Gas chromatographic–mass spectrometric identification and quantitation of benzyl alcohol in serum after derivatization with perfluorooctanoyl chloride: a new derivative

Benzyl alcohol is commonly used as an antibacterial agent in a variety of pharmaceutical formulations. Several fatalities in neonates have been linked to benzyl alcohol poisoning. Most methods for measuring benzyl alcohol concentrations in serum utilize direct extraction followed by high-performance liquid chromatography. We describe here a novel derivatization of benzyl alcohol using perfluorooctanoyl chloride after extraction from human serum for analysis by gas chromatography–mass spectrometry (GC–MS). The derivative was eluted at a significantly higher temperature respective to underivatized molecule and the method was free from interferences from more volatile components in serum and hemolyzed specimens. Another advantage of this derivatization technique is the conversion of low-molecular-mass benzyl alcohol (M_r 108) to a high-molecular-mass derivative (M_r 504). The positive identification of benzyl alcohol can be achieved by observing a distinct molecular ion at m/z 504 as well as the base peak at m/z 91. Quantitation of benzyl alcohol in human serum can easily be achieved by using 3,4-dimethylphenol as an internal standard. The within run and between run precisions (using serum standard of benzyl alcohol: 25 mg/l) were 2.7% (mean=24.1, S.D.=0.66 mg/l, n=8) and 4.2% (mean=24.3, S.D.=1.03 mg/l, n=8), respectively. The assay was linear for the serum benzyl alcohol concentrations of 2 mg/l to 200 mg/l and the detection limit was 0.1 mg/l. We observed no carry-over (memory effect) problem in our assay as when 2 μl ethyl acetate was injected into the GC–MS system after analyzing serum specimens containing 200 mg/l of benzyl alcohol, we observed no peak for either benzyl alcohol or the internal standard in the total ion chromatogram.     

Identification of a flunixin metabolite in camel by gas chromatography–mass spectrometry

A flunixin metabolite, a hydroxylated product, has been identified in camel urine and plasma samples using gas chromatography–mass spectrometry (GC–MS) and GC–MS–MS in the electron impact and chemical ionization modes. Its major fragmentation pattern has been verified by GC–MS–MS in daughter ion and parent ion scan modes. The method could detect flunixin and its metabolite in camel urine after a single intravenous dose of 2.2 mg of flunixin/kg body weight for 96 and 48 h, respectively, which increases the reliability of antidoping control analysis.     

Characteristics of mass spectrometric analyses coupled to gas chromatography and liquid chromatography for 22-oxacalcitriol, a vitamin D3 analog, and related compounds

The characteristics of the mass spectra of vitamin D₃ related compounds were investigated by GC–MS and LC–MS using 22-oxacalcitriol (OCT), an analog of 1,25-dihydroxyvitamin D₃, and related compounds. Fragmentation during GC–MS (electron impact ionization) of TMS-derivatives of OCT and the postulated metabolites gave useful structural information concerning the vitamin D₃-skeleton and its side-chain, especially with respect to the oxidation positions of metabolites. In contrast, few fragment ions were observed in LC–MS (atmospheric pressure chemical ionization), showing that LC–MS gave poor structural information, except for molecular mass. However, when comparing the signal-to-noise ratio (S/N) observed during GC–MS and LC–MS analysis for OCT in plasma extracts, the S/N in LC–MS was over ten-times greater than in GC–MS, possibly due to the low recovery on derivatization and thermal-isomerization in GC–MS. Furthermore, both the GC–MS and the LC–MS allowed the analysis of many postulated metabolites in a single injection without any prior isolation of target metabolites from biological fluids by LC. These results suggest that GC–MS and LC–MS analysis for vitamin D₃ related compounds such as OCT each have unique and distinct advantages. Therefore, the complementary use of both techniques enables the rapid and detailed characterization of vitamin D₃ related compounds.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A 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 1.5 ml of a diluted serum 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 the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

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%.     

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.     

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).     

