Quantification of isoflavones and lignans in serum using isotope dilution liquid chromatography/tandem mass spectrometry

Phytoestrogens (isoflavones and lignans) are receiving increasing attention due to a potential protective effect against a number of complex diseases. However, in order to investigate these associations, it is necessary to accurately quantify the levels of phytoestrogens in foods and biological fluids. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (O-desmethylangolensin and equol), and two lignans (enterodiol and enterolactone) in human serum using electrospray ionisation liquid chromatography/mass spectrometry (LC/MS) with selective reaction monitoring. A simple, highly automated sample preparation procedure requires only 200 microL of sample and utilises one solid-phase extraction stage. Limits of detection are in the region of 10 pg/mL for all analytes except equol, which had a limit of detection of approximately 100 pg/mL. The method developed is suitable for measuring the concentrations of phytoestrogens in blood samples collected from large epidemiological studies. The results of the analysis of serum samples from 300 men and women living in the UK are reported.     

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.     

Chromatographic analysis of lignans

Methods and procedures for analysis of lignans in trees and other plants are reviewed. The importance of cautious sample handling and pretreatment procedures to avoid contamination, loss of sample, and unwanted chemical reactions is discussed. Sequential extraction with a non-polar solvent followed by extraction with acetone or ethanol is recommended to separate the lignans from the plant matrix. An additional step of acid, alkaline, or enzymatic hydrolysis may be necessary for some plant matrixes. Flash chromatography is a convenient method for preparative separation and isolation of pure lignans from raw extracts. TLC is very suitable for qualitative screening of extracts and for monitoring of lignan isolation and purification steps. Trimethylsilyl ethers of lignans can be separated and quantified by GC even in the case of complex mixtures of lignans and other polyphenols, and the lignans can be identified by GC-MS in a routine manner. HPLC on reversed-phase columns is especially suited for analysis of lignans and their metabolites in biological matrixes. The recent development of HPLC-electrospray ionisation (ESI)-iontrap MS (MS(n)) and corresponding techniques with high sensitivity and selectivity has proven valuable in lignan analysis. Lignan enantiomers can be separated on chiral HPLC columns.     

Application of fast gas chromatography/mass spectrometry for the rapid screening of synthetic anabolic steroids and other drugs in anti-doping analysis

This paper describes a fast gas chromatographic/mass spectrometric (GC/MS) screening method for the detection, in urine, of 36 xenobiotics (30 synthetic anabolic steroids, four narcotics, one diuretic and one stimulant) excreted free or as glucuro-conjugates in urine and detectable as trimethylsilyl (TMS) derivatives. These drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid separation step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase and then assayed as TMS derivatives by GC/MS using electron ionisation (EI) and single ion monitoring (SIM) acquisition mode. The total time needed for the GC run is less than 8 min. Good reproducibility of the retention times (CV% <1) and the relative abundances of the diagnostic fragment ions (CV% <10) was observed for all target analytes. The sensitivity of the method is sufficient to match the requirements of the World Anti-Doping Agency (WADA) for the accredited laboratories, with limits of detection (LODs) that are lower than the corresponding WADA minimum required performance limits (MRPLs) for all target compounds.     

Analysis of estrogens in river sediments by liquid chromatography-electrospray ionisation mass spectrometry. Comparison of tandem mass spectrometry and time-of-flight mass spectrometry

A method has been developed and optimised for the determination of two natural estrogens, estrone (E1) and 17beta-estradiol (E2), and one synthetic estrogen, 17alpha-ethynylestradiol (EE2), in river sediments at the sub-ng/g level. This procedure includes microwave-assisted solvent extraction (MASE), solid-phase extraction and high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation. Using sediments spiked with the three estrogens at 10 ng/g wet weight, efficient extraction (>92%) of all the three analytes was achieved by MASE, and whole-procedure recoveries ranged from 82 to 98%. Optimisation of the LC separation allowed for substantial reduction of ionisation suppression in the electrospray source to a final level of <18% suppression. Time-of-flight mass spectrometry (TOF-MS) and MS/MS were compared for the analysis of sediment extracts, with the latter technique proving to be the most selective. The method detection limits achieved by LC-MS/MS were 15, 30 and 40 pg/g for E1, E2 and EE2, respectively, which were 13-fold lower than those obtained by LC-TOF-MS. Analysis of river sediments collected from the River Ouse, UK, showed the presence of the natural estrogens at sub-ng/g level. E1 levels ranged from 0.40 ng/g (dry weight) to 3.30 ng/g while E2 levels ranged from <0.03 to 1.20 ng/g and EE2 was never detected (<0.04 ng/g).     

