Phospholipids in liquid chromatography/mass spectrometry bioanalysis: comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids,the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects

Because plasma phospholipids may cause matrix effects in bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods, it is important to establish optimal mass spectrometric techniques to monitor the fate of phospholipids during method development and application. We evaluated three MS/MS techniques to monitor phospholipids using positive and negative electrospray ionization (ESI). The first technique is based on using positive precursor ion scan of m/z 184, positive neutral loss scan of 141 Da and negative precursor ion scan of m/z 153. The second technique is based on using class‐specific positive and negative selected reaction monitoring (SRM) transitions to monitor class‐representative phospholipids. The third technique, previously reported, utilizes in‐source collision‐induced dissociation (CID)‐based positive SRM of m/z 184 → 184. We recommend the all‐inclusive technique 1 for use in qualitative assessment of all classes of phospholipids and technique 2 for use in quantitative assessment of class‐representative phospholipids. Secondly, we evaluated the elution behaviors of the plasma phospholipids under different reversed‐phase mobile phase conditions. The phospholipid‐eluting strength of a mobile phase was mainly dependent on the type and amount (%) of the organic eluent and the strength increased in the order of methanol, acetonitrile and isopropyl alcohol. Under the commonly used gradient and isocratic elution schemes in LC/MS/MS bioanalysis, not all the phospholipids are eluted off the column. Thirdly, we investigated the association between phospholipids and matrix effects in positive and negative ESI using basic, acidic and neutral analytes. While the phospholipids caused matrix effects in both positive and negative ESI, the extent of ionization suppression was analyte‐dependent and was inversely related to the retention factor and broadness of the phospholipids peaks. The lysophospholipids which normally elute earlier in reversed‐phase chromatography are more likely to cause matrix effects compared to the later‐eluting phospholipids in spite of the larger concentrations of the latter in plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of triclosan metabolites by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry

Triclosan is a widely used broad‐spectrum antibacterial agent that acts by specifically inhibiting enoyl–acyl carrier protein reductase. An in vitro metabolic study of triclosan was performed by using Sprague‐Dawley (SD) rat liver S9 and microsome, while the in vivo metabolism was investigated on SD rats. Twelve metabolites were identified by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI‐ITMS) analysis. Compared to electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (MS/MS) that gave little fragmentation for triclosan and its metabolites, the in‐source fragmentation under APCI provided intensive fragmentations for the structural identifications. The in vitro metabolic rate of triclosan was quantitatively determined by using HPLC/ESI‐ITMS with the monitoring of the selected triclosan molecular ion. The metabolism results indicated that glucuronidation and sulfonation were the major pathways of phase II metabolism and the hydroxylated products were the major phase I metabolites. Moreover, glucose, mercapturic acid and cysteine conjugates of triclosan were also observed in the urine samples of rats orally administrated with triclosan. Copyright © 2010 John Wiley & Sons, Ltd.     

Stereochemical differentiation of C‐7 hydroxyltaxane isomers by electrospray ionization mass spectrometry

In this study, different electrospray ionization mass spectrometric (ESI‐MS) methods were utilized to analyze several pairs of taxane stereoisomers including paclitaxel and 7‐epi‐paclitaxel. Both ESI‐MS and tandem mass spectrometry (MS/MS) techniques provided stereochemically dependent mass spectra in negative‐ion mode, and all studied stereoisomers could be easily distinguished based on their characteristic ions or distinct fragmentation patterns. MS/MS experiments for several taxane analogues at various collision energies were performed to elucidate potential dissociation pathways. The gas‐phase deprotonation potentials were also calculated to estimate the most thermodynamically favorable deprotonation site using DFT B3LYP/6‐31G(d). The results of the theoretical studies agreed well with the fragmentation patterns of paclitaxel and 7‐epi‐paclitaxel observed from MS/MS experiments. In addition, it was found that liquid chromatography (LC)/ESI‐MS was a useful and sensitive technique for assignment of C‐7 taxane stereoisomers from realistic samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparison between charged aerosol detection and light scattering detection for the analysis of Leishmania membrane phospholipids

The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids.     

Synthesis and Electrospray Ionization Mass Spectra of N-（1,3, 2-Dioxaphosphorinan-2-ylmethyl）thiophosphoramidates

N-（1,3,2-Dioxaphosphorinan-2-ylmethyl）thiophosphoramidates were synthesized and determined by NMR spectra and positive ion electrospray ionization mass spectrometry （ESI-MS） in conjunction with tandem mass spectrometry （MS/MS）. The fragmentation pathways were investigated. The results show that these characteristic ions in ESI mass spectra are useful in the structural determination of N-（1,3,2-dioxaphosphorinan-2-ylmethyl）- thiophosphoramidates.     

