Mass spectrometric fragmentation of the tautomers of 1,3-diketones. A gas chromatographic/mass spectrometric study

The diketo and ketoenol tautomers of aliphatic 1,3-diketones can be easily separated by gas chromatography. The mass spectra of tautomers of pentane-2,4-diones substituted at C(l) and C(3), separated in this way, have been obtained. The fragmentation mechanisms are discussed. The mass spectra of the tautomers are quite different, and the main fragmentation pathways can be easily linked to the structures of the (non-interconverting!) tautomeric molecular ions. Furthermore, isomers differing by the position of the substituent can also be identified by their mass spectra.     

Comparison of gas chromatographic and gas chromatographic/mass spectrometric techniques for the analysis of TNT and related nitroaromatic compounds

The use of capillary column gas chromatography and gas chromatography/mass spectrometry for the analysis of a series of standard solutions (0.1 to 10 μg/ml) of 2,4,6-trinitrotoluene (TNT) and eight other nitroaromatic components was evaluated. The techniques included gas chromatography with electron capture detection (GC/ECD), full scan and selected ion monitoring gas chromatography/mass spectrometry with electron impact ionization (EI/FS and EI/SIM), full scan and selected ion monitoring gas chromatography/mass spectrometry with positive ion chemical ionization using methane reagent gas (PICI/FS and PICI/SIM), and full scan and selected ion monitoring gas chromatography/mass spectrometry with negative ion chemical ionization using methane reagent gas (NICI/FS and NICI/SIM). The performance of the techniques was comapared by determining the linear response range, precision, and detection limits of the analyses.     

Peralkylation of pentaerythritol for gas chromatographic— mass spectrometric analysis

Summary A rapid and convenient method for the full O-alkylation of pentaerythritol was investigated. The method involves an alkyl halide in a polar aprotic solvent, in the presence of sodium hydroxide. The optimal reaction conditions were studied for methyl iodide, ethyl iodide and bromide, n-propyl bromide and n-butyl bromide. Methyl and propyl derivatives were quantitatively obtained in a short reaction time. The method was applied for the gas chromatographic—mass spectrometric analysis of a commerical pentaerythritol sample.     

Solid-phase microextraction for gas chromatographic/mass spectrometric analysis of dimethoate in human biological samples

A new, simple and rapid procedure for the determination of dimethoate in urine and blood samples was developed using direct immersion solid-phase microextraction and gas chromatography/mass spectrometry. This technique required only 0.1 mL of sample, and ethion was used as internal standard. Two types of coated fibre were compared (100 microm polydimethylsiloxane, and 65 microm Carbowax/divinylbenzene). Other parameters, such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH, were optimized to enhance the sensitivity of the method. Limits of detection (LODs) and quantitation (LOQs) were 50 and 100 ng/mL for urine and 200 and 500 ng/mL for blood, respectively. The method was found to be linear between the LOQ and 40 microg/mL for urine, and between the LOQ and 50 microg/mL for blood, with correlation coefficients ranging from 0.9923-0.9996. Precision (intra- and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries of dimethoate were 1.24 and 0.50% for urine and blood, respectively. Because of its simplicity and the fact that small volumes of sample are used, the described method can be successfully used in the diagnosis of poisoning by this pesticide, namely in those situations where the sample volume is limited, as frequently occurs in forensic toxicology.     

Liquid chromatographic/tandem mass spectrometric method for the quantitation of tranilast in human plasma

An analytical method for the determination of tranilast in human plasma using tramadol as the internal standard has been developed based on liquid chromatography/tandem mass spectrometry. Sample preparation involved protein precipitation with methanol. Separation by reversed-phase high-performance liquid chromatography using methanol/10 mM ammonium acetate (70: 30, v/v) as mobile phase was complete in a run time of 2.4 min. Detection on a Q TRAP system used multiple reaction monitoring. The method was linear in the range 0.06-20 microg/mL with intra- and inter-day precisions (as relative standard deviation) of 2.2-2.6% and 2.3-2.9%, respectively. Accuracy (as relative error) was <-2.5%. The method was applied in a pharmacokinetic study in healthy volunteers treated with a single 80 mg oral dose of tranilast.     

