An algorithm for thorough background subtraction from high-resolution LC/MS data: application to the detection of troglitazone metabolites in rat plasma, bile, and urine

Interferences from biological matrices remain a major challenge to the in vivo detection of drug metabolites. For the last few decades, predicted metabolite masses and fragmentation patterns have been employed to aid in the detection of drug metabolites in liquid chromatography/mass spectrometry (LC/MS) data. Here we report the application of an accurate mass-based background-subtraction approach for comprehensive detection of metabolites formed in vivo using troglitazone as an example. A novel algorithm was applied to check all ions in the spectra of control scans within a specified time window around an analyte scan for potential background subtraction from that analyte spectrum. In this way, chromatographic fluctuations between control and analyte samples were dealt with, and background and matrix-related signals could be effectively subtracted from the data of the analyte sample. Using this algorithm with a +/- 1.0 min control scan time window, a +/- 10 ppm mass error tolerance, and respective predose samples as controls, troglitazone metabolites were reliably identified in rat plasma and bile samples. Identified metabolites included those reported in the literature as well as some that had not previously been reported, including a novel sulfate conjugate in bile. In combination with mass defect filtering, this algorithm also allowed for identification of troglitazone metabolites in rat urine samples. With a generic data acquisition method and a simple algorithm that requires no presumptions of metabolite masses or fragmentation patterns, this high-resolution LC/MS-based background-subtraction approach provides an efficient alternative for comprehensive metabolite identification in complex biological matrices. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Investigation of uremic analytes in hemodialysate and their structural elucidation from accurate mass maps generated by a multi‐dimensional liquid chromatography/mass spectrometry approach

Historically, structural elucidation of unknown analytes by mass spectrometry alone has involved tandem mass spectrometry experiments using electron ionization. Most target molecules for bioanalysis in the metabolome are unsuitable for detection by this previous methodology. Recent publications have used high‐resolution accurate mass analysis using an LTQ‐Orbitrap with the more modern approach of electrospray ionization to identify new metabolites of known metabolic pathways. We have investigated the use of this methodology to build accurate mass fragmentation maps for the structural elucidation of unknown compounds. This has included the development and validation of a novel multi‐dimensional LC/MS/MS methodology to identify known uremic analytes in a clinical hemodialysate sample. Good inter‐ and intra‐day reproducibility of both chromatographic stages with a high degree of mass accuracy and precision was achieved with the multi‐dimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) system. Fragmentation maps were generated most successfully using collision‐induced dissociation (CID) as, unlike high‐energy CID (HCD), ions formed by this technique could be fragmented further. Structural elucidation is more challenging for large analytes >270 Da and distinguishing between isomers where their initial fragmentation pattern is insufficiently different. For small molecules (<200 Da), where fragmentation data may be obtained without loss of signal intensity, complete structures can be proposed from just the accurate mass fragmentation data. This methodology has led to the discovery of a selection of known uremic analytes and two completely novel moieties with chemical structural assignments made. Copyright © 2009 John Wiley & Sons, Ltd.     

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3 ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.     

Metabolite identification of artemether by data-dependent accurate mass spectrometric analysis using an LTQ-Orbitrap hybrid mass spectrometer in combination with the online hydrogen/deuterium exchange technique

Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is a first-line antimalarial drug used in areas of multi-drug resistance. Artemisinin drugs can be metabolized extensively in vivo and this seems related to their autoinduction pharmacokinetics. In the present study, the metabolite identification of ARM was performed by the generic data-dependent accurate mass spectrometric analysis, using high-resolution (HR) liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange for rapid structural characterization. The LC separation was improved allowing the separation of ARM parent drugs and their metabolites from their diastereomers. A total of 77 phase I metabolites of ARM were identified in rat liver microsomal incubates and rat urine, including dihydroartemisinin and artemisinin. In rat bile, 12 phase II metabolites were found. Accurate mass data were obtained in both full scan and HR-MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC/HR-ESI-MS experiments provided additional evidence in differentiating dihydroxylated deoxy-ARM from mono-hydroxylated ARM. The results showed the main phase I metabolites of artemether are hydroxylated, dehydro, demethylated and deoxy products, and they will undergo subsequent phase II glucuronidation processes. Most metabolites were reported for the first time. This study also demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange LC/HR-MS(n) technique in rapid identification of drug metabolites.     

Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system

Acceleration of liquid chromatography/mass spectrometric (LC/MS) analysis for metabolite identification critically relies on effective data processing since the rate of data acquisition is much faster than the rate of data mining. The rapid and accurate identification of metabolite peaks from complex LC/MS data is a key component to speeding up the process. Current approaches routinely use selected ion chromatograms that can suffer severely from matrix effects. This paper describes a new method to automatically extract and filter metabolite-related information from LC/MS data obtained at unit mass resolution in the presence of complex biological matrices. This approach is illustrated by LC/MS analysis of the metabolites of verapamil from a rat microsome incubation spiked with biological matrix (bile). MS data were acquired in profile mode on a unit mass resolution triple-quadrupole instrument, externally calibrated using a unique procedure that corrects for both mass axis and mass spectral peak shape to facilitate metabolite identification with high mass accuracy. Through the double-filtering effects of accurate mass and isotope profile, conventional extracted ion chromatograms corresponding to the parent drug (verapamil at m/z 455), demethylated verapamil (m/z 441), and dealkylated verapamil (m/z 291), that contained substantial false-positive peaks, were simplified into chromatograms that are substantially free from matrix interferences. These filtered chromatograms approach what would have been obtained by using a radioactivity detector to detect radio-labeled metabolites of interest.     

Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS

A liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7-14 days. The method was also applied to two toxigenic fungi, A. flavus (200-138) and A. parasiticus (2999-465) grown on gamma radiation sterilised dried figs, for 7-14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)-TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5-6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.     

Discovering Sudan IV in Salty Eggs with LC–TOF-MS

This study evaluated the screening of Sudan dyes in salty egg using liquid chromatography/time-of-flight mass spectrometry (LC–TOF-MS). With this technology, accurate mass measurements were made to generate elemental compositions of ions and to provide complementary structural information. This approach is useful for the elucidation of organic pollutants at trace levels in complex food extracts and in the present study salty egg samples were analyzed for the presence of Sudan IV. TOF accurate mass elemental composition data of Sudan IV were found to be crucial for structural elucidation which was applied to the investigation of nine salty egg samples.

     

The combination of liquid chromatography/tandem mass spectrometry and chip-based infusion for improved screening and characterization of drug metabolites

An approach has been developed for drug metabolism studies of non-radiolabeled compounds using on-line liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with chip-based infusion following fraction collection. The potential of this approach, which improves the data quality compared with only LC/MS analysis, has been investigated for the analysis of in vitro metabolites of tolcapone and talinolol, two compounds with well-characterized metabolism. The information-dependent LC/MS/MS analysis enables the characterization of the major metabolites while the chip-based infusion is used to obtain good product ion spectra for lower level metabolites, to generate complementary MS information on potential metabolites detected in the LC/MS trace, or to screen for unexpected metabolites. Fractions from the chromatographic analysis are collected in 20 second steps, into a 96-well plate. The fractions of interest can be re-analyzed with chip-based infusion on a variety of mass spectrometers including triple quadrupole linear ion trap (QqLIT or Q TRAP) and QqTOF systems. Acquiring data for several minutes using multi-channel acquisition (MCA), or signal averaging while infusing the fractions at approximately 200 nL/min, permits about a 50 times gain in sensitivity (signal-to-noise) in MS/MS mode. A 5-10 microL sample fraction can be infused for more than 30 min allowing the time to perform various MS experiments such as MS(n), precursor ion or neutral loss scans and accurate mass measurement, all in either positive or negative mode. Through fraction collection and infusion, a significant gain in data quality is obtained along with a time-saving benefit, because the original sample needs neither to be re-analyzed by re-injection nor to be pre-concentrated. Therefore, a novel hydroxylated talinolol metabolite could be characterized with only one injection.     

