Development of capillary zone electrophoresis-electrospray ionization-mass spectrometry for the determination of lamotrigine in human plasma

A method of coupling capillary zone electrophoresis (CZE) with electrospray ionization-mass spectrometry (ESI-MS) detection has been developed for monitoring an antiepileptic drug, lamotrigine (LTG) in human plasma. The CZE-MS was developed in three stages: (i) CZE separation and ESI-MS detection of LTG and tyramine (TRM, internal standard) were simultaneously optimized by studying the influence of CZE background electrolyte (BGE) pH, BGE ionic strength, and nebulizer pressure of the MS sprayer; (ii) sheath liquid parameters, such as pH, ionic strength, organic modifier content, and flow rate of the sheath liquid, were systematically varied under optimum CZE-MS conditions developed in the first stage; (iii) MS sprayer chamber parameters (drying gas temperature and drying gas flow rate) were varied for the best MS detection of LTG. The developed assay was finally applied for the determination of LTG in plasma samples. The linear range of LTG in plasma sample assay was between 0.1-5.0 microg/mL with a limit of detection as low as 0.05 microg/mL and run time less than 6 min. Finally, the concentration-time profile of LTG in human plasma sample was found to correlate well when CZE-ESI-MS was compared to a more established method of high-performance liquid chromatography with ultraviolet detection.     

Characterization of gingerol-related compounds in ginger rhizome (Zingiber officinale Rosc.) by high-performance liquid chromatography/electrospray ionization mass spectrometry

This study sought to determine the utility of liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) coupled with diode array detection in identifying gingerol-related compounds from crude extracts of ginger rhizome. The fragmentation behaviors of compounds in both (-)- and (+)ESI-MS/MS were used to infer and confirm the chemical structures of several groups of compounds, including the gingerols, methylgingerols, gingerol acetates, shogaols, paradols, gingerdiols, mono- and diacetyl gingerdiols, and dehydrogingerdiones. Diode array detection at different wavelengths was used to confirm MS/MS-based identification. In total, 31 gingerol-related compounds were identified from the methanolic crude extracts of fresh ginger rhizome in this study. Three of these compounds were found to be new compounds. This study demonstrated that LC/ESI-MS/MS is a powerful on-line tool for identification of gingerol-related compounds, especially for thermally labile compounds that cannot be readily detected by GC/MS analysis.     

Dynamically coated capillaries improve the identification power of capillary zone electrophoresis for basic drugs in toxicological analysis

In systematic toxicological analysis (STA), analytical methods should have a high identification power. This can be suitably expressed by parameters such as mean list length (MLL) or discriminating power (DP). The reproducibility of a method has a great impact on its identification power, and should be as high as possible. In this study, two separation methods based on capillary zone electrophoresis (CZE) were evaluated towards STA applications. Besides a normal phosphate buffer, the commercially available buffer CElixir was used, which is a double-layer dynamic coating system. The coating stabilizes the endoosmotic flow, is independent of the pH, and is claimed to be more reproducible and faster at low pH than with normal buffers. A test set of 73 basic pharmaceutical compounds was analyzed by the two CZE methods. The total analysis time, including rinsing steps, was 8 min when the coating was used and 18 min without the coating. Effective mobilities were calculated and the reproducibilities were a factor of 2 better when the coating was used (between-days SD 0.020 and 0.040 m2/V s with and without the coating, respectively). MLL and DP were calculated for the two CZE methods and for combinations with standardized liquid and gas chromatography systems. CZE with CElixir coating clearly has a high potential for STA applications, as it was shown to have a higher identification power and shorter analysis times than normal CZE.     

