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Analysis of Hydrophilic Metabolites in Physiological Fluids by HPLC-MS using a Silica Hydride Based Stationary Phase
Abstract
Aqueous normal-phase chromatography was used for the analysis of metabolites in human saliva and urine samples. The column was packed with a silica hydride type separation material. Several gradients were tested with different mobile phase additives in order to produce retention for amino acids, small organic acids, and carbohydrates. Detection was done by TOF MS. In some cases the relative concentration levels of various metabolites in human saliva were compared for normal patients and patients with pancreatic cancer or pancreatitis. The reproducibility of retention of individual metabolites in these complex matrices was tested for several compounds.
... Succinic acid is determined in urine, as well as in plasma, serum and saliva. In order to isolate this acid from the complex matrix, like body fluids, LLE [40,42] and SPE [37] are used. Liquid chromatography permits an analysis of a larger number of samples per unit of time in comparison with gas chromatography [42]. ...
... Liquid chromatography permits an analysis of a larger number of samples per unit of time in comparison with gas chromatography [42]. In the case of GC analysis [38,42] the step of transforming analytes into their volatile derivatives can be avoided using liquid chromatography or capillary electrophoresis [36,40]. Fatty acids and related disorders are nowadays a very important issue in clinical chemistry. ...
... Adipic acid, suberic acid and azelaic acid are determined in urine, as well as in cerebrospinal fluid, blood plasma, serum and saliva. To isolate these acids from the matrix, LLE [35,[38][39][40]is usually used. SPE [35,37], mainly after prior acidification and salting, is less often used. ...
Chromatographic methods find application in the diagnostics and prognosis of diseases. They are used in finding new biomarkers, which may result in early medical intervention. Early diagnosis and intervention are especially important in the case of diseases of unknown etiology. One of these is autism. Autism is a neurodevelopmental disorder characterized by severe impairment in reciprocal social interaction and communication and a pattern of repetitive or stereotyped behavior. Organic acids are intermediate metabolites of all major groups of organic cellular components and can play a role in the pathogenesis of autism. This review presents information about abnormal levels of some organic acids observed in the urine of children with autism and determination of acids with the use of chromatographic techniques. 342 literature sources on frequency (2005-2012) of the use of chromatographic methods in the determination of organic compounds in various body fluids were searched.
... Since no single method currently exists that is sufficiently comprehensive to extract all metabolites, we developed a robust biphasic liquidliquid metabolite extraction and sample processing protocol that included three different pH-adjusted solvents to facilitate extraction of metabolites with pH-dependent solubilities [16] . Hydrophobic and amphipathic metabolites were readily separated by Reverse-Phase (RP) chromatography, while hydrophilic metabolites were recovered by Aqueous Normal Phase (ANP) chroma- tography [17,18]. We complemented LC analyses with GC/MS, primarily for sugars and volatile compounds, via retention time locked (RTL) matching to a spectral library of standards [19]. ...
... Aqueous Normal Phase (ANP) is a good choice for separation of hydrophilic metabolites on silica hydride columns. Prior sample derivatization is not required, it is compatible with LC/MS, and it exhibits rapid re-equilibration, and reproducible chromatography [17,18]. Two separate, general purpose ANP chromatographic methods were used to achieve retention and separation of compounds because ammonia was observed to permanently alter the physiochemical properties of the silica hydride surface. ...
Malaria is a global infectious disease that threatens the lives of millions of people. Transcriptomics, proteomics and functional genomics studies, as well as sequencing of the Plasmodium falciparum and Homo sapiens genomes, have shed new light on this host-parasite relationship. Recent advances in accurate mass measurement mass spectrometry, sophisticated data analysis software, and availability of biological pathway databases, have converged to facilitate our global, untargeted biochemical profiling study of in vitro P. falciparum-infected (IRBC) and uninfected (NRBC) erythrocytes. In order to expand the number of detectable metabolites, several key analytical steps in our workflows were optimized. Untargeted and targeted data mining resulted in detection of over one thousand features or chemical entities. Untargeted features were annotated via matching to the METLIN metabolite database. For targeted data mining, we queried the data using a compound database derived from a metabolic reconstruction of the P. falciparum genome. In total, over one hundred and fifty differential annotated metabolites were observed. To corroborate the representation of known biochemical pathways from our data, an inferential pathway analysis strategy was used to map annotated metabolites onto the BioCyc pathway collection. This hypothesis-generating approach resulted in over-representation of many metabolites onto several IRBC pathways, most prominently glycolysis. In addition, components of the "branched" TCA cycle, partial urea cycle, and nucleotide, amino acid, chorismate, sphingolipid and fatty acid metabolism were found to be altered in IRBCs. Interestingly, we detected and confirmed elevated levels for cyclic ADP ribose and phosphoribosyl AMP in IRBCs, a novel observation. These metabolites may play a role in regulating the release of intracellular Ca(2+) during P. falciparum infection. Our results support a strategy of global metabolite profiling by untargeted data acquisition. Untargeted and targeted data mining workflows, when used together to perform pathway-inferred metabolomics, have the benefit of obviating MS/MS confirmation for every detected compound.
... Indeed, salivary proteomics is an active research field where a variety of analytical techniques are being developed and applied to profile the proteomes of saliva samples. 5,[11][12][13][14] In contrast, there are only a few reports focusing on metabolome analyses of saliva using NMR [15][16][17][18][19] , gas chromatography mass spectrometry (GC-MS) 3,20,21 , liquid chromatography MS (LC-MS) 3,[22][23][24][25] , and capillary electrophoresis MS (CE-MS). 26 The major challenge in saliva metabolome profiling lies in the relatively low abundance of metabolites present in saliva. ...
... For example, recently reported metabolome profiles generated by LC-MS or CE-MS were generally composed of less than 100 metabolite peaks. 3,[22][23][24][25][26] To increase the probability of discovering specific biomarkers of diseases using salivary metabolomics, there is a clear need to develop more sensitive analytical tools to profile a large number of metabolites, i.e., to cast a bigger net to find the biomarkers. ...
Saliva is a readily available biofluid that may contain metabolites of interest for diagnosis and prognosis of diseases. In this work, a differential (13)C-/(12)C-isotope dansylation labeling method, combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), is described for quantitative profiling of the human salivary metabolome. New strategies are presented to optimize the sample preparation and LC-MS detection processes. The strategies allow the use of as little of 5 µL of saliva sample as a starting material to determine the concentration changes of an average of 1058 ion pairs or putative metabolites in comparative saliva samples. The overall workflow consists of several steps including acetone-induced protein precipitation, (12)C-dansylation labeling of the metabolites, and LC-UV measurement of the total concentration of the labeled metabolites in individual saliva samples. A pooled sample was prepared from all the individual samples and labeled with (13)C-dansylation to serve as a reference. Using this metabolome profiling method, it was found that compatible metabolome results could be obtained after saliva samples were stored in tubes normally used for genetic material collection at room temperature, -20(○)C freezer, and -80(○)C freezer over a period of one month, suggesting that many saliva samples already collected in genomic studies could become a valuable resource for metabolomics studies, although the effect of much longer term of storage remains to be determined. Finally, the developed method was applied for analyzing the metabolome changes of two different groups: normal healthy older adults and comparable older adults with mild cognitive impairment (MCI). Top-ranked 18 metabolites successfully distinguished the two groups, among which seven metabolites were putatively identified while one metabolite, taurine, was definitively identified.
... To meet this challenge, recent developments in high-performance liquid chromatography (LC) separations have provided promising platforms for the rapid resolution of thousands of metabolites in biological extracts [8][9][10][11][12][13]. Combined with advances in MS instrumentation that enable rapid acquisition of high-resolution accurate mass spectra and chemometric platforms for automated data analysis and interpretation, effective metabolomic strategies for profiling and identifying diverse metabolites have emerged [14][15][16][17]. ...
... The diluted samples were briefly vortexed and centrifuged for 5 min at 16,0006g to pellet precipitated proteins. For aqueous normal phase (ANP) chromatography [15], sample supernatants were directly transferred to autosampler vials for analysis by HPLC-MS. For reversed phase (RP) chromatographic separation, supernatants were dried down under vacuum, then resuspended with 5% ACN in ddH 2 O containing 0.1% formic acid before analysis by HPLC/MS. ...
A major challenge in systems biology is integration of molecular findings for individual enzyme activities into a cohesive high-level understanding of cellular metabolism and physiology/pathophysiology. However, meaningful prediction for how a perturbed enzyme activity will globally impact metabolism in a cell, tissue or intact organisms is precluded by multiple unknowns, including in vivo enzymatic rates, subcellular distribution and pathway interactions. To address this challenge, metabolomics offers the potential to simultaneously survey changes in thousands of structurally diverse metabolites within complex biological matrices. The present study assessed the capability of untargeted plasma metabolite profiling to discover systemic changes arising from inactivation of xanthine oxidoreductase (XOR), an enzyme that catalyzes the final steps in purine degradation. Using LC-MS coupled with a multivariate statistical data analysis platform, we confidently surveyed >3,700 plasma metabolites (50-1,000 Da) for differential expression in XOR wildtype vs. mice with inactivated XOR, arising from gene deletion or pharmacological inhibition. Results confirmed the predicted derangements in purine metabolism, but also revealed unanticipated perturbations in metabolism of pyrimidines, nicotinamides, tryptophan, phospholipids, Krebs and urea cycles, and revealed kidney dysfunction biomarkers. Histochemical studies confirmed and characterized kidney failure in xor-nullizygous mice. These findings provide new insight into XOR functions and demonstrate the power of untargeted metabolite profiling for systemic discovery of direct and indirect consequences of gene mutations and drug treatments.