Rapid identification of phase I and II metabolites of artemisinin antimalarials using LTQ‐Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange technique

Artemisinin drugs have become the first‐line antimalarials in areas of multi‐drug resistance. However, monotherapy with artemisinin drugs results in comparatively high recrudescence rates. Autoinduction of CYP‐mediated metabolism, resulting in reduced exposure, has been supposed to be the underlying mechanism. To better understand the autoinduction of artemisinin drugs, we evaluated the biotransformation of artemisinin, also known as Qing‐hao‐su (QHS), and its active derivative dihydroartemisinin (DHA) in vitro and in vivo, using LTQ‐Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high‐resolution (HR)‐LC/MS (mass spectrometry) for rapid structural characterization. The LC separation was improved allowing the separation of QHS parent drugs and their metabolites from their diastereomers. Thirteen phase I metabolites of QHS have been identified in liver microsomal incubates, rat urine, bile and plasma, including six deoxyhydroxylated metabolites, five hydroxylated metabolites, one dihydroxylated metabolite and deoxyartemisinin. Twelve phase II metabolites of QHS were detected in rat bile, urine and plasma. DHA underwent similar metabolic pathways, and 13 phase I metabolites and 3 phase II metabolites were detected. Accurate mass data were obtained in both full‐scan and MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC‐HR/MS experiments provided additional evidence in differentiating deoxydihydroxylated metabolites from mono‐hydroxylated metabolites. The results showed that the main phase I metabolites of artemisinin drugs are hydroxylated and deoxyl products, and they will undergo subsequent phase II glucuronidation processes. This study also demonstrated the effectiveness of online H/D exchange LC‐HR/MSⁿ technique in rapid identification of drug metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI‐HR‐MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague–Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O‐sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC‐MS/MS and MSⁿ experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of carbadox metabolites formed by liver microsomes from rats, pigs and chickens using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry

Carbadox (methyl-3-(2-quinoxalinylmethylene)-carbazate-N(1),N(4)-dioxide) is a chemotherapeutic growth promoter added to feed for starter pigs. In this work, the metabolism of carbadox in rat, pig and chicken liver microsomes has been studied firstly. The incubation mixtures were then processed and analyzed for metabolites with a sensitive and reliable method based on high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). With the help of chromatographic behavior and accurate mass measurements, it is possible to rapidly and reliably characterize the metabolites of carbadox. The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and fragment ions generated from precursor ions with those of parent drug. The present results showed that the metabolism of carbadox in liver microsomes had qualitative species-difference. A total of seven metabolites were identified in rat liver microsomes. Five metabolites (Cb1-Cb3, Cb5, Cb7) were observed in pig and chicken liver microsomes. In addition, metabolite Cb6 was also detected in chicken liver microsomes. The peak areas of the metabolites in the three species are different. For the formations of Cb1, Cb2, Cb5 and Cb6, the rank order was rat>chicken>pig; Cb3; pig~chicken>rat. Cb1, Cb2 and Cb3 have been previously reported, whereas the other four metabolites were novel. The N→O group reduction and hydroxylation followed by N→O group reduction were the main metabolic pathways for carbadox in the three species.     

Rapid metabolite identification with sub parts-per-million mass accuracy from biological matrices by direct infusion nanoelectrospray ionization after clean-up on a ZipTip and LTQ/Orbitrap mass spectrometry

Metabolite identification studies remain an integral part of pre-clinical and clinical drug development programs. Analysis of biological matrices, such as plasma, urine, feces and bile, pose challenges due to the large amounts of endogenous components that can mask a drug and its metabolites. Although direct infusion nanoelectrospray using capillaries has been used routinely for proteomic studies, metabolite identification has traditionally employed liquid chromatographic (LC) separation prior to analysis. A method is described here for rapid metabolite profiling in biological fluids that involves initial sample clean-up using pipette tips packed with reversed-phase material (i.e. ZipTips) to remove matrix components followed by direct infusion nanoelectrospray on an LTQ/Orbitrap mass spectrometer using a protonated polydimethylcyclosiloxane cluster ion for internal calibration. We re-examined samples collected from a prazosin metabolism study in the rat. Results are presented that demonstrate that sub parts-per-million accuracies can be achieved on molecular ions, facilitating identification of metabolites, and on product ions, facilitating structural assignments. The data also show that the high-resolution measurements (R = 100 000 at m/z 400) enable metabolites of interest to be resolved from endogenous components. The extended analysis times available with nanospray enables signal averaging for 1 min or more that is valuable when metabolites are present in low concentrations as encountered here in plasma and brain. Using this approach, the metabolic fate of a drug can be quickly obtained. A limitation of this approach is that metabolites that are structural isomers cannot be distinguished, although such information can be collected by LC/MS during follow-on experiments. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Identification of key metabolites of tectorigenin in rat urine by HPLC-MS(n)