Automated liquid chromatographic/tandem mass spectrometric method for screening beta-blocking drugs in urine

An automated liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method is presented for the screening and confirmation of 16 beta-blocking drugs in clinical and autopsy urine samples. The described method involved C(18) solid phase extraction, LC separation and MS analysis on a triple-stage quadrupole mass analyser. Samples were initially pre-screened for the presence of any beta-blocking drugs using LC/MS with selected ion monitoring. Any compounds tentatively identified as beta-blocking drugs on the basis of their LC retention time and protonated molecular ion were then automatedly subjected to a second analysis in which the relevant MS/MS product ion mass spectra were acquired. These product ion mass spectra were then automatically searched against a 400-substance mass spectral library containing previously acquired beta-blocking drugs. The results demonstrated that library search of beta-blocking drugs in urine with MS/MS product ion mass spectra was more reliable and produced fewer false negatives than library searching with mass spectra derived from single-stage quadrupole MS. The limits of identification in the MS/MS product ion scan ranged from 0.02 mg l(-1) for carvedilol to 1.2 mg l(-1) for pindolol, the majority of the values being below 0.2 mg l(-1).     

Development of a static headspace gas chromatographic/mass spectrometric method to analyze the level of volatile contaminants biodegradation

Volatile compound biodegradation analysis usually requires the time-consuming step of extraction of the analytes from the matrix using organic solvents or costly radioactive-compounds. Thus, it is desirable to have a simple and fast technique to generate a good evaluation of bacterial biodegradation. The goal of this research was to develop a methodology on the basis of static headspace-gas chromatography/mass spectrometry (HS-GC/MS) to evaluate the level of volatile contaminant biodegradation. The effects of the following parameters were studied: temperature and time of equilibration. The biodegradation experiments were carried out with bacteria inoculation in mineral media in presence of volatile hydrocarbon compounds (toluene, p-xylene, nonane and naphthalene). Autoclaved inoculates were used as control and reference sample. The optimal headspace conditions were observed when the vials were heated at 80 degrees C for 20 min, the syringe at 81 degrees C and an injection volume of 0.4 mL was used. This methodology has the advantage of being relative free from matrix effects.     

Gas chromatographic/mass spectrometric determination of flumioxazin extracted from soil and water

A method was developed for determining flumioxazin in soil and water. Recovery efficiencies for solid-phase extraction (SPE) of flumioxazin from deionized, well, and surface water were between 72 and 77%. SPE was superior to liquid-liquid extraction, using water-hexane and water-chloroform emulsions, which resulted in retrieval efficiencies of 25 and 22%, respectively. However, liquid-liquid extraction with ethyl acetate improved recovery of total flumioxazin to >64%. Extraction from soil samples by direct solvent/soil extraction methods recovered between 18 and 76% of applied flumioxazin, depending on the solvent combination used. However, the use of accelerated solvent extraction techniques resulted in a 106 +/- 8% recovery of flumioxazin from soil. In analysis by capillary gas chromatography with mass selective detection, flumioxazin had a calculated limit of detection of 9 ng/mL with a retention time of 16.66 min.     

6alpha-Methylandrostenedione: gas chromatographic mass spectrometric detection in doping control