... These provide a high degree of polar selectivity and are also able to retain non-polar metabolites. With this approach, organic acids, NTs, and amino acids were successfully analyzed [81][82][83], but applications in plants are still scarce. However, recently, Ns from Asparagus officinalis extracts were successfully separated from other polar metabolites using this technique [6]. ...
Nucleotides fulfill many essential functions in plants. Compared to non-plant systems, these hydrophilic metabolites have not been adequately investigated in plants, especially the less abundant nucleotide species such as deoxyribonucleotides and modified or damaged nucleotides. Until recently, this was mainly due to a lack of adequate methods for in-depth analysis of nucleotides and nucleosides in plants. In this review, we focus on the current state-of-the-art of nucleotide analysis in plants with liquid chromatography coupled to mass spectrometry and describe recent major advances. Tissue disruption, quenching, liquid–liquid and solid-phase extraction, chromatographic strategies, and peculiarities of nucleotides and nucleosides in mass spectrometry are covered. We describe how the different steps of the analytical workflow influence each other, highlight the specific challenges of nucleotide analysis, and outline promising future developments. The metabolite matrix of plants is particularly complex. Therefore, it is likely that nucleotide analysis methods that work for plants can be applied to other organisms as well. Although this review focuses on plants, we also discuss advances in nucleotide analysis from non-plant systems to provide an overview of the analytical techniques available for this challenging class of metabolites.
... Thus dilution is carried out mainly in water (1:1e1:9 v/v) or in highly polar organic solvents, such as methanol and/or acetonitrile [60,61]. When we expect to detect proteinuria, the dilution step has to be performed with at least three volumes of organic solvent to precipitate proteins or, alternatively, a molecular weight filtration step (centrifugal or syringe filters) can be included [43,62]. Salts are a frequent urine component that can damage both the LC and MS systems. ...
Metabolomics has become an invaluable tool for both studying metabolism and biomarker discovery. The great technical advances in analytical chemistry and bioinformatics have considerably increased the number of measurable metabolites, yet an important part of the human metabolome remains uncovered. Among the various MS hyphenated techniques available, LC-MS stands out as the most used. Here, we aimed to show the capabilities of LC-MS to uncover part of the metabolome and how to best proceed with sample preparation and LC to maximise metabolite detection. The analyses of various open metabolite databases served us to estimate the size of the already detected human metabolome, the expected metabolite composition of most used human biospecimens and which part of the metabolome can be detected when LC-MS is used. Based on an extensive review and on our experience, we have outlined standard procedures for LC-MS analysis of urine, cells, serum/plasma, tissues and faeces, to guide in the selection of the sample preparation method that best matches with one or more LC techniques in order to get the widest metabolome coverage. These standard procedures may be a useful tool to explore, at a glance, the wide spectrum of possibilities available, which can be a good starting point for most of the LC-MS metabolomic studies.
... Samples were stored at -80 o C until LC-MS analysis. LC-MS metabolomics was performed on non-diluted 505 samples, using a Diamond Hydride Type C column (Cogent) on an Agilent 1200 LC system coupled to an 506 Agilent Accurate Mass 6220 Time of Flight (TOF) spectrometer operating in the positive (amino acids) 507 and negative (keto acids) ionization mode, as described 62,63 . 508 ...
Organisms are defined by their genomes, yet many distinguishing features of a given organism are encoded by genes that are functionally unannotated. Mycobacterium tuberculosis (Mtb), the leading cause of death due to a single microbe, co-evolved with humans as its only known natural reservoir, yet the factors mediating Mtb's pathogenicity remain incompletely defined. rv3722c is a gene of unknown function predicted to encode a pyridoxal phosphate binding protein and to be essential for in vitro growth of Mtb. Using metabolomic, genetic and structural approaches, we show that Rv3722c is the primary aspartate aminotransferase of Mtb and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Together with the attenuation of Rv3722c-deficient Mtb in macrophages and mice, these results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in Mtb.
... Metabolites were extracted from duplicate samples and analyzed by LC-MS as described in de Carvalho et al. [107], Pesek et al. [108] and Brinsmade et al. [30]. Data analysis was as outlined in Brinsmade et al. [30]. ...
Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.
... μm pore size [22]. The use of special membranes with cutoff of 3-30 kDa has also been reported in order to cover hydrophilic metabolites [23]. Additionally, filtration also prevents bacterial growth during storage [24]. ...
Urine is a biological matrix that contains hundreds of metabolic end products which constitute the urinary metabolome. The development and advances on LC-MS/MS have revolutionized the analytical study of biomolecules by enabling their accurate identification and quantification in an unprecedented manner. Nowadays, LC-MS/MS is helping to unveil the complexity of urine metabolome, and the results obtained have multiple biomedical applications. This review focuses on the targeted LC-MS/MS analysis of the urine metabolome. In the first part, we describe general considerations (from sample collection to quantitation) required for a proper targeted metabolic analysis. In the second part, we address the urinary analysis and recent applications of four relevant families: amino acids, catecholamines, lipids and steroids.
... The determination of metabolite relative abundances by ontology mapping onto biochemical pathways was a major tool in a global, untargeted workflow and provided a protocol for rapidly assessing the response of S. cerevisiae to calcium and immunosuppressant drug treatment. A screening of metabolites in the urine and saliva of normal and cancer patients indicated key differences for a number of compounds [38]. The format outlined was rugged despite the complex nature of the matrices that are prone to cause column fouling. ...
... It is generally agreed that in HILIC the retention mechanism involves mostly partitioning between the largely organic mobile phase and a water-rich layer mobile phase on the surface of stationary phase [1,18]. However, some authors suppose that this effect is less important with the hydrosilated type silica stationary phases in comparison to the ordinary silica gel [22][23][24]. ...
In the present work, the adsorption of water was investigated in aqueous normal-phase liquid chromatography on Cogent Silica C and Cogent Phenyl hydride stationary phases at different temperatures by frontal analysis - using coulometric Karl Fischer titration - to compare the temperature dependence of adsorption of water from aqueous acetonitrile. The Cogent Silica-C and Cogent Phenyl Hydride columns have a silicon hydride surface (silica hydride) with less than 2% free silanol group; therefore, they do not have a strong association with water. The adsorption behavior of water on the mentioned stationary phases was modeled by Langmuir isotherm. The preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water slightly depends on the temperature. The adsorbed water may fill four to eight percent of the pore volume over the studied temperature range, which approximately corresponds to the equivalent of 0.24-0.68 water layer coverage of the adsorbent surface. The phenyl hydride stationary phase shows decreased water uptake in comparison to the Silica C stationary phase.
... Sample preparation and LC-HRMS were performed as described in the sup plementary Materials and methods (available as Supplementary data at JAC Online) using methods similar to those previously described. [9][10][11] LC-HRMS data processing and analysis Global metabolomics profiling was performed on a Thermo Q-Exactive Orbitrap mass spectrometer with a Dionex UHPLC and autosampler. All samples were analysed using positive and negative heated electrospray ionization with a mass resolution of 70 000 at m/z 200 as separate injections (2 lL for positive and 4 lL for negative). ...
Background:
Echinocandins are an important class of antifungal agents in the treatment of invasive candidiasis. However, little is known about the metabolomic effects of echinocandins on Candida We therefore performed LC-high-resolution MS (LC-HRMS)-based metabolomics profiling of the response of Candida albicans cells to increasing concentrations of micafungin to determine the metabolic response of Candida to micafungin subinhibitory injury.
Methods:
Isolates of C. albicans were cultured on nitrocellulose filters to mid-logarithmic phase of growth and micafungin (0-0.25 mg/L) was added. At mid-logarithmic phase, replicates were metabolically quenched. Intracellular metabolites were analysed by LC-HRMS. Changes in pool sizes of individual metabolites were analysed by Student's t-test adjusted for multiple hypothesis testing by Benjamini-Hochberg correction. Metabolites were ascribed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways database.
Results:
Among 3446 detected metabolites, 204 were identified by comparison against pure standard or comparison against a library of mass-retention-time pairs. Fifty had significantly altered abundances in response to increasing micafungin concentrations. Pool sizes of amino acids, nucleic acids and polyamine metabolism were significantly increased at subinhibitory concentrations, while exposure to inhibitory concentrations resulted in a precipitous decrease consistent with fungicidal activity.
Conclusions:
Micafungin induces a re-routing of metabolic pathways inhibiting protein synthesis and cell replication. These results shed light on new mechanisms of action of echinocandins.
... Recently, a new chromatographic mode, high-performance aqueous normal-phase chromatography (HP-ANPC) has been developed on stationary phases based on silica hydride surfaces [74][75][76][77]. Silica hydride stationary phases, which are based on high-purity, low-metal-content type-B silica, were developed 10 years ago to complement conventional silica materials. ...
