Simultaneous determination of local anesthetics including ester-type anesthetics in human plasma and urine by gas chromatography–mass spectrometry with solid-phase extraction

The present study describes the simultaneous determination of seven different kinds of local anesthetics and one metabolite by GC–MS with solid-state extraction: Mepivacaine, propitocaine, lidocaine, procaine (an ester-type local anesthetics), cocaine, tetracaine (an ester-type local anesthetics), dibucaine (Dib) and monoethylglycinexylidide (a metabolite of lidocaine) were clearly separated from each other and simultaneously determined by GC–MS using a DB-1 open tubular column. Their recoveries ranged from 73–95% at the target concentrations of 1.00, 10.0 and 100 μg/ml in plasma, urine and water. Coefficients of variation of the recoveries ranged from 2.3–13.1% at these concentrations. The quantitation limits of the method were approximately 100 ng/ml for monoethylglycinexylidide, propitocaine, procaine, cocaine, tetracaine and dibucaine, and 50 ng/ml for lidocaine and mepivacaine. This method was applied to specimens of patients who had been treated with drip infusion of lidocaine, and revealed that simultaneous determination of lidocaine and monoethylglycinexylidide in the blood and urine was possible.     

Gas chromatographic–mass spectrometric analysis of dichlorobenzene isomers in human blood with headspace solid-phase microextraction

Headspace solid-phase microextraction (HS-SPME) was utilized for the determination of three dichlorobenzene isomers (DCBs) in human blood. In the headspace at 30°C, DCBs were absorbed for 15 min by a 100-μm polydimethylsiloxane (PDMS) fiber. They were then analyzed by capillary column gas chromatography–mass spectrometry (GC–MS). By setting the initial column oven temperature at 20°C, the three isomers were resolved at the baseline level. p-Xylene-d₁₀ was used as the internal standard (I.S.). For quantitation, the molecular ion at m/z 146 for each isomer and the molecular ion at m/z 116 for I.S. were selected. For day-to-day precision, relative standard deviations in the range 3.2–10.7% were found at blood concentrations of 1.0 and 10 μg/ml. Each compound was detectable at a level of at least 0.02 μg per 1 g of whole blood (by full mass scanning). HS-SPME–GC–MS, when performed at relatively low temperatures, was found to be feasible in toxicological laboratories. Using this method, the plasma levels of one patient who had drunk a pesticide-like material were measured.     

Liquid chromatography–electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring

A simple and fast yet highly sensitive and specific method based on HPLC coupled to electrospray ionization mass spectrometry has been developed for the quantitation of corticosterone in rat plasma. After extraction of rat plasma (100 μl) with diethyl ether using 5-pregnen-3β-ol-20-one-16α-carbonitrile (Sigma) as internal standard, HPLC was performed on a short C₈ column (Zorbax-Eclipse, 50×4.6 mm I.D.) using a steep methanol–water gradient (methanol 54% to 90% in 6 min). Detection was performed on a single quadruple mass spectrometer in selected ion monitoring mode (m/z 369 for corticosterone and 364 for the internal standard). The detection limit of the assay was 9 fmol (3 pg) of corticosterone on column. In vitro data were subjected to curve fitting (cubic, r²=0.9999). Recovery of corticosterone after extraction ranged from 81 to 93%. The relative standard deviations for intra- and inter-assay precision ranged from 0.8 to 3.6% and 5.2 to 12.9%, respectively. Corticosterone did not undergo any appreciable degradation when stored in plasma at −20°C for 2 months. The assay is routinely used in our laboratory to examine corticosterone levels as a marker of stress in rats and may also be used for the determination of 18-hydroxy-11-deoxycorticosterone.     

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%).     

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.     

Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography–electron impact mass spectrometry

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography–mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.     

Determination of methadone and its metabolites EDDP and EMDP in human hair by headspace solid-phase microextraction and gas chromatography–mass spectrometry

A simple method for analysis of methadone and its two main metabolites EDDP and EMDP in hair was developed using automatic headspace solid-phase microextraction (HS-SPME) at a multipurpose sampler and gas chromatography – mass spectrometry with electron impact ionization and selected ion monitoring (GC–MS-SIM). The washed hair pieces were digested in the closed headspace vial in 1 ml 1 M NaOH containing 0.5 g NaCl and each 10 ng of the internal standards D₉-methadone and D₃-EDDP at 110°C for 20 min. Then the HS-SPME was performed with a 65 μm polydimethylsiloxan/divinylbenzene fiber at the same temperature in the same vial for another 20 min followed by the desorption in the GC injection port. The calibration curves were linear between 0.1 and 3 ng/mg (methadone and EMDP) and 10 ng/mg (EDDP) respectively, at higher concentrations a negative deviation from linearity was found. The detection limits were 0.03 ng/mg (methadone) and 0.05 ng/mg (EDDP and EMDP), and the reproducibility was 9.2% for methadone and 11.2% for EDDP (n=12). The method was applied to hair samples of 26 drug fatalities. 19 cases were positive with 0.36–11.8 ng/mg methadone and 0.19 –10.8 ng/mg EDDP. EMDP was found only in two cases with 0.18 and 0.84 ng/mg. The methadone concentration range was in agreement with previous data, but the EDDP/methadone concentration ratios (0.19–0.67) were definitely higher than those determined by other methods.     

