Metabolites In Human Samples Research Articles

Novel LC-MS/MS Method for Simultaneous Determination of Monoamine Neurotransmitters and Metabolites in Human Samples.

For the simultaneous determination of monoamine neurotransmitters (NTs) like dopamine, serotonin, noradrenaline, and epinephrine, and their metabolites (metanephrine, normetanephrine, 3-methoxytyramine, vanillylmandelic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid), a robust liquid chromatography method coupled with tandem mass spectrometry (LC-MS/MS) was introduced as the analytical method. This analytical method proved to be accurate for the simultaneous measurement of the amounts of 11 NTs and their metabolites in biological samples. The method proved to be more efficient and better than the previously reported method in terms of precision, recovery, sample requirement, and extraction procedure. The reported method requires only 100 μL of blood and 200 μL of urine, and the extraction procedure requires acetonitrile precipitation, filtration, drying, and reconstitution in water. The separation of all analytes was performed on an C18 column (4.6 mm × 150 mm and 1.8 μm). A 10 min gradient elution program with a mobile phase consisting of phase A (0.2% formic acid in water) and phase B (methanol) was used. The positive ionization mode was used for the detection of all analytes in multiple reaction monitoring (MRM). The proposed method was validated with an internal standard and yielded lower limits of detection and quantification ranges of 0.0182-0.0797 ng/mL and 0.0553-0.2415 ng/mL, respectively, with a good linearity (R2) between 0.9959 and 0.9994. The recoveries ranged from 73.37% to 116.63% in blood and from 80.9% to 115.33% in urine. For the NTs and metabolites, the intra- and interday % CV were 0.24-9.36 and 0.85-9.67, respectively. The developed LC-MS/MS method was successfully used for the determination of trace amounts of endogenous compounds in human blood and urine samples.

The first LC-MS/MS stereoselective bioanalytical methods to quantitatevely detect 9R- and 9S-hexahydrocannabinols and their metabolites in human blood, oral fluid and urine

A sensitive LC-MS/MS method for the simultaneous quantification of the (9R)- and (9S)- hexahydrocannabinols (HHCs), and their metabolites, in human urine, oral fluid (OF) and blood samples were developed, validated and used to the biological samples of volunteers. The analytes were extracted from 100µL human samples. An isocratic elution mode with methanol was used for chromatographic separation of (9R)- and (9S)-HHC on an immobilized amylose tris(3-chloro-5-methylphenylcarbamate)-based chiral column Lux i-Amylose-3. The flow-rate of the mobile phase was 0.5mL/min. An isocratic elution mode of methanol and water (80/20, v/v) was used for chromatographic separation of metabolites of (9R)- and (9S)-HHC on a Lux AMP chiral column (with a proprietary chiral selector) at a flow rate of 0.5mL/min. MS/MS analysis was performed in positive ionization mode for HHC epimers, while in negative ionization mode was used for metabolites of HHCs. The calibration curves for HHCs and their metabolites in human samples ranged from 0.25- 240ngmL-1 and 1 - 100ngmL-1, respectively, with determination coefficients (r2) of ≥ 0.99. All analytes were stable at room temperature, 4 °C, in the autosampler (+10°C) and −20 °C for 24h, after three freeze/thaw cycles, and when stored at −20 °C up to one week after quality control (QC) sample preparation (concentration differences less than 20% with respect to time zero response), in blood, urine and OF.

Contribution of Mass Spectrometry to the Advances in Risk Characterization of Marine Biotoxins: Towards the Characterization of Metabolites Implied in Human Intoxications.

A significant spread and prevalence of algal toxins and, in particular, marine biotoxins have been observed worldwide over the last decades. Marine biotoxins are natural contaminants produced during harmful algal blooms being accumulated in seafood, thus representing a threat to human health. Significant progress has been made in the last few years in the development of analytical methods able to evaluate and characterize the different toxic analogs involved in the contamination, Liquid Chromatography coupled to different detection modes, including Mass Spectrometry, the method of choice due to its potential for separation, identification, quantitation and even confirmation of the different above-mentioned analogs. Despite this, the risk characterization in humans is still limited, due to several reasons, including the lack of reference materials or even the limited access to biological samples from humans intoxicated during these toxic events and episodes, which hampered the advances in the evaluation of the metabolites responsible for the toxicity in humans. Mass Spectrometry has been proven to be a very powerful tool for confirmation, and in fact, it is playing an important role in the characterization of the new biotoxins analogs. The toxin metabolization in humans is still uncertain in most cases and needs further research in which the implementation of Mass Spectrometric methods is critical. This review is focused on compiling the most relevant information available regarding the metabolization of several marine biotoxins groups, which were identified using Mass Spectrometry after the in vitro exposition of these toxins to liver microsomes and hepatocytes. Information about the presence of metabolites in human samples, such as human urine after intoxication, which could also be used as potential biomarkers for diagnostic purposes, is also presented.

