Mixture designs for exploring class diversity and metabolite fingerprinting: an efficient column chromatographic strategy

The effects of four solvents, hexane, dichloromethane, ethyl acetate, methanol, and their mixtures on the separation of metabolites in crude extracts of Erythrina speciosa Andrews leaves were investigated using two strategies for open column chromatography. The classical extraction procedure was compared with mobile phases prepared according to a mixture design in order to explore the effects of solvent interactions on metabolite separations. Principal component analysis was used to compare the UV spectra obtained from RP-HPLC-DAD and to estimate the number of independent factors contained in the chromatographic data of the extracts. The results showed that, in addition to solvent polarity, solvent mixtures play an important role in metabolite separation. When pure solvents are used, larger groups of similar spectra are observed in the factor analysis score graphs indicating the same or a limited number of metabolite classes. In contrast solvent mixtures produced score graphs with a larger number of clusters indicating greater metabolic diversity. Besides resulting in more peaks than the pure solvents the chromatographic data of the design mixtures resulted in larger numbers of significant principal components confirming the greater chemical diversity of their extracts. Thus, if the objective of an analysis is to obtain metabolites of the same class, one should use pure solvents. On the other hand, binary and ternary solvent mixtures are recommended for more efficient investigations of class diversity and richer metabolite fingerprints.     

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.     

Liquid chromatography/mass spectrometry-based metabolic profiling to elucidate chemical differences of tobacco leaves between Zimbabwe and China

An approach was developed for extracting and analyzing the chemical components of tobacco leaves based on solvent extraction and rapid & resolution liquid chromatography/quadrupole time-of-flight mass spectrometry analysis. Two solvents with different polarities were used to extract hydrophilic components and hydrophobic components, respectively, the combined analytical data can provide a "global" view of metabolites. Based on the evaluation of parallel samples, it was found that this approach provided good repeatability, accurate and reliable profiling data, and is suitable for the metabolomics study of tobacco leaves. In order to find the chemical component differences of tobacco leaves, 56 samples from Zimbabwe and China were analyzed using the developed method. The metabolite data were processed by multivariate statistic technique; an obvious group classification between Zimbabwe and China was observed, 14 significantly changed compounds were found, and 9 of them were identified.     

Quantitative estimation of circulating metabolites without synthetic standards by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction

An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.     

基于液相色谱-质谱技术的肾脏代谢组学分析方法研究

建立了一种基于高效液相色谱-超高分辨质谱技术的肾脏代谢组学分析方法。肾脏组织于液氮中研磨成粉后,采用甲基叔丁基醚/甲醇/水溶剂体系进行提取,分别得到强极性部分(下层)和弱极性部分(上层)提取物,依次采用HILIC亲水色谱柱和反相C18色谱柱进行梯度洗脱分离后,进行高分辨质谱分析。采用电喷雾离子源(ESI),正、负离子模式检测,扫描方式为全扫描,扫描范围为100~1000 Da,质量分辨率为120000。结果表明,该方法灵敏度高,专属性强,稳定性良好,可同时获取肾脏组织中的强极性和弱极性代谢物信息,可为肾脏疾病和药物肾毒性生物标志物的发现提供一种新的方法。     

Multivariate approaches for efficient detection of potential metabolites from liquid chromatography/mass spectrometry data

This work describes a novel method for rapid screening of unknown metabolites in urine samples that narrows down the list of potential metabolites. Prior to analysis by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS), urine samples were prepared using solid-phase extraction (SPE). Automatic curve resolution was used for deconvolution of the LC/MS data, followed by peak alignment. Preprocessed data were then used for metabolite pattern recognition using principal component analysis (PCA), parallel factor analysis (PARAFAC), and multilinear partial least squares (N-PLS). This approach enabled the rapid detection of metabolites of citalopram in urine by maximizing the information extracted. The metabolites thus identified were compared with earlier studies on the metabolism of citalopram. In addition, new, unreported metabolites were found and characterized by LC/MS/MS and accurate mass measurements. A combination of data from positive and negative ionization enhanced the identification of metabolites.     