This is a report about the identification of key metabolites of tectorigenin in rat urine using high-performance liquid chromatography-electrospray ionization ion trap tandem mass spectrometric method (HPLC-ESI-MS(n)). Six healthy rats were administered a single dose (80 mg/kg) of tectorigenin by oral gavage. Urine was sampled for 0-24 h and centrifuged at 12,000 rpm for 10 min to obtain the supernatants, then the supernatants were purified by solid-phase extraction with a C(18) cartridge. The chromatographic separation was carried out on a reversed-phase C(18) column with a gradient elution program whereas acetonitrile-0.1% formic acid water was used as mobile phase. Mass spectra were acquired in negative ionization mode and a data-dependant scan was used for the identification of the key metabolites of tectorigenin in the urine samples. As a result, four phase II metabolites and the parent drug tectorigenin were found and identified in rat urine for the first time.     

Multiresidue pesticide analysis in ginseng and spinach by nontargeted and targeted screening procedures

Five different mass spectrometers interfaced to GC or LC were evaluated for their application to targeted and nontargeted screening of pesticides in two foods, spinach and ginseng. The five MS systems were capillary GC/MS/MS, GC-high resolution time-of-flight (GC/HR-TOF)-MS, TOF-MS interfaced with a comprehensive multidimensional GC (GCxGC/TOF-MS), an MS/MS ion trap hybrid mass (qTrap) system interfaced with an ultra-performance liquid chromatograph (UPLC-qTrap), and UPLC interfaced to an orbital trap high resolution mass spectrometer (UPLC/Orbitrap HR-MS). Each MS system was tested with spinach and ginseng extracts prepared through a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure. Each matrix was fortified at 10 and 50 ng/g for spinach or 25 and 100 ng/g for ginseng with subsets of 486 pesticides, isomers, and metabolites representing most pesticide classes. HR-TOF-MS was effective in a targeted search for characteristic accurate mass ions and identified 97% of 170 pesticides in ginseng at 25 ng/g. A targeted screen of either ginseng or spinach found 94-95% of pesticides fortified for analysis at 10 ng/g with GC/MS/MS or LC/MS/MS using multiple reaction monitoring (MRM) procedures. Orbitrap-MS successfully found 89% of 177 fortified pesticides in spinach at 25 ng/g using a targeted search of accurate mass pseudomolecular ions in the positive electrospray ionization mode. A comprehensive GCxGC/TOF-MS system provided separation and identification of 342 pesticides and metabolites in a single 32 min acquisition with standards. Only 67 or 81% of the pesticides were identified in ginseng and spinach matrixes at 25 ng/g or 10 ng/g, respectively. MS/MS or qTrap-MS operated in the MRM mode produced the lowest false-negative rates, at 10 ng/g. Improvements to instrumentation, methods, and software are needed for efficient use of nontargeted screens in parallel with triple quadrupole MS.     

Mass defect filter technique and its applications to drug metabolite identification by high‐resolution mass spectrometry

Identification of drug metabolites by liquid chromatography/mass spectrometry (LC/MS) involves metabolite detection in biological matrixes and structural characterization based on product ion spectra. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of metabolites using triple‐quadrupole and ion trap mass spectrometers. Recently, a novel mass defect filter (MDF) technique has been developed, which enables high‐resolution mass spectrometers to be utilized for detecting both predicted and unexpected drug metabolites based on narrow, well‐defined mass defect ranges for these metabolites. This is a new approach that is completely different from, but complementary to, traditional molecular mass‐ or MS/MS fragmentation‐based LC/MS approaches. This article reviews the mass defect patterns of various classes of drug metabolites and the basic principles of the MDF approach. Examples are given on the applications of the MDF technique to the detection of stable and chemically reactive metabolites in vitro and in vivo. Advantages, limitations, and future applications are also discussed on MDF and its combinations with other data mining techniques for the detection and identification of drug metabolites. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

A peptidomic approach for monitoring and characterising peptide cyanotoxins produced in Italian lakes by matrix-assisted laser desorption/ionisation and quadrupole time-of-flight mass spectrometry

