Characterization of boldione and its metabolites in human urine by liquid chromatography/electrospray ionization mass spectrometry and gas chromatography/mass spectrometry

Boldione (1,4-androstadiene-3,17-dione) is a direct precursor (prohormone) to the anabolic steroid boldenone (1,4-androstadiene-17beta-ol-3-one). It is advertised as a highly anabolic/androgenic compound promoting muscularity, enhancing strength and overall physical performance, and is available on the Internet and in health stores. This work was undertaken to determine and characterize boldione and its metabolites in human urine, using both liquid chromatography with electrospray ionization mass spectrometry and gas chromatography with mass spectrometry and derivatization. Boldione and its three metabolites were detected in dosed human urine after dosing a healthy volunteer with 100 mg boldione. The excretion studies showed that boldione and its metabolites were detectable in urine for 48 h after oral administration, with maximum excretion rates after 1.8 and 3.6 h (boldenone case). The amounts of boldione and boldenone excreted in urine from this 100 mg dose were 34.45 and 15.95 mg, respectively.     

Quantification of docetaxel and its main metabolites in human plasma by liquid chromatography/tandem mass spectrometry

Docetaxel is an antineoplastic agent widely used in therapeutics. The objective of this study was to develop and validate a routine assay, using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), for the simultaneous quantification of docetaxel and its main hydroxylated metabolites in human plasma. A structural analogue, paclitaxel, was used as the internal standard. Determination of docetaxel and four metabolites (M1, M2, M3 and M4) was achieved using only 100 microL of plasma. Liquid-liquid extraction was used for sample preparation, with extraction efficiency of at least 90% for all analytes. Detection used positive-mode electrospray ionization in selected reaction monitoring mode. The lower limit of quantification (LLOQ) was 0.5 ng/mL for all analytes. The assay was linear in the calibration curve range 0.5-1000 ng/mL and acceptable precision and accuracy (<15%) were obtained with concentrations above the LLOQ. This method was sufficiently selective and sensitive for quantification of metabolites in plasma from cancer patients receiving docetaxel chemotherapy, and is suitable for routine analyses during pharmacokinetic studies.     

Enantioselective analysis of zopiclone and its metabolites in plasma by liquid chromatography/tandem mass spectrometry

A high-performance liquid chromatographic method with triple-quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the first time for the simultaneous quantification of zopiclone and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid–liquid extraction and separated using a chiral stationary phase based on an amylose derivative, Chiralpak ADR-H column, and ethanol–methanol–acetonitrile (50:45:5, v/v/v) plus 0.025% diethylamine as the mobile phase, at a flow-rate of 1.0 mL min⁻¹. Moclobemide was used as the internal standard. The developed method was linear over the concentration range of 7.5–500 ng mL⁻¹. The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5, and 70.7 for zopiclone enantiomers, for N-desmethyl zopiclone enantiomers and for zopiclone-N-oxide enantiomers, respectively, and 75.9 for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15%). The method application in a pilot study of zopiclone kinetic disposition in rats showed that the levels of (+)-(S)-zopiclone were always higher than those of (−)-R-zopiclone. Higher concentrations were also observed for (+)-(S)-N-desmethyl zopiclone and (+)-(S)-N-oxide zopiclone, confirming the stereoselective disposition of zopiclone.     

Detection of sibutramine administration: a gas chromatography/mass spectrometry study of the main urinary metabolites

A gas chromatographic/mass spectrometric (GC/MS) study aimed at identifying the metabolites of sibutramine (1-(4-chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl)cyclobutanemethanamine) in urine is described. Urinary excretion of sibutramine metabolites following the oral administration of a single dose of sibutramine was followed by GC/MS analysis. After identification of the chromatographic signals corresponding to the six main urinary metabolites, the fragmentation pattern was studied in electron ionization (EI) mode after derivatization to the corresponding methyl and trimethylsilyl derivatives. Urine samples were pretreated according to a reference procedure (liquid/liquid separation, enzymatic hydrolysis, pre-concentration under a stream of nitrogen and derivatization, either under thermal incubation and by microwave irradiation). All sibutramine metabolites were excreted as glucuroconjugates, and retain the chiral carbon present in the sibutramine skeleton. The metabolites identified included mono-desmethylsibutramine (nor-sibutramine), bi-desmethylsibutramine (nor-nor-sibutramine), and the corresponding hydroxylated compounds, the hydroxylation taking place either on the cyclobutane or on the isopropyl chain. The excretion profiles of the different metabolites were also evaluated. From an analytical point of view, the method can be applied to different fields of forensic analytical toxicology, including anti-doping analysis. Although the lack of certified reference materials does not allow a precise determination of the limits of detection (LODs) of all the sibutramine metabolites, an estimation taking into account the response factor of similar compounds ensures that all metabolites are still clearly detectable in a range of concentrations between 10 and 50 ng/mL, thus satisfying the minimum required performance limits (MRPLs) of the World Anti-Doping Agency (WADA).     