Determination of a 'GW cocktail' of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry

A mass spectrometry based method for the simultaneous determination of an in vivo Greenford-Ware or 'GW cocktail' of CYP450 probe substrates and their metabolites in both human plasma and urine is described. The probe substrates, caffeine, diclofenac, mephenytoin, debrisoquine, chlorzoxazone and midazolam, together with their respective metabolites and stable isotope labelled internal standards, are simultaneously extracted from the biological matrix using solid phase extraction in 96-well microtitre plate format, automated by means of a custom built Zymark robotic system. The extracts are analysed by fast gradient high performance liquid chromatography (HPLC) with detection by tandem mass spectrometry (MS/MS) using thermally and pneumatically assisted electrospray ionisation in both positive and negative ion modes and selected reaction monitoring. The methods are specific, accurate and precise with intra- and inter-assay precision (%CV) of less than 15% for all analytes.     

Quantitative determination of four nitrofuran metabolites in meat by isotope dilution liquid chromatography-electrospray ionisation-tandem mass spectrometry

A confirmatory method based on isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the low-level determination of residues of four nitrofuran veterinary drugs in meat, e.g., furazolidone, furaltadone, nitrofurantoin, and nitrofurazone. The procedure entails an acid-catalysed release of protein-bound metabolites, followed by their in situ conversion into the 2-nitrobenzaldehyde (NBA) imine-type derivatives. Liquid-liquid extraction and clean-up on a polymeric solid phase extraction cartridge are then performed before LC-MS/MS analysis by positive electrospray ionisation (ESI) applying multiple reaction monitoring of three transition reactions for each compound. Reliable quantitation is obtained by using one deuterated analogue per analyte (d4-NBA derivative) as internal standard (IS). Validation of the method in chicken meat was conducted following the European Union (EU) criteria for the analysis of veterinary drug residues in foods. The decision limits (CCalpha) were 0.11-0.21 microg/kg, and the detection capabilities (CCbeta) 0.19-0.36 microg/kg, thus below the minimum required performance limit (MRPL) set at 1 microg/kg by the EU. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.     

A low blood volume LC-MS/MS assay for the quantification of fentanyl and its major metabolites norfentanyl and despropionyl fentanyl in children

Preterm and term neonates often require surgical procedures and analgesia. However, our knowledge about neonatal pharmacokinetics of fentanyl, the most commonly used drug for these procedures, and its metabolites is still incomplete. To facilitate pharmacokinetic studies of fentanyl and its metabolites in neonates and other children, we developed and validated an LC-MS/MS method based on minimally invasive, low blood volume sampling. LC-MS/MS was used for the simultaneous analysis of fentanyl, despropionyl fentanyl (DPF), and norfentanyl from dried blood samples (DBS) collected on filter paper. Positive ions were monitored using multiple reaction monitoring. Since the standard matrix for measuring fentanyl blood concentrations is plasma, the assay was developed and validated in plasma, whole blood, and then DBS. Our method was able to measure clinically relevant levels of fentanyl and its metabolites. In DBS, the lower limits of quantification were 100 pg/mL for fentanyl with a range of reliable response from 0.1 to 100 ng/mL (r(2)>0.99) and 250 pg/mL for both DPF and norfentanyl with a range of reliable response from 0.25 to 100 ng/mL (r(2)>0.99). In plasma and in DBS inter-day accuracy and precisions of fentanyl met predefined acceptance criteria and also indicated comparable assay performance in both matrices.     