Rapid screening and determination of 4‐chloroamphetamine in saliva by paper spray‐mass spectrometry and capillary electrophoresis‐mass spectrometry

A novel drug‐screening system, consisting of paper spray‐MS (PS‐MS) and a CE‐ESI‐MS method was developed. This system can be easily switched either to PS‐MS for rapidly screening samples or to the traditional CE‐ESI‐MS method for separation and to obtain detailed mass spectral information, while sharing the same mass spectrometer. In the former case, when a sharp (15°‐tip) chromatography paper was used, the optimized distance from the paper tip to the mass inlet was 7.7 mm, whereas the optimized distance for the CE‐ESI tip was ～13.5 mm. Using 4‐chloroamphetamine as a model compound, the LODs for PS‐MS and CE‐ESI‐MS were determined to ～0.1 and 0.25 ppm, respectively. Comparisons of results obtained using PS‐MS and CE‐ESI‐MS and the experimental conditions are described.     

Antioxidant Pathways and One‐Electron Oxidation of Dopamine and Cysteine in Electrospray and On‐Line Electrochemistry Electrospray Ionization Mass Spectrometry

Dopamine [DA]⁺ (m/z 154), DA dimer [2DA‐H]⁺ (m/z 307) and DA quinone [DAQ]⁺ (m/z 152) are detected in positive ion mode electrospray ionization mass spectrometry (ESI MS) of dopamine in 50/1/49 (vol%) water/acetic acid/methanol. H/D exchange experiments support a covalent structure of DA dimer. Thus, ESI of DA may involve 1e^?, 1H⁺ oxidation processes followed by rapid radical dimerization. The DA quinone signal is low in ESI MS, which indicates a low efficiency of the 2e^?, 2H⁺ oxidation reaction. On‐line electrochemistry ESI MS (EC/ESI MS) with low electrochemical cell voltage floated on high ES voltage increases electrospray current and improves sensitivity for DA. The DA quinone signal increases and DA dimer signal decreases. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity of ESI and EC/ESI MS measurements. A DA quinone‐cysteine adduct [DAQ+Cys]⁺ was detected in solutions of DA with cysteine (Cys). ESI MS and EC/ESI MS indicate formation of the DA quinone‐cysteine adduct by 1e^? pathway. Oxidation pathways in ESI MS are relevant to biological reactivity of DA and Cys.     

Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

Folic acid (FA) plays a vital role in central metabolism, including the one carbon cycle, nucleotide, and amino acid biosynthesis. The development of sensitive, accurate analytical methods to measure FA intermediates in tissues is critical to understand their biological roles in diverse physiological and pathological contexts. Here, we developed a highly sensitive method for the simultaneous quantification of FA intermediates in the nematode Caenorhabditis elegans as a model to dissect metabolic networks. The method was further validated by analyzing the worm folate pool upon RNAi knockdown of the dihydrofolate reductase gene dhfr‐1. Comparative mass spectrometry behavior of the FA analogs using two different ion sources, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), revealed ESI‐MS/MS to be more sensitive, but APCI‐MS provided more detailed structure inferences, which can elucidate chemical investigation and synthesis of FA analogs. Finally, we report on the use of in vitro oxidation coupled with high‐resolution mass spectrometry as a tool to discover new endogenous FA derivatives in the nematode.     

Mass spectrometry characterization of endophytic bacterium Curtobacterium sp. strain ER1/6 isolated from Citrus sinensis

The bacteria of the genus Curtobacterium are usually seen as plant pathogen, but some species have been identified as endophytes of different crops and could as such present a potential for disease control and plant growth promotion. We have therefore applied the desorption electrospray ionization mass spectrometry imaging (DESI‐MSI) in the direct analysis of living Curtobacterium sp. strain ER1/6 colonies to map the surface metabolites, and electrospray ionization tandem mass spectrometry (ESI‐MS/MS) for characterization of these compounds. Several colony‐associated metabolites were detected. The ESI‐MS/MS showed characteristic fragmentations for phospholipids including the classes of glycerophosphocholine, glycerophosphoglycerol, and glycerophosphoinositol as well as several fatty acids. Although a secure identification was not obtained, many other metabolites were also detected for this bacteria species. Principal component analysis showed that fatty acids were discriminatory for Curtobacterium sp. ER1/6 during inoculation on periwinkle wilt (PW) medium, whereas phospholipids characterize the bacterium when grown on the tryptic soy agar (TSA) medium.     