An ultrasensitive UHPLC-ESI-MS/MS method augmented with a controlled microwave derivatization reaction for quantitation of vitamin D3 and its major metabolites in COVID-19 patients

It is established that vitamin D deficiency is correlated with the disease severity in COVID-19 patients. However, the reliable and sensitive quantitation of vitamin D3 (D3) and its metabolites remains a difficult challenge. Herein, a novel ultrasensitive and reliable UHPLC-ESI-MS/MS method was developed and validated for the quantitation of D3 and its major metabolites in COVID-19 patients. The mass spectral sensitivity was augmented via controlled microwave-assisted derivatization reaction (CMDR) with 2-nitrosopyridine (Pyr-NO) at 65 °C for 2 min. CMDR hyphenation with UHPLC-MS/MS improves detection sensitivity while shortening separation and derivatization reaction times. The precursor to product ion transitions for D3, 25-hydroxy D3 (25(OH)D3), 1,25-dihydroxy D3 (1,25-(OH)2D3) and calcipotriol (CPT) as an internal standard were m/z 493.4 → 231.3, m/z 509.4 → 231.3, m/z 525.4 → 247.3, and m/z 521.4 → 247.3; respectively. The separation of the formed derivatives was conducted using a gradient elution mode with mobile phase A: formic acid (0.1%) in water and mobile phase B: formic acid (0.1%) in acetonitrile. The elution started with 40% (v/v) of B for 0.3 min then increased linearly to 90% (v/v) at 2 min on an Agilent EclipsePlus C18 (50 × 2.1 mm, 1.8 μm) column at a flow rate of 0.3 mL min−1. The method was validated using FDA standards for bioanalytical method validation over a concentration range of 0.02–50 ng mL−1 with correlation coefficient ≥0.9987 and the lower limit of quantitation (LLOQ) were 0.02–0.05 ng mL−1 in human plasma. The developed method has demonstrated excellent comparability to a well-established chemiluminescent immunoassay (CLIA) method for the analysis of D3 metabolites in human samples. The developed UHPLC-ESI-MS/MS method was implemented for routine and reliable quantitation of D3 and its major metabolites in COVID-19 patients.

Enzyme‐Based Chemical Identification for Metabolomics

Background The diversity of chemicals detectable in human samples by high-resolution mass spectrometry (HRMS) exceeds the number of readily available chemical standards, limiting our ability to identify minor metabolites of drugs and other xenobiotics in vivo. Standard workflows for identification of unknown metabolites typically begin by interpretation of ion dissociation spectra (MS/MS) and comparison with library spectra and/or authentic standards – many of which are not available and do not incorporate enzymatic precursor-product relationships as identification criteria. Objective To develop a high-throughput strategy to generate human biotransformation products from diverse xenobiotic and drugs for analysis with LC-HRMS. Hypothesis We hypothesize that unidentified metabolites are generated from enzymatic reactions of known metabolites. Therefore, strategies to generate and characterize metabolic products for diverse arrays of xenobiotics will facilitate identification of metabolites in vivo. Methods Here, we adopt a strategy to generate human biotransformation products of diverse xenobiotics in a high-throughput 96-well plate format using incubations with human liver S9 fractions. Extracts from these reactions were collected at 0 and 24 hour time points and analyzed using LC-HRMS (Thermo Scientific Fusion/High-Field Q-Exactive) to characterize metabolic products generated in a time-dependent manner. Data-dependent MS/MS was performed to collect MS/MS spectra. Expected biotransformation products were characterized by retention time, accurate mass m/z (MS1), MS/MS, and an increase in signal intensity for predicted or previously unreported biotransformation products. Incubations with stable-isotope precursors aided the identification of previously unreported biotransformation products. Results Our data show that known and previously unidentified Phase 1 and Phase 2 metabolites were produced from a range of xenobiotics in a 96-well format using human liver S9 fractions. S9 reaction extracts of 138 drug or xenobiotic precursors were analyzed generating a total of 502 validated metabolites. Selected metabolites were used to support their identification in mouse and human samples based on matched accurate mass MS1, retention time, and co-occurrence with the parent xenobiotic and/or related metabolites in the samples. Conclusion We have developed a scalable tool to generate metabolites from thousands of xenobiotics to facilitate identification of drug and other xenobiotic metabolites in human samples.

