Ultra-pressure liquid chromatography/tandem mass spectrometry targeted profiling of arachidonic acid and eicosanoids in human colorectal cancer

Cumulative evidence shows that eicosanoids such as prostaglandins, leukotrienes, thromboxanes and hydroxy eicosatetraenoic acids play an important role in associating inflammation with human colorectal cancer (CRC). In this study an ultra‐pressure liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the targeted profiling of eight relevant eicosanoids and the major metabolic precursor, arachidonic acid (AA), in human colon. Multiple reaction monitoring (MRM) experiments were performed in negative electrospray ionization mode. The metabolites were separated using a C₁₈ column consisting of 1.7 µm ethylene‐bridged hybrid particles (100 × 2.1 mm i.d.) and gradient elution (50 to 95% of solvent B) with a mobile phase comprising water (0.1% formic acid) [solvent A] and acetonitrile (0.1% formic acid) [solvent B] at a flow rate of 0.4 mL/min. The analysis time for each sample was 5.5 min. Our UPLC/MS/MS method demonstrated satisfactory validation results in terms of selectivity, sensitivity, matrix effect, linearity, extraction efficiency, intra‐ and inter‐day precision, accuracy and autosampler stability. The method was applied for the clinical profiling of matched pairs of cancerous and normal colon mucosae obtained from eight colorectal cancer patients. Endogenous levels of AA and selected eicosanoids such as prostaglandin E₂ (PGE₂), prostacyclin (PGI₂) [assayed as its stable hydrolytic product 6‐keto‐prostaglandin_1α (6‐k PGF_1α)] and 12‐hydroxy‐5Z,8Z,10E,14Z‐eicosatetraenoic acid (12‐HETE) were found to be significantly different (p <0.05; paired t‐test) between cancerous and normal mucosae. Copyright © 2011 John Wiley & Sons, Ltd.     

Metabolite analysis in Curcuma domestica using various GC‐MS and LC‐MS separation and detection techniques

The metabolic profile of polar (methanol) and non‐polar (hexane) extracts of Curcuma domestica, a widely used medicinal plant, was established using various different analytical techniques, including GC‐FID, GC‐MS, HR‐GC‐MS and analytical HPLC‐ESI‐MS/MS by means of LTQ‐Orbitrap technology. The major non‐volatile curcuminoids curcumin, demethoxycurcumin and bisdemethoxycurcumin were identified when their chromatographic and precursor ion masses were compared with those of authentic standard compounds. In this paper we describe for the first time a GC/MS‐based method for metabolic profiling of the hydrophilic extract. We also identified 61 polar metabolites as TMS derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

Joint MS‐based platforms for comprehensive comparison of rat plasma and serum metabolic profiling

Metabolomics is a rapidly growing field in the comprehensive understanding of cellular and organism‐specific responses associated with perturbations induced by medicines, chemicals and environment. Blood matrices are frequently used in clinical and biological studies. In this study, we compared metabolic profiling between rat plasma and serum using complementary platforms of gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography–quadruple time‐of‐flight–mass spectrometry (LC‐QTOF‐MS). The sample types that were tested included plasma prepared with K₂EDTA and serum collected using venous blood collection protocols. The results of peak area variation for each detected metabolite/feature in the quality control samples showed a good reproducibility in LC‐QTOF‐MS and better reproducibility in GC‐MS. In GC‐MS analysis: (a) 25.8% of the defined metabolites differed serum from plasma profiling (t‐test, p < 0.05); and (b) serum possessed higher sensitivity than plasma for its generally higher peak intensity in the metabolic profiling. In LC‐QTOF‐MS analysis, 13 (in positive ion mode) and seven (in negative ion mode) important metabolites were identified as mainly contributing to the separation between serum and plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of different mass spectrometry techniques in the measurement of L‐[ring‐13C6]phenylalanine incorporation into mixed muscle proteins

Precise measurement of low enrichment of stable isotope labeled amino‐acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L‐[ring‐¹³C₆]phenylalanine and a bolus dose of L‐[ring‐¹³C₆]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC‐combustion‐isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring‐¹³C₆]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R² = 0.9962 and R² = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra‐assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter‐assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L‐[ring‐¹³C₆]phenylalanine tracer enrichment in low abundance and in small quantity samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabotyping of human colorectal cancer using two-dimensional gas chromatography mass spectrometry