Determination of organochlorine pesticides and polychlorinated biphenyls in human serum using headspace solid-phase microextraction and gas chromatography-electron capture detection

A simple procedure for the determination of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum using headspace solid-phase microextraction (HS-SPME) was developed. The analysis was carried out by gas chromatography (GC) equipped with electron capture detector (ECD). A 2(7-4) Plackett-Burman reduced factorial design for screening and a central composite design for optimizing the significant variables were applied. A 100 microm PDMS fiber, 3/5 headspace ratio (3 ml in 5 ml vial), 85 degrees C extraction temperature, 50 min extraction time, and 1 ml of acidic solution (pH 3) added to 1 ml of diluted serum (1:1) were chosen for the best response in HS extraction mode. The detection limits found were from 1 pg/ml (PCB 167) to 52 pg/ml (beta-HCH), the relative standard deviation for the procedure varied from 3% (PCB 52) to 12% (PCB 189) and the accuracy was checked by using validated solid-phase extraction (SPE) procedure. The method that avoids the use of clean-up steps and the hazardous solvents enabled reliable determinations of the OCPs and the PCBs except beta-HCH. The method was applied to the analysis of 33 human serum samples. The most abundant target compound was p-p'-DDE (range, 0.3-8.0 ng/ml; median value, 2.1 ng/ml). Among the PCBs the prevalent congeners were 138, 153 and 180.     

Stereoselective determination of mepivacaine in human serum using a brush-type chiral stationary phase and solid-phase extraction

A stereoselective high-performance liquid chromatography assay method was developed for the quantitation of R-(+)- and S_-(−)-mepivacaine in human serum. The assay uses a Pirkle brush-type. ((S)-tert.-leucine, (R)-(-naphthyl)ethylamine stationary phase (Sumichiral OA-4700, 250×4 mm I.D.) at ambient temperature with a mobile phase of hexane-ethylenedichloride-absolutte methanol (85:10:5, v/v) for the separation of R-(+) and (S)-(−)-mepivacaine. The eluents were monitored using UV detection at 220 nm. Isolation of the analytes from serum was performed using a 1-ml C₁₈ solid-phase extraction cartridge with high recovery and selectivity. The detection limits were 100 ng/ml for each enantiomer and the limits of quantitation were 150 ng/ml for both enantiomers. Linear calibration curves in the 150–2400 ng/ml range showed good correlation coefficients (r>0.9994, N=3). Precision and accuracy of the method were within 2.1–5.3 and 2.0–3.6%, respectively, for (R)-(+)-mepivacaine and 2.7–5.7% and 1.7–4.2%, respectively, for S-(−)-mepivacaine.     

Headspace solid-phase microextraction and gas chromatographic–mass spectrometric screening for volatile hydrocarbons in blood

Optimization for headspace solid-phase microextraction (SPME) was studied with a view to performing gas chromatographic–mass spectrometric (GC–MS) screening of volatile hydrocarbons (VHCs) in blood. Twenty hydrocarbons comprising aliphatic hydrocarbons ranging from n-hexane to n-tridecane, and aromatic hydrocarbons ranging from benzene to trimethylbenzenes were used in this study. This method can be used for examining a burned body to ascertain whether the victim had been alive or not when the burning incident took place. n-Hexane, n-heptane and benzene, the main indicators of gasoline components, were found as detectable peaks through the use of cryogenic oven trapping upon SPME injection into a GC–MS instrument. The optimal screening procedure was performed as follows. The analytes in the headspace of 0.2 g of blood mixed with 0.8 ml of water plus 0.2 μg of toluene-d₈ at −5°C were adsorbed to a 100-μm polydimethylsiloxane (PDMS) fiber for 30 min, and measured using the full-mass-scanning GC–MS method. The lower detection limits of all the compounds were 0.01 μg per 1 g of blood. Linearities (r²) within the range 0.01 to 4 μg per 1 g of blood were only obtained for the aromatic hydrocarbons at between 0.9638 (pseudocumene) and 0.9994 (toluene), but not for aliphatic hydrocarbons at between 0.9392 (n-tridecane) and 0.9935 (n-hexane). The coefficients of variation at 0.2 μg/g were less than 8.6% (n-undecane). In conclusion, this method is feasible for the screening of volatile hydrocarbons from blood in forensic medicine.     