Profiling ABA metabolites in Nicotiana tabacum L. leaves by ultra-performance liquid chromatography-electrospray tandem mass spectrometry

We have developed a simple method for extracting and purifying (+)-abscisic acid (ABA) and eight ABA metabolites - phaseic acid (PA), dihydrophaseic acid (DPA), neophaseic acid (neoPA), ABA-glucose ester (ABAGE), 7′-hydroxy-ABA (7′-OH-ABA), 9′-hydroxy-ABA (9′-OH-ABA), ABAaldehyde, and ABAalcohol - before analysis by a novel technique for these substances, ultra-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS). The procedure includes addition of deuterium-labelled standards, extraction with methanol-water-acetic acid (10:89:1, v/v), simple purification by Oasis^® HLB cartridges, rapid chromatographic separation by UPLC, and sensitive, accurate quantification by MS/MS in multiple reaction monitoring modes. The detection limits of the technique ranged between 0.1 and 1 pmol for ABAGE and ABA acids in negative ion mode, and 0.01-0.50 pmol for ABAGE, ABAaldehyde, ABAalcohol and the methylated acids in positive ion mode. The fast liquid chromatographic separation and analysis of ABA and its eight measured derivatives by UPLC-ESI-MS/MS provide rapid, accurate and robust quantification of most of the substances, and the low detection limits allow small amounts of tissue (1-5 mg) to be used in quantitative analysis. To demonstrate the potential of the technique, we isolated ABA and its metabolites from control and water-stressed tobacco leaf tissues then analysed them by UPLC-ESI-MS/MS. Only ABA, PA, DPA, neoPA, and ABAGE were detected in the samples. PA was the most abundant analyte (ca. 1000 pmol/g f.w.) in both the control and water-stressed tissues, followed by ABAGE and DPA, which were both present at levels ca. 5-fold lower. ABA levels were at least 100-fold lower than PA concentrations, but they increased following the water stress treatment, while ABAGE, PA, and DPA levels decreased. Overall, the technique offers substantial improvements over previously described methods, enabling the detailed, direct study of diverse ABA metabolites in small amounts of plant tissue.     

Mass spectrometric confirmation criterion for product-ion spectra generated in flow-injection analysis. Environmental application

The suitability of a confirmation criterion recently recommended in the Netherlands for gas chromatography with mass spectrometric detection (GC–MS), was evaluated for flow-injection analysis (FIA) with atmospheric pressure chemical ionisation MS–MS detection. The main feature of the criterion is that the relative ion abundances of the four diagnostic ions are taken into account. That is, for lower-intensity peaks, relative standard deviations may be higher; this is an advantage with chemical ionisation MS procedures. A series of triazines and their degradation products were used as test compounds. Tap and surface water samples spiked at 0.33 μg/l were analysed by means of a selected reaction monitoring MS–MS procedure. For all analytes but hydroxysimazine (3 transitions), 4–9 transitions could be selected which invariably met the demands of the criterion. Some of the transitions used originate from the ³⁷Cl isotopic mass of the parent compounds which provides additional structural information. Data for twenty surface water samples analysed by means of FIA–MS–MS as well as GC–MS and liquid chromatography with diode array UV and MS–MS detection gave essentially the same results over the 0.1–1.0 μg/l range. In two samples desethylatrazine was reported by FIA–MS–MS whereas this compound was not detected by GC–MS. For a first test, this is a promising result.     

Elucidation of diazinon metabolites in rice plants by liquid ‎chromatography ion-trap mass spectrometry

ABSTRACT

A fast, selective and sensitive reversed-phased liquid chromatography coupled to ion-trap mass spectrometry has been developed to elucidate and confirm the diazinon metabolites with a wide range of polarity in the rice plant samples. Sample extraction and purification were performed with a QuEChERS-based (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure. To boost all metabolism sensitivities, all rice extracts were concentrated under vacuum to near dryness and taken up into initial mobile phase. Careful optimisation of the LC–MS/MS parameters were achieved in order to attain a fast separation with the best sensitivity. The detection was carried out on an ion-trap mass spectrometer by electrospray ionisation in positive ion mode (ESI+) with multiple reaction monitoring.     