Colorectal cancer (CRC) is the fourth most common cause of death from cancer in the world. The limitations of the currently available methods and biomarkers for CRC management highlight the necessity of finding novel markers. Metabonomics can be used to search for potential markers that can provide molecular insight into human CRC. The emergence of two-dimensional gas chromatography time of flight mass spectrometry (GC?×?GC/TOFMS) has comprehensively enhanced the metabolic space coverage of conventional GC/MS. In this study, a GC?×?GC/TOFMS was developed for the tissue-based global metabonomic profiling of CRC. A Pegasus GC?×?GC/TOFMS (Leco Corp., St. Joseph, MI, USA) system comprising an Agilent 7890 GC and Pegasus IV TOFMS was used for this purpose. An Agilent DB-1 (30 m?×?250 μm?×?0.25 μm) fused silica capillary column and a Restek Rxi®-17 (1 m?×?100 μm?×?0.10 μm) fused silica capillary column were used as the primary and secondary columns, respectively. The method was applied for global metabonomic profiling of matched CRC and normal tissues (n?=?63) obtained from 31 CRC patients during surgery. An attempt was also made to compare GC?×?GC/TOFMS with GC/MS and NMR in similar application. The results showed that the metabotype associated with CRC is distinct from that of normal tissue and led to the identification of chemically diverse marker metabolites. Metabolic pathway mapping suggested deregulation of various biochemical processes such as glycolysis, Krebs cycle, osmoregulation, steroid biosynthesis, eicosanoid biosynthesis, bile acid biosynthesis, lipid, amino acid and nucleotide metabolism.

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Fig Workflow of GCGC/TOFMS metabonomic profiling of human colorectal cancer

     

Determination of human muscle protein fractional synthesis rate: an evaluation of different mass spectrometry techniques and considerations for tracer choice

In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring‐¹³C₆]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically labeled amino acid will be incorporated within the few hours of a typical laboratory experiment. GC combustion isotope ratio MS (GC‐C‐IRMS) has thus far been considered the ‘gold’ standard for the precise measurements of these low enrichment levels. However, advances in liquid chromatography‐tandem MS (LC‐MS/MS) and GC‐tandem MS (GC‐MS/MS) have made these techniques an option for human muscle FSR measurements. Human muscle biopsies were freeze dried, cleaned, and hydrolyzed, and the amino acids derivatized using either N‐acetyl‐n‐propyl, phenylisothiocyanate, or N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide (MTBSTFA) for GC‐C‐IRMS, LC‐MS/MS, and GC‐MS/MS analysis, respectively. A second derivative, heptafluorobutyric acid (HFBA), was also used for GC‐MS/MS analysis as an alternative for MTBSTFA. The machine reproducibility or the coefficients of variation for delta tracer‐tracee‐ratio measurements (delta tracer‐tracee‐ratio values around 0.0002) were 2.6%, 4.1%, and 10.9% for GC‐C‐IRMS, LC‐MS/MS, and GC‐MS/MS (MTBSTFA), respectively. FSR determined with LC‐MS/MS compared well with GC‐C‐IRMS and so did the GC‐MS/MS when using the HFBA derivative (linear fit Y = 1.08 ± 0.10, X + 0.0049 ± 0.0061, r = 0.89 ± 0.01, P < 0.0001). In conclusion, (1) IRMS still offers the most precise measurement of human muscle FSR, (2) LC‐MS/MS comes quite close and is a good alternative when tissue quantities are too small for GC‐C‐IRMS, and (3) If GC‐MS/MS is to be used, then the HFBA derivative should be used instead of MTBSTFA, which gave unacceptably high variability. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic Profiling of Human Colorectal Cancer Using High Resolution 1H Nuclear Magnetic Resonance Spectroscopy

Colorectal cancer (CRC) is the third commonest malignancy cancer worldwide. Clear understandings of global metabolic profiling of the normal mucosa and cancer tissues are vitally important to aid optimizing the clinical management strategy and understanding CRC biology. We studied metabolic characteristics of 20 CRC and 20 distant normal mucosa tissues extracts from 20 patients using high resolution ¹H NMR spectroscopy in conjunction with multivariate analyses, such as principal component analysis (PCA). Compared with distant normal mucosa tissues, lactate, taurine, ornithine and polyamine were present at significantly higher levels in CRC tissue extracts whereas myo‐inositol was present at significantly lower level. Two metabolites ratios such as myo‐inositol/taurine and myo‐inositol/(ornithine+polyamine) appear to be the most valuable biomarkers for the differentiation CRC from normal mucosa tissues. Our data suggested that HR ¹H NMR spectroscopy combined with multivariate analyses is a potentially useful technology for detecting malignant changes in the normal mucosa tissues, the technique may be further exploited for future CRC biomarker research or identification of targets for therapeutic manipulations.     