High-performance liquid chromatography (HPLC) is a fully automated form of liquid chromatography that allows separations of high speed, high resolution, and high sensitivity. HPLC, when coupled to mass spectrometry, can be used for the separation, detection, identification and quantification of a very large number of individual components in a sample, thus enabling challenging peptide and protein analyses in complex matrices. In this chapter, the major separation modes, including size-exclusion, reversed-phase, normal-phase, hydrophilic interaction, aqueous normal phase, hydrophobic interaction, ion-exchange, and affinity chromatography, and their applications to the analysis of peptides and proteins, are described. Method development approaches for analytical HPLC procedures and their upscaling to a preparative level are discussed in the context of analytical “green chemistry” and process analytical technologies. Various options for multidimensional HPLC, involving different separation modes in sequence for the efficient analysis and purification complex peptide and protein mixtures, are presented. Recognition of the opportunities for HPLC in systems biology, medical diagnostics, personalized medicine, and in the manufacture of biotherapeutics are currently the drivers of interdisciplinary research in sorbent development, surface chemistry, mathematical modeling of chromatographic processes, protein engineering, and bioinformatics.
... Other notable features of silica hydride include a very thin water shell (∼0.5 monolayer) on the surface [23], rapid equilibration of the surface solvation layer after gradients and highly reproducible retention for polar compounds in the aqueous normal-phase mode. These properties have been used advantageously for the analysis of metabolites [24][25][26][27][28], nucleotides [29,30], pharmaceuticals [31][32][33][34], food and beverage components [35][36][37][38][39], lipophilic interferences [40] and chiral compounds [41]. ...
... Analyses of the prepared serum samples were performed using an Agilent 1200 series high performance liquid chromatography (HPLC) system connected to an Agilent 6520 Quadrupole Time-of-Flight (Q-TOF) MS fitted with a dual electrospray ionization (ESI) source (Agilent Technologies, Palo Alto, CA). Metabolites were separated using a Cogent hydrophilic type-C silica diamond-hydride column (particle size 4μm, pore size 100 Å, 2.1 mm x 150 mm) with a Cogent diamond hydride guard column (size 2.0 mm x 20 mm) (Microsolv Technology Corporation, NJ) [45,46]. A 5-μl aliquot of each processed serum sample was applied to the column that had been equilibrated with 5% solvent A (0.1% formic acid in H 2 O) and 95% solvent B HPLC column eluent was directly introduced into the Q-TOF instrument for metabolite detection. ...
Background:
Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and clinical care of dengue patients throughout the tropical and subtropical world. The ability to predict severe dengue disease outcomes (DHF/DSS) using acute phase clinical specimens would be of enormous value to physicians and health care workers for appropriate triaging of patients for clinical management. Advances in the field of metabolomics and analytic software provide new opportunities to identify host small molecule biomarkers (SMBs) in acute phase clinical specimens that differentiate dengue disease outcomes.
Methodology/principal findings:
Exploratory metabolomic studies were conducted to characterize the serum metabolome of patients who experienced different dengue disease outcomes. Serum samples from dengue patients from Nicaragua and Mexico were retrospectively obtained, and hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) identified small molecule metabolites that were associated with and statistically differentiated DHF/DSS, DF, and non-dengue (ND) diagnosis groups. In the Nicaraguan samples, 191 metabolites differentiated DF from ND outcomes and 83 differentiated DHF/DSS and DF outcomes. In the Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes. The structural identities of 13 metabolites were confirmed using tandem mass spectrometry (MS/MS). Metabolomic analysis of serum samples from patients diagnosed as DF who progressed to DHF/DSS identified 65 metabolites that predicted dengue disease outcomes. Differential perturbation of the serum metabolome was demonstrated following infection with different DENV serotypes and following primary and secondary DENV infections.
Conclusions/significance:
These results provide proof-of-concept that a metabolomics approach can be used to identify metabolites or SMBs in serum specimens that are associated with distinct DENV infections and disease outcomes. The differentiating metabolites also provide insights into metabolic pathways and pathogenic and immunologic mechanisms associated with dengue disease severity.
... Lysates were clarified by centrifugation at 14 g for 10 min at 48C and extracted 50:50 into acetonitrile with 0.2% formic acid. Intracellular metabolites were analysed by LC/MS as previously described 11,13 with the modification that 0.2% formic acid was used in place of 0.2% acetic acid for the mobile phase. Each fluconazole concentration was tested in eight replicates and the drug-free replicates were used as controls. ...
Background:
Little is known about the effects of fluconazole on the metabolism of Candida albicans. We performed LC/MS-based metabolomic profiling of the response of C. albicans cells to increasing doses of fluconazole.
Methods:
C. albicans cells were cultured to mid-logarithmic growth phase in liquid medium and then inoculated in replicate on to nitrocellulose filters under vacuum filtration. Organisms were cultured to mid-logarithmic growth phase and treated with 0-4 mg/L fluconazole. Following metabolic quenching at mid-logarithmic growth phase, intracellular metabolites were extracted and analysed by LC/MS. Changes in pool sizes of individual metabolites were verified by Student's t-test, adjusted for multiple hypothesis testing by Benjamini-Hochberg correction. Distribution of metabolites was analysed by the Kyoto Encyclopedia of Genes and Genomes metabolic pathways database.
Results:
We reproducibly detected 64 metabolites whose identities were confirmed by comparison against a pure standard and a library of accurate mass-retention time pairs. These 64 metabolites were broadly representative of eukaryotic central metabolic pathways. Among them 12 had their mean abundance significantly altered in response to increasing fluconazole concentrations. Pool sizes of four intermediates of central carbon metabolism (α-ketoglutarate, glucose-6-phosphate, phenylpyruvate and ribose-5-phosphate) and mevalonate were increased by 0.5-1.5-fold (P ≤ 0.05). Five amino acids (glycine, proline, tryptophan, aminoisobutanoate and asparagine) and guanine were decreased by 0.5-0.75-fold (P ≤ 0.05).
Conclusions:
Fluconazole treatment of C. albicans resulted in increased central carbon and decreased amino acid synthesis intermediates, suggesting a rerouting of metabolic pathways. The function of these metabolomic changes remains to be elucidated; however, they may represent previously unrecognized mechanisms of metabolic injury induced by fluconazole against C. albicans.
... Perhaps the most extensively studied area by ANP is metabolites [9]. These investigations include both physiological fluids and bacteria culture media [10][11][12][13][14][15][16][17][18][19][20]. Small polar molecules constitute a significant fraction of the pharmaceutical compounds in use today. ...
... In addition, extensive sample preparation protocols involving extraction methods are usually necessary [18][19][20]. However, when using ANP relatively little sample preparation is needed except for protein precipitation for physiological samples [21] or filtration for solid samples after analyte extraction [22]. The importance of thiopurine and thiopurine-related analysis has already been established for practical applications such as clinical analysis [23]. ...
The chromatography of several thiopurines is investigated using aqueous normal phase (ANP) conditions in conjunction with a silica hydride-based column. Both isocratic and gradient elution modes are tested. Detection of higher concentration samples is done by UV to demonstrate feasibility in this format while lower concentration samples utilize mass spectrometry (MS). Repeatability of successive runs is also tested with particular attention to gradient methods where the equilibration time of the stationary phase can be evaluated.
... The hydride surface responsible for hydrophilic interaction provides the greatest degree of polar selectivity; whereas the small amount of n-alkyl carbon results in better peak shape (Pesek et al. 2013). The capability of this column for the separation of polar compounds has been demonstrated for amino acids, small organic acids and carbohydrates (Pesek et al. 2008(Pesek et al. , 2009, as well as nucleotides (Matyska et al. 2010). Callahan et al. (2009) used the CDH column to profile metabolites in human urine. ...
Metabolite analysis or metabolomics is an important component of systems biology in the post-genomic era. Although separate liquid chromatography (LC) methods for quantification of the major classes of polar metabolites of plants have been available for decades, a single method that enables simultaneous determination of hundreds of polar metabolites is possible only with gas chromatography-mass spectrometry (GC-MS) techniques. The rapid expansion of new LC stationary phases in the market and the ready access of mass spectrometry in many laboratories provides an excellent opportunity for developing LC-MS based methods for multi-target quantification of polar metabolites. Although various LC-MS methods have been developed over the last 10 years or so with the aim to quantify one or more classes of polar compounds in different matrices, currently there is no consensus LC-MS method that is widely used in plant metabolomics studies. The most promising methods applicable to plant metabolite analysis will be reviewed in this paper and the major problems encountered highlighted. The aim of this review is to provide plant scientists, with limited to moderate experience in analytical chemistry, with up-to-date and simplified information regarding the current status of polar metabolite analysis using LC-MS techniques.
... The benefits of silica hydride-based stationary phases are becoming well-known in the bioanalytical industry [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Alongside a characteristic rapid chemical re-equilibration allowing expedition of gradient elution methods, of particular note among these benefits is the well-established ability of such phases to operate in the aqueous normal phase (ANP) mode, the reversed-phase (RP) as well as the organic normal phase [3]. ...
