Use of ion trap gas chromatography–tandem mass spectrometry for detection and confirmation of anabolic substances at trace levels in doping analysis

A procedure for detecting and confirming 23 anabolic substances and/or metabolites has been developed using a GC–MS–MS ion trap system in full-scan mode. The process used to select the precursor ion, and the optimization of the system parameters used to obtain the daughter ion spectra, are explained. Urine samples were prepared using solid-phase extraction and enzymatic hydrolysis, and after TMS derivatives had been formed, they were injected into the mass spectrometer. This method permits confirmation of the presence of anabolic substances at low ng ml⁻¹ levels without the need of further purification procedures on the samples. This procedure has been used on more than 2000 urine samples collected from sporting competitions and has made it possible to confirm more than 45 true positive cases which could not have been confirmed using routine GC–MS methods.     

Determination of nandrolone and metabolites in urine samples from sedentary persons and sportsmen

Metabolites of nandrolone were determined in the urine of several sportsmen, sedentary and post-menopausal women by capillary gas chromatography–mass spectrometry quadrupole (GC–MS) and capillary gas chromatography mass–mass spectrometry ion trap (GC–MS–MS) methods. The method employed was GC–EI-MS with 17α-methyltestosterone as internal standard with ethyl ether extraction prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405 and 420 for the nandrolone metabolites, and 418 and 403 for nandrolone derivative. Recovery for nandrolone, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) was 97.20, 94.17 and 95.54%, respectively. Detection limits for nandrolone, 19-NA and 19-NE were 0.03, 0.01 and 0.06 ng/ml. Metabolites of nandrolone (19-NA and 19-NE) were found in 12.5% (n=40) of sportsmen and 40% (n=10) of post-menopausal women.     

Testing hair for pharmaceuticals

More than hundred pharmaceuticals, drugs of abuse or doping agents have been reported to be detectable in human hair. This article reviews the analysis of 90 drugs and drug metabolites by chromatographic procedures, including the pretreatment steps, the extraction methods, the reported limits of detection and the measured concentrations in real human hair samples. Some progress is observed in the detection of low dose drugs, like fentanyl or flunitrazepam. The general tendency in the last years, to highly sophisticated techniques (GC–MS–NCI, HPLC–MS, GC–MS–MS) illustrates well this constant fight for sensitivity. Some new findings, based on the recent experience of the authors, are also added.     

Determination of dichloroanilines in human urine by GC–MS, GC–MS–MS, and GC–ECD as markers of low-level pesticide exposure

Methods for the determination of 3,4-dichloroaniline (3,4-DCA) and 3,5-dichloroaniline (3,5-DCA) as common markers of eight non-persistent pesticides in human urine are presented. 3,5-DCA is a marker for the exposure to the fungicides vinclozolin, procymidone, iprodione, and chlozolinate. Furthermore the herbicides diuron, linuron, neburon, and propanil are covered using their common marker 3,4-DCA. The urine samples were treated by basic hydrolysis to degrade all pesticides, metabolites, and their conjugates containing the intact moieties completely to the corresponding dichloroanilines. After addition of the internal standard 4-chloro-2-methylaniline, simultaneous steam distillation extraction (SDE) followed by liquid–liquid extraction (LLE) was carried out to produce, concentrate and purify the dichloroaniline moieties. Gas chromatography (GC) with mass spectrometric (MS) and tandem mass spectrometric (MS–MS) detection and also detection with an electron-capture detector (ECD) after derivatisation with heptafluorobutyric anhydride (HFBA) were employed for separation, detection, and identification. Limit of detection of the GC–MS–MS and the GC–ECD methods was 0.03 and 0.05 μg/l, respectively. Absolute recoveries obtained from a urine sample spiked with the internal standard, 3,5-, and 3,4-DCA, ranged from 93 to 103% with 9–18% coefficient of variation. The three detection techniques were compared concerning their performance, expenditure and suitability for their application in human biomonitoring studies. The described procedure has been successfully applied for the determination of 3,4- and 3,5-DCA in the urine of non-occupationally exposed volunteers. The 3,4-DCA levels in these urine samples ranged between 0.13 and 0.34 μg/g creatinine or 0.11 and 0.56 μg/l, while those for 3,5-DCA were between 0.39 and 3.33 μg/g creatinine or 0.17 and 1.17 μg/l.     

