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.     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

High-temperature solid-phase microextraction procedure for the detection of drugs by gas chromatography–mass spectrometry

High-temperature headspace solid-phase microextraction (SPME) with simultaneous (“in situ”) derivatisation (acetylation or silylation) is a new sample preparation technique for the screening of illicit drugs in urine and for the confirmation analysis in serum by GC–MS. After extraction of urine with a small portion of an organic solvent mixture (e.g., 2 ml of hexane–ethyl acetate) at pH 9, the organic layer is separated and evaporated to dryness in a small headspace vial. A SPME-fiber (e.g., polyacrylate) doped with acetic anhydride–pyridine (for acetylation) is exposed to the vapour phase for 10 min at 200°C in a blockheater. The SPME fiber is then injected into the GC–MS for thermal desorption and analysis. After addition of perchloric acid and extraction with n-hexane to remove lipids, the serum can be analysed after adjusting to pH 9 as described for urine. Very clean extracts are obtained. The various drugs investigated could be detected and identified in urine by the total ion current technique at the following concentrations: amphetamines (200 μg/l), barbiturates (500 μg/l), benzodiazepines (100 μg/l), benzoylecgonine (150 μg/l), methadone (100 μg/l) and opiates (200 μg/l). In serum all drugs could be detected by the selected ion monitoring technique within their therapeutic range. As compared to liquid–liquid extraction only small amounts of organic solvent are needed and larger amounts of the pertinent analytes could be transferred to the GC column. In contrast to solid-phase extraction (SPE), the SPME-fiber is reusable several times (as there is no contamination by endogenous compounds). The method is time-saving and can be mechanised by the use of a dedicated autosampler.     

Determination of butorphanol in horse race urine by immunoassay and gas chromatography–mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC–MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC–MS system. The ELISA test (20 μl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC–MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC–MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10–250 ng/ml), recovery (±100%), intra-assay (4.1–14.9%) and inter-assay (9.3–45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

Determination of olanzapine in human breast milk by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic–electrochemical assay was developed and validated for the quantification of olanzapine in human breast milk. The assay involved a solid-phase extraction (SPE) of olanzapine and its internal standard on a Bond Elut Certify LRC mixed-mode cartridge. After conditioning of the SPE cartridge, human milk (1 ml) was passed through the cartridge. The cartridge was washed with five separate washing steps to remove endogenous compounds, and the analytes were eluted with ethyl acetate–ammonium hydroxide (98:2, v/v) solution. The eluate was evaporated to dryness (gentle stream of nitrogen at 40°C), and the residue was dissolved in mobile phase. The extract was injected onto a YMC basic column (150 mm×4.6 mm I.D., 5 μm particle size) at a flow-rate of 1 ml/min. A mixture of 75 mM phosphate buffer, pH 7.0–acetonitrile–methanol (48:26:26, v/v/v) was used as the mobile phase. Standard curves with a lower limit of quantitation of 0.25 ng/ml of olanzapine were linear (r²≥0.9992) over a range of 0.25–100 ng/ml. Based on the analysis of quality control (QC) samples, the average inter-day accuracy (RE) was 99.0% with an average precision (CV) of 6.64% over the entire range. The stability of olanzapine in human milk was established after three freeze–thaw–heat cycles and storage at −70°C for 10 months. The validated method was used to measure olanzapine concentrations in human milk during a clinical trial.     

Simple and sensitive assay of zonisamide in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A rapid and sensitive method for the assay of zonisamide in serum was developed using a solid-phase extraction technique followed by high-performance liquid chromatography. A 20-μl volume of human serum was first purified with a Bond-Elut cartridge column. Then, the methanol eluate was injected onto a reversed-phase HPLC column with a UV detector. The mobile phase was acetonitrile—methanol—distilled water (17:20:63, v/v) and the detection wavelength was 246 nm. The detection limit was 0.1 μg/ml in serum. The coefficients of variation were 4.2–5.6% and 5.1–9.1% for the within-day and between-day assays, respectively. This method can be used for clinical pharmacokinetic studies of zonisamide in serum even in infant patients with epilepsy.     