Single-step transesterification with simultaneous concentration and stable isotope analysis of fatty acid methyl esters by gas chromatography-combustion-isotope ratio mass spectrometry

Gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) is increasingly applied to food and metabolic studies for stable isotope analysis (δ¹³C), with the quantification of analyte concentration often obtained via a second alternative method. We describe a rapid direct transesterification of triacylglycerides (TAGs) for fatty acid methyl ester (FAME) analysis by GC‐C‐IRMS demonstrating robust simultaneous quantification of amount of analyte (mean r² = 0.99, accuracy ±2% for 37 FAMEs) and δ¹³C (±0.13‰) in a single analytical run. The maximum FAME yield and optimal δ¹³C values are obtained by derivatizing with 10% (v/v) acetyl chloride in methanol for 1 h, while lower levels of acetyl chloride and shorter reaction times skewed the δ¹³C values by as much as 0.80‰. A Bland‐Altman evaluation of the GC‐C‐IRMS measurements resulted in excellent agreement for pure oils (±0.08‰) and oils extracted from French fries (±0.49‰), demonstrating reliable simultaneous quantification of FAME concentration and δ¹³C values. Thus, we conclude that for studies requiring both the quantification of analyte and δ¹³C data, such as authentication or metabolic flux studies, GC‐C‐IRMS can be used as the sole analytical method. Copyright © 2011 John Wiley & Sons, Ltd.     

Semi‐quantitative and structural metabolic phenotyping by direct infusion ion trap mass spectrometry and its application in genetical metabolomics

The identification of quantitative trait loci (QTL) for plant metabolites requires the quantitation of these metabolites across a large range of progeny. We developed a rapid metabolic profiling method using both untargeted and targeted direct infusion tandem mass spectrometry (DIMSMS) with a linear ion trap mass spectrometer yielding sufficient precision and accuracy for the quantification of a large number of metabolites in a high‐throughput environment. The untargeted DIMSMS method uses top‐down data‐dependent fragmentation yielding MS² and MS³ spectra. We have developed software tools to assess the structural homogeneity of the MS² and MS³ spectra hence their utility for phenotyping and genetical metabolomics. In addition we used a targeted DIMS(MS) method for rapid quantitation of specific compounds. This method was compared with targeted LC/MS/MS methods for these compounds. The DIMSMS methods showed sufficient precision and accuracy for QTL discovery. We phenotyped 200 individual Lolium perenne genotypes from a mapping population harvested in two consecutive years. Computational and statistical analyses identified 246 nominal m/z bins with sufficient precision and homogeneity for QTL discovery. Comparison of the data for specific metabolites obtained by DIMSMS with the results from targeted LC/MS/MS analysis showed that quantitation by this metabolic profiling method is reasonably accurate. Of the top 100 MS¹ bins, 22 ions gave one or more reproducible QTL across the 2 years. Copyright © 2009 John Wiley & Sons, Ltd.     

Low resolution and high resolution MS for studies on the metabolism and toxicological detection of the new psychoactive substance methoxypiperamide (MeOP)