Background:
An investigation was carried out into the chromatographic behavior, on a silica hydride-based phase and a comparator silica-based phase, of an important group of lipids endogenous to human plasma, which are associated with matrix effect and in the context of quantitative peptide analysis.
Results:
The propensity for aqueous normal phase (ANP) retention on the silica hydride-based phase was strong and extensive in comparison with the silica-based comparator, and the lipophilic interferences in question were readily eluted using the ANP mode, a contrast to over-retention issues with accompanying implications for method ruggedness typically found with silica-based phases.
Conclusion:
The silica hydride-based phase, with ANP operation, offered selectivity conducive to rapid lipophilic interferent elimination and the bimodal retention involved in suitable gradient elution was appropriate for general peptide analytical application.
... A broad range of separation capabilities have been demonstrated for the analysis of hydrophilic compounds, particularly metabolites. [6,[15][16][17][18] Carotenoids are typically red, yellow, or orange colored pigments that are widely distributed in nature. Most carotenoids are isomers of 40 carbon chain atoms, and it is estimated 100 million tons of carotenoids are naturally produced each year. ...
A triacontyl (C30) stationary phase has been synthesized on a silica hydride surface for use in preparative applications. The success of bonding via hydrosilation was monitored by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and elemental (carbon) analysis. A semi-preparative column was packed in order to test this material for potential applications. The test compounds selected for this evaluation were carotenoids. A number of organic solvents applicable to either normal or reversed-phase phase retention were used as binary mobile phase components. The effect of flow rate on resolution was also determined.
... The mechanism of separation on HILIC is based on partition of solutes between a water-enriched layer at the surface of hydrophilic bed and the bulk of the aqueous /organic mobile phase [8]. However, HILIC analyses provide only a partial solution to the total number of analytes that must be separated [9]. Aqueous normal phase chromatography is typically used when partition is not the main retention mechanism of hydrophilic compounds, but where interactions may involve adsorption and ion and/or ligand exchange. ...
An aqueous normal phase (ANP) liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (ANP-LC-micrOTOFQ) method was used for the determination of zanamivir in human serum. Zanamivir was extracted with methanol from protein-precipitated human serum samples and further purified with SCX solid-phase extraction cartridges. Scherzo SM-C18, Agilent Zorbax SB-Aq, Cogent Diamond Hydride, Cogent Bidentate and Luna HILIC columns were compared and optimized for the retention and separation of zanamivir and the Luna HILIC and Diamond Hydride columns exhibited the best retention of zanamivir. The former provided a shorter retention time, a sharper peak and relatively high sensitivity, whereas the latter exhibited a longer retention time and less matrix interference. The analytical range of the calibration curve was between 5 and 1000ng/mL.
In this article, we review the preparation methods of silica and silica hybrid materials, as well as their surface modification and application in liquid chromatography (LC). First we discuss the synthesis and properties of silica and silica hybrids. In the second part, we present the modification methods leading to their stationary phases. The current state of the art of the stationary phase's synthesis during silanization, hydrosilation, and coating is discussed. A detailed characterization of both native silica and its modifications is provided. The most common stationary phases for reversed‐phase, normal‐phase, and hydrophilic interaction LC are featured. Unconventional stationary phases, for example, with bonded cholesterol molecules, are also included. The potential applications of the described stationary phases are presented.
Metabolomics, the global characterisation of the small molecule complement involved in metabolism, has evolved into a powerful suite of approaches for understanding the global physiological and pathological processes occurring in biological organisms. The diversity of metabolites, the wide range of metabolic pathways and their divergent biological contexts require a range of methodological strategies and techniques. Methodologies for Metabolomics provides a comprehensive description of the newest methodological approaches in metabolomic research. The most important technologies used to identify and quantify metabolites, including nuclear magnetic resonance and mass spectrometry, are highlighted. The integration of these techniques with classical biological methods is also addressed. Furthermore, the book presents statistical and chemometric methods for evaluation of the resultant data. The broad spectrum of topics includes a vast variety of organisms, samples and diseases, ranging from in vivo metabolomics in humans and animals to in vitro analysis of tissue samples, cultured cells and biofluids.
Metabolomics, the global characterisation of the small molecule complement involved in metabolism, has evolved into a powerful suite of approaches for understanding the global physiological and pathological processes occurring in biological organisms. The diversity of metabolites, the wide range of metabolic pathways and their divergent biological contexts require a range of methodological strategies and techniques. Methodologies for Metabolomics provides a comprehensive description of the newest methodological approaches in metabolomic research. The most important technologies used to identify and quantify metabolites, including nuclear magnetic resonance and mass spectrometry, are highlighted. The integration of these techniques with classical biological methods is also addressed. Furthermore, the book presents statistical and chemometric methods for evaluation of the resultant data. The broad spectrum of topics includes a vast variety of organisms, samples and diseases, ranging from in vivo metabolomics in humans and animals to in vitro analysis of tissue samples, cultured cells and biofluids.
Metabolomics, the global characterisation of the small molecule complement involved in metabolism, has evolved into a powerful suite of approaches for understanding the global physiological and pathological processes occurring in biological organisms. The diversity of metabolites, the wide range of metabolic pathways and their divergent biological contexts require a range of methodological strategies and techniques. Methodologies for Metabolomics provides a comprehensive description of the newest methodological approaches in metabolomic research. The most important technologies used to identify and quantify metabolites, including nuclear magnetic resonance and mass spectrometry, are highlighted. The integration of these techniques with classical biological methods is also addressed. Furthermore, the book presents statistical and chemometric methods for evaluation of the resultant data. The broad spectrum of topics includes a vast variety of organisms, samples and diseases, ranging from in vivo metabolomics in humans and animals to in vitro analysis of tissue samples, cultured cells and biofluids.
Metabolomics relies on a variety of bioinformatics tools aiming to derive information from data. Data acquisition with chromatography‐coupled mass spectrometry yields high volumes of raw data that need to be processed to achieve de‐noised and meaningful biochemical data that subsequently can be presented for statistics and pathway analysis in a coherent manner. A variety of new tools and algorithms have recently been developed that help in this process. Nevertheless, a surprisingly low number of metabolic signals can be unambiguously identified. If cutting‐edge methods are used, we can confidently interpret signals that can neither be identified by reference standards nor annotated by database matching as ‘novel metabolites’. Such methods may comprise high‐resolution, high‐accurate mass data in conjunction with good data alignment programs that perform peak picking and deconvolution, calculation of elemental formulas, database queries and constraining hit lists by interpreting mass spectral fragmentations and retention‐based metabolomic libraries. In an effort to improve standardizations for metabolomic reports, we propose that not only metabolites must be named but also, for all reported metabolites, identifiers or structure codes (InChI keys) from public (bio)chemical databases need to be detailed. Machine‐readable structures will vastly accelerate our knowledge of the magnitude and importance of the metabolome in various plant species, organs and physiological conditions. We detail how well‐annotated metabolome data can then be used to visualize and interrogate biochemical pathways by matching information to the broad plant databases that currently exist.
Reversed-phase liquid chromatography (RPLC) has become the method of choice for the chromatographic separation of low-molecular weight organic compounds due to its broad applicability. Today, the most rational way to address the separation of very hydrophilic and uncharged compounds in HPLC is by utilizing hydrophilic interaction liquid chromatography (HILIC), which is considered to be a variant of normal-phase chromatography. According to McCalley, stationary phases used for HILIC can be classified into five different groups:silica gel, neutral bonded phases, charged bonded phases, zwitterionic phases and mixed reversed-phase/HILIC phases. These applications contribute to the fields of pharmaceutical chemistry, metabolomics, glycomics, medical sciences, and agricultural and food chemistry. HILIC separations can be easily combined with a large number of spectrometric detection techniques such as UV/Vis, fluorescence, and mass spectrometry as well as nonspecific detection techniques such as RI, ELSD, and Corona CAD.
A potent, non-cytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This non-cytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above MIC for 24 hours were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate dependent, effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.
An analytical strategy employing multi-dimensional sample extraction together with optimal analytical platforms was established. The proposed workflow was based on the fact that most biological samples are complex in terms of molecular species as well as their properties, e.g. hydrophobicity. A few of new methods based on the methyl tert-butyl ether (MTBE) extraction which was developed for lipid extraction and managed to perform a lipidomic study on the upper layer fraction of the MTBE extraction system and a metabolomics analysis by mixing the upper and lower fractions from the same sample were developed acquire more information from samples, and in the present work we established a similar method employing the MTBE procedure to separate samples into polar and apolar fractions, then associated each with an adapted subsequent analysis technique. The polar fraction was silylanized for GC/MS analysis, and apolar lipids were analyzed by reverse phase liquid chromatography (RPLC) on a T3 column. The proposed strategy was applied to investigate acute human pancreatitis and was compared with results obtained by conventional LC/MS-only and GC/MS-only analysis. Features obtained in our LC-GC/MS data outnumbered either the LC/MS-only or GC/MS-only data. Furthermore, with the aid of multivariate analysis, differential metabolites from pancreatitis vs normal pancreas, mainly amino acids and phospholipids, were identified. This work demonstrates that the proposed analytical strategy is a promising tool to perform metabolomics. In addition, it improved our understanding of the pathogenesis of AP and provided potential biomarkers for AP diagnosis.
