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

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

Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry

Olaquindox (N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide) is a growth-promoting feed additive for food-producing animals. Its toxicity is closely related to the metabolism. The complete metabolic pathways of olaquindox are not revealed. To improve studies of the metabolism and toxicity of olaquindox, its biotransformation in rat liver microsomes and the structure of its metabolites using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) were investigated. When olaquindox was incubated with an NADPH-generating system and rat liver microsomes, ten metabolites (M1-M10) were detected. The structures of these metabolites were identified from mass spectra and comparison of their changes in their accurate molecular masses and fragment ions with those of the parent drug. With the high resolution and good mass accuracy achieved by this technique, the elemental compositions of the metabolites and their fragment ions were exactly determined. The results indicate that the N --> O group reduction is the main metabolic pathway of olaquindox metabolism in rat liver microsomes, because abundant 1-desolaquindox (M2), 4-desolaquindox (M1) and bisdesoxyolaquindox (M9) were produced during the incubation step. Seven other minor metabolites were revealed which were considered to be hydroxylation metabolites, based on the position of the quinoxaline ring or 3-methyl group and a carboxylic acid derivative on the side chain at position 2 of the quinoxaline ring. Among the identified metabolites, five new hydroxylated metabolites (M3-M7) were found for the first time in rat liver microsomes. This work will conduce to complete clarification of olaquindox metabolism, and improve the in vivo metabolism of olaquindox in food animals.     

Profiling of ecdysteroids in complex biological samples using liquid chromatography/ion trap mass spectrometry

A sensitive method using high-performance liquid chromatography coupled to a mass spectrometer with electrospray ionization source (HPLC/ESI-MS) was developed for detection of ecdysteroids in biological samples. We report here for the first time that ecdysteroids can be classified into three groups based on ESI full-scan mass spectra: group 1 (ecdysone (E), 2-deoxyecdysone (2dE), 2,22-dideoxyecdysone (3beta5beta-KT), and 3alpha5alpha[H]-dihydroxycholest-7-en-6-one (3alpha5alpha-KD)), in which loss of one molecule of water from the protonated molecular ion ([M+H](+)) represents the dominant ion; group 2 (20-hydroxyecdysone (20E), makisterone A (MakA), 3beta5beta-KD, and 3beta5alpha-KD), in which [M+H](+) is a major ion but some water loss is observed; and group 3 (muristerone A (MurA) and ponasterone A (PonA)), in which [M+H](+) is the dominant ion with no water loss observed. Based on the analytical procedure in combination with structural information from the group classification and with the application of source-induced dissociation, we identified free ecdysteroids in biological samples: 20,26-dihydroxyecdysone and ecdysonic acid in the larval hemolymph, and the progressive metabolism of 26-hydroxyecdysone (26E) to 3alpha-26E from day-1 to day-3 embryos of the tobacco hornworm Manduca sexta.     

Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression

The effect of liquid chromatography separation on liquid chromatography-tandem mass spectrometry (LC-MS-MS) signal response for the characterization of low-molecular-mass compounds in a complex matrix was investigated. Matrix induced signal suppression appears throughout the entire LC-MS-MS analysis of wheat forage extract, with greatest suppression occurring at early retention times. Experimental results show that co-elution of matrix components and analytes from the LC column may be most strongly attributed to column overloading rather than similar analyte and matrix retention behavior. As a result, two-dimensional (LC-LC) separation can be a highly effective approach to address signal suppression effects for the quantitative LC-MS-MS analysis of complex matrix samples.     

Structure elucidation of three isomeric metabolites of SYN-2836, a novel antifungal agent, in dogs via liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry methodologies.

This paper presents liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) approaches for the rapid characterization of three urinary isomeric metabolites and their two precursor metabolites of SYN-2836, a novel antifungal agent, in dogs administered multiple oral doses of the agent (30 mg kg(-1) day(-1)). A collection of correlative data regarding the SYN-2836 metabolites was obtained by LC/MS and LC/MS/MS performed under complementary conditions such as the columns (C(18) vs cyano type), the mobile phase systems (acetonitrile-water-formic acid vs acetonitrile-water-ammonium acetate) and the electrospray ionization modes (positive vs negative). Metabolite identification was accomplished based on not only the LC/MS/MS data (product ion spectra) but also the LC/MS data indicating chromatographic behaviors of the metabolites. SYN-2836 and SYN-2869, an analog of the former, showed almost the same metabolic pathways following the same multiple-dose administration of the individual agents to the dogs. Therefore, correlation analysis in product ion spectra between corresponding metabolites of SYN-2836 and SYN-2869, and also in metabolic pathways between the two agents, was strategically used to facilitate the identification of the SYN-2836 (and SYN-2869 if necessary) metabolites. For the reason that various elucidation strategies were used complementarily, the chemical structures of the metabolites were unambiguously attained and the isomeric metabolites were explicitly differentiated without the use of other analytical methods. The methodologies used in this study may be applicable to metabolite screening of several structurally related agents simultaneously, promoting lead finding and optimization of drug candidates using a metabolism-based approach.     