In 2013, the new psychoactive substance methoxypiperamide (MeOP) was first reported to the European Monitoring Centre for Drug and Drug Addiction. Its structural similarity to already controlled piperazine designer drugs might have contributed to the decision to offer MeOP for online purchase. The aims of this work were to identify the phase I/II metabolites of MeOP in rat urine and the human cytochrome P450 (CYP) isoenzymes responsible for the initial metabolic steps. Finally, the detectability of MeOP in rat urine by gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography coupled with multistage mass spectrometry (LC‐MSⁿ) standard urine screening approaches (SUSAs) was evaluated. After sample preparation by cleavage of conjugates followed by extraction for elucidating phase I metabolites, the analytes were separated and identified by GC‐MS as well as liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). For detection of phase II metabolites, the analytes were separated and identified after urine precipitation followed by LC‐HR‐MS/MS. The following metabolic steps could be postulated: hydrolysis of the amide, N‐oxide formation, N‐ and/or O‐demethylation, oxidation of the piperazine ring to the corresponding keto‐piperazine, piperazine ring opening followed by oxidation of a methylene group to the corresponding imide, and hydroxylation of the phenyl group. Furthermore, N‐acetylation, glucuronidation and sulfation were observed. Using human CYPs, CYP1A2, CYP2C19, CYP2D6, and/or CYP3A4 were found to catalyze N‐oxide formation and N‐, O‐demethylation and/or oxidation. Mostly MeOP and N‐oxide‐MeOP but to a minor degree also other metabolites could be detected in the GC‐MS and LC‐MSⁿ SUSAs. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of metabolites in rat plasma after intravenous administration of salvianolic acid A by liquid chromatography/time‐of‐flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Salvianolic acid A (SalA) is one of the main active constituents in Salvia miltiorrhiza (Danshen). Although the pharmacokinetics of SalA in rats after intravenous (i.v.) administration of Danshen injection has been reported, the information relevant to the metabolites of SalA in vivo is absent so far. In this study, by means of liquid chromatography with time‐of‐flight mass spectrometry (LC/TOFMS) and liquid chromatography with ion trap mass spectrometry (LC/MSⁿ) techniques, the unknown metabolites of SalA in rat plasma after i.v. administration of the purified SalA at the dose of 20 mg/kg body weight were identified. A liquid‐liquid extraction method was established to separate the metabolites from the plasma and the chromatographic separations were performed on a Xterra MS C₁₈ column (100 mm × 4.6 mm i.d., 3.5 µm) with acetonitrile/methanol/water/formic acid (20.5:19.5:64: 0.05, v/v/v/v) as the mobile phase at a constant flow rate of 0.2 mL/min. Based on the data obtained from the LC/TOFMS determination (the total ion chromatograms, MS spectra and extracted ion chromatograms), in combination with the characteristic fragment ions acquired from the LC/MSⁿ determination, five metabolites were identified as SalA‐monoglucuronide, monomethyl‐SalA‐monoglucuronide, mono‐methyl‐SalA, dimethyl‐SalA and dimethyl‐SalA‐monoglucuronide, and the possible chemical structures were deduced. The results indicated that SalA might mainly undergo two metabolic pathways in vivo in rats, which were methylation and glucuronidation. The present studies have laid a solid foundation for the metabolic mechanism of SalA in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of terbinafine in human plasma using UPLC–MS/MS: Application to a bioequivalence study in healthy subjects

A high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the determination of terbinafine in human plasma. The method employed liquid–liquid extraction of terbinafine and terbinafine‐d7 (used as internal standard) from 100 μL human plasma with ethyl acetate–n‐hexane (80:20, v/v) solvent mixture. Chromatography was performed on a BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using acetonitrile–8.0 mm ammonium formate, pH 3.5 (85:15, v/v) under isocratic elution. For quantitative analysis, MS/MS ion transitions were monitored at m/z 292.2/141.1 and m/z 299.1/148.2 for terbinafine and terbinafine‐d7, respectively, using electrospray ionization in the positive mode. The method was validated according to regulatory guidance for selectivity, sensitivity, linearity, recovery, matrix effect, stability, dilution reliability and ruggedness with acceptable accuracy and precision. The method shows good linearity over the tested concentration range from 1.00 to 2000 ng/mL (r² ≥ 0.9984). The intra‐batch and inter‐batch precision (CV) was 1.8–3.2 and 2.1–4.5%, respectively. The method was successfully applied to a bioequivalence study with 250 mg terbinafine in 32 healthy subjects. The major advantage of this method includes higher sensitivity, small plasma volume for processing and a short analysis time.     

Monitoring of Polychlorinated Biphenyls in Surface Water Using Liquid Extraction,GC/MS,and GC/ECD

Abstract

A rapid and reliable analytical method, at trace level concentration was developed and validated for monitoring polychlorinated biphenyls (PCBs) in Jordanian surface water. The method combines the advantage of liquid extraction together with gas chromatography‐mass spectrometry (GC/MS) and gas chromatography‐electron capture detector (GC/ECD). The performance of the method was evaluated by analyzing certified reference material (CRM) of the analytes and applied on real water samples collected from different sites in Jordan. A mixture of 60∶40 dichloromethan‐petroleum ether was chosen as a convenient binary solvent for liquid–liquid extraction. The GC conditions for GC/MS were optimized using He as a carrier gas, temperature programming, and chlorpropham as an internal standard (IS).

