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.     

Simultaneous determination of two human urinary metabolites of N,N-dimethylformamide using gas chromatography–thermionic sensitive detection with mass spectrometric confirmation

Two human urinary metabolites of the industrial solvent N,N-dimethylformamide (DMF), N-hydroxymethyl-N-methylformamide (HMMF) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), were assayed using a new analytical method (gas chromatography and thermionic sensitive detection). Clean-up of urine samples includes a liquid–liquid extraction step followed by a solid-phase extraction step to separate HMMF and AMCC from other urine components. During clean-up, AMCC is converted into ethyl-N-methylcarbamate (EMC), and during gas chromatography, HMMF is degraded in the injector to N-methylformamide (NMF). All the validation data necessary for a quantitative procedure are given. The method was applied to urine samples from workers exposed to DMF and from the general population. The results were confirmed by mass spectrometric determination. For this purpose a further liquid–liquid extraction step was introduced in the clean-up procedure. Background levels of AMCC in the general population were identified.     

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.     

Determination of an arylether antiarrhythmic and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates using liquid chromatography–tandem mass spectrometry

A method was developed and validated for the quantification of (±)-trans-[2-morpholino-1-(1-naphthaleneethyloxy]cyclohexane monohydrochloride (RSD1070) and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography coupled with positive ion electrospray ionization and detection by tandem mass spectrometry. The assay was linear from 2.5 to 100 ng/ml and this range was used for validation. Inter- and intra-assay variability (n=6), extraction recovery, and stability in plasma were assessed. The estimated limit of quantitation was in the range 2.5–3 ng/ml for both analytes in rat plasma. The analytical method was used in a pharmacokinetic study of RSD1070 in rats after a single i.v. bolus of 12 mg/kg.     

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.     

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

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

Simultaneous determination of fifteen low-dosed benzodiazepines in human urine by solid-phase extraction and gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, -hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and -hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard. The urine sample preparation involves enzymatic hydrolysis of the conjugated metabolites with Helix pomatia β-glucuronidase for 1 h at 56°C followed by solid-phase extraction on a phenyl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitivity. The extraction efficiency exceeded 80% for all compounds except for oxazepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60%. The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SIM mode. The within-day and day-to-day relative standard deviations at three different concentrations never exceeded 15%.     

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.     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

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%.     

Identification and quantification of sophorolipid analogs using ultra-fast liquid chromatography–mass spectrometry

An ultra-fast liquid chromatographic method combined with atmospheric pressure chemical ionization mass detection (UHPLC/APCI-MS) has been developed for the separation and quantification of sophorolipid analogs produced by the yeast Candida bombicola. The sophorolipid mixture was produced by growing the yeast in the presence of glucose and oleic acid under higher aeration. It was found that more than 95% of the analogs are lactonic sophorolipids and all the produced sophorolipids produced were either mono- or di-acetylated. Also observed was a sophorolipid analog with a tri-unsaturated fatty acid, which has not been reported previously.     

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS–MS). The first procedure was based on the manual liquid–liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.     

High-throughput screening of corticosteroids and basic drugs in horse urine by liquid chromatography-tandem mass spectrometry

This paper describes two high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the screening of two important classes of drugs in equine sports, namely corticosteroids and basic drugs, at low ppb levels in horse urine. The method utilized a high efficiency reversed-phase LC column (3.3 cm L x 2.1 mm i.d. with 3 microm particles) to provide fast turnaround times. The overall turnaround time for the corticosteroid screen was 5 min and that for the basic drug screen was 8 min, inclusive of post-run and equilibration times. Method specificity was assessed by analysing a total of 35 negative post-race horse urine samples. No interference from the matrices at the expected retention times of the targeted masses was observed. Inter-day precision for the screening of 19 corticosteroids and 48 basic drugs were evaluated by replicate analyses (n = 10) of a spiked sample on 4 consecutive days. The results demonstrated that both methods have acceptable precision to be used on a routine basis. The performance of these two methods on real samples was demonstrated by their applications to drug administration and positive post-race urine samples.     