In recent years products containing 6alpha-methylandrost-4-ene-3,17-dione have appeared on the sport supplement market. Scientific studies have proven aromatase inhibition and anabolic and mild androgenic properties; however, no preparation has been approved for medical use up to now. In sports 6alpha-methylandrost-4-ene-3,17-dione has to be classified as a prohibited substance according to the regulations of the World Anti-Doping Agency (WADA). For the detection of its misuse the metabolism was studied following the administration of two preparations obtained from the Internet (Formadrol and Methyl-1-Pro). Several metabolites as well as the parent compounds were synthesized and the structures of 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, 6alpha-methylandrost-4-ene-3,17-dione, and 5beta-dihydromedroxyprogesterone were confirmed by nuclear magnetic resonance (NMR) spectroscopy. The main metabolite, 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, was found to be excreted as glucuronide and was still detectable in microg/mL amounts until urine collection was terminated (after 25 h). Additionally, samples from routine human sports doping control had already tested positive for the presence of metabolites of 6alpha-methylandrost-4-ene-3,17-dione. Screening analysis can be easily performed by the existing screening procedure for anabolic steroids using 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one as target substance (limit of detection <10 ng/mL). Its discrimination from the closely eluting drostanolone metabolite, 3alpha-hydroxy-2alpha-methyl-5alpha-androstan-17-one, is possible as the mono-TMS derivative.     

Extraction, quantitative gas chromatographic determination and gas chromatographic/mass spectrometric detection of ecgonine for identification of cocaine and its metabolites in urine

Summary Quantitative extraction studies from urine were carried out by addition of cocaine, benzoylecgonine, ecgonine methyl ester and ecgonine to urine samples. After hydrolysis to ecgonine the compounds were analyzed together. Ecgonine was isolated by a cation-exchange resin and purified by an anion-exchange resin. The quantitative determination was performed by GC after silylation with MSTFA. The recovery was 77% at a concentration of 150 g ecgonine/ml urine. A qualitative determination of ecgonine by GC/MS was possible up to the detection limit of 20 ng/ ml. The method can be applied for the detection of cocaine abuse.

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Extraktion, quantitative gas-chromatographische Bestimmung und gas-chromatographischer massenspektrometrischer Nachweis von Ecgonin zur Identifizierung von Cocain und seinen Metaboliten in Urin

Zusammenfassung Es wurden quantitative Untersuchungen zur Extraktion von Urin durchgeführt, dem Cocain, Benzoylecgonin, Ecgoninmethylester und Ecgonin zugesetzt wurden. Die Summe dieser Verbindungen kann über eine vorgeschaltete Hydrolyse zu Ecgonin indirekt bestimmt werden. Nach Hydrolyse wird Ecgonin an einem Kationenaustauscher isoliert und der Extrakt an einem Anionenaustauscher gereinigt. Die quantitative Bestimmung erfolgt gaschromatographisch nach Silylierung mit MSTFA. Dabei beträgt die Wiederfindungsrate 77% bei Konzentrationen von 150 g Ecgonin/ml Urin. Der qualitative Nachweis von Ecgonin durch GC/MS ist bis zu einer Nachweisgrenze von 20 ng/ml möglich. Damit eignet sich die Methode zum Nachweis einer Cocain-Einnahme im Urin.

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From the dissertation by U. Zerell, University of Bonn, 1986 (in preparation)     

A new reversed-phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism

Seventy-six compounds of biological interest for the diagnosis of inherited disorders of amino acids (AA) metabolism have previously been demonstrated to be detectable in positive mode electrospray ionisation tandem mass spectrometry (ESI-MS/MS), after separation by ion-pairing reversed-phase liquid chromatography (RPLC). The separation method used tridecafluoroheptanoic acid as ion-pairing agent, and a gradient of acetonitrile for the elution of the most retained compounds. This method had previously been demonstrated to be suitable for the qualitative diagnosis of many AA disorders, and for the quantitative measurement of 16 AA in biological fluids, using their stable isotope labelled (SIL) AA as internal standard. For quantification of the other AA, an internal standard was chosen among the available SIL-AA, as close as possible to the analyte to be measured, in terms of structural analogy, and of retention time in the chromatographic system. The performances of the quantitative analysis of the other AA to be measured are reported here. They show validated results for several AA, allowing their accurate quantification, with another SIL-AA as internal standard. For some other AA, quantitative results were not accurate, allowing only semi-quantitative or qualitative determination for these parameters.     