The conditions for GC/ECD were performed using N₂ gas and a temperature program from 160 to 280°C with different increasing rates. The method of GC/MS in the selective ion mode (SIM) gave linear relationships for all PCBs tested between 0.60–6.0 µg/l with R ²=0.9934 (n=7×18). Recoveries from spiked water samples ranged between 87.6 and 91.4%. The mean accuracy and precision obtained were 4.9% and 2.16%, respectively. The mean of detection limit was 0.14±0.04 µg/l. In GC/ECD, linear relationships for all PCBs examined over the range of 0.3–2.4 µg/l was verified as characterized by a linear regression equation and correlation coefficient, R ²=0.9915 (n=12). The average precision and accuracy were 4.86% and 5.21%, respectively. Analyses results clarified that none of the examined Jordanian water samples contained any of the searched for PCBs within the detection limit achieved.     

Bioanalytical method development and validation of moxidectin in plasma by LC–MS/MS: Application to in vitro metabolism

Moxidectin (MOX) has recently been approved by the US Food and Drug Administration for the treatment of river blindness in select populations. It is also being evaluated as an alternative for the use of ivermectin, widespread resistance to which is becoming a global health issue. Moreover, MOX is becoming increasingly used as a prophylactic antiparasitic in the cattle industry. In this study, we developed and validated an LC–MS/MS method of MOX in human, monkey and mouse plasma. The separation was achieved on an ACE C₁₈ (50 × 3.0 mm, 3 μm) column with isocratic elution using 0.1% acetic acid and methanol–acetonitrile (1:1, v/v) as mobile phase. MOX was quantitated using MS/MS with an electrospray ionization source operating in negative multiple reaction monitoring mode. The multiple reaction monitoring precursor ion → product ion transitions for MOX and abamectin (IS) were m/z 638.40 → 236.30 and m/z 871.50 → 565.35 respectively. The MS/MS response was linear over the concentration range 0.1–1000 ng/mL in plasma with a correlation coefficient (r²) of 0.997 or better. The within‐ and between‐day precision (relative standard deviation, RSD) and accuracy were within the acceptable limits per US Food and Drug Administration guidelines. The method was successfully applied to an in vitro metabolic stability study of MOX.     

Determination of d‐limonene in mice plasma and tissues by a new GC–MS/MS method: Comparison of the pharmacokinetics and tissue distribution by oral and inhalation administration in mice

The aim of the present study was to develop a method based on gas chromatography–tandem mass spectrometry (GC–MS/MS) to determine and quantify the d ‐limonene in mouse plasma and tissue samples. This new method was validated for the quantification of d ‐limonene with the linearity ranges 1.0–1000.0 ng/mL (r² > 0.9952) for plasma samples and 5.0–5000.0 ng/g (r² > 0.9940) for tissue samples. The intra‐ and inter‐day assay of precisions in plasma and tissues were <13.4% and the accuracies were within 91.1–105.8%. In the oral/inhalation administration pharmacokinetics and tissue distribution studies, the main pharmacokinetic parameters were the peak concentration = (97.150 ± 34.450)/(4336.415 ± 1142.418) ng/mL, the area under the curve = (162.828± 27.447)/(2085.721 ± 547.787) h ng/mL and the half‐life = (3.196 ± 0.825)/(0.989 ± 0.095) h. The tissue distribution of d ‐limonene in mice after oral/inhalation administration demonstrated a decreasing tendency in different tissues (liver > kidney > heart > lung > spleen).     

Acidic metabolite profiling analysis of catecholamine and serotonin as O‐ethoxycarbonyl/tert‐butyldimethylsilyl derivatives by gas chromatography–mass spectrometry

A novel derivatization method was developed for the simultaneous determination of six acidic metabolites of catecholamine and serotonin by gas chromatography–mass spectrometry (GC‐MS). The metabolites were converted to O‐ethoxycarbonyl/tert‐butyldimethylsilyl (EOC/TBDMS) derivatives for the direct GC‐MS analysis in selected ion monitoring mode. Their mass spectral pattern as EOC/TBDMS derivatives showed characteristic fragment ions of [M – 15]⁺ and [M – 57]⁺, which permitted rapid and accurate structural confirmation of acidic metabolites. The present method was linear (r ≥ 0.998), reproducible (percentage relative standard deviation = 1.0–10.0) and accurate (% relative error = ?9.7–9.8) with detection limits of 0.001–4.7 ng/mL. When applied to human urine samples, the method allowed simultaneous determination of six acidic metabolites of catecholamine and serotonin. Copyright © 2012 John Wiley & Sons, Ltd.     

New cathinone‐derived designer drugs 3‐bromomethcathinone and 3‐fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS and their detectability in urine

3‐Bromomethcathinone (3‐BMC) and 3‐Fluoromethcathinone (3‐FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so‐called “bath salts” or “plant feeders.” The aim of the present study was to identify for the first time the 3‐BMC and 3‐FMC Phase I and II metabolites in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS (HR‐MS) and to test for their detectability by established urine screening approaches using GC–MS or LC–MS. Furthermore, the human cytochrome‐P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drug–drug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GC–MS and by LC–HR‐MS. The main metabolic steps were N‐demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GC–MS could be confirmed by LC–HR‐MS. Furthermore, corresponding Phase II metabolites were identified using the LC–HR‐MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GC–MS and LC–MSⁿ after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N‐demethylation of 3‐BMC and 3‐FMC after in vitro–in vivo extrapolation. Copyright © 2012 John Wiley & Sons, Ltd.     

GC‐MS of amaryllidaceous galanthamine‐type alkaloids

Galanthamine‐type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC‐EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high‐resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC‐MS/MS) and accurate mass measurements (GC‐HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC‐MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine‐N‐oxide and N‐formylnorgalanthamine, the galanthamine‐type compounds showed abundant [M]^+. and [M‐H]⁺ ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine‐type alkaloids, including 3‐O‐(2′‐butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC‐MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel liquid–liquid extraction and stable isotope dilution NCI‐GC‐MS method for quantitation of agmatine in postmortem brain cortex

The group of biologically important amines includes putrescine, spermidine and spermine, as well as agmatine, which is a guanidino‐amine. There is considerable evidence supporting a role of these amines in the etiology and pathology of mental disorders. We have previously developed a quantitative GC‐MS method for simultaneous measurement of three major polyamines to support our studies linking polyamines to mental disorders. However, a unique GC‐MS method is required for agmatine. To efficiently extract agmatine from postmortem brain tissues, we developed an isopropanol based liquid–liquid extraction protocol using potassium carbonate as a salting‐out agent which showed a much greater recovery than n‐butanol used in earlier methods. The GC‐MS analysis employed hexafluoroacetylacetone as derivatization reagent and was carried out using negative chemical ionization with total ion and selected ion monitoring. ¹⁵N₄‐Agmatine was synthesized from ¹⁵N₄‐L ‐arginine and used as internal standard in a conventional stable isotope dilution assay. This method accurately measures the level of agmatine from very small quantities (10–20 mg) of postmortem brain tissue, with a quantitation limit down to 1 ng/g of wet tissue. The limit of detection is 0.01 ng/g of wet tissue. Copyright © 2010 John Wiley & Sons, Ltd.     

The impact of blood on liver metabolite profiling – a combined metabolomic and proteomic approach

Metabolomics has entered the well‐established omic sciences as it is an indispensable information resource to achieve a global picture of biological systems. The aim of the present study was to estimate the influence of blood removal from mice liver as part of sample preparation for metabolomic and proteomic studies. For this purpose, perfused mice liver tissue (i.e. with blood removed) and unperfused mice liver tissue (i.e. containing blood) were compared by two‐dimensional gas chromatography time of flight mass spectrometry (GC × GC‐TOFMS) for the metabolomic part, and by liquid chromatography tandem mass spectrometry (LC‐MS/MS) for the proteomic part. Our data showed significant differences between the unperfused and perfused liver tissue samples. Furthermore, we also observed an overlap of blood and tissue metabolite profiles in our data, suggesting that the perfusion of liver tissue prior to analysis is beneficial for an accurate metabolic profile of this organ. Copyright © 2013 John Wiley & Sons, Ltd.