Characterization of phenolic compounds in the crude extract of Hedysarum multijugum by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

The fragmentation behavior of four types of phenolic compounds: coumestans, pterocarpenes, benzofurans and isoflavones, were studied using electrospray ionization multistage mass spectrometry (ESI-MS(n)) (n = 2-5) in negative ion mode. Losses of methyl (15 Da), CO (28 Da), CO(2) (44 Da), isopropyl (43 Da) and isobutenyl (55 Da) were dominating fragmentation patterns. Different positions and numbers of the substituents also led to the different fragmentation behavior. These fragmentation rules were applied for the identification of constituents in methanolic extracts of Hedysarum multijugum, in which 29 compounds were characterized, including nine new compounds. The method established in this study could be applied to the comprehensive quality control of the herb and its formulations. It could also be applied to the biological and pharmacological research of traditional Chinese medicines.     

Analysis of synthetic chemical drugs in adulterated Chinese medicines by capillary electrophoresis/electrospray ionization mass spectrometry

Sixteen synthetic chemical drugs, often found in adulterated Chinese medicines, were studied by capillary electrophoresis/UV absorbance (CE/UV) and capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS). Only nine peaks were detected with CZE/UV, but on-line CZE/MS provided clear identification for most compounds. For a real sample of a Chinese medicinal preparation, a few adulterants were identified by their migration times and protonated molecular ions. For coeluting compounds, more reliable identification was achieved by MS/MS in selected reaction monitoring mode. Micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) provided better separation than capillary zone electrophoresis (CZE), and, under optimal conditions, fourteen peaks were detected using UV detection. In ESI, the interference of SDS was less severe in positive ion mode than in negative ion mode. Up to 20 mM SDS could be used in direct coupling of MEKC with ESI-MS if the mass spectrometer was operated in positive ion mode. Because of better resolution in MEKC, adulterants can be identified without the use of MS/MS.     

Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis

Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mM ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 microM and a limit of quantitation of 12 microM for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing with control cell samples.     

Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography-tandem mass spectrometry

A method for quantitation of six prenylflavonoids (xanthohumol, isoxanthohumol, desmethylxanthohumol, 6- and 8-prenylnaringenins and 6-geranylnaringenin) in hops and beer by HPLC-tandem mass spectrometry has been developed. The method allows direct analysis of beer and crude methanolic extracts of hops. After HPLC separation, prenylflavonoids were detected by positive ion multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with a heated nebulizer--atmospheric pressure chemical ionization interface. The accuracy and precision were evaluated by replicate analyses of (spiked) samples. Thirteen commercial beers were analysed with the method. Isoxanthohumol, formed by isomerization of xanthohumol during the brewing process, was the most abundant flavonoid in hopped beers, ranging from 0.04 to 3.44 mg/l.     

Chemotaxonomic markers of organic, natural, and genetically modified soybeans detected by direct infusion electrospray ionization mass spectrometry

Summary The crude methanolic extracts of a single bean from samples of organic, natural or genetically modified (GM) soybeans [Glycine max. (Merrill) L.] were analyzed by direct infusion electrospray ionization mass spectrometry (ESI-MS). These extracts, containing the most polar natural products of soybeans (free aglycones, monoglucosides, diglucosides and esters including isoflavones and flavones) provide characteristic fingerprinting mass spectra owing to different proportions or sets of components. Spectra distinctiveness is confirmed by chemometric multivariate analysis of the ESI-MS data, which place the three-types of beans into well-defined groups. When ESI-MS is applied, these polar components constitute therefore unique chemotaxonomic markers able to provide fast soybean typification.     

Pressurized liquid extraction-capillary electrophoresis-mass spectrometry for the analysis of polar antioxidants in rosemary extracts