Detection of new propofol metabolites in human urine using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry techniques

Using hyphenated analytical techniques, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), a study on minor propofol metabolites in human urine was conducted. These techniques allowed identification of two new phase I metabolites (2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol). In addition, their four corresponding conjugates (three glucuronides and one sulphate) were detected. Thus in human urine at least eight conjugate metabolites are produced, derived from four different aglycones (propofol; 2, 6-diisopropyl-1,4-quinol; 2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol).     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Determination of nandrolone metabolites in human urine: comparison between liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry

Nandrolone (19‐nortestosterone) is an androgenic anabolic steroid illegally used as a growth‐promoting agent in animal breeding and as a performance enhancer in athletics. Therefore, its use was officially banned in 1974 by the Medical Commission of the International Olympic Committee (IOC). Following nandrolone administration, the main metabolites in humans are 19‐norandrosterone, 19‐norethiocolanolone and 19‐norepiandrosterone, and their presence in urine is the basis of detecting its abuse. The present work was undertaken to determine, in human urine, nandrolone metabolites (phase I and phase II) by developing and comparing multiresidue liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) methods. A double extraction by solid‐phase extraction (SPE) was necessary for the complete elimination of the interfering compounds. The proposed methods were also tested on a real positive sample, and they allow us to determine the conjugated/free fractions ratio reducing the risk of false positive or misleading results and they should allow laboratories involved in doping control analysis to monitor the illegal use of steroids. The advantages of LC/MS/MS over GC/MS (which is the technique mainly used) include the elimination of the hydrolysis and derivatization steps: it is known that during enzymatic hydrolysis several steroids can be converted into related compounds and deconjugation is not always 100% effective. The validation parameters for the two methods were similar (limit of quantification (LOQ) <1 ng/mL and percentage coefficient of variance (CV%) <16.4), and both were able to confirm unambiguously all the analytes, thus confirming the validity of both techniques. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of triacetone triperoxide by gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry by electron and chemical ionization

The explosive triacetone triperoxide (TATP) has been analyzed by gas chromatography/mass spectrometry (GC/MS) and sub-nanogram detection limits are reported by ammonia positive ion chemical ionization (PICI), electron ionization (EI) and methane negative ion chemical ionization (NICI). Analysis by methane PICI and ammonia NICI gave detection limits in the low nanogram range. Analyses were carried out on (linear) quadrupole and ion trap instruments. Analysis of TATP by PICI using ammonia reagent gas is the preferred analytical method, producing low limits of detection as well as an abundant (greater than 60% of base peak) diagnostic adduct ion at m/z 240 corresponding to [TATP + NH4]+. Isolation of the [TATP + NH4]+ ion with subsequent collision-induced dissociation (CID) produces extremely low abundance product ions at m/z values greater than 60, and the m/z 223 ion corresponding to [TATP + H]+ was not observed. Density functional theory (DFT) calculations at the B88LYP/DVZP level indicate that dissociation of the complex to form NH4+ and TATP occurs at energies lower than peroxide bond dissociation, while protonation of TATP leads to cleavage of the ring structure. These results provide a method for pico-gram detection levels of TATP using commercial instrumentation commonly available in forensic laboratories. As a point of comparison, a detection limit of 15 ng was obtained by flame ionization detection.     

Simultaneous enantioselective determination of fenbuconazole and its main metabolites in soil and water by chiral liquid chromatography/tandem mass spectrometry

A novel and sensitive method was developed for the simultaneous determination of fenbuconazole and its main metabolites enantioselectively using chiral liquid chromatography coupled with tandem mass spectrometry. The separation and determination were performed using reversed-phase chromatography on a cellulose chiral stationary phase, a Chiralcel OD-RH (150 mm×4.6 mm) column, under isocratic conditions at 0.5 mL/min flow rate. The effects of three cellulose-based columns and three amylose-based columns on the separation were also investigated. The elution orders of the eluting enantiomers were identified by an optical rotation detector. The QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) method and solid-phase extraction (SPE) were used for the extraction and clean-up of the soil and water samples, respectively. Parameters including the matrix effect, linearity, precision, accuracy and stability were evaluated. Under optimal conditions, the mean recoveries for all enantiomers from the soil samples were 82.5-104.1% with 2.7-9.5% intra-day relative standard deviations (RSD) and 5.7-11.2% inter-day RSD at 5, 25 and 50 μg/kg levels; the mean enantiomer recoveries from the water samples were 81.8-104.6% with 2.6-11.4% intra-day RSD and 5.3-10.4% inter-day RSD at 0.25, 0.5 and 2.5 μg/L levels. Coefficients of determination R2≥0.9991 were achieved for each enantiomer in the soil and water matrix calibration curves within the range of 1.0-125 μg/L. The limits of detection (LOD) for all enantiomers in the soil and water were less than 0.8 μg/kg, whereas the limit of quantification (LOQ) did not exceed 2.5 μg/kg. The results of the method validation confirm that this proposed method is convenient and reliable for the enantioselective determination of the enantiomers of fenbuconazole and its main metabolites in soil and water.     