Evaluation of four mass spectrometric methods for the gas chromatographic analysis of polychlorinated n-alkanes

The suitability of four mass spectrometric methods for the gas chromatographic analysis of polychlorinated n-alkanes (PCAs, also called chlorinated paraffins) was evaluated and compared using spiked and fish liver samples. Electron ionization tandem mass spectrometry (EI-MS/MS) as well as electron capture negative ionization (ECNI) combined with low and high resolution mass spectrometry and CH4/CH2Cl2-negative ion chemical ionization (NICI) low resolution mass spectrometry were investigated. All methods showed an accuracy of <21% for the analysis of spiked fish samples. However, the analysis of real samples showed deviations of up to 46% between the four mass spectrometric methods. The influence of the selected reference standard on quantification was also evaluated. The use of a quantification standard with a degree of chlorination deviating from that of the sample can result in differences of > 100% for the ECNI methods. EI-MS/MS and CH4/CH2Cl2-NICI led to errors of maximum 17% and 33%, respectively, independent from the degree of chlorination of the used reference standard.     

A validated liquid chromatographic/tandem mass spectrometric method for the determination of phencyclidine in microliter samples of rat serum

A liquid chromatographic/tandem mass spectrometric method is described for the determination of phencyclidine (PCP) in small volumes of rat serum (e.g. 50 microl). Samples were extracted using a mixed-mode strong cation-exchange column and then separated isocratically using a narrow-bore (2.1 mm i.d.) 3 microm Hypersil phenyl column and a mobile phase consisting of an ammonium formate buffer (pH 2.7) with 60% (v/v) methanol. Detection was accomplished using positive ion electrospray ionization in the multiple reaction monitoring mode. Mass spectra were obtained and peaks were observed at an m/z (% abundance) of 244 (100), 159 (25), and 86 (89). Tandem mass spectra were also obtained from the m/z 244 precursor ion with peaks observed at m/z 159 (100), 86 (96), and 91 (11). Optimum serum PCP sensitivity and precision were obtained at a transition of m/z 244 --> 159. Matrix-associated ion suppression did not significantly affect the accuracy (100-112%) or precision (CV < or =8%) of the assay. The lower limit of quantitation was 1 ng ml(-1) in 50 microl of serum. The method was used to study the serum pharmacokinetics of PCP in rats after an intravenous bolus dose of PCP.     

Liquid chromatographic/mass spectrometric determination of desmedipham and phenmedipham and their metabolites in soil

A simple method is described for the simultaneous determination of residues of 2 carbamate herbicides (phenmedipham and desmedipham) and related metabolites (m-aminophenol, aniline, and m-toluidine) in soil. The analytes are extracted from spiked soils with methanol. The solvent/soil suspension is centrifuged, and the supernatant is directly injected, without any further cleanup, into a reversed-phase liquid chromatography/mass spectrometry apparatus equipped with a TurbolonSpray interface. The method was tested on 5 soils having different physicochemical properties. Recoveries from the soil types, spiked over the range of 50-200 ppb, were essentially quantitative for each analyte. The detection limits of the method are < or = 25 ng/g.     

Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.     

Screening and diagnosis of beta-ureidopropionase deficiency by gas chromatographic/mass spectrometric analysis of urine