Improved sample preparation method for selected persistent organochlorine pollutants in human serum using solid-phase disk extraction with gas chromatographic analysis

An improved solid-phase extraction (SPE) method was developed to isolate and concentrate trace levels of selected POPs (persistent organochlorine pollutants) in human serum prior to GC–MS in SIM mode or GC–ECD quantitation. The extraction involves denaturation of serum proteins with formic acid, SPE using C₁₈ Empore™ disk cartridges, followed by elimination of lipid interferences using a sulfuric acid wash of the eluate. Use of the SPE disk improved assay throughput and gave a cleaner analytical matrix compared with previously reported solid-phase and liquid–liquid extraction techniques. The extraction method provided consistent recoveries at three fortification levels using ¹³C₁₂ PCB 149 as internal standard. Recoveries ranged from 48 to 140% for organochlorine pesticides (6.25, 12.5 and 25 ng/ml) and 71 to 126% for polychlorinated biphenyls (0.625, 1.25 and 2.5 ng/ml).     

Gas chromatographic–mass spectrometric determination of 4-nonylphenols and 4-tert-octylphenol in biological samples

A simple and rapid method is described for the GC–MS determination of 4-nonylphenols (NOs) and 4-tert-octylphenol (OC) in biological samples. The NOs and OC in the sample are extracted with acetonitrile and the lipid in the sample extract is eliminated by partitioning between hexane and acetonitrile. After Florisil PR column clean-up, the sample extract is analyzed by GC–MS in the selected ion monitoring (SIM) mode. Average recoveries in pale chub (fish) and corbicula (shellfish) are 86.0 and 93.4% for NOs, and 95.8 and 96.4% for OC, respectively, spiked at the levels of 1.0 μg of NOs and 0.1 μg of OC per 5 g of fish and shellfish samples. The detection limits are 20 ng/g for NOs and 2 ng/g for OC.     

Simple analysis of local anaesthetics in human blood using headspace solid-phase microextraction and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring

A simple method for analysis of five local anaesthetics in blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring (GC–MS–EI-SIM). Deuterated lidocaine (d₁₀-lidocaine) was synthesized and used as a desirable internal standard (I.S.). A vial containing a blood sample, 5 M sodium hydroxide and d₁₀-lidocaine (I.S.) was heated at 120°C. The extraction fiber of the SPME system was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC–MS system. The calibration curves showed linearity in the range of 0.1–20 μg/g for lidocaine and mepivacaine, 0.5–20 μg/g for bupivacaine and 1–20 μg/g for prilocaine in blood. No interfering substances were found, and the time for analysis was 65 min for one sample. In addition, this proposed method was applied to a medico–legal case where the cause of death was suspected to be acute local anaesthetics poisoning. Mepivacaine was detected in the left and right heart blood samples of the victim at concentrations of 18.6 and 15.8 μg/g, respectively.     

High-performance liquid chromatographic procedures for the quantitative determination of paclitaxel (Taxol) in human urine

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the quantitative determination of paclitaxel in human urine. A comparison is made between solid-phase extraction (SPE) and liquid-liquid extraction (LLE) as sample pretreatment. The HPLC system consists of an APEX octyl analytical column and acetonitrile-methanol-0.2 μM ammonium acetate buffer pH 5 (4:1:5, v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The SPE procedure involves extraction on Cyano Bond Elut columns. n-Butylchloride is the organic extraction fluid used for the LLE. The recoveries of paclitaxel in human urine are 79 and 75% for SPE and LLE, respectively. The accuracy for the LLE and SPE sample pretreatment procedures is 100.4 and 104.9%, respectively, at a 5 μg/ml drug concentration. The lower limit of quantitation is 0.01 μg/ml for SPE and 0.25 μg/ml for LLE. Stability data of paclitaxel in human urine are also presented.     

Study of the solid-phase extraction of diclofenac sodium, indomethacin and phenylbutazone for their analysis in human urine by liquid chromatography

A selective semi-automated solid-phase extraction (SPE) of the non-steroidal anti-inflammatory drugs diclofenac sodium, indomethacin and phenylbutazone from urine prior to high-performance liquid chromatography was investigated. The drugs were recovered from urine buffered at pH 5.0 using C₁₈ Bond-Elut cartridges as solid sorbent material and mixtures of methanol–aqueous buffer or acetonitrile–aqueous buffer as washing and elution solvents. The extracts were chromatographed on a reversed-phase ODS column using 10 mM acetate buffer (pH 4.0)–acetonitrile (58:42, v/v) as the mobile phase, and the effluent from the column was monitored at 210 nm with ultraviolet detection. Absolute recoveries of the anti-inflammatory drugs within the range 0.02–1.0 μg/ml were about 85% for diclofenac and indomethacin, and 50% for phenylbutazone without any interference from endogenous compounds of the urine. The within-day and between-day repeatabilities were in all cases less than 5% and 10%, respectively. Limits of detection were 0.007 μg/ml for diclofenac sodium and indomethacin and 0.035 μg/ml for phenylbutazone, whereas limits of quantitation were 0.02 μg/ml for diclofenac and indomethacin and 0.1 μg/ml for phenylbutazone.     