Identification and confirmation of 3-hydroxy metabolite of ketoprofen in camels by gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy

The metabolites of ketoprofen were investigated in five camels following intravenous administration of a dose of 2.0 mg/kg body weight. Two metabolites were identified. The first one was purified with thin-layer chromatography. It was identified by gas chromatography–mass spectrometry (GC–MS) in comparison with authenticated reference standard and was found to be hydroxyketoprofen due to reduction of the ketone group of ketoprofen. The second metabolite was purified by high-performance liquid chromatography. It was identified with GC–MS and nuclear magnetic resonance spectroscopy as 3-hydroxybenzolketoprofen resulting from oxidation of the aromatic ring.     

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.     

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.     

Screening of chlorpropamide in horse plasma by high-performance liquid chromatography with ultraviolet absorbance detection, and confirmation by gas chromatography–mass spectrometry

A chromatographic method was developed to detect and confirm the presence of chlorpropamide (I) in horse plasma samples, for antidoping control. The plasma sample (1 ml) was extracted with dichloromethane and screened by high-performance liquid chromatography, and confirmation of the drug's presence was accomplished by using gas chromatography–mass spectrometry (GC–MS). The limit of detection was found to be 3.5 ng/ml at a signal-to-noise ratio of three. Derivatization of I with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane allowed for highly stable, accurate and sensitive GC–MS analysis. Plasma samples collected after the administration of diabinese were positive for I (one–five days) in all samples analysed.     

Short column gas chromatography–mass spectrometry and principal component analysis for the identification of coeluted substances in doping control analysis

The identification of four doping control substances in an artificial mixture, using short column gas chromatography–mass spectrometry (GC–MS) analysis was examined. Two chromatographic peaks were recorded in the chromatogram, using a short capillary column (1.8 m) at an oven temperature of 180°C. The first peak was associated with a mixture of a solvent derivative and an artifact. The second one corresponded to the mixture of four control substances. Principal component analysis was applied on a selected GC–MS data set of the latter peak to determine clear full spectra of pure substances from mixture spectra. The time of GC–MS analysis was significantly reduced to less than 1 min from 30 min which is a typical GC–MS analysis time, using standard methods of doping control analysis.     

Urinary organic acid screening by solid-phase microextraction of the methyl esters

We developed a new sample preparation method for profiling organic acids in urine by GC or GC–MS. The method includes derivatisation of the organic acids directly in the aqueous urine using trimethyloxonium tetrafluoroborate as a methylating agent, extraction of the organic acid methyl esters from the urine by solid-phase microextraction, using a polyacrylate fiber with a thickness of 85 μm and transfer of the methyl esters into the GC or the GC–MS instrument. Desorption of the analytes takes place in the heated injection port. The proposed sample preparation is very simple. There is no need for any evaporation step and for the use of an organic solvent. The risk of contamination and the loss of analytes are minimized. The total sample preparation time prior to GC or GC–MS analysis is about 40 min, and therefore more rapid than other sample preparation procedures. The urinary organic acids are well separated by GC and 29 substances are identified by GC–MS.     

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.     

Measurement of azacyclonol in urine and serum of humans following terfenadine (Seldane) administration using gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric (GC—MS) method is presented for the analysis of azacyclonol (AZA), a metabolite of terfenadine in serum and urine specimens. Following an alkaline extraction, AZA and an internal standard were derivatized using heptafluorobutyric anhydride. Fourier transform infrared spectrometry suggested that two sites on the AZA molecule were derivatized. GC—MS of the extracts had a limit of quantitation (LOQ) of 1 ng/ml and linear range of 1–1000 ng/ml in urine. Four volunteers were administered a therapeutic regimen of terfenadine followed by urine and serum specimen collection(s) during the next seven days. The results indicated that following a 60-mg dose of terfenadine each 12 h for five days, (1) AZA appears in urine within 2 h, (2) urine AZA concentrations were above the LOQ 72 h following the last dose, (3) peak urine concentrations were as high as 19 000 ng/ml, and (4) mean serum concentration following the ninth dose was 59 ng/ml.     