The sample preparation method plays a critical role in untargeted profiling of biological fluids and tissues for biomarker discovery. In this chapter, commonly used sample preparation methods in global metabolomics by nuclear magnetic resonance and mass spectrometry, such as solvent precipitation, ultrafiltration, and solid-phase extraction, are critically discussed with an emphasis on how well they meet the requirements of an ideal metabolomic method, including (1) depth of metabolite coverage, (2) simplicity and minimal handling to prevent metabolite loss and/or degradation and reduce introduction of extraneous contaminants, (3) reproducibility, and (4) incorporation of a metabolism quenching step in order to reflect the true metabolome composition at the time of sampling. Recent developments in this field are also examined including in vivo methods such as microdialysis and solid-phase microextraction, turbulent flow chromatography, and dried blood spot sampling. Finally, recent comprehensive studies comparing the performance of various sample preparation methods are highlighted to aid in the selection of the most appropriate method for a given application.
Urine metabolomics has recently emerged as a prominent field for the discovery of non-invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non-biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid-liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4°C) and repetitive freeze-thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high-performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)-MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC-MS, other MS-based technologies have been utilized in urine metabolomics. These include direct injection (infusion)-MS, capillary electrophoresis-MS and gas chromatography-MS. In this article, the current progresses of different MS-based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed. © 2015 Wiley Periodicals, Inc. Rapid Commun. Mass Spectrom. 9999: XX-XX, 2015.
© 2015 Wiley Periodicals, Inc.
Complex biological samples include thousands of metabolites that range widely in both physiochemical properties and concentration. Simultaneously analyzing metabolites with different properties using a single analytical method is very challenging. The analytical process for metabolites comprises multiple steps including sampling, quenching, sample preparation, separation and detection. Each step can have a significant effect on the reliability and precision of ultimate analytic results. The aim of review is a discussion of considerations and challenges for the simultaneous analysis of metabolites using LC- and GC-MS systems. The review discusses available methodology for each analytical step, and presents the limitations and advantages of each method for the large-scale targeted metabolomics analysis of human and animal biological samples.
Excess adsorption of water from aqueous acetonitrile mobile phases was investigated on 16 stationary phases using the frontal analysis method and coulometric Karl–Fischer titration. The stationary phases include silica gel and silica-bonded phases with different polarities, octadecyl and cholesterol, phenyl, nitrile, pentafluorophenylpropyl, diol and zwitterionic sulfobetaine and phosphorylcholine ligands bonded on silica, hybrid organic-silica and hydrosilated matrices. Both fully porous and core-shell column types were included. Preferential uptake of water by the columns can be described by Langmuir isotherms. Even though a diffuse rather than a compact adsorbed discrete layer of water on the adsorbent surface can be formed because of the unlimited miscibility of water with acetonitrile, for convenience, the preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water strongly depends on the polarity and type of the column. Less than one monomolecular water layer equivalent was adsorbed on moderate polar silica hydride-based stationary phases, Ascentis Express F5 and Ascentis Express CN column at the saturation capacity, while on more polar stationary phases, several water layer equivalents were up-taken from the mobile phase. The strongest affinity to water was observed on the ZIC cHILIC stationary phases, where more than nine water layer equivalents were adsorbed onto its surface at its saturation capacity. Columns with bonded hydroxyl and diol ligands show stronger water adsorption in comparison to bare silica. Columns based on hydrosilated silica generally show significantly decreased water uptake in comparison to stationary phases bonded on ordinary silica. Significant correlations were found between the water uptake and the separation selectivity for compounds with strong polarity differences.
Free silanols on the surface of silica are the “villains”, which are responsible for detrimental interactions of those compounds
and the stationary phase (i.e., bad peak shape, low efficiency) as well as low thermal and chemical stability. For these reasons,
we began this review describing new silica and hybrid silica stationary phases, which have reduced and/or shielded silanols.
At present, in liquid chromatography for the majority of analyses, reversed-phase liquid chromatography is the separation
mode of choice. However, the needs for increased selectivity and increased retention of hydrophilic bases have substantially
increased the interest in hydrophilic interaction chromatography (HILIC). Therefore, stationary phases and this mode of separation
are discussed. Then, non-silica stationary phases (i.e., zirconium oxide, titanium oxide, alumina and porous graphitized carbon),
which afford increased thermal and chemical stability and also selectivity different from those obtained with silica and hybrid
silica, are discussed. In addition, the use of these materials in HILIC is also reviewed.
A high throughput LC–MS/MS method for quantification of metformin substrate uptake enables conversion of radiometric transporter inhibition assays for multidrug and toxin extrusion transporters (MATE 1 and 2) and organic cation transporter 2 (OCT2) to a nonradioactive format. Such conversion greatly simplifies assay complexity and reduces assay costs. The development of a quantitative LC–MS/MS method for metformin in support of the high throughput transporter inhibition assays faced specific challenges of achieving both adequate chromatographic retention and rapid analytical turnaround. Here we report a method that circumvents both challenges. The utilization of a porous graphitic carbon column (Hypercarb) ensured adequate retention of highly polar metformin in biological samples. The combined employment of a ballistic gradient on a 3 mm × 30 mm, 5 μm Hypercarb column, and dual staggered chromatography coupled with multiple injection chromatography acquisition, yielded a fast injection-to-injection cycle time of 30 s. The method demonstrated good accuracy, precision and excellent robustness for high throughput applications, and has been successfully implemented in the development and validation of the nonradioactive transporter inhibition assays for MATEs and OCT2.
This review focuses on the application of various silica hydride stationary phases under conditions that enable the advantages of reversed-phase, aq. normal phase and org. normal phase selectivity to be gained. Here, an overview is given related to the characteristics of the most frequently used silica hydride stationary phases, including those involving short n-alkyl chain (Diamond Hydride), phenyl, octyl, octadecyl and cholesterol phases. Explanations are provided to account for their unique retention properties, which enable their versatile application in the anal. of biol. and pharmaceutical samples under conditions significantly different to those employed with conventional silica-based reversed phase or hydrophilic interaction stationary phase materials.
Aqueous normal-phase chromatography utilizing silica-hydride-based stationary phases provides unique selectivities for the separation of polar and non-polar compounds and offers new avenues to solve difficult analytical separations. By varying the amount of water in the mobile phase, all silica-hydride-based separation materials can operate in the reversed-phase mode, the normal-phase mode or, in some instances, a combination of both.
This review describes some of the fundamental properties of silica-hydride materials and applications that encompass a broad range of separation challenges. Moreover, exemplars are provided that illustrate many of the prominent features that lead to the selection of such stationary phases for solving a particular analytical problem.
IntroductionBasic Terms and Concepts in ChromatographyChemical Structure of Peptides and ProteinsHPLC Separation Modes in Peptide and Protein AnalysisMethod Development from Analytical to Preparative Scale Illustrated for HP-RPCMultidimensional HPLCConclusions
References
In this study, the zeta potentials of type-B silica, bare silica hydride, the so-called Diamond Hydride™ and phenyl substituted silica hydride stationary phases have been measured in aqueous-organic media and correction procedures developed to account for the more negative zeta potential values in media containing different acetonitrile contents. Retention studies of 16 basic, acidic and neutral compounds were also performed with these four stationary phases with mobile phases containing 0.1% (v/v) formic acid and various acetonitrile-water compositions ranging from 0-90% (v/v) acetonitrile. The retention properties of these analytes were correlated to the corrected stationary phase zeta potentials measured under these different mobile phase conditions with R(2) values ranging from 0.01 to 1.00, depending on the stationary phase and analyte type. Using linear solvation energy relationships, stationary phase descriptors for each stationary phase have been developed for the different mobile phase conditions. Very high correlations of the zeta potentials with the ionic interaction descriptors were obtained for the type-B silica and the Diamond Hydride™ phases and good correlation with bare silica hydride material whilst there was no correlation observed for the phenyl substituted silica hydride phase. The nature of the retention mechanisms which gives rise to these different observations is discussed. The described methods represent a useful new approach to characterize and assess the retention properties of silica-hydride based chromatographic stationary phases of varying bonded-phase coverage and chemistries, as would be broadly applicable to other types of stationary phase used in the separation sciences.
The Identification of a glutathione-fumarate conjugate, dicarboxyethyl glutathione (GSF), formed during the non-enzymatic succination of glutathione by fumarate was confirmed in fumarate hydratase (FH) deficient cells using a product ion scan approach followed by hydrophilic interaction chromatography (HILIC) coupled with MS/MS. Glutathione (GSH) and its conjugates, including GSF and glutathione disulfide (GSSG), were successfully separated on a zwitterionic stationary phase and detected by MS/MS operated under negative ESI mode. The relative quantitation of the analytes in cell extracts was carried out and a correction model was established to determine correction factors under matrix effects and the response mismatch between the analytes. These factors were calculated and iteratively used to measure all analytes in cell extracts, based on calibration curves constructed in neat solution. The model was a closed-loop calculation, consisting of two sides with each side of the loop presenting a calculation pathway. Deviation of the correction factors obtained from these pathways manifested the model accuracy. The model was evaluated and there was no significant difference between the two pathways. This article is protected by copyright. All rights reserved.
