for 5 min. The SPME fiber (65 μm carbonwax–divinylbenzene fiber) was exposed to the headspace and then inserted into a GC–MS instrument in which a DB-WAX capillary column (30 m×0.32 mm, film thickness 0.5 μm) was installed. The total analysis time was 15 min. The inter-assay and intra-assay coefficients of variation were 10.07 and 5.79%, respectively. The calibration curve demonstrated good linearity throughout concentrations ranging from 1 to 10 000 nM. The headspace SPME method exhibits high sensitivity and requires a short analysis time as well as the previous method. We conclude that this method is useful to measure urinary acrolein.     

Fast detection of fluoroacetamide in body fluid using gas chromatography-mass spectrometry after solid-phase microextraction

A novel method for fast determination of fluoroacetamide, a kind of organic fluorine pesticide, in blood and urine samples was developed with acetamide as an internal standard using gas chromatography/mass spectrometry (GC/MS) after solid-phase microextraction (SPME) technique. The SPME was performed by immersing a PDMS fiber of 100 microm coating thickness in a sample solution for 25 min at 70 degrees C with (CH(3)CH(2))(4)NBr to improve the extraction efficiency. After a GC sample injection, the extracted fluoroacetamide was desorbed from the fiber for 4 min to perform the GC/MS detection with a HP-PLOT Q capillary column. The analytical conditions were optimized by examining systematically, the effects of experimental parameters on the ratio of characteristic ion peak areas of fluoroacetamide to acetamide. Under optimal conditions, the ratio was proportional to the concentration of fluoroacetamide ranging from 5.0 to 90 microg/ml with a detection limit of 1.0 microg/ml. The average recovery of fluoroacetamide in blood sample was 92.2%. The established method could be used for the fast and convenient measurement of fluoroacetamide in poisoned sample.     

Analysis of cantharidin in false blister beetles (Coleoptera: Oedemeridae) by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) method was developed to determine a type of terpenoid named as cantharidin in the false blister beetles, family Oedemeridae. The experimental parameters for HS-SPME method were optimized. Six commercial fibers for HS-SPME method development were tested and the divinylbenzene/carboxene/polydimethylsiloxane fiber was selected to provide the best detection of analyzed compound. The calibration curve showed linearity in the range of 0.1-50 μg mL(-1), correlation coefficient (R(2)=0.992), limit of detection (0.01 ng mL(-1)) and quantitation (0.04 ng mL(-1)) were obtained for the proposed method. The relative standard deviations of intra-day and inter-day assays were 7.8 and 3.4%, respectively. The recovery values, obtained after spiking the beetle samples by three concentration levels of standard solution, were higher than 87%. The results indicated the successful application of the proposed method on the analysis of cantharidin from the false blister beetles.     

Simultaneous determination of selenomethionine enantiomers in biological fluids by stable isotope dilution gas chromatography-mass spectrometry

A method for the stereoselective determination of D- and L-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS-SIM). DL-[(2)H(3,)(82)Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for D- and L-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for D-selenomethionine and 6% and 3% for L-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for D-selenomethionine and 2% and 1% for L-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.     

Determination of cocaine, benzoylecgonine and cocaethylene in human hair by solid-phase microextraction and gas chromatography-mass spectrometry

The present work describes a highly precise and sensitive method developed to detect cocaine (COC), benzoylecgonine (BE, its main metabolite) and cocaethylene (CE, transesterification product of the coingestion of COC with ethanol) in human head hair samples. The method was based on an alkylchloroformate derivatization of benzoylecgonine and the extraction of the analytes by solid-phase microextraction (SPME). Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify the analytes in selected ion monitoring mode (SIM). The limits of quantification and detection (LOQ and LOD) were: 0.1 ng/mg for COC and CE, and 0.5 ng/mg for BE. Good inter- and intra-assay precision was observed. The dynamic range of the assay was 0.1-50 ng/mg. The method is not time consuming and was shown to be easy to perform.     

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.     