Supersonic gas chromatography/mass spectrometry

A new gas chromatography/mass spectrometry (GC/MS) system was designed and evaluated which we have named 'Supersonic GC/MS'. It is based on a modification of a commercially available GC/MS system to include a supersonic molecular beam (SMB) MS interface. In this system the standard electron ionization (EI) ion source was replaced with a fly-through EI ion source mounted in the path of the SMB. A hyperthermal surface ionization (HSI) ion source combined with a 90 degrees ion mirror (for the EI-produced ions) was also added, and placed inside the quadrupole mass analyzer in place of its original EI ion source. The 'Supersonic GC/MS' system requires 18 cm added bench space plus the addition of an air-cooled 60 L/s diffusion pump and a 537 L/min rotary pump. The system is user friendly since all the gas flow rates, heated zones, sampling and data analysis are performed the same way as the original system and are computer-controlled via the original software. Similar EI sensitivity was obtained as with the original system for hexachlorobenzene and octafluoronaphthalene, while improved EI detection limits were demonstrated for methyl stearate and eicosane due to the significant enhancement of their molecular ion abundances. A GC/MS detection limit of 500 ag for pyrene was demonstrated using HSI. Good supersonic expansion cooling was achieved with large alkanes, despite the use of a rotary pump at the nozzle chamber instead of a diffusion pump. High temperature GC/MS analysis was demonstrated for large polycyclic aromatic hydrocarbons (PAHs) including ovalene and decacyclene (ten rings). Library searches with EI mass spectra are demonstrated, and it is explained why the enhancement of the molecular ion actually improves the library search in most cases. The analysis of large phthalate esters is also described, and the improvement obtained is shown to originate from their enhanced molecular and high mass fragment ions.     

Direct analysis of selected N-acyl-L-homoserine lactones by gas chromatography/mass spectrometry

A rapid, simple and selective method involving direct separation by gas chromatography (GC) with electron ionization mass spectrometry (EI-MS) was employed to determine some N-acylhomoserine lactones (AHLs). Using GC/EI-MS, simultaneous separation and characterization of AHLs were possible without prior derivatization. Informative fragmentation patterns were obtained to identify the structures of N-acyl chains of AHLs. Electron ionization resulted in a common fragmentation pattern with the most abundant ion at m/z 143 and other minor peaks at m/z 71, 57, and 43. The presence of AHLs in extracts of Burkholderia cepacia strains was achieved in selected ion monitoring mode by using the prominent fragment at m/z 143.     

Early gas chromatography/mass spectrometry

Spectroscopy Laboratory, The Dow Chemical Company, Midland, Michigan, USA In December 1955 or thereabouts, the authors coupled a homemade gas chromatograph to a research time-of-flight mass spectrometer constructed by W. C. Wiley, I. H. McLaren, and D. B. Harrington. This unique gas chromatography/mass spectrometry (GC/MS) instrument generated mass spectra at a lo-kHz rate for display on an oscilloscope; eluted gas chromate graphic components, such as methanol, acetone, benzene, toluene, and carbon tetrachloride, could be visually identified immediately from the oscilloscope display. Many years of further research and development in many laboratories worldwide were necessary, however, to make continuous on-line GC/MS the uniquely valuable analytical tool that it is today.     

Quantitation of the enantiomers of rimantadine and its hydroxylated metabolites in human plasma by gas chromatography/mass spectrometry.

A gas chromatographic/mass spectrometric procedure has been developed for the quantitation in human plasma of the enantiomers of rimantadine and its three hydroxylated metabolites. The assay utilized derivatization of all analytes with the optically active reagent S-alpha-methyl-alpha-methoxy(pentafluorophenyl)acetic acid, selective ion monitoring, methane negative ion chemical ionization mass spectrometry and stable isotope dilution techniques. This method has been used to measure plasma concentrations of the enantiomers of rimantadine, m-hydroxyrimantadine and p-hydroxyrimantadine (equatorial and axial epimers) in the ranges 2.5-250, 2.5-50, 1.25-62.5 and 1.25-62.5 ng/mL, respectively, in six subjects given a single 200 mg dose of racemic rimantadine. Although there are no significant differences in the concentration-time profiles of R- and S-rimantadine, large stereospecific differences in the disposition of their metabolites are observed.     