We investigated the retention behavior of phenolic acids in nonaqueous normal phase LC with buffered methanol/acetonitrile mobile phases on hydrosilated silica-based stationary phases. The silica hydride, Diamond hydride, Bidentate C18 and Cholesterol columns showed a higher retention of phenolic acids in the nonaqueous mobile phases than in aqueous normal-phase mobile phases. There are some selectivity differences between the aqueous and nonaqueous mobile phases, but generally the resolution and selectivity are better in the aqueous systems. The retention of the phenolic acids tested decreased with increasing concentration of methanol in the mobile phase, up to 20% v/v methanol. At increased temperatures, the retention factors and peak widths decrease in both normal-phase modes, showing linear ln k versus 1/T plots, due to a single retention mechanism over the temperature range from 25°C up to the column stability limit, however, the best separations are achieved at low temperatures. The enthalpic and entropic contributions to the retention were determined, and the differences between the aqueous and nonaqueous modes are possibly due to the adsorbed water layer. This article is protected by copyright. All rights reserved.
The retention behaviour of amino acids was studied in hydrophilic liquid chromatography on zwitterionic stationary phases. Evaluation of the influences of acetonitrile/water content, ammonium acetate concentration and mobile phase pH values were performed. 14 amino acids were tested and they were all retained to varying extents, with poorer retention in high water content eluents. The linear relationship between the logarithm of retention factor and log (water content) indicated that adsorption dominated or at least was partly involved in the separation mechanism. Electrostatic and hydrophilic interactions also contributed to the retention of these amino acids under different separation conditions with various mobile phase pH values and ammonium acetate concentrations. Thus, the overall retention mechanism could be explained as a combination of adsorption, electrostatic and hydrophilic interactions. The magnitude and contribution of each mechanism is dependent on the nature of the analyte and the separation conditions applied. This article is protected by copyright. All rights reserved.
Metabolomics offers a revolutionary framework for phenotyping individuals at a molecular level that is needed for new breakthroughs in cell biology and personalized medicine. Separation science plays several critical roles in metabolomics for reliable quantification and improved identification of metabolites in complex sample mixtures. Separation efficiency and peak capacity are two important parameters for determining the performance of a separation in terms of the total number of unique compounds that are baseline resolved prior to detection. Higher peak capacity within a wider separation window reduces solute coelution that can complicate relative quantification or spectral matching for unknown identification. The use of a wider bore/thicker nonpolar stationary phase film as the second dimension column enhances separation performance by increasing sample loadability that is important when quantifying low abundance metabolites in complex biological samples.
The possibility of applying a theoretical model in the prediction of the retention of phenolic acids on hydrosilated silica, in aqueous normal phase mode was studied. The actual gradient of the aqueous component in acetonitrile may fluctuate from the pre-set program, as even the gradient-grade acetonitrile contains some water. Hence, the actual concentration of water during the gradient run is higher than pre-set by the gradient program, which leads to lower than expected sample retention. Furthermore, the actual gradient profile may be affected by an increase in water uptake on a polar column during the gradient run. These effects were investigated using the using frontal analysis method and Karl-Fischer titration, for the determination of water in the initial mobile phase, and in the column effluent. Preferential adsorption of water on the Silica hydride, Diamond hydride, UDC Cholesterol, Bidentate C18, and Phenyl hydride columns can be described by Langmuir isotherms. At the column saturation capacity, less than one monomolecular water layer is adsorbed, with a further decrease in coverage density for modified materials. Parameters of semi-logarithmic and logarithmic model equations, describing the dependence of retention factor on the concentration of water, were determined under isocratic conditions. These parameters and linear gradient profiles corrected for the actual water concentrations were used in calculation of gradient retention data. The corrections for the actual water concentration greatly improved the agreement between the experiment and the predicted gradient elution volumes. Generally, the semi-logarithmic model provides slightly better prediction of the gradient data, with respect to the logarithmic retention model.
Metabolomics relies on a variety of bioinformatics tools aiming to derive information from data. Data acquisition with chromatography-coupled mass spectrometry yields high volumes of raw data that need to be processed to achieve de-noised andmeaningful biochemical data that subsequently can be presented for statistics and pathway analysis in a coherent manner. A variety of new tools and algorithms have recently been developed that help in this process. Nevertheless, a surprisingly low number of metabolic signals can be unambiguously identified. If cutting-edgemethods areused, we can confidently interpret signals that can neither be identified by reference standards nor annotated by databasematching as ‘novel metabolites’. Such methods may comprise high-resolution, high-accurate mass data in conjunction with good data alignment programs that perform peak picking and deconvolution, calculation of elemental formulas, database queries and constraining hit lists by interpreting mass spectral fragmentations and retention-based metabolomic libraries. In an effort to improve standardizations formetabolomic reports, we propose that not only metabolites must be named but also, for all reported metabolites, identifiers or structure codes (InChI keys) from public (bio)chemical databases need to be detailed. Machine-readable structures will vastly accelerate our knowledge of the magnitude and importance of the metabolome in various plant species, organs and physiological conditions. We detail how well-annotated metabolome data can then be used to visualize and interrogate biochemical pathways by matching information to the broad plant databases that currently exist.
In this paper, trend analyses were performed to compare the different ‘omic’ technologies and the different analytical platforms
and biological matrices exploited in metabonomic studies. While common and differential marker metabolites had been identified
using various analytical platforms in metabonomics, little research was directed to review and consolidate marker metabolites
in each disease state. A systematic review of metabonomics-derived marker metabolites in different cancers was performed to
understand the significance of metabonomics in elucidating cancer biochemistry. The biological pathways associated with the
cancer marker metabolites were further correlated to the pathology of cancers. Our trend analyses indicated that metabonomic
publications increased exponentially in recent years, with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography/mass
spectrometry (LC/MS) being the most popular analytical platforms while blood, urine and tissue are the most commonly profiled
biological matrices. Based on the consolidated cancer marker metabolites, it is reinforced that different cancers possess
some common and yet distinct metabolic phenotypes, exhibiting numerous perturbed biochemical pathways related to their needs
to support cell growth and proliferation and facilitate cancer cell survival.
KeywordsMetabonomics–Metabolomics–Clinical oncology–Cancer–Metabolic profiling
A key unmet need in metabolomics is the ability to efficiently quantify a large number of known cellular metabolites. Here we present a liquid chromatography (LC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) method for reliable measurement of 141 metabolites, including components of central carbon, amino acid, and nucleotide metabolism. The selected LC approach, hydrophilic interaction chromatography with an amino column, effectively separates highly water soluble metabolites that fail to retain using standard reversed-phase chromatography. MS/MS detection is achieved by scanning through numerous selected reaction monitoring events on a triple quadrupole instrument. When applied to extracts of Escherichia coli grown in [12C]- versus [13C]glucose, the method reveals appropriate 12C- and 13C-peaks for 79 different metabolites.
Silica hydride, where Si‐H groups replace 95% of the silanols on the surface, is rapidly being developed as a chromatographic support material for HPLC. This chemical structure results in changes of fundamental properties compared to ordinary silica, as well as the bonded stationary phases produced by the further modification of the hydride surface. Several unique chromatographic capabilities of hydride based phases are described, as well as some general application areas where these bonded materials may be used in preference to, or have advantages not available from current commercial stationary phases. The fabrication, properties and applications of etched chemically modified capillaries for electrophoretic analysis are also reviewed. The etching process creates a surface that is fundamentally different in comparison to a bare fused silica capillary. The new surface matrix produces altered electroosmotic flow properties and is more compatible with basic and biological compounds. After chemical modification of the surface, the bonded organic moiety (stationary phase) contributes to the control of migration of solutes in the capillary. Both electrophoretic and chromatographic processes take place in the etched, chemically modified capillaries leading to a variety of experimental variables that can be used to optimize separations. Representative examples of separations on these capillaries are described.
The chromatographic properties of two hydride-based stationary phases (a material with a hydride surface and one with a hydride surface modified by the addition of C18) are tested with several types of compounds that are typically retained in the organic normal phase (ONP) mode. Both phases display ONP retention, but to differing degrees. Various mobile phases are investigated, as well as some gradient elution profiles. Efficiency, peak shape, and reproducibility for the several solutes in both isocratic and gradient elution, as well as the effects of water in the mobile phase, are assessed.
The electrospray ionisation–ion trap mass spectrometry (ESI–MSn) of selected hypnotic drugs, i.e. zopiclone, zolpidem, flunitrazepam and their metabolites have been investigated. Sequential product ion fragmentation experiments (MSn) have been performed in order to elucidate the degradation pathways for the [M+H]+ ions and their predominant fragment ions. These MSn experiments show certain characteristic fragmentations in that functional groups are generally cleaved from the ring systems as neutral molecules such as H2O, CO, CO2, NO2, amines and HF. When an aromatic entity is present in a drug molecule together with a nitrogen-containing saturated ring structure as with zopiclone and its N-desmethyl metabolite fragmentation initially occurs at the latter ring with the former being resistant to fragmentation. The structures of fragment ions proposed for ESI–MSn can be supported by electrospray ionisation–quadrupole time-of-flight mass spectrometry (ESI–QTOF–MS).These molecules can be identified and determined in mixtures at low ng/ml concentrations by the application of liquid chromatography (LC)–ESI–MSn which can be used for their analysis in saliva samples.This paper includes a tabulation of mass losses/signals at low m/z values for these hypnotic drugs and many others in recent publications which will be of value in the characterisation of drug metabolites of unknown structure and also natural product pharmaceuticals isolated from plants, etc.