Development of rapid methodologies for the isolation and quantitation of drug metabolites by differential mobility spectrometry – mass spectrometry

Clinical and forensic toxicology laboratories are inundated with thousands of samples requiring lengthy chromatographic separations prior to mass spectrometry. Here, we employ differential mobility spectrometry (DMS) interfaced to nano-electrospray ionization-mass spectrometry to provide a rapid ion filtration technique for the separation of ions in gas phase media prior to mass spectral analysis on a DMS-integrated AB SCIEX API 3000 triple-quadrupole mass spectrometer. DMS is efficient at the rapid separation of ions under ambient conditions and provides many advantages when used as an ion filtration technique in tandem with mass spectrometry (MS) and MS/MS. Our studies evaluated DMS-MS/MS as a rapid, quantitative platform for the analysis of drug metabolites isolated from urine samples. In targeted applications, five metabolites of common drugs of abuse were effectively and rapidly separated using isopropanol and ethyl acetate as transport gas modifiers, eliminating the gas chromatography or liquid chromatography-based separations commonly employed in clinical and forensic toxicology laboratories. Calibration curves were prepared for the selected drug metabolites utilizing deuterated internal standards for quantitative purposes. The feasibility of separating and quantitating drug metabolites in a rapid fashion was evaluated by compensation voltage stepping followed by multiple reaction monitoring (MRM) detection. Rapid profiling of clinical and forensic toxicology samples could help to address an urgent need within the scientific community by developing high-throughput analytical methodologies, which could reduce significant case backlogs present within these laboratories.     

MSE with mass defect filtering for in vitro and in vivo metabolite identification

Metabolite identification studies involve the detection and structural characterization of the biotransformation products of drug candidates. These experiments are necessary throughout the drug discovery and development process. The use of high-resolution chromatography and high-resolution mass spectrometry together with data processing using mass defect filtering is described for in vitro and in vivo metabolite identification studies. Data collection was done using UPLC coupled with an orthogonal hybrid quadrupole time-of-flight mass spectrometer. This experimental approach enabled the use of MS(E) data collection (where E represents collision energy) which has previously been shown to be a powerful approach for metabolite identification studies. Post-acquisition processing with a prototype mass defect filtering program was used to eliminate endogenous interferences in the study samples, greatly enhancing the discovery of metabolites. The ease of this approach is illustrated by results showing the detection and structural characterization of metabolites in plasma from a preclinical rat pharmacokinetic study.     

Rapid and efficient purification of cimetropium bromide and mifentidine drug metabolite mixtures derived from microsomal incubates for analysis by mass spectrometry.

A comparative study of the use of organic solvent extraction versus Sep-Pak C18 cartridges in the recovery and analysis of phase I (unconjugated) drug metabolites using mass spectrometry is presented. Standard mixtures of putative metabolites of the anticholinergic drug cimetropium bromide and the H2-antagonist mifentidine were purified from inactivated liver microsomal preparations using both methods, and subsequently the recovery of each compound was quantitated. In general, the percentage recovery and degree of purification were greater when using Sep-Pak C18 cartridges compared with organic solvent extraction. Even more efficient recovery was achieved when zinc sulphate precipitation of proteins in the liver microsomal mixtures was carried out prior to analysis. Also, the HPLC-grade solvents used in this study contained a variety of ultraviolet-inactive, hydrophobic components. This leads to problems of suppression in fast atom bombardment mass spectrometric analysis. Using Sep-Pak C18 cartridges directly prior to analysis by fast atom bombardment with single or tandem mass spectrometry leads to far superior mass spectral results compared with organic solvent extraction.     

Quantitative determination of valproic acid and 14 metabolites in serum and urine by gas chromatography/mass spectrometry.