Comparison of two arylsulfatases for targeted mass spectrometric analysis of microbiota-derived metabolites

Sulfation of metabolites is the second highest phase II modification in humans, which plays a critical role in the xenobiotics clearance process and gut microbiota-host co-metabolism. Besides the main function to remove xenobiotics from the body, sulfated metabolites have also been linked to inflammation, bacterial pathogenesis and metabolic disorders. A better understanding of how these metabolites impact the human body has turned into an important research area. Analytical methods for selective identification of this metabolite class are scarce. We have recently developed an assay utilizing the arylsulfatase from Helix pomatia due to a high substrate promiscuity combined with state-of-the-art metabolomics bioinformatic analysis for the selective identification of O-sulfated metabolites in human samples. This enzyme requires a multistep purification process as highest purity is needed for the developed mass spectrometric assay. In this study, we have utilized a new and recombinant overexpressed arylsulfatase (ASPC) for the selective identification of organic sulfate esters in human urine samples. We have compared the substrate conversion in urine samples and substrate specificity of this enzyme with purified arylsulfatase from Helix pomatia. Our analysis of urine samples revealed that both enzymes can be utilized for the selective analysis and discovery of sulfated metabolites with high promiscuity as demonstrated by equal hydrolysis of 108 substrates including sulfated conjugates of 27 metabolites of microbial origin. Importantly, we also identified 21 substrates in human urine samples that are exclusively hydrolyzed by ASPC and application of this enzyme increases the discovery of unknown sulfated metabolites with a higher scaffold diversity.

Comparative dietary sulfated metabolome analysis reveals unknown metabolic interactions of the gut microbiome and the human host

The gut microbiome converts dietary compounds that are absorbed in the gastrointestinal tract and further metabolized by the human host. Sulfated metabolites are a major compound class derived from this co-metabolism and have been linked to disease development. In the present multidisciplinary study, we have investigated human urine samples from a dietary intervention study with 22 individuals collected before and after consumption of a polyphenol rich breakfast. These samples were analyzed utilizing our method combining enzymatic metabolite hydrolysis using an arylsulfatase and mass spectrometric metabolomics. Key to this study is the validation of 235 structurally diverse sulfated metabolites. We have identified 48 significantly upregulated metabolites upon dietary intervention including 11 previously unknown sulfated metabolites for this diet. We observed a large variation in subjects based on their potential to sulfate metabolites, which may be the foundation for classification of subjects as high and low sulfate metabolizers in future large cohort studies. The reported sulfatase-based method is a robust tool for the discovery of unknown microbiota-derived metabolites in human samples.

The key points in the pre-analytical procedures of blood and urine samples in metabolomics studies.

Metabolomics provides measurement of numerous metabolites in human samples, which can be a useful tool in clinical research. Blood and urine are regarded as preferred subjects of study because of their minimally invasive collection and simple preprocessing methods. Adhering to standard operating procedures is an essential factor in ensuring excellent sample quality and reliable results. In this review, we summarize the studies about the impacts of various preprocessing factors on metabolomics studies involving clinical blood and urine samples in order to provide guidance for sample collection and preprocessing. Clinical information is important for sample grouping and data analysis which deserves attention before sample collection. Plasma and serum as well as urine samples are appropriate for metabolomics analysis. Collection tubes, hemolysis, delay at room temperature, and freeze-thaw cycles may affect metabolic profiles of blood samples. Collection time, time between sampling and examination, contamination, normalization strategies, and storage conditions may alter analysis results of urine samples. Taking these collection and preprocessing factors into account, this review provides suggestions of standard sample preprocessing.