Simultaneous determination of 186 fungal and bacterial metabolites in indoor matrices by liquid chromatography/tandem mass spectrometry

This paper describes the application of a previously published multi-mycotoxin method for food and feed matrices based on liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) to the analysis of microbial metabolites in indoor matrices. The range of investigated analytes has been extended by 99 fungal and bacterial metabolites to cover now 186 compounds overall. The method is based on a single extraction step using an acidified acetonitrile/water mixture (which has been determined to be preferable to methanol and ethyl acetate) followed by analysis of the diluted crude extract. The analytical signal of one third of the investigated analytes was reduced by more than 50% due to matrix effects in a spiked extract of house dust, whereas the other investigated materials were less critical in that aspect. For determination of method performance characteristics, a spiked reference material for house dust was chosen as a model sample for an extremely complex matrix. With few exceptions, coefficients of variation of the whole procedure of <10% and limits of detection of <50 μg kg⁻¹ were obtained. The apparent recoveries were below 50% for half of the investigated analytes due to incomplete extraction and/or detection-related matrix effects. The application of the method to 14 samples from damp buildings revealed the presence of 20 different analytes at concentrations of up to 130 mg kg⁻¹. Most of these compounds have never been identified before in real-world samples, although they are known to be produced by indoor-relevant fungi. This underlines the great value of the described method for the on-site determination of microbial metabolites.      

Identification of bupropion urinary metabolites by liquid chromatography/mass spectrometry

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

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

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

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

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

Structure elucidation of four related substances in gramicidin with liquid chromatography/mass spectrometry

A selective reversed phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of related substances in commercial gramicidin samples. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray interface operated in the positive and the negative ion mode. The LCQ is ideally suited for identification of related substances because it provides on-line LC/MS(n) capability. Compared with UV detection the main advantage of this hyphenated LC/MS(n) technique is the efficient identification of novel related substances without time-consuming isolation and purification procedures. Using this method four novel related substances were separated and identified in a commercial sample.     

Quantitation of human glutathione S-transferases in complex matrices by liquid chromatography/tandem mass spectrometry with signature peptides

Direct quantitation of glutathione S-transferase (GST) isoforms [alpha (GST-A) and micro (GST-M)] in human liver cytosol was achieved by liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) analysis of signature peptides of GST-A and GST-M and their corresponding stable isotopic peptide internal standards via multiple reaction monitoring (MRM). The selection of signature peptides was performed via trypsin digestion of commercially available cDNA-expressed GST-A1 and GST-M1, followed by LC/ESI-MS/MS with an ion trap mass spectrometer and sequencing with the TurboSEQUEST application. Quantitative analysis of the selected signature peptides in the multi-reaction monitoring (MRM) mode was performed using a triple-quadruple mass spectrometer. A series of human cytosol samples was quantitatively analyzed for levels of GST-A and GST-M. The total level of GST-A and GST-M obtained from this LC/ESI-MS/MS method was well correlated with the total level of GST determined by the 1-chloro-2,4-dinitrobenzene (CDNB) method.     

Rapid structural elucidation of composite bacterial hopanoids by atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry

Bacteriohopanepolyols (BHPs) are membrane lipids produced by a wide range of eubacteria. Their use, however, as molecular markers of bacterial populations and processes has until recently been hampered by the lack of a suitable rapid method for fingerprinting their composition in complex environmental matrices. New analytical procedures employing ion trap mass spectrometry now allow us to investigate the occurrence of BHPs in diverse biological and environmental samples including bacterial cultures, soils, and recent and ancient sediments. Here, we describe the structural characterisation using atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry (APCI-LC/MS(n)) of a number of previously identified but less commonly occurring BHPs such as adenosylhopane and ribonylhopane. Many of the structures described here have previously only been reported in one or just a small number of cultured organisms having been isolated from large amounts of cellular mass (4-26 g) and identified by nuclear magnetic resonance (NMR) techniques after purification of individual compounds. Now, having established characteristic APCI fragmentation patterns, it is possible to rapidly screen many more bacterial cultures using only small amounts of material (<50 mg) as well as environmental samples for these atypical structures and a rapidly growing suite of novel structures.     

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

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

Comparison of a two-dimensional liquid chromatography/mass spectrometry approach with a chip-based nanoelectrospray device for structural elucidation of metabolites in a human ADME study using a quadrupole time-of-flight mass spectrometer

The study of the metabolic fate of drugs is essential for the safety assessment of new compounds in the drug development process. However, the characterization and structural elucidation of metabolites from in vivo experiments is still a very challenging task. In this paper, we compare a two-dimensional liquid chromatography/mass spectrometry (LC/MS) approach using either a capillary LC/MS system or the recently introduced chip-based nanoelectrospray/MS system (Nanomate) as the second dimension for structural elucidation of metabolites by MS. More than 30 radioactive fractions of a chromatographic separation from a human urine sample were analyzed and 54 metabolites could be identified. The long persisting and stable nanoelectrospray enabled the search for unknown metabolites by precursor-ion scanning experiments followed by product-ion scanning experiments of potential metabolites using a quadrupole time-of-flight (qTOF) mass spectrometer. The number of fragments produced by nanoelectrospray with product-ion scanning was significantly higher compared to LC/MS experiments with in-source fragmentation. Therefore, the assignment of possible modifications in metabolites to certain moieties of the drug could be investigated with higher accuracy. The capillary LC/MS system for the second dimension was more sensitive in the case of low abundant metabolites. These metabolites could not be detected by direct nanoelectrospray infusion, which limits the application of the Nanomate for trace metabolites.     