A method based on capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS) was developed to qualitatively characterize natural antioxidants from rosemary (Rosmarinus officinalis L.) in different fractions obtained by pressurized liquid extraction (PLE) using subcritical water. The parameters of CE-ESI-MS were adjusted allowing the separation and characterization of different compounds from rosemary in the PLE fractions. These parameters for CE are kind, pH and concentration of the separation buffer, parameters for ESI-MS are dry gas temperature and flow, nebulizing gas pressure, and make-up flow. The following analytical conditions were found most favorable: aqueous CE buffer (40 mM ammonium acetate/ammonium hydroxide, pH 9); sheath liquid containing 2-propanol-water (60:40, v/v) and 0.1% (v/v) triethylamine at a flow rate of 0.24 mL/h; drying gas flow rate equal to 7 L/min at 350 degrees C, nebulizing gas pressure of 13.8 kPa (2 psi), using a compound stability of 50%. Different antioxidant compounds (e.g., rosmarinic acid and carnosic acid) could be detected in the rosemary extracts by CE-ESI-MS without any additional treatment, enabling the determination of variations in the extract composition caused by the different PLE conditions (i.e., 60 and 100 degrees C). The results provide complementary information to HPLC analysis.     

Bioactive compounds, RP-HPLC analysis of phenolics, and antioxidant activity of some Portuguese shrub species extracts

In the ecosystem of Serra Da Estrela, some plant species have the potential to be used as raw material for extraction of bioactive products. The goal of this work was to determine the phenolic, flavonoid, tannin and alkaloid contents of the methanolic extracts of some shrubs (Echinospartum ibericum, Pterospartum tridentatum, Juniperus communis, Ruscus aculeatus, Rubus ulmifolius, Hakea sericea, Cytisus multiflorus, Crataegus monogyna, Erica arborea and Ipomoea acuminata), and then to correlate the phenolic compounds and flavonoids with the antioxidant activity of each extract. The Folin-Ciocalteu's method was used for the determination of total phenols, and tannins were then precipitated with polyvinylpolypyrrolidone (PVPP); a colorimetric method with aluminum chloride was used for the determination of flavonoids, and a Dragendorff's reagent method was used for total alkaloid estimation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and beta-carotene bleaching tests were used to assess the antioxidant activity of extracts. The identification of phenolic compounds present in extracts was performed using RP-HPLC. A positive linear correlation between antioxidant activity index and total phenolic content of methanolic extracts was observed. The RP-HPLC procedure showed that the most common compounds were ferulic and ellagic acids and quercetin. Most of the studied shrubs have significant antioxidant properties that are probably due to the existence of phenolic compounds in the extracts. It is noteworthy to emphasize that for Echinospartum ibericum, Hakea sericea and Ipomoea acuminata, to the best of our knowledge, no phytochemical studies have been undertaken nor their use in traditional medicine been described.     

On-line coupling of capillary isotachophoresis and zone electrophoresis for the assay of phenolic compounds in plant extracts

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column coupling configuration was optimized in a mode where the electrolyte for the CZE step is different from the leading and terminating ITP electrolytes. Two colored markers, picric acid and 1-nitroso-2-naphthol, were used for exact timing of the transfer of isotachophoretically stacked analyte zones into the CZE column and for the control of the residual amount of the leading and terminating ITP electrolytes entering the CZE capillary together with the analytes, thus controlling the duration of transient ITP migration in the CZE capillary and ensuring good separation of the analytes and reproducibility of the migration times (relative standard deviations 1%). ITP-CZE was applied to the simultaneous assay of several cinnamic acid derivatives and flavonoids in methanolic extracts of Sambucus flowers and Crataegus leaves and flowers. The preconcentrating and cleansing effect of the ITP step allowed injection of relatively large sample volumes (30 microL). The limits of detection were approximately 20-50 ng x mL(-1) and 100 ng x mL(-1) for the acids and flavonoids, respectively ( thick similar 200-times lower compared to conventional CE) with spectrophotometric detection at 254 nm. The ITP-CZE exhibited satisfactory linearity and precision when using CZE buffer of pseudo "pH" 9.0; 1-nitroso-2-naphthol was employed as the internal standard. The separation took approximately 35 min. The ITP-CZE results for rutin, hyperoside, and vitexin-2-O"-rhamnoside were in good accordance with those obtained previously by high-performance liquid chromatography.     