Monitoring quinolone antibacterial residues in bovine tissues: extraction with hot water and liquid chromatography coupled to a single- or triple-quadrupole mass spectrometer

A rapid and sensitive procedure for determining residues of seven quinolone antibacterials in bovine muscle, kidney and liver is presented. The method is based on the matrix solid-phase dispersion (MSPD) technique with hot water as extractant followed by liquid chromatography/single quadrupole mass spectrometry (LC/MS) or triple-quadrupole mass spectrometry (LC/MS/MS). After dispersing tissue samples on hydrazine sulfate treated sand, target compounds were eluted from the MSPD column by passing through it 4 mL of water heated at 100 degrees C. After pH adjustment and filtration, 200 and 5 microL of the aqueous extracts were respectively injected into the LC/MS and LC/MS/MS instruments. With the former instrument, MS data were acquired in the three-ion selected ion monitoring mode, while MS/MS data acquisition was performed in the multi-reaction monitoring mode by selecting two precursor ion to product ion transitions for each target compound. Hot water appeared to be an efficient extracting medium, since absolute recoveries of the analytes were 84-102%. Using norfloxacin (a quinolone not used in veterinary medicine) as surrogate internal standard, the accuracy of the method at three concentration levels equal to 0.5, 1 and 1.5 times the maximum residue limits (MRLs) set by the european union was 88-109% with relative standard deviations (RSDs) not higher than 7%. The use of LC/MS/MS allowed detection and quantification of the analytes in any tissue considered to be performed at concentrations by far lower than half of their MRLs. Vice versa, the single-quadrupole MS arrangement, while succeeding in monitoring quinolones in muscle tissue at the 0.5 MRL level, showed to be not sufficiently selective for unambiguous identification of some quinolones in kidney and liver.     

Determination of azaspiracids in shellfish using liquid chromatography/tandem electrospray mass spectrometry.

Azaspiracid (AZA1), a recently discovered marine toxin, is responsible for the new human toxic syndrome, azaspiracid poisoning (AZP), which is caused by the consumption of contaminated shellfish. A new, sensitive liquid chromatography/mass spectrometry (LC/MS) method has been developed for the determination of AZA1 and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3). Separation of these toxins was achieved using reversed-phase LC and coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer. Spectra showed the protonated molecules, [M + H]+, and their major product ions, due to the sequential loss of two water molecules, [M + H - H2O]+, [M + H - 2H2O]+, in addition to fragment ions that are characteristic of these cyclic polyethers. A highly specific and sensitive LC/MS(3) analytical method was developed and, using shellfish extracts containing AZA1, the detection limit (S/N = 3) was 4 pg on-column, corresponding to 0.8 ng/mL. Using the protocol presented here, this is equivalent to 0.37 ng/g shellfish tissue and good linear calibrations were obtained for AZA1 in shellfish extracts (average r2 = 0.9988). Good reproducibility was achieved with % RSD values (N = 5) ranging from 1.5% (0.75 microg/mL) to 4.2% (0.05 microg/mL). An efficient procedure for the extraction of toxins from shellfish aided the development of a rapid protocol for the determination of the three predominant azaspiracids.     

Method validation of microcystins in water and tissue by enhanced liquid chromatography tandem mass spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) method has been developed and validated to identify and quantify trace levels of cyanotoxins or microcystins (MC) in water, bivalves and fish tissue with enhanced sensitivity and specificity. The method enables confirmation and quantification of six MCs (MC-LA, LF, LR, LW, RR and YR) with a single chromatographic run. The applied chromatography also allows determination of certain MC metabolites (Desmethyl-LR and -RR). By using LC-ESI–MS/MS in multiple reaction monitoring (MRM) mode, the limit of detection and quantitation for the microcystins studied, were determined to be between 0.2 and 1 pg on column (5:1 S/N ratio). These values are below the 2 pg detection limits found in the available literature.     

Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that are growing on agricultural commodities. Their frequent presence in food and their severe toxic, carcinogenic and estrogenic properties have been recognised as potential threat to human health. A reliable risk assessment of mycotoxin contamination for humans and animals relies basically on their unambiguous identification and accurate quantification in food and feedstuff. While most screening methods for mycotoxins are based on immunoassays, unambiguous analyte confirmation can be easily achieved with mass spectrometric methods, like gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). Due to the introduction of atmospheric pressure ionisation (API) techniques in the late 80s, LC/MS has become a routine technique also in food analysis, overcoming the traditional drawbacks of GC/MS regarding volatility and thermal stability. During the last few years, this technical and instrumental progress had also an increasing impact on the expanding field of mycotoxin analysis. The aim of the present review is to give an overview on the application of LC-(API)MS in the analysis of frequently occurring and highly toxic mycotoxins, such as trichothecenes, ochratoxins, zearalenone, fumonisins, aflatoxins, enniatins, moniliformin and several other mycotoxins. This includes also the investigation of some of their metabolites and degradation products. Suitable sample pre-treatment procedures, their applicability for high sample through-put and their influence on matrix effects will be discussed. The review covers literature published until July 2006.     