A method for the determination of the antiepileptic drug valproic acid and 14 of its metabolites in serum and urine by gas chromatography/mass spectrometry with selected ion monitoring of the trimethylsilylated derivatives has been developed. Sample preparation, including hydrolysis of VPA-conjugates and removal of urea in urine is carried out at pH 5.0 and is rapid and simple. The samples are extracted with ethyl acetate and the concentrated extracts are trimethylsilylated. Analysis with adequate separation of metabolites is achieved with a DB 1701 fused silica (Megabore) capillary column. The method exhibits high recovery and reproducibility and is sufficiently sensitive and selective for analysis of small sample volumes. Application of the method for screening patient serum and urine samples for unusual metabolite patterns, with possible predictive value for early detection of liver injury, is presented.     

基于气相色谱-质谱联用技术的水稻代谢轮廓分析方法的建立

开展了基于衍生化气相色谱-质谱联用的水稻代谢物分析方法的研究。采用D-最优试验设计对代谢物的提取溶剂进行优化,考察了水、甲醇、乙腈和异丙醇的提取效率,通过多元统计分析评价提取效能和溶剂配比的相关性,最终确立以80%(v/v)甲醇/水作为代谢物提取的最适溶剂。在此基础上对该方法的分析性能进行评价,发现绝大多数代谢物(＞90%)具有良好的精密度、重现性和稳定性(相对标准偏差小于30%),且占总峰面积88.0%的代谢物的响应值与其浓度间呈线性关系(相关系数＞0.9)。采用气相色谱-质谱联用方法从水稻种子中共鉴定出86个代谢物,涵盖糖、氨基酸、有机酸、甾体等多类浓度差异大的物质,适合于水稻的代谢表型差异研究。     

化学衍生用于代谢物异构体质谱分析

内源性代谢物是机体生命活动的中间体和终产物,对其进行定性和定量分析在生命科学研究中具有重要意义.质谱能够同时提供化合物的定性和定量信息,已经成为一种通用的内源性代谢物分析技术.由于质谱是通过检测离子质荷比获取化合物组成信息,区分生物体内复杂多样代谢物同分异构体仍然是质谱分析亟待解决的难题之一.化学衍生通过放大同分异构体...     

Combining desorption electrospray ionization mass spectrometry and nuclear magnetic resonance for differential metabolomics without sample preparation

Desorption electrospray ionization mass spectrometry (DESI-MS) and nuclear magnetic resonance (NMR) spectroscopy are used to provide data on urine examined without sample preparation to allow differentiation between diseased (lung cancer) and healthy mice. Principal component analysis (PCA) is used to shortlist compounds with potential for biomarker screening which are responsible for significant differences between control urine samples and samples from diseased animals. Similar PCA score plots have been achieved by DESI-MS and NMR, using a subset of common detected metabolites. The common compounds detected by DESI and NMR have the same changes in sign of their concentrations thereby indicating the usefulness of corroborative analytical methods. The effects of different solvents and surfaces on the DESI mass spectra are also evaluated and optimized. Over 80 different metabolites were successfully identified by DESI-MS and tandem mass spectrometry experiments, with no prior sample preparation.     

Coated Blade Spray-Mass Spectrometry as a New Approach for the Rapid Characterization of Brain Tumors

Brain tumors are neoplasms with one of the highest mortality rates. Therefore, the availability of methods that allow for the quick and effective diagnosis of brain tumors and selection of appropriate treatments is of critical importance for patient outcomes. In this study, coated blade spray-mass spectrometry (CBS-MS), which combines the features of microextraction and fast ionization methods, was applied for the analysis of brain tumors. In this approach, a sword-shaped probe is coated with a sorptive material to enable the extraction of analytes from biological samples. The analytes are then desorbed using only a few microliters of solvent, followed by the insertion of the CBS device into the interface on the mass spectrometer source. The results of this proof-of-concept experiment confirmed that CBS coupled to high-resolution mass spectrometry (HRMS) enables the rapid differentiation of two histologically different lesions: meningiomas and gliomas. Moreover, quantitative CBS-HRMS/MS analysis of carnitine, the endogenous compound, previously identified as a discriminating metabolite, showed good reproducibility with the variation below 10% when using a standard addition calibration strategy and deuterated internal standards for correction. The resultant data show that the proposed CBS-MS technique can be useful for on-site qualitative and quantitative assessments of brain tumor metabolite profiles.     