Determination of Phenolic Acids in Herba Epilobi by ITP–CE in the Column-Coupling Configuration

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column-coupling configuration has been optimized in a mode in which the background electrolyte employed in the CZE step was different from the leading and terminating electrolytes of the ITP step. The optimum composition of the electrolyte system was 0.01 M HCl, 0.02 M IMI, 0.2% HEC, pH 7.2 (leading electrolyte), 0.01 M HEPES, pH 8.2 (terminating electrolyte), and 25 mM MES, 50 mM TRIS, 30 mM boric acid, 0.2% HEC, pH 8.3 (background electrolyte). All solutions contained 20% methanol. The timing of the transfer of isotachophoretically stacked analyte zones into the CZE column was also optimized. An ITP–CZE method with UV detection at 270 nm was developed for separation of nine phenolic acids (protocatechuic, syringic, vanillic, cinnamic, ferulic, caffeic, ρ-coumaric, chlorogenic, and gentisic acids) in a model mixture and used for assay of some of these acids in a methanolic extract of herba epilobi. Application of ITP–CZE resulted in 100-fold better sensitivity than conventional CZE; limits of detection ranged between 10 and 60 ng mL⁻¹. When MES–TRIS–borate-based buffer, pH 8.3, was used in the CZE separation step the linearity of the ITP–CZE response was satisfactory (correlation coefficients were from 0.9937 to 0.9777). Repeatability was also satisfactory (RSD values ranged between 0.77% and 1.28% for migration times and between 1.65% and 13.69% for peak area). Revised: 23 March and 27 April 2006     

Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: effect of instrumental parameters

Electrospray ionization mass spectrometry (ESI-MS) is being increasingly employed in the study of metal-ligand equilibria in aqueous solution. In the present work, the ESI-MS spectral changes due to different settings of the following instrumental parameters are analyzed: the solution flow rate (F(S)), the nebulizer gas flow rate (F(G)), the sprayer potential (E), and the temperature of the entrance capillary (T). Twenty-eight spectra were obtained for each of six samples containing aluminum(III) and 2,3-dihydroxypyridine at various pH, in the absence or in the presence of a buffer and of sodium ions. Among the considered instrumental parameters, T produced the largest effects on the ionic intensities. F(S) and F(G) affected the ESI-MS spectra to a lower extent than T. In the investigated conditions E had the weakest effects on the spectra.The correlations observed between the ionic intensities and these instrumental parameters were interpreted considering the presence of three kinds of perturbations occurring in the ESI-MS ion source: formation of some dimers in the droplets, different transfer efficiencies from the droplets to the gas phase for different complexes (according to their surface activity), and subsequent partial thermal decomposition of the dimers and of one of the monomeric complexes in the gas phase. Our results show that the evaluation of the effects produced in the ESI-MS spectra by a change of instrumental parameters can allow to identify the perturbations occurring when metal-ligand solutions are studied by ESI-MS.     

Capillary electrophoresis coupled to inductively coupled plasma mass spectrometry (CE/ICP-MS) and to electrospray ionization mass spectrometry (CE/ESI-MS): An approach for maximum species information in speciation of selenium

On-line hyphenations consisting of a separation and a detection step are one of the most efficient techniques for identification and characterization of metal containing species. The high resolution power of capillary electrophoresis (CE) is used for the separation of three selenium species, whereas either electrospray ionization mass spectrometry (ESI-MS) or inductively coupled plasma mass spectrometry (ICP-MS) are taken for molecule or element specific detection. This work gives an overview about the possibilities and limitations, when using the two hyphenated systems for speciation investigations. In order to show the power of the two complementary techniques, a CZE method using 5% acetic acid as background electrolyte was applied to the separation of selenomethionine (SeM), selenocystine (SeC) and selenocystamine (SeCM). Depending on the species and the element, the detection limits of the CE/ICP-MS hyphenated system are up to 10² to 10³ times better than that for the CE/ESI-MS system.     