A new strategy for non-targeted screening of phthalate metabolites using liquid chromatography-high resolution mass spectrometry

The ubiquity and potential toxicity of phthalates make their exposure on humans a matter of great concern. Measuring the targeted phthalate metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a commonly used approach for human biomonitoring of phthalates. However, the targeted LC-MS/MS methods have been unable to meet the desire to comprehensively and rapidly screen and identify for as many known and potentially unknown phthalate metabolites in human samples. Instead, non-targeted screening methods, allowing the rapid detection and identification of unknown compounds in a wide variety of environmental and human samples are complementary to the targeted methods. In this study, 23 phthalate metabolites were analyzed using liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) for development of a non-targeted screening method. The fragmentation patterns of the 23 phthalate metabolites were investigated using collision-induced dissociation experiments at 10, 20, and 40 eV. And three common specific product ions at m/z 121.0297, m/z 147.0088, and m/z 165.0193 corresponding to C7H5O2-, C8H3O3-, and C8H5O4-, respectively were observed in their mass spectra. Based on the three common product ions, a new strategy was proposed for non-targeted screening of the new phthalate metabolites as exposure biomarkers for human biomonitoring. It can greatly reduce the time spent on data processing for potential precursor ions. Additionally, the correlation of the retention time and logKow of the phthalate metabolites was explored, which provides a useful tool to reduce the number of identified candidates in the non-targeted screening.

Coupled Enzymatic Treatment and Mass Spectrometric Analysis for Identification of Glucuronidated Metabolites in Human Samples.

Glucuronidation is the most common phase II modification and plays an important role in human clearance metabolism. Glucuronidated metabolites have also been linked to disease development and microbiota-host co-metabolism. Although many of these compounds have been identified, the total number of unknown glucuronides and their impact on the human host's physiology can only be estimated. Herein, we describe the combination of an untargeted metabolomics analysis and enzymatic metabolic conversion for the selective detection of glucuronide conjugates by using UPLC-MS/MS in human urine samples. Our study demonstrates that this powerful strategy can be used for the selective identification of glucuronidated molecules and to discover unknown natural metabolites. In total, we identified 191 metabolites in a single sample including microbiota-derived compounds as well as previously unidentified molecules.

New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites.

Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.

Quantitative profiling of bile acids in blood, adipose tissue, intestine, and gall bladder samples using ultra high performance liquid chromatography-tandem mass spectrometry.

An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed for the determination of 33 target and 28 unknown bile acids (BAs) in biological samples. Sixty-one BAs could be measured in 20min using only a small amount of sample and with a simple sample preparation. The method proved to be very sensitive (limit of detection 5-350pg/mL, lower limit of quantitation 0.1-2.6ng/mL), linear (R(2) > 0.99) and reproducible (typically CV <15% in biological matrixes). The method was used to analyze human adipose tissue, plasma, and serum (from same subjects) and mouse serum, gall bladder, small intestine, and colon samples (from same animals). Cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid, deoxycholic acid, and their conjugates (mainly glycine, but also taurine conjugates) were the main metabolites in human samples, and cholic acid, glycine cholic acid, and several taurine conjugates in mouse samples. Using the method, 28 unknown BAs could also be detected. UHPLC-MS/MS spectra, accurate mass, and tissue distribution suggested that nine of the unknown bile acids were taurine conjugates, 13 were glycine conjugates, and six were intact BAs, respectively. To our knowledge, this was the first time BAs were detected in adipose tissue. Results showed that 17 targeted BAs were found at ng/g level in human adipose tissue. Our findings give a novel insight ofthe endogenous role of BAs in adipose tissue and their role as biomarkers (e.g., in metabolic diseases).

Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples

Over the past decade, the number of known eicosanoids has expanded immensely and we have now developed an ultra-performance liquid chromatography–electrospray ionization triple quadrupole mass spectrometric (UPLC-QTRAP/MS/MS) method to monitor and quantify numerous eicosanoids. The UPLC-QTRAP/MS/MS approach utilizes scheduled multiple reaction monitoring (MRM) to optimize sensitivity, number of metabolites that can be analyzed and the time requirement of the analysis. A total of 184 eicosanoids including 26 deuterated internal standards can be separated and monitored in a single 5min UPLC run. To demonstrate a practical application, human plasma samples were analyzed following solid-phase extraction (SPE) and the recovery rate and matrix effects were determined for the 26 deuterated internal standards added to the plasma. The method was validated and shown to be sensitive with the limit of quantitation at pg levels for most compounds, accurate with recovery rates of 70–120%, and precise with a CV<30 for all compounds. Also, the method showed a linear response over a range spanning several orders of magnitude. In a QC human plasma sample, we identified and rigorously quantified over 120 eicosanoids.