Identification of the major metabolites of quinocetone in swine urine using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Quinocetone (QCT), 3‐methyl‐2‐cinnamoylquinoxaline‐1,4‐dioxide, is a quinoxaline‐N,N‐dioxide used in veterinary medicine as a feed additive. QCT is broadly used in China to promote animal growth, but few studies have been performed to reveal the metabolism of QCT in animals until now. In the present study, the metabolites of QCT in swine urine were investigated using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight mass spectrometry (UPLC/ESI‐QTOF‐MS). Multiple scans of metabolites in MS and MS/MS modes and accurate mass measurements were performed simultaneously through data‐dependent acquisition. Most measured mass errors were less than ±5 mDa for both protonated molecules and product ions using external mass calibration. The structures of metabolites and their product ions were easily and reliably characterized based on the accurate MS² spectra and known structure of QCT. As expected, extensive metabolism was observed in swine urine. Thirty‐one metabolites were identified in swine urine, most of which were reported for the first time. The results reveal that the N‐O group reduction at position 1 and the hydroxylation reaction occurring at the methyl group, the side chain or on the benzene ring are the main metabolic pathways of quinocetone in swine urine. There was abundant production of 1‐desoxyquinocetone and hydroxylation metabolites of 1‐desoxyquinocetone. The proposed metabolic pathway of quinocetone in vivo can be expected to play a key role in food safety evaluations. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of salsolinol enantiomers by stable isotope dilution liquid chromatography with tandem mass spectrometric detection

Salsolinol, 1‐methyl‐6,7‐dihydroxy‐2,3,4,5‐tetrahydroisoquinoline (SAL), is a precursor of a Parkinsonian neurotoxin, N‐methysalsolinol (N‐methyl‐SAL). Previous studies have shown that individual enantiomers of N‐methyl‐SAL possess distinct neurotoxicological properties. In this work, a chiral high‐performance liquid chromatography (HPLC) method with electrospray ionization tandem mass spectrometric (ESI‐MS/MS) detection was developed for the quantification of (R/S)‐SAL enantiomers. Enantioseparation was achieved on a β‐cyclodextrin‐bonded silica gel column, and the resolved enantiomers were detected by ESI‐MS/MS operated in positive ion mode. The ESI collision‐induced dissociation (CID) mass spectrum of SAL was studied together with that of its deuterium‐labeled analog (i.e. salsolinol‐α,α,α,1‐d₄, SAL‐d₄) so that the fragmentation pathways could be elucidated. Further, using SAL‐d₄ as internal standard in HPLC/MS/MS analysis of SAL improved significantly assay accuracy and reliability. Determination of (R/S)‐SAL enantiomers present in food samples such as dried banana chips was demonstrated. Copyright © 2008 John Wiley & Sons, Ltd.     

Metabolic profiling study on potential toxicity and immunotoxicity‐biomarker discovery in rats treated with cyclophosphamide using HPLC‐ESI‐IT‐TOF‐MS

Despite the recent advances in understanding toxicity mechanism of cyclophosphamide (CTX), the development of biomarkers is still essential. CTX‐induced immunotoxicity in rats by a metabonomics approach was investigated using high‐performance liquid chromatography coupled with ion trap time‐of‐flight mass spectrometry (HPLC‐ESI‐IT‐TOF‐MS). The rats were orally administered CTX (30 mg/kg/day) for five consecutive days, and on the fifth day samples of urine, thymus and spleen were collected and analyzed. A significant difference in metabolic profiling was observed between the CTX‐treated group and the control group by partial least squares‐discriminant analysis (PLS‐DA), which indicated that metabolic disturbances of immunotoxicity in CTX‐treated rats had occurred. One potential biomarker in spleen, three in urine and three in thymus were identified. It is suggested that the CTX‐toxicity mechanism may involve the modulation of tryptophan metabolism, phospholipid metabolism and energy metabolism. This research can help to elucidate the CTX‐influenced pathways at a low dose and can further help to indicate the patients' pathological status at earlier stages of toxicological progression after drug administration. Copyright © 2014 John Wiley & Sons, Ltd.     