Rapid and simultaneous determination of polychlorinated biphenyls and their main metabolites (hydroxylated and methyl sulfonyl) by gas chromatography coupled to mass spectrometry: Comparison of different ionisation modes

Instrumental methods based on gas chromatography coupled to mass spectrometry (GC–MS) have been developed and compared using two different MS ionisation modes, electron impact (EI) and electron capture negative ionisation (ECNI), for the fast, quantitative and simultaneous determination of polychlorinated biphenyls (PCBs) and their main metabolites (hydroxylated PCBs, OH-PCBs, and methyl sulfone PCBs, MeSO₂-PCBs). Parameters affecting chromatographic separation and MS detection were evaluated in order to achieve the highest selectivity and sensitivity for both operation modes. The analytical characteristics of the developed methods were studied and compared in terms of linear range, limits of detection (LODs), limits of quantification (LOQs), and instrumental precision (repeatability and intermediate precision). Both ionisation methods showed similar precision, being relative standard deviations (RSD, %) lower than 9% and 14% for repeatability and intermediate precision, respectively. However, better LODs (from 0.01 to 0.14 pg injected for the three families of congeners studied) were achieved using ECNI-MS as ionisation mode. The suitability of the developed method was demonstrated through their application to fish liver oil samples.     

Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.     

LC-MS/MS profiling for detection of endogenous steroids and prostaglandins in tissue samples

Roles of steroid hormones, and compounds that can influence their levels in cells, are of increasing interest in e.g. cancer research, partly because resistance to hormone therapies often complicates treatment. To elucidate the processes involved, the hormones and related compounds need to be accurately measured. Reversed-phase liquid chromatography with dynamic multiple reaction monitoring mass spectrometric detection in electrospray mode is capable of providing such measurements. Therefore, LC-MS/MS was developed for sensitive, selective analysis of 11 steroid hormones, cholesterol and two prostaglandins. The effects of the tissue matrix, and solid-phase extraction (SPE) sample clean-up, on the LC-MS/MS signals of the hormones were also investigated. The results show that the developed LC-MS/MS method, following SPE clean-up to reduce matrix interference, can detect selected steroids in extracts of mouse tissues. The method provides linear measurements of the steroids at concentrations up to few ng/μL, and limits of detection in the range 0.03-0.2 pg/μL (for some compounds lower than those of previously reported methods).     

Detection, characterization, and quantification of resveratrol glycosides in transgenic arabidopsis over-expressing a sorghum stilbene synthase gene by liquid chromatography/tandem mass spectrometry

Transgenic Arabidopsis plants were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) to investigate the glycosylation patterns of resveratrol derived from expression of a sorghum stilbene synthase gene. In negative ionization mode, the different resveratrol derivatives fragmented to yield the diagnostic deprotonated resveratrol ion at m/z 227.2. The use of precursor ion scanning led to the identification of precursor ions for different resveratrol glycosides through rapid differentiation from other phytochemical constituents. Structural information was generated simultaneously from the low-collision-energy product ion spectra using hybrid linear ion-trap mass spectrometry. Three additional resveratrol-related metabolites - a resveratrol diglucoside (M1) and trans- and cis-resveratrol acetylhexosides (M2 and M3) - were detected in the crude plant extracts. The identities of M1, M2, and M3 were confirmed by accurate mass analysis on a quadrupole time-of-flight mass spectrometer as well as beta-glucosidase digestion or UV-induced isomerization. Quantitative analyses by LC/MS in multiple reaction monitoring mode revealed that resveratrol diglucoside and cis-resveratrol acetyhexoside accumulated up to 2.79 and 10.38 microg/g, respectively, while trans-resveratrol acetylhexoside was barely detectable. This study demonstrated the power of the hybrid linear ion-trap technology for simultaneous profiling and structural characterization of stilbene-related metabolites, which would be useful to understand how resveratrol is modified in sorghum and other plants.     

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies.