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 ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

Confirmation of a dopamine metabolite in parkinsonian brain tissue by gas chromatography—mass spectrometry

Gas chromatography—mass spectrometry was used to identify a dopamine metabolite isolated from the substantia nigra of parkinsonian brain tissue. Incubation of dopamine with monoamine oxidase B gave the same product which was identified as 3,4-dihydroxyphenylacetaldehyde. The structure of the compound was established by chemical synthesis, metastable ion measurement and high-resolution mass spectrometry.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Determination of nitrate in blood by gas chromatography and gas chromatography–mass spectrometry

We devised a sensitive and simple method for determining nitrate in whole blood, using an extractive alkylation technique. Nitrate in whole blood was reduced to nitrite by hydrazine sulfate in the presence of Cu²⁺ and Zn²⁺ as catalysts, and alkylated with pentafluorobenzyl bromide using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst. The obtained derivative was analyzed qualitatively by gas chromatography–mass spectrometry and quantitatively by gas chromatography with electron-capture detection. The detection limit of nitrate in whole blood was 0.01 mM. The calibration curve was linear over the concentration range from 0.02 to 1.0 mM for nitrate in whole blood. The accuracy and precision of the method were evaluated and the relative standard deviations were found to be within 10%. Using this method, the blood nitrate levels of two victims who committed suicide by inhaling automobile exhaust gas were determined.     

Determination of flunarizine in rat brain by liquid chromatography–electrospray mass spectrometry

A rapid liquid chromatography–electrospray mass spectrometry (LC–ES-MS) assay for the determination of flunarizine (FZ) in rat brain has been developed. A C₁₈ column and an isocratic elution were employed for the separation. Using post-column split, 64% of the eluent was introduced into the ES-MS system for detection. The [M+H]⁺ (m/z 406) and a fragmented ion (m/z 203) were detected using selected ion monitoring. The linear range of this assay was good, ranging from 0.05 to 5 μM (r²=0.99). The intra- and inter-day precisions showed relative standard deviations ranging from 1.4% to 2.0% and 1.3% to 2.9%, respectively. The application of this newly developed method was demonstrated by examining the pharmacokinetics of FZ in rat brain.     

Determination of metabolites of pirimicarb in human urine by gas chromatography–mass spectrometry

The analytical method described permits the determination of 2-dimethylamino-5,6-dimethyl-4-hydroxypyrimidine (DDHP), 2-methylamino-5,6-dimethyl-4-hydroxypyrimidine (MDHP) and 2-amino-5,6-dimethyl-4-hydroxypyrimidine (ADHP) in human urine. These hydroxypyrimidines are metabolites of pirimicarb (2-dimethylamino-5,6-dimethylpyrimidin-4-yldimethylcarbamate) which is applied as insecticide. The analytes are extracted into a mixture of diethyl ether and acetonitrile. Pentafluorobenzyl bromide serves as derivatising reagent. The derivatives are analysed using capillary gas chromatography with mass selective detection. 2-Amino-4-hydroxy-6-methylpyrimidine and 4-hydroxy-6-trifluoromethylpyrimidine are used as internal standards. The detection limits are 0.5 μg/l (DDHP), 1 μg/l (MDHP) and 4 μg/l (ADHP), respectively. The method was used for analysing seven urine samples collected from workers who had applied pirimicarb. The three metabolites were found in every sample in concentrations up to 60 μg/l.     

Validation of an analytical method for a potent antitumor agent, TZT-1027, in plasma using liquid chromatography–mass spectrometry

A sensitive and specific analytical method for a potent antitumor agent, TZT-1027, in plasma has been developed using liquid chromatography–mass spectrometry (LC–MS) with [²H₄]TZT-1027 as an internal standard (I.S.). A plasma sample was purified by solid-phase extraction on a C₁₈ cartridge, followed by solvent extraction with diethyl ether. The extract was then injected into the LC–MS system. Chromatography was carried out on a C₁₈ reversed-phase column using acetonitrile–0.05% trifluoroacetic acid (TFA) (55:45) as a mobile phase. Mass spectrometric analysis was performed in atmospheric pressure chemical ionization (APCI) mode with positive ion detection, and the protonated molecular ions ([M+H]⁺) of TZT-1027 and I.S. were monitored to allow quantitation. The method was applied to the determination of TZT-1027 in human, monkey, dog, rat and mouse plasma. As far as the sample preparation was concerned, good recoveries (73.5–99.1%) were obtained. The calibration curves were linear over the range of 0.25–100 ng per 1 ml of human, dog and rat plasma, per 0.5 ml of monkey plasma, and per 0.1 ml of mouse plasma. From the intra- and inter-day accuracy and precision, the present method satisfies the accepted criteria for bioanalytical method validation. TZT-1027 was stable when stored below −15°C for 6 months in human plasma and for 3 weeks in plasma from other species. TZT-1027 was also stable in plasma through at least three freeze–thaw cycles.     

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.     

Analysis of rat brain microdialysate by gas chromatography—high-resolution selected-ion monitoring mass spectrometry

Gas chromatography—high-resolution selected-ion monitoring mass spectrometry was used to analyze catecholamine metabolites in rat brain microdialysate. Dialysate samples were collected in vials containing stable isotope analogues of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and 5-hydroxyindoleacetic acid (5HIAA) and analyzed as their trimethylsilyl derivatives. The metabolite levels were monitored at 20-min intervals throughout the time course of the experiment, beginning immediately after surgery and implantation of the dialysis probe and ending 4 h after amphetamine treatment. The levels of HVA were observed to decrease after amphetamine treatment, while those of MHPG and 5HIAA did not change significantly.     

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).     

Measurement of testosterone and pregnenolone in nails using gas chromatography–mass spectrometry

An efficient method for the determination of testosterone and pregnenolone in human nails using gas chromatography–mass spectrometry (GC–MS) with d₃-testosterone as an internal standard is described. The method involves alkaline digestion and liquid–liquid extraction, with subsequent conversion to mixed pentafluoropenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected-ion monitoring (SIM) mode. The limit of detection (LOD) and limit of quantification (LOQ) were lowered to 0.1 and 0.2 pg/g, respectively, when 100 mg of nail-clippings were used. The mean recoveries of testosterone and pregnenolone were 89.8 and 86.7%, respectively, while good overall precision (% C.V.; 4.5–9.5) and accuracy (% bias; 3.9–8.4) were demonstrated. Linearity as a correlation coefficient was 0.9913 (testosterone) and 0.9965 (pregnenolone). When applied to fingernail and toenail samples from seven healthy men and nine healthy women, testosterone and pregnenolone were positively detected in the concentration range of 0.24–5.80 ng/g. The levels of two steroids studied in the nails were found to be higher in the male subjects than in the female subjects, and except for the toenails of the females, the levels of testosterone were higher than those of pregnenolone.     

Determination of cyclophosphamide in urine by gas chromatography—mass spectrometry

A sensitive gas chromatographic method for the determination of cyclophosphamide in urine is presented. After liquid—liquid extraction with diethyl ether and derivatization with trifluoroacetic anhydride, cyclophosphamide was identified and quantified with mass spectrometry. The method is suitable for the determination of cyclophosphamide at concentrations of more than 0.25 ng/ml, which enables the uptake of cyclophosphamide during occupational activities, such as the preparation and administration of antineoplastic agents in hospitals, to be measured. Simple preparation makes the method appropriate for routine analysis.     

Simultaneous determination of dimethylamphetamine and its metabolites in rat hair by gas chromatography–mass spectrometry

In order to study the disposition of dimethylamphetamine (DMAP) and its metabolites, DMAP N-oxide, methamphetamine (MA) and amphetamine (AP), from plasma to hair in rats, a simultaneous determination method for these compounds in biological samples using gas chromatography–mass spectrometry with selected ion monitoring (GC–MS-SIM) was developed. As DMAP N-oxide partially degrades to DMAP and MA during GC–MS analysis, it was necessary to avoid conditions which co-extract the N-oxide in the sample preparation so as to assure no contribution of artifactual products from DMAP N-oxide in the detection of the other compounds. For confirmation of the satisfactory separation of DMAP N-oxide from the others, the internal standards used for quantification were labeled with different numbers of deuterium atoms. Determination of unchanged DMAP was performed without any derivatization, that of DMAP N-oxide was carried out after conversion into trifluoroacetyl-MA by reaction with trifluoroacetic anhydride, and MA and AP were quantified after trifluoroacetyl-derivatization.After intraperitoneal administration of DMAP HCl to pigmented hairy rats (5 mg kg⁻¹ day⁻¹, 10 days, n=3), concentrations of DMAP and its metabolites in urine, plasma and hair were measured by GC–MS-SIM. The area under the concentration versus time curves (AUCs) of DMAP, DMAP N-oxide, MA and AP in the plasma were 397.2±97.5, 279.7±68.3, 18.4±1.2 and 15.9±2.2 μg min ml⁻¹, while their concentrations in the hair newly grown for 4 weeks after administration were 4.82±0.67. 0.45±0.09, 3.25±0.36 and 0.89±0.05 ng mg⁻¹, respectively. This fact suggested that the incorporation tendency of DMAP N-oxide from plasma into hair was distinctly low in comparison with the other compounds.     

Determination of perfluorodecalin and perfluoro-N-methylcyclohexylpiperidine in rat blood by gas chromatography–mass spectrometry

A gas chromatography–mass spectrometry method (SIM mode) was developed for the determination of perfluorodecalin (cis and trans isomers, 50% each) (FDC), and perfluoromethylcyclohexylpiperidine (3 isomers) (FMCP) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Analysis was performed by electronic impact ionization. The ions m/z 293 and m/z 181 were selected to quantify FDC and FMCP due to their abundance and to their specificity, respectively. The ion m/z 295 was selected to monitor internal standard. Before extraction, blood samples were stored at −30°C for at least 24 h in order to break the emulsion. The sample preparation procedure involved sample clean-up by liquid–liquid extraction. The bis(F-butyl)ethene was used as the internal standard. For each perfluorochemical compound multiple peaks were observed. The observed retention times were 1.78 and 1.87 min for FDC, and 2.28, 2.34, 2.48 and 2.56 min for FMCP. For each compound, two calibration curves were used; assays showed good linearity in the range 0.0195–0.78 and 0.78–7.8 mg/ml for FDC, and 0.00975–0.39 and 0.39–3.9 mg/ml for FMCP. Recoveries were 90 and 82% for the two compounds, respectively with a coefficient of variation <8%. Precision ranged from 0.07 to 15.6%, and accuracy was between 89.5 and 111.4%. The limits of quantification were 13 and 9 μg/ml for FDC and FMCP, respectively. This method has been used to determine the pharmacokinetic profile of these two perfluorochemical compounds in blood following administration of 1.3 g of FDC and 0.65 g of FMCP per kg body weight, in emulsion form, in rat.     

Simultaneous determination of methionine and total homocysteine in human plasma by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method for the simultaneous determination of methionine and total homocysteine in human plasma is described. dl-[²H₄]Methionine and dl-[²H₈]homocystine were used as internal standards. The method involved reduction of the disulfide bond with dithiothreitol, purification by cation-exchange chromatography using a BondElut SCX cartridge and derivatization with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of N(O,S)-isobutyloxycarbonyl ethyl ester (IBC-OEt) derivatives for methionine and [²H₄]methionine, respectively, and the fragment ions ([M+H–COOisoBu–COOEt]⁺) for IBC-OEt derivatives for homocysteine and [²H₄]homocysteine, respectively. The sensitivity, specificity, accuracy and precision of the method were demonstrated to be satisfactory for measuring concentrations of methionine and total homocysteine in human plasma.     

Determination of trace amounts of ginkgolic acids in Ginkgo biloba L. leaf extracts and phytopharmaceuticals by liquid chromatography–electrospray mass spectrometry

Ginkgolic acids (GAs) are toxic phenolic compounds present in the fruits and leaves of Ginkgo biloba L. (Ginkgoacae). Their maximum level in phytopharmaceuticals containing ginkgo extracts has been recently restricted to 5 μg/g by the Commission E of the former Federal German Health Authority. In order to detect ginkgolic acids at these low levels, a sensitive and selective analytical method, based on liquid chromatography–electrospray mass spectrometry (LC–ES-MS) has been developed. The three main phenolic acids (1–3) of the chloroform fruit extract were isolated and used as standards for quantification. In the LC–ES-MS negative ion mode, calibration curves with good linearities (r=0.9973, n=6) were obtained in the range of 0.5–10 μg/g for compounds 1, 2 and between 0.1 and 7.5 μg/g (r=0.9949, n=6) for ginkgolic acid 3. The detection limits at a S/N ratio of 3 were 0.1 (3) and 0.25 μg/g (1, 2). Recoveries were around 101% at 5 μg/g for the substances detected in the leaf extracts. Good precision was achieved with relative standard deviations of less than 4% (n=6). The optimised method was applied to verify whether the amount of gingkolic acids was below 5 μg/g in a standardised leaf extract which is a constituent of a phytopreparation.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.