N‐terminal valine adduct from the anti‐HIV drug abacavir in rat haemoglobin as evidence for abacavir metabolism to a reactive aldehyde in vivo

The aim of this study was to obtain evidence for the activation of the nucleoside reverse transcriptase inhibitor abacavir to reactive aldehyde metabolites in vivo. Protein haptenation by these reactive metabolites may be a factor in abacavir-induced toxic events. The formation of N-terminal valine adducts from the abacavir-derived aldehydes was investigated in the haemoglobin of Wistar rats treated with eight daily doses (120 mg·kg(-1)) of abacavir. The analyses were conducted by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry upon comparison with synthetic standards. An N-terminal valine haemoglobin adduct derived from an α,β-unsaturated aldehyde metabolite of abacavir was identified in vivo for the first time. This preliminary work on abacavir metabolism provides the first unequivocal evidence for the formation of an α,β-unsaturated aldehyde metabolite in vivo and of its ability to form haptens with proteins. The methodology described herein can be used to assess the formation of this metabolite in human samples and has the potential to become a valuable pharmacological tool for mechanistic studies of abacavir toxicity. In fact, the simplicity of the method suggests that the abacavir adduct with the N-terminal valine of haemoglobin could be used to investigate abacavir-induced toxicity for accurate risk/benefit estimations.

Lipid peroxidation and DNA adduct formation in lymphocytes of premenopausal women: Role of estrogen metabolites and fatty acid intake

A diet high in linoleic acid (an ω-6 PUFA) increased the formation of miscoding etheno (ε)--DNA adducts in WBC-DNA of women, but not in men (Nair et al., Cancer Epidemiol Biomark Prev 1997;6:597-601). This gender specificity could result from an interaction between ω-6 PUFA intake and estrogen catabolism, via redox-cycling of 4-hydroxyestradiol (4-OH-E(2) ) and subsequent lipid peroxidation (LPO). In this study, we investigated whether in premenopausal women LPO-derived adducts in WBC-DNA are affected by serum concentration of 2- and 4-hydroxyestradiol metabolites and by fatty acid intake. DNA extracted from buffy coat and plasma samples, both blindly coded from healthy women (N = 124, median age 40 year) participating in the EPIC-Heidelberg cohort study were analyzed. Three LPO-derived exocyclic DNA adducts, εdA, εdC and M(1) dG were quantified by immuno-enriched (32) P-postlabelling and estradiol metabolites by ultra-sensitive GC-mass spectrometry. Mean M(1) dG levels in WBC-DNA were distinctly higher than those of εdA and εdC, and all were positively and significantly interrelated. Serum levels of 4-OH-E(2) , but not of 2-OH-E(2) , metabolites were positively related to etheno DNA adduct formation. Positive correlations existed between M(1) dG levels and linoleic acid intake or the ratios of dietary linoleic acid/oleic acid and PUFA/MUFA. Aerobic incubation of 4-OH-E(2) , arachidonic acid and calf thymus DNA yielded two to threefold higher etheno DNA adduct levels when compared with assays containing 2-OH-E(2) instead. In conclusion, this study is the first to compare M(1) dG and etheno-DNA adducts and serum estradiol metabolites in human samples in the same subjects. Our results support a novel mechanistic link between estradiol catabolism, dietary ω-6 fatty acid intake and LPO-induced DNA damage supported by an in vitro model. Similar studies in human breast epithelial tissue and on amplification of DNA-damage in breast cancer patients are in progress.