Fast separation and determination of carnosic acid and rosmarinic acid in different rosemary (Rosmarinus officinalis) extracts by capillary zone electrophoresis with ultra violet-diode array detection

Summary Carnosol, carnosic acid, rosmarinic acid and other not identified phenolic compounds were separated by capillary zone electrophoresis (CZE) using a 40-cm long capillary and a 20 mM tetraborate buffer (pH 9.0), within 3 min. A UV-diode array detector was employed to collect spectra of phenolic compounds. The effect of some separation parameters on peak resolution and migration time of phenolic species present in a refined rosemary extract was studied. The repeatability of the method was also investigated: the intraday relative standard deviation on total peak area was less than 4%, while the intraday relative standard deviation on migration time was less than 0.6%. Moreover the CZE method showed good sensitivity (0.0007 μg mL¹ for carnosic acid and rosmarinic acid). Carnosic acid and rosmarinic acid have been quantified in different commercial extracts of rosemary. Finally, the optimized method was also applied to evaluate the recovery of these two compounds when different organic solvents were employed during the extraction procedure.     

Analysis of Carbaryl and carbofuran in tobacco samples by HRGC,HPLC, and CZE

Determination of carbamate residues in tobacco samples was carried out by three different methods, two having been described in the literature and one developed for the purpose of this work. The extracts were analyzed by HRGC-FID (using derivatization), by HPLC-RP with photodiode array detection and by Capillary Zone Electrophoresis (CZE) with UV detection. The extraction method developed used in conjunction with capillary zone electrophoresis showed to be the most suitable for carbamate residue analysis in real tobacco samples. The developed method presented better recovery results in relation to the others. CZE allowed the determination of carbamate residue in real tobacco sample while HPLC and HRGC could not do this, due to co-elution problems.     

Capillary electrophoresis-electrospray ionization-mass spectrometry method to determine the phenolic fraction of extra-virgin olive oil

We describe the first analytical method involving SPE and CZE coupled to ESI-IT MS (CZE-ESI-MS) used to identify and characterize phenolic compounds in olive oil samples. The SPE, CZE and ESI-MS parameters were optimized in order to maximize the number of phenolic compounds detected and the sensitivity of their determination. To this end we have devised a detailed method to find the best conditions for CE separation and the detection by MS of the phenolic compounds present in olive oil using a methanol-water extract of Picual extra-virgin olive oil (VOO). Electrophoretic separation was carried out using an aqueous CE buffer system consisting of 60 mM NH(4)OAc at pH 9.5 with 5% of 2-propanol, a sheath liquid containing 2-propanol/water 60:40 v/v and 0.1% v/v triethylamine. This method offers to the analyst the chance to study important phenolic compounds such as phenolic alcohols (tyrosol (TY), hydroxytyrosol (HYTY) and 2-(4-hydroxyphenyl)ethyl acetate), lignans ((+)-pinoresinol and (+)-1-acetoxypinoresinol), complex phenols (ligstroside aglycon (Lig Agl), oleuropein aglycon, their respective decarboxylated derivatives and several isomeric forms of these (dialdehydic form of oleuropein aglycon, dialdehydic form of ligstroside aglycon, dialdehydic form of decarboxymethyl elenolic acid linked to HYTY, dialdehydic form of decarboxymethyl elenolic acid linked to TY) and 10-hydroxy-oleuropein aglycon) and one other phenolic compound (elenolic acid) in extra-VOO by using a simple SPE before CE-ESI-MS analysis.     

Speciation of inorganic and methylated arsenic compounds by capillary zone electrophoresis with indirect UV detection. Application to the analysis of alkali extracts of As2S2 (realgar) and As2S3 (orpiment)

A capillary zone electrophoresis (CZE) method with indirect UV detection was developed to simultaneously separate inorganic and organic arsenic compounds including arsenite (iAsIII), arsenate (iAsV), monomethylarsonate and dimethylarsenic acid (DMAV). 2,6-Pyridinedicarboxylic acid (PDC) and n-hexadecyltrimethylammonium hydroxide (CTAOH) were selected to compose a background electrolyte (BGE), where PDC was used as chromophore and CTAOH functioned as electroosmotic flow (EOF) modifier to reduce/eliminate EOF. The choice of detection wavelength, the optimization of BGE pH, and effects of applied electric field strength and temperature on separation were further investigated. The limits of detection for the targeted analytes were between 0.19 and 0.23 ppm as molecule. Good linearity of more than three orders of magnitude was obtained. Repeatability of migration times and peaks areas were 0.8-1.7 and 3.4-6.9% R.S.D.; whereas reproducibility were 1.2-2.2 and 3.6-7.1% R.S.D., respectively. The established CZE method was then applied to analyze the alkali extracts of realgar (As2S2) and orpiment (As2S3). The main components in both alkali extracts were identified to be iAsIII and iAsV.     

CZE for the speciation of arsenic in aqueous soil extracts

We developed two separation methods using CZE with UV detection for the determination of the most common inorganic and methylated arsenic species and some phenylarsenic compounds. Based on the separation method for anions using hydrodynamic sample injection the detection limits were 0.52, 0.25, 0.27, 0.12, 0.37, 0.6, 0.6, 1.2 and 1.0 mg As/L for phenylarsine oxide (PAO), p-aminophenylarsonic acid (p-APAA), o-aminophenylarsonic (o-APAA), phenylarsonic acid (PAA), 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenite or arsenious acid (As(III)) and arsenate (As(V)), respectively. These detection limits were improved by large-volume sample stacking with polarity switching to 32, 28, 14, 42, 22, 27, 26 and 27 microg As/L for p-APAA, o-APAA, PAA, roxarsone, MMA, DMA, As(III) and As(V), respectively. We have applied both methods to the analysis of the arsenic species distribution in aqueous soil extracts. The identification of the arsenic species was validated by means of both standard addition and comparison with standard UV spectra. The comparison of the arsenic species concentrations in the extracts determined by CZE with the total arsenic concentrations measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) indicated that CZE is suited for the speciation of arsenic in environmental samples with a high arsenic content. The extraction yield of phenylarsenic compounds from soil was derived from the arsenic concentrations of the aqueous soil extracts and the total arsenic content of the soil determined by ICP-AES after microwave digestion. We found that 6-32% of the total amount of arsenic in the soil was extractable by a one-step extraction with water in dependence on the type of arsenic species.     

Use of the bromine isotope ratio in HPLC-ICP-MS and HPLC-ESI-MS analysis of a new drug in development

A combination of inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization mass spectrometry (ESI-MS) was deployed for the metabolite profiling and metabolite identification of a new antituberculosis compound (R207910, also known as TMC207) that is currently in drug development. R207910 contains one bromine atom, allowing the detection by ICP-MS. Fluctuations in the Br sensitivity caused by the HPLC gradient were counteracted by the use of species-unspecific isotope dilution. In order to evaluate the method developed, the results obtained were compared with those acquired via radioactivity detection. HPLC-ESI-MS was used for the structural identification of R207910 and its metabolites. The ⁷⁹Br/⁸¹Br isotope ratio is also valuable in the search for metabolites in the complex background of endogenous compounds obtained using HPLC-ESI-MS analyses. Data-dependent scanning using isotope recognition with an ion trap mass spectrometer or processing of Q-Tof data provides HPLC-ICP-MS-like “bromatograms”. The combination of accurate mass measurements and the fragmentation behavior in the MS² spectra obtained using the Q-Tof Ultima mass spectrometer or MSⁿ spectra acquired using the LTQ-Orbitrap allowed structural characterization of the main metabolites of R207910 in methanolic dog and rat faeces extracts taken 0–24 h post-dose. Figure Analyses of a rat faeces extract taken 0–24 h post-dose: a HPLC-ICP-MS using isotope dilution, b corresponding Br mass flow chromatogram, c radio-HPLC, d Q-Tof ESI-MS TIC, e Q-Tof ESI-MS bromatogram after Br stripping, f LTQ-Orbitrap ESI-MS² TIC obtained with isotopic-data-dependent scanning