Analysis of 2-(3-methyl-4-aminophenyl)-benzothiazole (NSC 674495) in plasma by gas chromatography with mass-selective detection

Certain naturally occurring isoflavonoids have been shown to inhibit protein-tyrosine kinases, and this has led to investigations of ring-modified structural analogs. Most recently, 2-(3-methyl-4-aminophenyl)-benzothiazole (MAB: NSC 674495) was shown to possess significant activity against certain breast cell cancer lines in vitro and in vivo. Our efforts thus focussed on developing a simple and sensitive method for quantitating MAB in plasma using GC–MS. The GC–MS assay was found to be linear over the range of 0.050 to 5.0 μg/ml, and was applied to monitor the plasma concentration of MAB in a rat dosed with 25 mg/kg as a 1 min intravenous infusion. Plasma was collected at intervals from 3 through 180 min, and concentrations of MAB were determined. Non-linear regression analysis of the plasma concentration-time data revealed that levels declined from a maximum at 3 min of 18 μg/ml to 1 μg/ml at 3 h in a biphasic manner. In another investigation, significant plasma concentrations of a major metabolite was detected and determined to be mono-N-acetylated MAB.     

Simultaneous determination of anabolic and catabolic steroid hormones in meat by gas chromatography–mass spectrometry

A rapid and economical method for the determination in meat of androgens, estrogens, progestogens and corticoids, including some precursors and metabolites, has been developed. The extracted steroids are separated in a polar, a neutral, and a phenolic fraction by C₈-SPE followed by a liquid–liquid extraction of the phenolates. Each fraction is separately purified by normal-phase SPE. The different steroid fractions can be analysed either together to obtain a comprehensive hormone pattern in one step or separately to enhance detection selectivity and sensitivity. Using a universally applicable silylation of the hydroxyl and keto groups, detection limits of 0.02–0.1 μg/kg are reached by GC–MS (EI) in the selected ion monitoring mode.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

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.     

Application of solid-phase microextraction and gas chromatography–mass spectrometry for the determination of chlorophenols in urine

This study investigated the feasibility of applying solid-phase microextraction (SPME) combined with gas chromatography–mass spectrometry to analyze chlorophenols in urine. The SPME experimental procedures to extract chlorophenols in urine were optimized with a polar polyacrylate coated fiber at pH 1, extraction time for 50 min and desorption in GC injector at 290°C for 2 min. The linearity was obtained with a precision below 10% R.S.D. for the studied chlorophenols in a wide range from 0.1 to 100 μg/l. In addition, sample extraction by SPME was used to estimate the detection limits of chlorophenols in urine, with selected ion monitoring of GC–MS operated in the electron impact mode and negative chemical ionization mode. Detection limits were obtained at the low ng/l levels. The application of the methods to the determination of chlorophenols in real samples was tested by analyzing urine samples of sawmill workers. The chlorophenols were found in workers, the urinary concentration ranging from 0.02 μg/l (PCP) to 1.56 μg/l (2,4-DCP) depending on chlorophenols. The results show that trace chlorophenols have been detected with SPME–GC–MS in the workers of sawmill where chlorophenol-containing anti-stain agents had been previously used.     

Systematic analysis of acid, neutral and basic drugs in horse plasma by combination of solid-phase extraction, non-aqueous partitioning and gas chromatography–mass spectrometry

A sample preparation method for mass chromatographic detection of doping drugs from horse plasma is described. Bond Elut Certify (1 g/6 ml) is used for the extraction of 4 ml of horse plasma. Fractionation is performed with 6 ml of CHCl₃–Me₂CO (8:2) and 5 ml of 1% TEA–MeOH according to its property. Simple and effective clean-up based on non-aqueous partitioning is adopted to remove co-eluted contaminants in both acid and basic fractions. Two kinds of 1-(N,N-diisopropylamino)-n-alkanes are co-injected with the sample into the GC–MS system for the calculation of the retention index. Total recoveries of 107 drugs are examined. Some data of post administration plasma are presented. This procedure achieves sufficient recoveries and clean extracts for GC–MS analysis. The method is able to detect ng/ml drug levels in horse plasma.     

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.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.