Determination of amphetamines in human urine by headspace solid-phase microextraction and gas chromatography

Solid-phase microextraction (SPME) is under investigation for its usefulness in the determination of a widening variety of volatile and semivolatile analytes in biological fluids and materials. Semivolatiles are increasingly under study as analytical targets, and difficulties with small partition coefficients and long equilibration times have been identified. Amphetamines were selected as semivolatiles exhibiting these limitations and methods to optimize their determination were investigated. A 100- micro m polydimethylsiloxane (PDMS)-coated SPME fiber was used for the extraction of the amphetamines from human urine. Amphetamine determination was made using gas chromatography (GC) with flame-ionization detection (FID). Temperature, time and salt saturation were optimized to obtain consistent extraction. A simple procedure for the analysis of amphetamine (AMP) and methamphetamine (MA) in urine was developed and another for 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methamphetamine (MDMA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) using headspace solid-phase microextraction (HS-SPME) and GC-FID. Higher recoveries were obtained for amphetamine (19.5-47%) and methamphetamine (20-38.1%) than MDA (5.1-6.6%), MDMA (7-9.6%) and MDEA (5.4-9.6%).     

Sensitive headspace gas chromatography-mass spectrometry determination of trihalomethanes in urine

A sensitive and straightforward method for the determination of trihalomethanes (THMs) in urine by using headspace extraction technique has been developed. Chemical and instrumental variables were studied in order to optimize the method for sensitivity: an excess of KCl (4 g per 12 ml of urine), an oven temperature of 85 degrees C and an equilibration time of 30 min were selected. The use of the mass spectrometer in selected ion monitoring mode allows achieving linear ranges between 10 and 5000 ng/l and detection limits from 3 to 10 ng/l, for 12 ml of urine. The stability of the urine sample during storage at 4 and -20 degrees C was also evaluated: THMs remained stable for up to 2 days and 2 months, respectively. Finally, the method was successfully applied to study the THM uptake from swimmers of an indoor swimming pool, as well as non-swimmers. This study revealed that the concentrations of THMs in urine increased approximately three times for chloroform and bromodichloromethane after swimming activity. In addition, THMs in unchanged form were mainly excreted within 2-3h after the end of exposure.     

Analysis of Rhioxma Curcumae Aeruginosae volatiles by solid-phase microextraction with gas chromatography-mass spectrometry

In this paper, a headspace solid-phase microextraction (HS-SPME) method was applied to analyse the volatile compounds in a traditional Chinese medicine (TCM), Rhioxma Curcumae Aeruginosae. SPME parameters such as fibers, extraction temperature, extraction time and desorption time were investigated. Thirty-five volatile compounds were separated and identified. Relative standard deviations (RSDs) were less than 8.4%, showing that the method has a good reproducibility. The volatile constituents were also analyzed by steam distillation (SD) and thirty-seven compounds were identified. The similar results obtained by the two methods showed that SPME is a good alternative for the analysis of volatile constituents in Rhioxma Curcumae Aeruginosae samples and it is a relatively simple, rapid and solvent-free method.     

Determination of trichloroethylene in biological samples by headspace solid-phase microextraction gas chromatography/mass spectrometry

A simple, rapid and sensitive method for determination of trichloroethylene (TCE) in rat blood, liver, lung, kidney and brain, using headspace solid-phase microextraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS), is presented. A 100-microm polydimethylsiloxane (PDMS) fiber was selected for sampling. The major analytical parameters including extraction and desorption temperature, extraction and desorption time, salt addition, and sample preheating time were optimized for each of the biological matrices to enhance the extraction efficiency and sensitivity of the method. The lower limits of quantitation for TCE in blood and tissues were 0.25ng/ml and 0.75ng/g, respectively. The method showed good linearity over the range of 0.25-100ng TCE/ml in blood and 0.75-300ng TCE/g in tissues, with correlation coefficient (R(2)) values higher than 0.994. The precision and accuracy for intra-day and inter-day measurements were less than 10%. The relative recoveries of TCE respect to deionized water from all matrices were greater than 55%. Stability tests including autosampler temperature and freeze and thaw of specimens were also investigated. This validated method was successfully applied to study the toxicokinetics of TCE following administration of a low oral dose.     