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–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.     

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: II. Validation of a direct injection/on-line guard cartridge extraction–tandem mass spectrometry method for CYP2D6 inhibition assessment

A highly efficient direct injection/on-line guard cartridge extraction–tandem mass spectrometry (DI/GCE–MS–MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2D6 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE–MS–MS analysis. Due to the novel use of an extremely short C₁₈ guard cartridge, this method exhibits several advantages, such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI/GCE–MS–MS method demonstrates acceptable accuracy and precision for the quantification of dextrorphan, a marker metabolite of dextromethorphan mediated by CYP2D6, in microsomal incubations. The CYP2D6 inhibition assay has been validated using quinidine as a known selective inhibitor of the isoform. The IC₅₀ value (0.20 μM) measured by the new method is in good agreement with the literature value (0.22 μM).     

Novel liquid chromatographic–tandem mass spectrometric methods using silica columns and aqueous–organic mobile phases for quantitative analysis of polar ionic analytes in biological fluids

Use of silica stationary phase and aqueous–organic mobile phases could significantly enhance LC–MS–MS method sensitivity. The LC conditions were compatible with MS detection. Analytes with basic functional groups were eluted with acidic mobile phases and detected by MS in the positive ion mode. Analytes with acid functional groups were eluted with mobile phases at neutral pH and detected by MS in the negative ion mode. Analytes poorly retained on reversed-phase columns showed good retention on silica columns. Compared with reversed-phase LC–MS–MS, 5–8-fold sensitivity increases were observed for basic polar ionic compounds when using silica columns and aqueous–organic mobile phase. Up to a 20-fold sensitivity increase was observed for acidic polar ionic compounds. Silica columns and aqueous–organic mobile phases were used for assaying nicotine, cotinine, and albuterol in biological fluids.     

Determination of seven prostanoids in 1 ml of urine by gas chromatography—negative ion chemical ionization triple stage quadrupole mass spectrometry

In an isotope dilution assay, prostaglandin (PG) E₂, 6-keto-PGF_1α, thromboxane (Tx) B₂ and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF_1α, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.     

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.     

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.     

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.     

Validated method for the therapeutic drug monitoring of flunitrazepam in human serum using liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry with an ion trap detector

An HPLC–MS–MS method for the quantitative analysis of flunitrazepam in human serum was established. The method features a very simple liquid–liquid extraction, the use of a standard 4-mm HPLC column, isocratic elution using a buffer-free solvent, short retention times in connection with good peak resolution and the sensitivity and selectivity of an ion trap MS–MS detector. The procedure enables unambiguous identification of analytes by their product ion spectra, as well as sensitive quantitation (limit of quantitation for flunitrazepam=0.5 ng/ml). This feature was used for the confirmation of HPLC–UV results for nitrazepam.     

Liquid chromatography–tandem mass spectrometric quantitation of cyclophosphamide and its hydroxy metabolite in plasma and tissue for determination of tissue distribution

Cyclophosphamide (CP) and its metabolite, hydroxycyclophosphamide (OH-CP) have been quantitated in mouse plasma and tissue by derivatization combined with liquid chromatography–tandem mass spectrometry (LC–MS–MS). The derivatization was conducted immediately upon sample collection, to trap the OH-CP metabolite intermediate prior to further conversion to phosphoramide mustard or other reaction products. This simple and straightforward derivatization procedure, combined with sample extraction via protein precipitation, allowed quantitation of CP and the oxime derivative of OH-CP in plasma for concentrations ranging from approximately 12.5–3333 ng/ml, and in spleen tissue for concentrations of 1250–50 000 ng/g. The short cycle time (2.5 min) of the LC–MS–MS method allowed high throughput analysis with minimal matrix interference. Mouse plasma levels were quantitated for doses of 40, 65 and 120 mg/kg; spleen concentrations were determined for mice dosed at 120 mg/kg. The CP and oxime plasma levels correlated well with dose amounts. The CP levels in the spleen and plasma were similar while the oxime levels in the spleen were significantly lower than the plasma.     