In order to perform metabonomic investigations, it is necessary to generate comprehensive “global” metabolite profiles from complex samples, such as biofluids and tissues/tissue extracts. Analytical technologies based on liquid chromatography coupled to mass spectrometry (LC-MS) are destined to become a major source of global metabolite profiling data. Here, we describe and compare the utility of various analytical strategies and techniques currently used for LC-MS (capillary, high-performance and ultra-performance). We describe current challenges in the application of LC-MS-based techniques for metabonomic analysis.
The use of silica hydride-based stationary phases for the retention and analysis of nucleotides has been investigated. Both reversed-phase columns with a hydride surface underneath as well as those with an unmodified or a minimally modified hydride material were tested. With these systems, an aqueous normal-phase mode was used with high organic content mobile phases in combination with an additive to control pH for the retention of the hydrophilic nucleotides. Isocratic and gradient elution formats have been used to optimize separations for mixtures containing up to seven components. All conditions developed are suitable for methods that utilize mass spectrometry detection.
Uric acid (UA) can be directly converted to allantoin enzymatically by uricase in most mammals except humans or by reaction with superoxide. UA can react directly with nitric oxide to generate 6-aminouracil and with peroxynitrite to yield triuret; both of these metabolites have been identified in biological samples. We now report a validated high-performance liquid chromatography and tandem mass spectrometry method for the determination of these urinary UA metabolites. Urine samples were diluted 10-fold, filtered and directly injected onto HPLC for LC-MS/MS analysis. The urinary metabolites of UA were separated using gradient HPLC. Identification and quantification of UA urinary metabolites was performed with electrospray in positive ion mode by selected-reaction monitoring (SRM). Correlation coefficients were 0.991-0.999 from the calibration curve. The intra- and inter-day precision (R.S.D., %) of the metabolites ranged from 0.5% to 13.4% and 2.5-12.2%, respectively. In normal individuals (n=21), urinary allantoin, 6-aminouracil and triuret, were 15.30 (+/-8.96), 0.22 (+/-0.12), and 0.12 (+/-0.10) microg/mg of urinary creatinine (mean (+/-S.D.)), respectively. The new method was used to show that smoking, which can induce oxidative stress, is associated with elevated triuret levels in urine. Thus, the method may be helpful in identifying pathways of oxidative stress in biological samples.
An LC/MS/MS method was developed for the analysis of twelve cytochrome P450 (CYP)-specific probe metabolites and their nine parent drugs from human urine. CYP-specific metabolites of melatonin (CYP1A2), nicotine (CYP2A6), bupropion (CYP2B6), repaglinide (CYP2C8), losartan (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1) and midazolam (CYP3A4) were all analyzed using the same LC/MS/MS method with a single analytical run, either after a one-at-a-time dose or cocktail-type dosing of the parent drugs. Ultra performance liquid chromatography (UPLC) with a 1.7 microm particle size column was utilized, providing 1.5-3-fold increase in sensitivity, decrease of analysis time to one third and clearly better chromatographic peak shapes when comparing it with the method using traditional high performance liquid chromatography for the same metabolites. In addition, the method was applied for the analysis of the metabolites from human urine samples collected at multiple time points after single and N-in-one dosing of each of the drugs, showing that the use of both the analytical method and these probe metabolites as CYP-specific markers is feasible in in vivo drug-drug interaction or phenotyping studies.
A novel silica hydride-based stationary phase was used to evaluate the retention behavior in the aqueous normal-phase (ANP) mode of standards representing three classes of metabolites. The effects on retention behavior of amino acids, carbohydrates and small organic acids were examined by altering the column temperature, and by adding different additives to both the mobile phase and sample solvent. Gradient mode results revealed the repeatability of retention times to be very stable for these compound classes. At both 15 and 30 degrees C, excellent RSD values were obtained with less than 1% variation for over 50 injections of an amino acid mixture. The ability to separate the 19 nonderivatized amino acid standards, organic acids and carbohydrates was demonstrated as well as the potential for this material to separate polar metabolites in complex fluids such as urine.
The capillary electrophoretic separation and immunochemical recognition of the two naturally fluorescing, cationic diastereomers quinine (QN) and quinidine (QD), their hydroderivatives and two major QD metabolites (3-hydroxyquinidine and quinidine-N-oxide) was investigated. Plain aqueous phosphate buffers and an alkaline buffer containing dodecyl sulfate micelles are shown to be incapable of resolving the two diastereomers. However, incorporation of an additional chemical equilibrium (with beta-cyclodextrin) in the case of capillary zone electrophoresis (CZE) and the presence of a small amount of an organic solvent as buffer modifier (2-propanol) in dodecyl sulfate based micellar electrokinetic capillary chromatography (MECC), were found to provide separation media which lead to complete resolution of QN, QD and the other compounds of interest. Furthermore, for MECC- and CZE-based immunoassay formats, a commercially available antibody against QD was found to be a perfect discriminator between QD and QN. It was determined to recognize QD and the two QD metabolites (cross reactivity of 20--30%) but not QN. MECC and CZE with laser induced fluorescence (LIF) detection are shown to be suitable to determine QD and metabolites in urine and plasma (quinidine-N-oxide only) collected after single dose intake of 50 mg QD sulfate and of QN in urine, saliva and serum samples that were collected after self-administration of 0.5 l of quinine water (25 mg of QN). With direct injection of a body fluid, MECC with LIF was found to provide 10 ng/ml detection limits for QD and QN. This ppb sensitivity is comparable to that obtained in HPLC assays that are based upon drug extraction. Furthermore, MECC and CZE assays with UV detection are shown to provide the ppm sensitivity required for therapeutic drug monitoring and clinical toxicology of QD and QN.
In nano-ESI MS, the qualitative and quantitative characteristics of mass spectra vary considerably upon the use of different spraying conditions, i.e., aperture of the spraying needle and the voltage applied. The major parameters affected by the aperture size is the liquid flow rate which determines the initial droplet size and the current emitted upon the spray process, as described by different models of the ESI process. In the present study, the effect of flow rate on ion signals was studied systematically using mixtures of compounds with different physicochemical properties (i.e., detergent/oligosaccharide and oligosaccharide/peptide). For these model systems, the functional dependence of certain analyte-ion ratios upon the flow rate can be correlated to changes in analyte partition during droplet fission prior to ion release. Analyte suppression is practically absent at minimal flow rates below 20 nL/min.
Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile components in food. However, polar low-molecular-weight compounds such as hydrophilic amino acids, di- and tripeptides, and organic acids are often not sufficiently retained and represent a challenge for RPLC. Hydrophilic interaction liquid chromatography in combination with electrospray mass spectrometry (HILIC-ESI-MS) on a carbamoyl-derivatized stationary phase was successfully employed to separate free amino acids and small polar peptides in complex food matrixes such as wheat gluten hydrolysate and Parmesan cheese. Glutamyl dipeptides were separated in a sequence-specific order with peptides with N-terminal glutamic acid residues eluting prior to their reverse sequence analogues. ESI-MSn detection in the positive ionization mode provided the necessary information to unambiguously identify isobaric peptides due to their characteristic fragmentation patterns. The technique also proved useful to separate and identify glycoconjugates between amino acids and reducing sugars (Amadori compounds). The investigation of organic acids present in food used a mobile phase comprising ammonium acetate buffer at pH 7 and mass spectrometric detection in the negative ionization mode.
Application of C18 monolithic silica capillary columns in HPLC coupled to ion trap mass spectrometry detection was studied for probing the metabolome of the model plant Arabidopsis thaliana. It could be shown that the use of a long capillary column is an easy and effective approach to reduce ionization suppression by enhanced chromatographic resolution. Several hundred peaks could be detected using a 90-cm capillary column for LC separation and a noise reduction and automatic peak alignment software, which outperformed manual inspection or commercially available mass spectral deconvolution software.
A highly sensitive bioanalytical method based on a simple liquid/liquid extraction and hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) analysis has been developed, validated and transferred for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific nitrosamine metabolite. Deuterated NNAL (NNAL-d(4)) was synthesized and used as the internal standard. This method can be used for the analysis of free and total NNAL (free NNAL plus NNAL-gluc) in K(3)-EDTA human plasma. Free NNAL and NNAL-d(4) are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K(3)-EDTA human plasma is treated with beta-glucuronidase to deconjugate the NNAL-gluc; the total NNAL and internal standard are then extracted using liquid/liquid extraction. After drying down under nitrogen, the residue is reconstituted with acetonitrile and analyzed using positive ion electrospray and HILIC/MS/MS at a flow rate of 1.0 mL/min. The chromatographic run time is 1.0 min per injection, with retention time for both NNAL and NNAL-d(4) of 0.75 min with a capacity factor (k') of 2. The standard curve range for this assay is from 5.00-1000 pg/mL for both free and total NNAL, using a total plasma sample volume of 1.0 mL. The interday precision and accuracy of the quality control (QC) samples demonstrated <7.6% relative standard deviation (RSD) and <3.3% relative error (RE) for free NNAL. For total NNAL, the interday precision and accuracy of the QC samples demonstrated <11.7% RSD and <2.8% RE. Optimization of enzyme hydrolysis of NNAL-gluc is discussed in detail. The overall recoveries for free and total NNAL and IS were 68.2 and 71.5% (free) and 70.7 and 65.5% (total). No adverse matrix effects were noticed for this assay.