Conjugation of Synthetic Cannabinoids JWH-018 and JWH-073, Metabolites by Human UDP-Glucuronosyltransferases

K2, a synthetic cannabinoid (SC), is an emerging drug of abuse touted as "legal marijuana" and marketed to young teens and first-time drug users. Symptoms associated with K2 use include extreme agitation, syncope, tachycardia, and visual and auditory hallucinations. One major challenge to clinicians is the lack of clinical, pharmacological, and metabolic information for the detection and characterization of K2 and its metabolites in human samples. Information on the metabolic pathway of SCs is very limited. However, previous reports have shown the metabolites of these compounds are excreted primarily as glucuronic acid conjugates. Based on this information, this study evaluates nine human recombinant uridine diphosphate-glucuronosyltransferase (UGT) isoforms and human liver and intestinal microsomes for their ability to glucuronidate hydroxylated metabolites of 1-naphthalenyl-1(1-pentyl-1H-indol-3-yl)-methanone (JWH-018) and (1-butyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-073), the two most common SCs found in K2 products. Conjugates were identified and characterized using liquid chromatography/tandem mass spectrometry, whereas kinetic parameters were quantified using high-performance liquid chromatography-UV-visible methods. UGT1A1, UGT1A3, UGT1A9, UGT1A10, and UGT2B7 were shown to be the major enzymes involved, showing relatively high affinity with K(m) ranging from 12 to 18 μM for some hydroxylated K2s. These UGTs also exhibited a high metabolic capacity for these compounds, which indicates that K2 metabolites may be rapidly glucuronidated and eliminated from the body. Studies of K2 metabolites will help future development and validation of a specific assay for K2 and its metabolites and will allow researchers to fully explore their pharmacological actions.

Focus on analytical science in Spain (JAI 2008)

Every 3 years Barcelona celebrates the EXPOQUIMIA, the largest chemical exhibition in southern Europe. On this occasion a major Conference on Instrumental Analysis (Jornadas de Analisis Instrumental, JAI) is held that takes advantage of the synergies generated by the simultaneous presence of scientists and technical staff working in analytical chemistry, commercial exhibitors of instrumentation, and companies whose main activities are related to chemistry. From October 21 to October 23, 2008, JAI celebrated its 12th anniversary. It was organized by the Spanish Society of Chromatography and Related Techniques (SECyTA), the Society of Analytical Chemistry, the Society of Mass Spectrometry (SEEM), and Society of Proteomics and Optics, the Society of Applied Spectroscopy, and the Association of Environmental Sciences and Techniques. The conference was attended by 580 registered participants. A total of 444 communications were given, 71 in oral form and 373 as posters. A large proportion of these covered environmental applications (122) and food quality and safety (107). Others were related to developments in instrumental analysis (41), pharmaceutical products (21), clinical analysis (21), metal speciation (17), chemometrics and theoretical developments (12), nanotechnology (10), and several other topics. In addition, interesting lectures were given by invited speakers such as Kurt Wuthrich, a Nobel laureate in chemistry (2002), from the Scripps Research Institute (La Jolla, California, USA) and the Institute of Molecular Biology and Biophysics (ETH Zurich, Switzerland), Yoshinobu Baba from the Plasma Nanotechnology Research Center of Nagoya University (Japan), Detlef Gunther from the Institute of Inorganic Chemistry (ETH Zurich, Switzerland), Philip J. Marriott from the School of Applied Sciences of RMIT University (Melbourne, Australia), and Cameron McLeod from the Centre for Analytical Sciences of the University of Sheffield (UK). Several awards were given to the participating students. There were 11 best poster prizes, some of them for specific subjects and others without topic restriction. As customary during previous JAI meetings, Analytical and Bioanalytical Chemistry was present and two ABC poster prizes (on bioanalytics and on chemical speciation) were awarded on behalf of the journal. There were also two awards for the two best oral communications in chromatography (awarded by SECyTA) and for the two best papers in mass spectrometry (awarded by SEEM). The special issue is a collection of a few of the presentations given at the 12th JAI conference, devoted to instrumental analysis, for example, gas chromatography– mass spectrometry (GC-MS) and liquid chromatography– mass spectrometry (LC-MS) applications to determinations of trace organic compounds in environmental samples and technical formulas. Applications of GC-MS, LC-MS, and selective electrodes to the analysis of drugs and metabolites in human samples are covered. Studies of atomic fluorescence spectrometry for total determinations and speciation of hydride-forming elements in garlic still show continuing Anal Bioanal Chem (2009) 394:1515–1516 DOI 10.1007/s00216-009-2845-2

Simultaneous Analysis of Phosphorus-containing Amino Acid Type Herbicides and Their Metabolites in Human Samples Using N-Acetyl,O-Methyl Derivatives by LC/MS