Atmospheric pressure chemical ionisation normal phase liquid chromatography mass spectrometry and tandem mass spectrometry of chlorophyll a allomers

Four oxidation products formed during the allomerisation reaction of chlorophyll a in methanol have been separated and identified using normal phase high-performance liquid chromatography (NP-HPLC) coupled with atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry (MS/MS) and photodiode array detection. Fragmentation pathways of the main stereoisomers of the four major products, Mg-purpurin-7 dimethyl phytyl ester, 15(1)-MeO-lact-chl a, 13(2)-MeO-chl a and 13(2)-HO-chl a, have been assigned using APCI-MS(n) aided by examination of deuteriated counterparts of the purpurin and the lactone. Diagnostic fragmentations have been determined, including those for Mg-purpurin-7 dimethyl phytyl ester and 15(1)-MeO-lact-chl a, which display very similar full MS and MS/MS spectra. The use of NP-HPLC offers a rapid and efficient method which, combined with MS/MS, reduces reliance on precursor ion mass for assignment of structure in the allomerisation reaction where major and minor components are often isobaric.     

Gas‐phase fragmentation study of a novel series of synthetic 2‐oxo‐2H‐benzopyrano[2,3‐d]pyrimidine derivatives using electrospray ionization tandem mass spectroscopy measured with a quadrupole orthogonal time‐of‐flight hybrid instrument

The fragmentation patterns of a series of six novel synthesized benzopyranopyrimidine derivatives 1–6, possessing the same 2‐oxo‐2H‐benzopyrano[2,3‐d]pyrimidine backbone structure, were investigated by electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (MS/MS) techniques using a quadrupole orthogonal time‐of‐flight (QqToF)‐hybrid instrument. The series of six pure benzopyranopyrimidine compounds contained three constitutional isobaric isomers (compounds 4–6). A simple methodology, based on the use of ESI (positive ion mode) and increasing the declustering potential in the atmospheric pressure/vacuum interface resulting in collision‐induced dissociation (CID), was used to enhance the formation of the product ions. In general, the novel synthetic benzopyranopyrimidine derivatives 1–6 afforded exact accurate masses for the protonated molecules. This led to the confirmation of both molecular masses and chemical structures of the studied compounds. The breakdown routes of the protonated molecules were rationalized by conducting low‐energy CID‐MS/MS analyses. It was shown that the MS/MS fragmentation routes for the protonated molecules 1 and 2 were similar, and that the MS/MS fragmentations of the constitutional isobaric protonated molecules 5 and 6 were identical. It was also shown that the gas‐phase CID fragmentations of 5 and 6 were different from that of their constitutional isomer 4. Finally, the ESI‐MS and CID‐MS/MS analyses of the protonated molecules that were obtained from the monodeuterated benzopyranopyrimidine derivatives 1–6 confirmed the values obtained for the exact masses, the precise structural assignments of all product ions and all the pathways described in the proposed CID fragmentations. Copyright © 2008 John Wiley & Sons, Ltd.     

Identification of intact oxidation products of glycerophospholipids in vitro and in vivo using negative ion electrospray iontrap mass spectrometry

Free radical‐induced oxidation products of polyunsaturated fatty acids esterified to phospholipids have been implicated in a number of human diseases including atherosclerosis and neurodegenerative diseases. Some of these phospholipid oxidation products have potent biological activities and likely contribute to human pathophysiological conditions. Oxidation products have also been used as markers of oxidative stress in vivo. Identification and quantification of phospholipid oxidation products are often performed by analyzing the oxidized free fatty acid moieties after hydrolysis from the phospholipids head groups by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS). We now describe the definitive identification of intact oxidized products of glycerophospholipids including glycerophosphatidylcholine (GPC), glycerophosphatidylethanolamine (GPE), and glycerophosphatidylserine (GPS) in vitro and in vivo using iontrap MS. For these analyses, the negative ions of the oxidation products of phospholipids are fragmented to MSⁿ and unequivocal structural characterization is obtained based on collision‐induced dissociation (CID) of the sn‐2 carboxylate ion. This technique overcomes the need to hydrolyze fatty acids from phospholipids in the analysis. The method has been used to identify a number of oxidation products of glycerophospholipids including hydroxyeicosatetraenoates (HETEs) and isoprostanes (IsoPs) esterified to different classes of glycerophospholipids in vitro and in vivo. These studies thus provide a new approach to identify the intact oxidation products of glycerolphospholipids. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrospray ionization mass spectrometric analysis of newly synthesized α,β‐unsaturated γ‐lactones fused to sugars

Knowledge of the fragmentation mechanisms of lactones and their behaviour under electrospray ionization (ESI) conditions can be extended to larger and more complex natural products that contain an α,β‐unsaturated γ‐lactone moiety in their structure. Moreover, little is known about the gas‐phase behaviour of α,β‐unsaturated γ‐lactones linked or fused to sugars. Therefore, five α,β‐unsaturated γ‐lactones (butenolides) fused to a pyranose ring, recently synthesized compounds with potential relevance regarding their biological properties, were investigated using ESI‐MS and ESI‐MS/MS in both positive and negative ion modes. Their fragmentation mechanisms and product ion structures were compared. It was observed that two isomers could be unambiguously distinguished in the negative ion mode by the fragmentation pathways of their deprotonated molecules as well as in the positive ion mode by the fragmentation pathways of either the protonated or the sodiated molecule. Fragmentation mechanisms are proposed taking into account the MS/MS data and semi‐empirical calculations using the PM6 Hamiltonean. The semi‐empirical calculations were also very useful in determining the most probable protonation and cationization sites. Copyright © 2010 John Wiley & Sons, Ltd.     

Combined use of tandem mass spectrometry and computational chemistry to study 2H‐chromenes from Piper aduncum

Natural 2H‐chromenes were isolated from the crude extract of Piper aduncum (Piperaceae) and analyzed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) applying collision‐induced dissociation. Density functional theory (DFT) calculations were used to explain the preferred protonation sites of the 2H‐chromenes based on thermochemical parameters, including atomic charges, proton affinity, and gas‐phase basicity. After identifying the nucleophilic sites, the pathways were proposed to justify the formation of the diagnostic ions under ESI‐MS/MS conditions. The calculated relative energy for each pathway was in good agreement with the energy‐resolved plot obtained from ESI‐MS/MS data. Moreover, the 2H‐chromene underwent proton attachment on the prenyl moiety via a six‐membered transition state. This behavior resulted in the formation of a diagnostic ion due to 2‐methylpropene loss. These studies provide novel insights into gas‐phase dissociation for natural benzopyran compounds, indicating how reactivity is correlated to the intrinsic acid‐base equilibrium and structural aspects, including the substitution pattern on the aromatic moiety. Therefore, these results can be applied in the identification of benzopyran derivatives in a variety of biological samples.     

Electrospray Ionization Mass Spectrometry: A Powerful Platform for Noble‐Metal Nanocluster Analysis

Electrospray ionization mass spectrometry (ESI‐MS) is an analytical technique that measures the mass of a sample through “soft” ionization. Recent years have witnessed a rapid growth of its application in noble‐metal nanocluster (NC) analysis. ESI‐MS is able to provide the mass of a noble‐metal NC analyte for the analysis of their composition (n, m, q values in a general formula [M_nL_m]^q), which is crucial in understanding their properties. This review attempts to present various developed techniques for the determination of the composition of noble metal NCs by ESI‐MS. Additionally, advanced applications that use ESI‐MS to further understand the reaction mechanism, complexation behavior, and structure of noble metal NCs are introduced. From the comprehensive applications of ESI‐MS on noble‐metal NCs, more possibilities in nanochemistry can be opened up by this powerful technique.     

Electron ionization and electrospray ionization mass spectrometric study of a series of isomeric N‐chloro(or bromo)benzyl‐substituted (E)‐2′(3′‐ or 4′)‐hydroxy‐4‐stilbazole halides

The electron ionization (EI) mass spectra and electrospray ionization (ESI) mass spectra of a series of isomeric N‐chlorobenzyl‐ and N‐bromobenzyl‐substituted (E)‐2′(3′ or 4′)‐hydroxy‐4‐stilbazole chlorides and bromides (1–12) were recorded. The fragmentation pathways of all of the compounds and the characteristic fragment ions formed by EI‐MS were studied by means of B/E and B²/E constant linked‐scanning techniques. The formation of ions originating from preionization reactions, characteristic of quaternary halides under EI‐MS conditions, such as the elimination of chloro‐ or bromobenzyl halides, dehydrohalogenation or substitution reactions, is explained. As soft ionization methods cause no such degradation reactions, the ESI‐MS spectra of the studied compounds were also obtained for comparison. We thus demonstrated the applicability of EI‐MS even in cases when preionization takes place, as long as such secondary processes are properly accounted for. Copyright © 2010 John Wiley & Sons, Ltd.     

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.