The reproducible acquisition of comparative liquid chromatography/tandem mass spectrometry data from complex biological samples

An in-depth study of the reproducibility of data acquired for comparative proteomics analysis using a prototype two-stage heated laminar flow chamber fitted to a commercial high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) instrument was undertaken. The study is based on 24 replicate samples from four independent membrane preparations derived from two matched breast cancer cell lines. Variation and reproducibility in the data were evaluated at several levels highlighting the relative efficiency and variability of the acquisition routines used. Specifically, variation in the number and relative intensities of chromatographic peaks eluted from the LC column, precursor ion selection and sequence identification were evaluated. On average, approximately 6500 chromatographic peaks were generated for each acquisition with a corresponding coefficient of variance (CV) of less than 20%. Precursor ion selection and sequence identification averaged 1380 and 780 events per acquisition sample, respectively, with corresponding CVs of less than 10% for each. The reproducibility in the precursor ion selection was typically better than 60% between similar replicates. Using protein and peptide internal standards, it was found that the CV in retention time across the gradient between two acquisition pairs was typically less than 5%, whereas the average intensity ratio was 1.0 (expected) with a CV approaching 20%. An evaluation of the intensity ratios calculated from endogenous peptide sequences, identified across the acquisition set, indicated a CV of approximately 30%. Similarly, the CV associated with the top 1000 peptides indicated a mean and median of 28.4 and 26.95%. For a given acquisition pair it was also found that approximately 11% of the chromatographic peaks eluting from the column were linked to a sequence or identified. For these experiments, less than 10% of the peak pairs had absolute ratios greater than 2.0 and of those only approximately 10% had sequences linked to them. For each matched acquisition set on average 406 proteins were identified with a CV of less than 10%. Of the proteins that were identified approximately 30% had at least one predicted trans-membrane domain, indicating a four-fold increase over a crude homogenate sample with only minor enrichment. During these experiments it was found that the interface did not significantly alter the relative charge state distribution of ions, nor did it introduce significant interference from background ions. The interface was capable of 24-hour acquisition cycles.     

Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry

A phospholipid mixture extracted from cultured cells was directly analyzed by capillary (Cap) liquid chromatography (LC)/electrospray ionization (ESI) mass spectrometry (MS). Using a quadrupole mass spectrometer, we analyzed positive molecular ions, negative molecular ions, positive fragment ions and negative fragment ions under four different functions. In the analysis of the elution patterns of the phospholipids, a two-dimensional map, in which the first dimension is elution time and the second dimension is mass, proved useful. Consequently, four different maps can be obtained by each of four different functions. Among them, from negative fragment ions at high cone voltage in the negative ion mode, ions that originated from acyl fatty acid and phosphorylcholine, phosphorylethanolamine and cyclic inositol phosphate can be detected at specific elution times. The map from positive fragment ions at high cone voltage in the positive ion mode indicated ions such as diradylglycerol and derivatives of 1-alkyl or 1-alkenyl cyclic phosphatidic acid from phosphatidylethanolamine (PE), and phosphorylcholine from choline-containing phospholipids. The map produced from positive molecular ions indicated choline-containing phospholipids such as phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and PE. The map of negative molecular ions effectively indicated acidic phospholipids such as phosphatidylinositol. We were able to obtain more than 500 molecular species of phospholipids by this method within a few hours immediately after extraction from culture cells using a mixture of chloroform and methanol (2:1). In this context, we concluded that the combination of Cap-LC and ESIMS seems to be very effective in the analysis of phospholipid classes and their molecular species.     

Structural elucidation of in vitro metabolites of emodin by liquid chromatography-tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was employed to investigate the in vitro metabolism of emodin. Emodin was incubated with rat liver microsomes in the presence of a NADPH-generating system, followed by extraction with ethyl acetate. After separation on a reversed-phase C18 analytical column with a linear gradient elution of methanol and 0.1% formic acid in water, negative electrospray ionization tandem mass spectrometry experiments were performed. As a result, the parent drug and its six metabolites were detected from rat liver microsomal incubations. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. Besides three mono-hydroxylated metabolites (omega-hydroxyemodin, 2-hydroxyemodin, 4-hydroxyemodin), three other metabolites were identified, which were emodic acid, 3-carbomethoxy-6-methoxy-1,8-dihydroxyanthraquinone and physcion, respectively.