A rapid screening method for simultaneous determination of phosphorus-containing amino acid type herbicides such as glyphosate (GLYP), glufosinate (GLUF), bialaphos (BIAL) and their major metabolites, AMPA and MPPA in human samples using N-Acetyl,O-Methyl derivatives with high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was developed. Serum sample with deporoteinization with 10% trifluoroacetic acid and urine sample without pretreatment was evaporated, and then the drid analytes were directly derivatized by trimethyl orthoacetate and acetic acid at 100°C for 30 minutes. LC analyses were perfomed on a reverse phase column (Symmetry C8) in the gradient solvent system of 5 mM ammonium formate and acetonitrile. Calibration curves of GLYP, GLUF, BIAL, AMPA and MPPA showed good linearity in the range of 0.1-5, 0.1-5, 0.2-5, 0.2-5 and 0.1-5 μg/ml in serum, and 0.1-5, 0.1-5, 0.5-5, 0.5-5 and 0.1-5 μg/ml in urine, respectively. The detection limits of GLYP, GLUF, BIAL, AMPA and MPPA in the selected ion monitoring (SIM) mode were 0.05, 0.03, 0.1, 0.2 and 0.05 μg/ml in serum, and were 0.05, 0.05, 0.4, 0.2 and 0.05 μg/ml in urine, respectively.

Determination of triethylenetetramine (TETA) and its metabolites in human plasma and urine by liquid chromatography–mass spectrometry (LC–MS)

A liquid chromatography–mass spectrometry (LC–MS) method has been developed to measure triethylenetetramine (TETA) and its metabolites in human samples. We identified two metabolites of TETA, N 1-acetyltriethylenetetramine (MAT) and N 1, N 10-diacetyltriethylenetetramine (DAT), the latter being novel. We further developed this LC–MS method for the measurement of TETA and these metabolites in human plasma and urine in a single injection. Separation of analytes was achieved on a cyano column using 15% acetonitrile, 85% water (18 MΩ), and 0.1% heptafluorobutyric acid as the mobile phase. Simultaneous MS detection was performed at [M + H] + values of 147, 189, 231 and 245, corresponding to TETA, MAT, DAT, and N 1-acetylspermine as the internal standard, respectively. This method was successfully applied to measure TETA, MAT and DAT in plasma and urine of humans receiving oral drug treatment.

STRUCTURAL ELUCIDATION OF HUMAN OXIDATIVE METABOLITES OF MURAGLITAZAR: USE OF MICROBIAL BIOREACTORS IN THE BIOSYNTHESIS OF METABOLITE STANDARDS

Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor activator, is currently in clinical development for treatment of type 2 diabetes. This study describes the structural elucidation of the human oxidative metabolites of muraglitazar through the use of a combination of microbial bioreactors, NMR and accurate mass analyses, and organic synthesis. Plasma, urine, and feces were collected from six healthy subjects following oral administration of 14C-labeled muraglitazar (10 mg, 100 microCi) and pooled samples were analyzed. Approximately 96% of the recovered radioactive dose was found in the feces and 3.5% in the urine. The parent compound represented >85% of the radioactivity in plasma. The fecal radioactivity was distributed among 16 metabolites (M1-M12, M14-M16, and M8a) and the parent drug, of which hydroxylation and O-demethylation metabolites (M5, M10, M11, M14, and M15) represented the prominent human metabolites. The urinary radioactivity was distributed into several peaks including muraglitazar glucuronide (M13) and the parent drug. Low concentrations of metabolites in human samples prevented direct identification of metabolites beyond liquid chromatographic (LC)-mass spectrometric analysis. Microbial strains Cunninghamella elegans and Saccharopolyspora hirsuta produced muraglitazar metabolites that had the same high performance liquid chromatography retention times and the same tandem mass spectrometric (MS/MS) properties as the corresponding human metabolites. The microbial metabolites M9, M10, M11, M14, M15, and M16 were isolated and analyzed by NMR. Based on these LC-MS/MS and NMR analyses, and organic synthesis, the structures of 16 human oxidative metabolites were identified. The oxidative metabolism of muraglitazar was characterized by hydroxylation, O-demethylation, oxazolering opening, and O-demethylation/hydroxylation, as well as O-dealkylation and carboxylic acid formation. This study demonstrated the utility of microbial bioreactors for the identification of metabolites.