Simultaneous determination of bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry

We devised a sensitive and simple method to simultaneously determine bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry. Bromvalerylurea and allylisopropylacetylurea were extracted using an Extrelut column with an internal standard, 2-bromohexanoylurea, followed by derivatization with heptafluorobutyric anhydride. The derivatized extract was submitted to GC-MS analysis of EI-SIM mode. The calibration curves of both compounds were linear in the concentration range from 0.01 to 10 microg/ml in both blood and urine samples. The lower limits of detection of bromvalerylurea and allylisopropylacetylurea were 0.005 and 0.005 microg/ml, respectively. This method proved most useful in accurately identifying these drugs in blood and urine from an autopsied individual.     

Simultaneous determination of amphetamine and its analogs in human whole blood by gas chromatography-mass spectrometry

A sensitive and specific gas chromatography-mass spectrometry (GC-MS) method for the determination of amphetamine (AM), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA) and methylenedioxyethylamphetamine (MDEA) in whole blood was designed, using the respective pentadeuterated analogs of the analytes as internal standards (I.S.). After alkalinisation of blood samples, the amphetamines were extracted using diethyl ether, derivatized with heptafluorobutyric anhydride, then purified by successive washings with deionized water and 4% NH₄OH. Extraction recoveries were 85.2% for AM, 90.9% for MA, 76.5% for MDA, 84.1% for MDMA and 63.6% for MDEA. Chromatographic separation was performed on a non-polar 30 m×0.32 mm HP 5 MS capillary column using a temperature program. Detection was carried out in the electron-impact, selected ion-monitoring mode, using three mass-to-charge ratios for each analyte and one for each I.S. Limits of detection ranged from 0.5 to 8 ng/ml and limits of quantification were 10 ng/ml for AM, MDMA and MDEA; 20 ng/ml for MA; and 50 ng/ml for MDA. The method was linear from this limit up to 1000 ng/ml for all analytes, with good intra-assay precision and good intermediate precision and accuracy over these ranges. There was no interferences from other sympathomimetic drugs such as ephedrine, norephedrine or methoxyphenamine. This method is thus suitable for clinical and forensic toxicology, as well as for doping control.     

Rapid screening procedure based on headspace solid-phase microextraction and gas chromatography-mass spectrometry for the detection of many recreational drugs in hair

An increasing number of synthetic drugs are appearing on the illicit market and on the scene of drug use by youngsters. Official figures are underestimated. In addition, immunochemical tests are blind to many of these drugs and appropriate analytical procedures for routine clinical and epidemiological purposes are lacking. Therefore, the perceived increasing abuse of recreational drugs has not been proved yet. In a previous paper, we proposed a procedure for the preliminary screening of several recreational substances in hair and other biological matrices. Unfortunately, this procedure cannot apply to cocaine. Consequently, we performed a new headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) procedure for the simultaneous detection of cocaine, amphetamine (A), methamphetamine (MA), methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE), N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), ketamine, and methadone in human hair. Hair was washed with water and acetone in an ultrasonic bath. A short acid extraction with 1M hydrochloric acid was needed; the fiber was exposed to a 5 min absorption at 90 degrees C and thermal desorption was performed at 250 degrees C for 3 min. The procedure was simple, rapid, required small quantities of sample and no derivatization. Good linearity was obtained over the 0.1-20.0 ng/mg range for the target compounds. Sensitivity was good enough: limits of detection (LOD) were 0.7 ng/mg of hair for the majority of substances. The intra-day precision ranged between 7 and 20%. This paper deals with the analytical performance of this procedure and its preliminary application to hair samples obtained on a voluntary basis from 183 young people (138 males and 45 females) in the Rome area.     

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography-mass spectrometry

A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography-mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax/divinylbenzene (CW/DVB 65 microm) and polydimethylsiloxane (PDMS 100 microm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 microL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 microg/mL for blood, and 0.01 to 50 microg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within +/-15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.