In recent years, the occurrence of cyanobacterial blooms in eutrophic freshwaters has been described all over the world, including most European countries. Blooms of cyanobacteria may produce mixtures of toxic secondary metabolites, called cyanotoxins. Among these, the most studied are microcystins, a group of cyclic heptapeptides, because of their potent hepatotoxicity and activity as tumour promoters. Other peptide cyanotoxins have been described whose structure and toxicity have not been thoroughly studied. Herein we present a peptidomic approach aimed to characterise and quantify the peptide cyanotoxins produced in two Italian lakes, Averno and Albano. The procedure was based on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry mass spectrometry (MALDI-TOF-MS) analysis for rapid detection and profiling of the peptide mixture complexity, combined with liquid chromatography/electrospray ionisation quadrupole time-of- flight tandem mass spectrometry (LC/ESI-Q-TOF-MS/MS) which provided unambiguous structural identification of the main compounds, as well as accurate quantitative analysis of microcystins. In the case of Lake Averno, a novel variant of microcystin-RR and two novel anabaenopeptin variants (Anabaenopeptins B(1) and Anabaenopeptin F(1)), presenting homoarginine in place of the commonly found arginine, were detected and characterised. In Lake Albano, the peculiar peptide patterns in different years were compared, as an example of the potentiality of the peptidomic approach for fast screening analysis, prior to fine structural analysis and determination of cyanotoxins, which included six novel aeruginosin variants. This approach allows for wide range monitoring of cyanobacteria blooms, and to collect data for evaluating possible health risks to consumers, through the panel of the compounds produced along different years.     

Herbal medicine analysis by liquid chromatography/time-of-flight mass spectrometry

The fact that the effects of herbal medicines (HMs) are brought about by their chemical constituents has created a critical demand for powerful analytical tools performing the chemical analysis to assure their efficacy, safety and quality. Liquid chromatography coupled to mass spectrometry (LC–MS) is an excellent technique to analyze multi-components in complex herbal matrices. Due to its inherent characteristics of accurate mass measurements and high resolution, time-of-flight (TOF) MS is well-suited to this field, especially for qualitative applications. The purpose of this article is to provide an overview on the potential of TOF, including the hybrid quadrupole- and ion trap-TOF (QTOF and IT-TOF), hyphenated to LC for chemical analysis in HMs or HM-treated biological samples. The peculiarities of LC–(Q/IT)TOF-MS for the analysis of HMs are discussed first, including applied stationary phase, mobile-phase selection, accurate mass measurements, fragmentation and selectivity. The final section is devoted to describing the applicability of LC–(Q/IT)TOF-MS to routine analysis of multi-components, including target and non-target (unknown) compounds, in herbal samples, emphasizing both the advantages and limitations of this approach for qualitative and quantitative purposes. The potential and future trends of fast high-performance liquid chromatography (HPLC) (e.g. rapid resolution LC and ultra-performance LC) coupled to (Q)TOF-MS for chemical analysis of HMs are highlighted.     

Identification of metabolites of Danggui Buxue Tang in rat urine by liquid chromatography coupled with electrospray ionization time‐of‐flight mass spectrometry

A method coupling liquid chromatography with electrospray ionization time‐of‐flight mass spectrometry (LC/ESI‐TOF/MS) has been developed for rapid and sensitive analysis of rat urinary metabolite profile of Danggui Buxue Tang (DBT), a well‐known Chinese herbal formula. After oral administration of DBT, urine samples were collected during 0–24 h, and then pretreated by solid‐phase extraction. A total of 68 compounds including 13 parent compounds and 55 metabolites were detected in the drug‐containing urines compared with blank urines. The total analytical time was less than 20 min. Metabolites of DBT were identified using dynamic adjustment of the fragmentor voltage to produce structure‐relevant fragment ions. By using this approach, the mass accuracy of precursor and fragment ions was typically within ±5 ppm of the theoretical values, and enabled the identification of 43 metabolites including 27 isoflavanoid and 16 phthalide metabolites. Our results indicated that glucuronidation and sulfation were the major metabolic pathways of isoflavonoids, while glutathione conjugation, glucuronidation and sulfation were the main metabolic pathways of phthalides. No saponin‐related metabolites were detected. The results of the present study provided important structural information relating to the metabolism of DBT. Furthermore, this work demonstrated the potential of the LC/ESI‐TOF/MS approach for identification of metabolites from Chinese herbal medicines in urine. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time‐consuming processes. In this study, a novel multiple‐stage tandem mass spectrometric method (MSⁿ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β‐lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data‐dependent LC/MSⁿ screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks?). The proposed data‐dependent LC/MSⁿ method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling. Copyright © 2010 John Wiley & Sons, Ltd.