Determination of isoprostaglandin F2α type III in human urine by gas chromatography–electronic impact mass spectrometry. Comparison with enzyme immunoassay

F₂-Isoprostanes are stable lipid peroxidation products of arachidonic acid, the quantification of which provides an index of oxidative stress in vivo. We describe a method for analysing isoprostaglandin F_2α type III (15-F_2t-IsoP) in biological fluids. The method involves solid-phase extraction on octadecyl endcapped and aminopropyl cartridges. After conversion to trimethylsilyl ester trimethylsilyl ether derivatives, isoprostaglandin F_2α type III is analysed by mass spectrometry, operated in electronic impact selected ion monitoring mode. We have compared enzyme immunoassay (EIA; Cayman, Ann Arbor, MI, USA) to this method with 30 human urine aliquots following the same extraction procedure in order to determine the agreement between both methods. Isoprostaglandin F_2α type III concentrations determined with gas chromatography–mass spectrometry (GC–MS) did not agree with those determined with EIA. Our results suggest that GC–MS and EIA do not measure the same compounds. As a consequence, comparison of clinical results using GC–MS and EIA should be avoided.     

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.     

Determination of carnitine and acylcarnitines in biological samples by capillary electrophoresis–mass spectrometry

We present fast LC–MS–MS analyses of multicomponent mixtures containing flavones, sulfonamides, benzodiazepines and tricyclic amines. Using a short microbore HPLC column with small particle size, five to eight compounds were partially resolved within 15 to 30 s. TurboIonSpray and atmospheric pressure chemical ionization interfaces were well suited to tolerate the higher eluent flow-rates of 1.2 to 2 ml/min. The methods were applied to biological sample matrices after clean-up using solid-phase or liquid–liquid extraction. Good precision and accuracy (average 8.9 and 97.7%, respectively) were achieved for the determination of tricyclic amines in human plasma. Benzodiazepines were determined in human urine with average precision of 9% and average accuracy of 95% for intra- and inter-assay. Detection limits in the low ng/ml range were obtained. An example for 240 injections per hour of demonstrated the feasibility of rapid LC–MS–MS analysis.     

Application of gas chromatography—mass spectrometry with selected ion monitoring to the urinanalysis of 4-pyridoxic acid

An analytical protocol has been developed for the analysis of urinary 4-pyridoxic acid (4-PA) by gas chromatography—mass spectrometry (GC—MS) for use in metabolic studies. Aliquots of urine were deproteinised and fractionated by isocratic reversed-phase high-performance liquid chromatography. The eluent fraction containing the 4-PA was collected, freeze-dried and silylated using N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. Derivatisation produced the mono-tert.-butyldimethylsilyl derivative of 4-PA lactone. This derivative was readily amenable to GC—MS analysis in the electron ionisation (70 eV) mode, yielding a prominent fragment ion at m/z 222 ([M — 57]⁺; base peak). A heavy isotope-labelled derivative of pyridoxine [dideuteriated pyridoxine; 3-hydroxy-4-(hydroxymethyl)-5-[hydroxymethyl-²H₂]-2-methylpyridine] has been synthesised and is being employed to determine the kinetics of labelling of the body pools of vitamin B₆. Kinetic measurements are based on the determination of the relative proportions of metabolically produced deuterium-labelled and non-labelled 4-PA in urine, obtained from stable isotope ratios determined by low-resolution selected ion monitoring using a bench-top quadrupole GC—MS system.     

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.