Two-dimensional liquid chromatography/mass spectrometry/mass spectrometry separation of water-soluble metabolites

Off-line two-dimensional liquid chromatography with tandem mass spectrometry detection (2D-LC/MS-MS) was used to separate a set of metabolomic species. Water-soluble metabolites were extracted from Escherichia coli and Saccharomyces cerevisae cultures and were immediately analyzed using strong cation exchange (SCX)-hydrophilic interaction chromatography (HILIC). Metabolite mixtures are well-suited for multidimensional chromatography as the range of components varies widely with respect to polarity and chemical makeup. Some currently used methods employ two different separations for the detection of positively and negatively ionized metabolites by mass spectrometry. Here we developed a single set of chromatographic conditions for both ionization modes and were able to detect a total of 141 extracted metabolite species, with an overall peak capacity of ca. 2500. We show that a single two-dimensional separation method is sufficient and practical when a pair or more of unidimensional separations are used in metabolomics.     

Regioisomeric differentiation of 2,3- and 3,4-methylenedioxy ring-substituted phenylalkylamines by gas chromatography/tandem mass spectrometry

Numerous abused drugs of the 3,4-methylenedioxymetamphetamine (MDMA; Ecstasy; N-methyl-1-(3,4-methylenedioxyphenyl)-2-propaneamine) type and various alkyl chain- and aromatic ring-substituted isomers give very similar electron ionization (EI) mass spectra. This seriously affects the analysis of especially ring regioisomeric drug variants. Using collision-induced dissociation (CID) (argon) under EI and chemical ionization, the mass spectra of 18 2,3- and 3, 4-methylenedioxy ring-substituted phenylethylamines were recorded. These techniques permitted an unequivocal differentiation of all studied ring regioisomeric methylenedioxyphenylethylamines. CID mass spectrometry therefore appear to be a reliable tool to establish the kind of ring substitution pattern in regioisomeric methylenedioxyphenalkylamines. Copyright 2000 John Wiley & Sons, Ltd.     

Study on the photostability of guaiazulene by high-performance liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

The photostability of guaiazulene (1,4-dimethyl-7-isopropylazulene; GA), a natural azulenic compound used in cosmetic and health-care products, as well as in pharmaceutical preparations, was investigated in solution (methanol, ethanol, acetonitrile), by different techniques: gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry and UV detection (LC/APCI-MS and HPLC/UV). A solar simulator (xenon-arc lamp) was used as UV-A radiation source. The study involved: monitoring compound decomposition, identifying products of photodegradation (PPs), assessing the role of oxygen and evaluating the kinetics of the process. Minor PPs are volatile compounds and were characterized by GC/MS, while oligomeric polyoxygenated compounds, tentatively characterized on the basis of MS and MS/MS spectra, were found to be the main photoproducts. The photodegradation was found to be enhanced by the presence of oxygen; nevertheless, determination of the singlet oxygen quantum yield for GA gave a lower value than that for the reference standard Rose Bengal. The obtained results and the developed stability-indicating methods (GC/MS and LC/MS) are of interest for stability studies and/or quality control purposes of GA as raw material or cosmetic products.     

The analysis of thiocarbamides by gas chromatography/negative-ion chemical-ionization mass spectrometry.

Thiocarbamides were converted to their di-N-pentafluorobenzyl (PFB) derivatives and analysed by gas chromatography/negative-ion chemical-ionization mass spectrometry with methane as reagent gas. The PFB derivatives of the 2-thiouracils gave mass spectra in which the ion current was carried largely by an ion arising from [M-PFB]-. The derivative was used in the determination of the uptake and metabolism of thiocarbamides by cultures of melanoma cells.     

Identification of bupropion urinary metabolites by liquid chromatography/mass spectrometry

Human urinary metabolism of the antidepressant bupropion was studied using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). A total of 20 metabolites were detected and identified. The phase I metabolism included formation of morpholinohydroxybupropion, threo- and erythrohydrobupropion, aromatic hydroxylation, butyl group hydroxylation with ketone hydrogenation and dihydroxylation. These metabolites were detected either as the free form or as glucuronide and/or sulphate conjugates. In addition also m-chlorohippuric acid was detected. Of the phase I metabolites, a dihydroxylation to the aromatic ring and to the methyl group in the middle of the substrate molecule was reported here for the first time, as well as eight of the glucuronide conjugates (to hydroxy, dihydroxy, hydroxy and hydrogenation metabolites) and three of the sulphate conjugates (to aromatic hydroxy and hydroxy and hydrogenation metabolites).