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography–mass spectrometry

We report here the development and validation of an LC–MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C₁₈ reversed-phase column using an acetonitrile–water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS–MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477→266), DMLOP (m/z 463→252) and A-LOP (m/z 519→266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3±1.50% (range: 70.7–73.7%) and 79.4±12.8% (64.9–88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

Investigations of animal blood samples after fragrance drug inhalation by gas chromatography/mass spectrometry with chemical ionization and selected ion monitoring.

The fragrance compounds linalool (1) and linalyl acetate (2) could be detected, identified and quantified (1: 7-9 ng ml-1; and 2: 1-2 ng ml-1 and 4-5 ng ml-1 as free linalool) in blood samples after inhalation in animal experiments (mice) by gas chromatography/mass spectrometry (GC/MS) with chemical ionization (CI) (ammonia); selected ion monitoring (SIM) mode (1: m/z 81, 137 and 154; 2: 47, 57 and 137) and GC/flame ionization detection (FID). The inhalation of these monoterpenes in concentrations of 5 mg l-1 air leads to a significant reduction of the motility of the test animals down to 30-40% with respect to the control group.     

Development of headspace solid-phase microextraction with on-fiber derivatization for determination of hexanal and heptanal in human blood

Hexanal and heptanal in human blood have been regarded as potential biomarkers of lung cancer. Owing to their high volatilities and activities, it is difficult to accurately measure the two biomarkers. In the current work, headspace solid-phase microextraction (HS-SPME) with on-fiber derivatization technique was developed for quantitative analysis of hexanal and heptanal in human blood. In the proposed method, the two aldehydes in blood were headspace extracted by using a poly (dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber with O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine (PFBHA) at 60 degrees C for 8 min. The aldehyde oximes formed on the fiber were desorbed and analyzed by gas chromatography-mass spectrometry (GC-MS). The method validations including detection limit, recovery and precision were studied. It was found that the method provided low detection limits of 0.006 nM for hexanal and 0.005 nM for heptanal, recoveries from 89% to 95% and R.S.D. values less than 8.5%. The present method was applied to quantitative analysis of hexanal and heptanal in normal blood and lung cancer blood. Hexanal concentrations from 7.33 to 15.23 microM and heptanal concentrations from 2.47 to 9.23 microM were found in the lung cancer blood, while both hexanal and heptanal in the control blood were lower than 0.6 microM. This further demonstrated that hexanal and heptanal might be the biomarkers of lung cancer. The experimental results showed that GC-MS and HS-SPME with on-fiber derivatization is a simple, rapid, sensitive and solvent-free method for determination of in hexanal and heptanal human blood.     

Simultaneous determination of selegiline and desmethylselegiline in human body fluids by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A method for the simultaneous determination of selegiline and its metabolite, desmethylselegiline, in human whole blood and urine is presented. The method, which combines a fiber-based headspace solid-phase microextraction (SPME) technique with gas chromatography-mass spectrometry (GC-MS), required optimization of various parameters (e.g., salt additives, extraction temperatures, extraction times and the extraction properties of the SPME fiber coatings). Pargyline was used as the internal standard. Extraction efficiencies for both selegiline and desmethylselegiline were 2.0-3.4% for whole blood, and 8.0-13.2% for urine. The regression equations for selegiline and desmethylselegiline extracted from whole blood were linear (r(2)=0.996 and 0.995) within the concentration ranges 0.1-10 and 0.2-20 ng/ml, respectively. For urine, the regression equations for selegiline and desmethylselegiline were linear (r(2)=0.999 and 0.998) within the concentration ranges 0.05-5.0 and 0.1-10 ng/ml, respectively. The limit of detection for selegiline and desmethylselegiline was 0.01-0.05 ng/ml for both samples. The lower and upper limits of quantification for each compound were 0.05-0.2 and 5-20 ng/ml, respectively. Intra- and inter-day coefficients of variation for selegiline and desmethylselegiline in both samples were not greater than 8.7 and 11.7%, respectively. The determination of selegiline and desmethylselegiline concentrations in Parkinson's disease patients undergoing continuous selegiline treatment is presented and is shown to validate the present methodology.     

Valproic acid analysis in saliva and serum using selected ion monitoring (electron ionization) of the tert.-butyldimethylsilyl derivatives

A highly sensitive ion monitoring method for the determination of valproic acid in saliva and in serum has been developed based on the gas chromatographic—mass spectrometric analysis of the tert.-butyldimethylsilyl derivatives. Extraction methods are simple and the techniques for derivatization are rapid and convenient. Selected ion monitoring was carried out using electron ionization conditions and a common ion m/z 201 (M⁺ − 57) present in valproic acid and the internal standard octanoic acid. The lower limit of sensitivity that has acceptable precision for assay purposes is 0.1 mg/l based on a 200-μl sample size. The ion monitoring method (derivatized) was compared to a gas chromatographic method (underivatized) for serum valproate assays and found to be essentially identical.The assay methodology was used in a kinetic study of valproic acid in two normal subjects. Saliva levels of drug were found to give reasonably good correlations with serum total and with serum free concentrations of drug in both individuals.