Screening strategy for the rapid detection of in vitro generated glutathione conjugates using high‐performance liquid chromatography and low‐resolution mass spectrometry in combination with LightSight® software for data processing

The knowledge of drug metabolism in the early phases of the drug discovery process is vital for minimising compound failure at later stages. As chemically reactive metabolites may cause adverse drug reactions, it is generally accepted that avoiding formation of reactive metabolites increases the chances of success of a molecule. In order to generate this important information, a screening strategy for the rapid detection of in vitro generated reactive metabolites trapped by glutathione has been developed. The bioassay incorporated the use of native glutathione and its close analogue the glutathione ethyl ester. The generic conditions for detecting glutathione conjugates that undergo constant neutral loss of 129 Da were optimised using a glutathione‐based test mix of four compounds. The final liquid chromatography/tandem mass spectrometry constant neutral loss method used low‐resolution settings and a scanning window of 200 amu. Data mining was rapidly and efficiently performed using LightSight® software. Unambiguous identification of the glutathione conjugates was significantly facilitated by the analytical characteristics of the conjugate pairs formed with glutathione and glutathione ethyl ester, i.e. by chromatographic retention time and mass differences. The reliability and robustness of the screening strategy was tested using a number of compounds known to form reactive metabolites. Overall, the developed screening strategy provided comprehensive and reliable identification of glutathione conjugates and is well suited for rapid routine detection of trapped reactive metabolites. This new approach allowed the identification of a previously unreported diclofenac glutathione conjugate. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Ion-pairing reversed-phase liquid chromatography fractionation in combination with isotope labeling reversed-phase liquid chromatography-mass spectrometry for comprehensive metabolome profiling

We report a novel two-dimensional (2D) separation strategy aimed at improving the detectability of liquid chromatography mass spectrometry (LC-MS) for metabolome analysis. It is based on the use of ion-pairing (IP) reversed-phase (RP) LC as the first dimension separation to fractionate the metabolites, followed by isotope labeling of individual fractions using dansylation chemistry to alter the physiochemical properties of the metabolites. The labeled metabolites having different hydrophobicity from their unlabeled counterparts are then separated and analyzed by on-line RPLC Fourier-transform ion-cyclotron resonance mass spectrometry (FTICR-MS). This off-line 2D-LC-MS strategy offers significant improvement over the one-dimensional (1D) RPLC MS technique in terms of the number of detectable metabolites. As an example, in the analysis of a human urine sample, 3564 13C-/12C-dansylated ion pairs or metabolites were detected from seven IP RPLC fractions, compared to 1218 metabolites found in 1D-RPLC-MS. Using a library of 220 amine- and phenol-containing metabolite standards, 167 metabolites were positively identified based on retention time and accurate mass matches, which was about 2.5 times the number metabolites identified by 1D-RPLC-MS analysis of the same urine sample.     

Characterizing ionic liquids on the basis of multiple solvation interactions

Room-temperature ionic liquids (RTILs) are useful in many chemical applications. Recent publications have attempted to determine the polarity of RTILs using empirical solvent polarity scales. The results have indicated that most RTILs have similar polarities. Nevertheless, RTILs are capable of behaving quite differently when used as solvents in organic synthesis, matrixes in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, liquid-liquid extraction, and as stationary phases in gas chromatography. The work presented in this study uses a linear free energy approach to characterize 17 RTILs on the basis of their distinct multiple solvation interactions with probe solute molecules. This model provides data that can be used to help identify the interactions and properties that are important for specific chemical applications.