In order to establish a fast screening method for the determination of the CYP2D6 metabolic phenotype a sensitive LC-MS/MS assay to quantify dextromethorphan (DEX) and its O-demethylated metabolite dextrorphan (DOR) in human saliva was developed with limits of quantitation of 1 pmol/ml. Saliva was provided by 170 medical students 2h after oral ingestion of 30 mg (81 micromol) dextromethorphan hydrobromide. Individual ratios of the concentrations DEX/DOR (metabolic ratio, MR(DEX/DOR)) varied more than 25,000-fold (0.03-780). Two groups comprising 156 'Extensive' and 14 'Poor Metabolizers' were clearly distinguished. For the investigation of individual differences in N-demethylation and glucuronidation, four additional metabolites of DEX, 3-methoxymorphinan (MOM), 3-hydroxymorphinan (HOM), and the two O-glucuronides (DORGlu and HOMGlu) were measured by LC-MS/MS analysis of 6-h urine of 24 volunteers. The N-demethylation reactions DEX-to-MOM and DOR-to-HOM defined by the respective MR were significantly correlated. The same holds for the glucuronidation pathways (MR(DOR/DORGlu) versus MR(HOM/HOMGlu)). The three poor CYP2D6 metabolizers excreted relatively high amounts of the parent compound DEX (up to 7 micromol), but only low amounts of glucuronides (DORGlu: 0.4-1.0 micromol; HOMGlu: 0.2-0.7 micromol). For the 21 'Extensive Metabolizers', the two glucuronides were the most abundant, with relatively little interindividual variation (DORGlu: 10-44 micromol; HOMGlu: 5-17 micromol). For the excretion of the glucuronides, two normal distributions provided the best fit, indicating that the determination of the glucuronides alone could allow assignment of the CYP2D6 metabolic phenotype.
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.
The sensitivity of proteomics measurements using liquid chromatography (LC) separations interfaced with electrospray ionization-mass spectrometry (ESI-MS) improves approximately inversely with liquid flow rate (for the columns having the same separation efficiency, linear velocity, and porosity), making attractive the use of smaller inner diameter LC columns. We report the development and initial application of 10 microm i.d. silica-based monolithic LC columns providing more sensitive proteomics measurements. A 50-microm-i.d. micro solid-phase extraction precolumn was used for ease of sample injection and cleanup prior to the reversed-phase LC separation, enabling the sample volume loading speed to be increased by approximately 50-fold. Greater than 10-fold improvement in sensitivity was obtained compared to analyses using more conventional capillary LC, enabling e.g. the identification of >5000 different peptides by MS/MS from 100-ng of a Shewanella oneidensis tryptic digest using an ion trap MS. The low nL/min LC flow rates provide more uniform responses for different peptides, and provided improved quantitative measurements compared to conventional separation systems without the use of internal standards or isotopic labeling. The improved sensitivity allowed LC-MS measurements of immunopurified protein phosphatase 5 that were in good agreement with quantitative Western blot analyses.
We have developed an analytical method, consisting of ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IP-LC-ESI-MS), for the simultaneous quantitative analysis of several key classes of polar metabolites, like nucleotides, coenzyme A esters, sugar nucleotides, and sugar bisphosphates. The use of the ion-pair agent hexylamine and optimization of the pH of the mobile phases were critical parameters in obtaining good retention and peak shapes of many of the above-mentioned polar and acidic metabolites that are impossible to analyze using standard reversed-phase LC/MS. Optimum conditions were found when using a gradient from 5 mM hexylamine in water (pH 6.3) to 90% methanol/10% 10 mM ammonium acetate (pH 8.5). The IP-LC-ESI-MS method was extensively validated by determining the linearity (R2 > 0.995), sensitivity (limit of detection 0.1-1 ng), repeatability, and reproducibility (relative standard deviation <10%). The IP-LC-ESI-MS method was shown to be a useful tool for microbial metabolomics, i.e., the comprehensive quantitative analysis of metabolites in extracts of microorganisms, and for the determination of the energy charge, i.e., the cellular energy status, as an overall quality measure for the sample workup and analytical protocols.
The diagnosis of cancer by examination of the urine has the potential to improve patient outcomes by means of earlier detection. Due to the fact that the urine contains metabolic signatures of many biochemical pathways, this biofluid is ideally suited for metabolomic analysis, especially involving diseases of the kidney and urinary system. In this pilot study, we test three independent analytical techniques for suitability for detection of renal cell carcinoma (RCC) in urine of affected patients. Hydrophilic interaction chromatography (HILIC-LC-MS), reversed-phase ultra performance liquid chromatography (RP-UPLC-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) all were used as complementary separation techniques. The combination of these techniques is best suited to cover a very large part of the urine metabolome by enabling the detection of both lipophilic and hydrophilic metabolites present therein. In this study, it is demonstrated that sample pretreatment with urease dramatically alters the metabolome composition apart from removal of urea. Two new freely available peak alignment methods, MZmine and XCMS, are used for peak detection and retention time alignment. The results are analyzed by a feature selection algorithm with subsequent univariate analysis of variance (ANOVA) and a multivariate partial least squares (PLS) approach. From more than 2000 mass spectral features detected in the urine, we identify several significant components that lead to discrimination between RCC patients and controls despite the relatively small sample size. A feature selection process condensed the significant features to less than 30 components in each of the data sets. In future work, these potential biomarkers will be further validated with a larger patient cohort. Such investigation will likely lead to clinically applicable assays for earlier diagnosis of RCC, as well as other malignancies, and thereby improved patient prognosis.
Two stationary phases attached to a silica hydride surface, cholesterol and bidentate C18, are investigated with a number of pharmaceutically related compounds in order to illustrate the various retention mechanisms that are possible for these bonded materials. The test solutes range from hydrophilic to hydrophobic based on log P (octanol/water partition coefficient) and pKa values. The mobile phases consist of acidified (formic and perchloric acid) water/methanol or water/ACN mixtures. Of particular interest are the high organic content mobile phase compositions where the retention would increase if the bonded material was operating in the aqueous normal phase (ANP) mode. Plots of retention factor (k) versus mobile phase composition are used to elucidate the retention mechanism. A number of examples are presented where solutes are retained based on RP, ANP, or dual retention mechanisms. The silica hydride-based stationary phases can also retain compounds in the organic normal phase.
High-performance liquid chromatography (LC) coupled to mass spectrometry (MS) is increasingly being used for urinary metabonomic studies. Most studies utilize reversed-phase separation techniques, which are not suited to retaining highly polar analytes. Metabonomic studies should encompass a representative "fingerprint" that contains the largest amount of information possible. In this work, we have analyzed human urine samples with LC-MS, comparing traditional reversed-phase separation with hydrophilic interaction chromatography (HILIC), using both positive and negative electrospray ionization modes. The resulting data were analyzed using principal components analysis and partial least-squares-discriminant analysis. Discriminant models were developed for the response variables gender, diurnal variation, and age and were evaluated using external test sets to classify their predictive ability. The developed models using both positive and negative ionization mode data for reversed-phase and HILIC separations were very comparable, indicating that HILIC is a suitable method for increasing the fingerprint coverage for LC-MS metabonomic studies.
Stimulants are banned by the World Anti-Doping Agency (WADA) if used "in competition". Being the analysis of stimulants presently carried out on urine samples only, it might be useful, for a better interpretation of analytical data, to discriminate between an early intake of the substance and an administration specifically aimed to improve the sport performance. The purpose of the study was to investigate the differences, in terms of excretion/disappearance of drugs, between urine and oral fluid, a sample that can reflect plasmatic concentrations. Oral fluid and urine samples were collected following oral administration of the following stimulants: modafinil (100 mg), selegiline (10 mg), crotetamide/cropropamide (50 mg each), pentetrazol (100 mg), ephedrine (12 mg), sibutramine (10 mg), mate de coca (a dose containing about 3mg of cocaine); analysis of drugs/metabolites was carried out by gas chromatography/mass spectrometry (GC/MS) in both body fluids. Our results show that both the absolute concentrations and their variation as a function of time, in urine and in oral fluid, are generally markedly different, being the drugs eliminated from urine much more slowly than from oral fluid. Our results also suggest that the analysis of oral fluid could be used to successfully complement the data obtained from urine for "in competition" anti-doping tests; in all those cases in which the metabolite(s) concentration of a substance in urine is very low and the parent compound is not detected, it is indeed impossible, relying on urinary data only, to discriminate between recent administrations of small doses and remote administrations of higher doses.
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