Dihydropyrimidine dehydrogenase (DHPDase), dihydropyrimidinase (DHPase) and beta-ureidopropionase (betaUPase) are the enzymes that catalyze the first, second, and third steps of the degradation of pyrimidines, respectively. beta-Ureidopropionate (betaUP) and beta-ureidoisobutyrate (betaUIB) are increased in the urine of patients with betaUPase deficiency. The original case in which betaUPase deficiency was discovered by NMR spectroscopy was an 11-month-old patient who presented with hypotonia and dystonic movement. We detected a second but asymptomatic case during a pilot study of neonatal screening with filter-paper urine, urease pretreatment and gas chromatography/mass spectrometry (GC/MS). The urease pretreatment of urine without fractionation resulted in a high recovery of these polar ureide compounds and allowed the highly sensitive GC/MS detection and diagnosis of betaUPase deficiency. betaUP and betaUIB were identified using GC/MS techniques. In the urine of the neonate with betaUPase deficiency, betaUP and betaUIB were persistently increased. Thymine, 5,6-dihydrothymine and 5,6-dihydrouracil were increased only moderately but significantly. It is known that thymine and uracil increase markedly in DHPDase deficiency, and 5,6-dihydrothymine and 5,6-dihydrouracil increase in DHPase deficiency. Therefore, betaUPase deficiency can be differentially diagnosed from the first and second enzyme deficiencies. Application of this specific and sensitive diagnostic procedure will lead to an understanding of the clinical heterogeneity of betaUPase deficiency. Furthermore, the identification of patients with defects in pyrimidine metabolism will enable doctors to avoid cancer chemotherapy with pyrimidine analogues such as 5-fluorouracil, which could be dangerous for these patients.     

Liquid chromatographic/tandem mass spectrometric method for the determination of the tobacco-specific nitrosamine metabolite NNAL in smokers' urine

A specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for NNAL, a metabolite of the tobacco-specific nitrosamine metabolite NNK. The metabolite was detected in smokers' urine with a limit of quantitation (LOQ) of 20 pg ml(-1) and a linear range up to 1000 pg ml(-1). The method features a single solid-phase extraction step and MS/MS monitoring following electrospray ionization. Fragmentation pathways for the protonated molecular ion are proposed. The sample preparation is simpler than that for gas chromatographic methods reported in the literature and maintains sensitivity adequate for determining NNAL in smokers' urine. By using enzyme hydrolysis to determine total NNAL in urine, the amount of NNAL-glucuronide was calculated. A standard pooled smokers' urine sample used for development gave values of 176 +/- 8 pg ml(-1) free NNAL and 675 +/- 26 pg ml(-1) total NNAL following enzyme hydrolysis. The method was applied to a group of seven smokers; the free NNAL level for the group was 101-256 pg ml(-1) with NNAL-glucuronides at 247-566 pg ml(-1). The ratio of conjugated to free NNAL was in the range 0.98-2.95. The variability in total daily amount of NNAL excreted (ng per 24 h) had RSDs of 6-21% for free NNAL, 7-22% for conjugated NNAL and 6-20% for total NNAL excreted. When normalized to the number of cigarettes smoked, the amounts of NNAL excreted per cigarette smoked were in the range of amounts of NNK yields reported for cigarettes in the literature.     

Comparison of different mass spectrometric detection techniques in the gas chromatographic analysis of pyrethroid insecticide residues in soil after microwave-assisted extraction

A comparative study was carried out for the simultaneous determination of 11 pyrethroid insecticides in soil by gas chromatography (GC) - ion trap (IT)- mass spectrometry (MS), by means of two different ionization modes; electron impact and negative chemical ionization and three data acquisition procedures; full scan, selected ion monitoring and MS/MS. Pyrethroids investigated were tetramethrin, bifenthrin, phenothrin, λ-cyhalothrin, permethrin, cyfluthrin, cypermethrin, flucythrinate, esfenvalerate, fluvalinate and deltamethrin. Soil samples were treated with toluene/water by microwave-assisted extraction for 9 min at 700 W and a cleaning up with florisil was performed. Clean soil samples were spiked with pyrethroids at a spiking level of 10, 25 and 50 ng/g. The method employed provides a concentration factor of 10. The ionization gas employed in the negative chemical ionization mode was methane. The use of MS/MS acquisition, in electron impact ionization, provided the best results, due to its high selectivity and sensitivity, giving very low limits of detection from 0.08 to 0.54 ng/g. In negative chemical ionization full scan and selected ion monitoring methods detection limits from 0.12 to 1.40 ng/g were found. The proposed methods were applied to several levels from 10 to 50 ng/g of spiked soils, being electron impact MS/MS method which minimizes matrix spectrum interferences and provided recovery average values from 84% to 120% with relative standard deviations which varied from 3.2 to 7.2%.