High-performance liquid chromatographic determination of [C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide in mouse plasma and tissue and in human plasma

The high-performance liquid chromatographic determination of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide ([¹¹C]PK 11195) is described. The method was successfully applied for plasma and tissue analysis after i.v. injection of [¹¹C]PK 11195 in mice and for plasma analysis after administration of [¹¹C]PK 11195 to humans. Separation is effected on a RP-C₁₈ column, using a mixture of acetonitrile–water–triethylamine (65:35:0.5, v/v). Quantitative measurements of radioactivity are performed on a one-channel γ-ray spectrometer equipped with a 2×2 in. NaI(Tl) detector. For humans rapid metabolisation of [¹¹C]PK 11195 was observed. At 5, 20 and 35 min post injection 5%, 22% and 32%, respectively, of the plasma activity consisted of at least two more polar metabolites. Despite the extensive metabolisation rate in mice (up to 42% at 10 min post injection of [¹¹C]PK 11195), no ¹¹C-labelled metabolites could be detected in the extracts of brain and heart.     

Electrospray ionisation–mass spectrometric characterisation of selected anti-psychotic drugs and their detection and determination in human hair samples by liquid chromatography–tandem mass spectrometry

Electrospray ionisation quadrupole ion-trap mass spectrometric (ESI–MS) characterisation of the anti-psychotic drugs chlorpromazine, trifluoperazine, flupenthixol, risperidone and the antidepressant/internal standard trimipramine is presented and possible mechanisms for the observed MSⁿ fragmentation patterns proposed. A validated liquid chromatography (LC)–MS–MS method is then applied to the detection and determination of these drugs in the hair of a patient under clinical treatment for schizophrenia. Chlorpromazine, trifluoperazine and flupenthixol are identified and determined in this hair sample following alkaline degradation of the matrix, solvent extraction and LC–MS–MS using trimipramine as internal standard.     

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.     

Assay method for the carboxylic acid metabolite of clopidogrel in human plasma by gas chromatography–mass spectrometry

This paper describes a GC–MS method for the analysis of the carboxylic acid metabolite (SR26334, II) of methyl (+)-(S)--(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate hydrogensulfate (clopidogrel, SR 25990, I) in plasma and serum. The analytical procedure involves a robotic liquid–liquid extraction with diethyl ether followed by a solid–liquid extraction on C₁₈ cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and -bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue (III) of II, was used as internal standard. The 1/X²; weighted calibration curve obtained in the range 5–250 ng/ml was well described by a quadratic equation. The extraction efficiency was better than 48% over the range studied; for the internal standard it averaged 51% at 50 ng/ml. Precision ranged from 3.6 to 15.8%, and accuracy was between 92 and 114%. Dilution has no influence on the performance of the method which could then be used to quantitate plasma samples containing up to 25 000 ng/ml. The limit of quantification was 5 ng/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay methods for use in pharmacokinetic studies.     

Optimization and performance of a rapid gas chromatography–mass spectrometry analysis for methylmalonic acid determination in serum and plasma

We have developed a rapid and sensitive GC–MS assay for methylmalonic acid determination in serum and plasma utilizing an anion exchange solid-phase extraction and trimethylsilyl derivatization. Each step of the procedure was optimized by the experimental design methods to assure the assay reliable performance. The limit of detection and limit of quantitation were 0.025 and 0.1 μmol/l. The total coefficient of variation for the method was 9.8, 4.4, and 4.6% at the concentration of 0.2, 3.1, and 6.2 μmol/l methylmalonic acid concentration, respectively. The assay are linear up to 9.0 μmol/l, and showed good correlation with a reference method. The method has proven to be reliable in routine production, producing clean chromatography, unique ion fragments, and consistent ion mass ratio.     

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.     

Simultaneous determination of thioTEPA, TEPA and a novel, recently identified thioTEPA metabolite, monochloroTEPA, in urine using capillary gas chromatography

An assay for the simultaneous quantitative determination of thioTEPA, TEPA and the recently identified metabolite N,N′-diethylene-N″-2-chloroethylphosphoramide (monochloroTEPA) in human urine has been developed. MonochloroTEPA was synthesized by incubation of TEPA with sodium chloride at pH 8. Thus, with this assay monochloroTEPA is quantified as TEPA equivalents. Analysis of the three analytes in urine was performed using gas chromatography with selective nitrogen–phosphorous detection after extraction with a mixture of 1-propanol and chloroform from urine samples. Diphenylamine was used as internal standard. Recoveries ranged between 70 and 100% and both accuracy and precision were less than 15%. Linearity was accomplished in the range of 25–2500 ng/ml for monochloroTEPA and 25–5000 ng/ml for thioTEPA and TEPA. MonochloroTEPA proved to be stable in urine for at least 4 weeks at −80°C. ThioTEPA, TEPA and monochloroTEPA cummulative urinary excretion from two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples and that the excreted amount of monochloroTEPA exceeded that of thioTEPA on day 2 to 5 of urine collection.