Gas chromatographic–tandem mass spectrometric quantification of human plasma and urinary nitrate after its reduction to nitrite and derivatization to the pentafluorobenzyl derivative

Gas chromatography–mass spectrometry (GC–MS) of nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode is a useful analytical tool to quantify accurately and sensitively nitrite and nitrate after its reduction to nitrite in various biological fluids. In the present study we demonstrate the utility of GC–tandem MS to quantify nitrate in human plasma and urine. Our present results verify human plasma and urine levels of nitrite and nitrate measured previously by GC–MS.     

Qualitative and quantitative analysis of some synthetic, chemically acting laxatives in urine by gas chromatography—mass spectrometry

A method for the qualitative and quantitative simultaneous analysis of dioxyanthraquinone, desacetyl-Bisacodyl, phenolphthalein and Oxyphenisatin in human urine using gas chromatography—mass spectrometry (GC—MS) has been developed. The compounds were extracted from urine at pH 7.5 with diethyl ether using Extrelut extraction columns, followed by evaporation and trimethylsilylation.The method used electron beam ionization GC—MS employing a computer-controlled multiple-ion detector (mass fragmentography). The recovery from urine for the various compounds was between 80% and 100%. The detection limit for these compounds was in the range 0.01–0.05 μg/ml of urine.The method proved to be suitable for measuring urine concentrations for at least four days after administration of a single oral low therapeutic dose of the laxatives to sixteen healthy volunteers.     

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.     

Measurement of dexfenfluramine metabolism in rat liver microsomes by gas chromatography–mass spectrometry

A specific and useful method was developed for the determination of dexfenfluramine metabolism by microsomal systems utilising GC–MS. The synthesis of two metabolites 1-(3-trifluoromethylphenyl)propan-2-ol (‘alcohol') and 1-(3-trifluoromethylphenyl)-1,2-propanediol (‘diol') via straightforward routes, were confirmed by MS and NMR spectra. The conditions for extraction from alkalinised microsomal mixtures of the metabolites nordexfenfluramine, 1-(3-trifluoromethylphenyl)propan-2-one (‘ketone'), alcohol and diol, their conversion to trifluoroacetate derivatives and analysis by GC–MS–SIM are described. Calibration curves were constructed between 48 and 9662 nM and fitted to quadratic equations (r²>0.999). The method precision was good over low (121 nM) medium (2415 nM) and above medium (9662 nM) concentrations for all metabolites; the within- and day-to-day coefficients of variation ranged between 2.5–12.4% and 6.7–17.5%, respectively. The accuracy, measured as bias, was very good both within- and day-to-day (range: −0.4–12.6%, 0.8–18.9%). For most metabolites, the C.V. for the assay and bias increased at 121 nM. Dexfenfluramine metabolism by rat liver microsomes was investigated using the assay method and showed a concentration dependent increase in nordexfenfluramine and ketone metabolites over the substrate range of 5–200 μM.     

Simultaneous determination of imipramine and its metabolite desipramine in human plasma by capillary gas chromatography with mass-selective detection

An analytical method for the simultaneous determination of imipramine (IMI) and its N-desmethyl metabolite, desipramine (DIMI) in human plasma by capillary gas chromatography–mass selective detection (GC–MS), with D4-imipramine (D4-IMI) and D4-desipramine (D4-DIMI) as internal standards, was developed and validated. After addition of the internal standards, the compounds were extracted from plasma at basic pH into n-heptane–isoamyl alcohol (99:1, v/v), back-extracted into acidic aqueous solution and re-extracted at basic pH into toluene. Desipramine and D4-desipramine were converted into their pentafluoropropionyl derivatives. The compounds were determined by gas chromatography using a mass selective detector at m/z 234 for IMI, m/z 238 for D4-IMI, m/z 412 for DIMI and m/z 416 for D4-DIMI. The method was applied to clinical samples.