Evidence of nerve agent VX exposure in rat plasma by detection of albumin-adducts in vitro and in vivo.

VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys34(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys448(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys34(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys34(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.

Skin sensitizing effects of sulfur mustard and other alkylating agents in accordance to OECD guidelines.

Vesicants cause a multitude of cutaneous reactions like erythema, blisters and ulcerations. After exposure to sulfur mustard (SM) and related compounds, patients present dermal symptoms typically known for chemicals categorized as skin sensitizer (e.g. hypersensitivity and flare-up phenomena). However, although some case reports led to the assumption that SM and other alkylating compounds represent sensitizers, a comprehensive investigation of SM-triggered immunological responses has not been conducted so far. Based on a well-structured system of in chemico and in vitro test methods, the Organization for Economic Co-operation and Development (OECD) established procedures to categorize agents on their skin sensitizing abilities. In this study, the skin sensitizing potential of SM and three related alkylating agents (AAs) was assessed following the OECD test guidelines. Besides SM, investigated AAs were chlorambucil (CHL), nitrogen mustard (HN3) and 2-chloroethyl ethyl sulfide (CEES). The methods are described in detail in the EURL ECVAM DataBase service on ALternative Methods to animal experimentation (DB-ALM). In accordance to OECD recommendations, skin sensitization is a pathophysiological process starting with a molecular initiating step and ending with the in vivo outcome of an allergic contact dermatitis. This concept is called adverse outcome pathway (AOP). An AOP links an adverse outcome to various key events which can be assayed by established in chemico and in vitro test methods. Positive outcome in two out of three key events indicates that the chemical can be categorized as a skin sensitizer. In this study, key event 1 "haptenation" (covalent modification of epidermal proteins), key event 2 "activation of epidermal keratinocytes" and key event 3 "activation of dendritic cells" were investigated. Covalent modification of epidermal proteins measured by using the DPRA-assay provided distinct positive results for all tested substances. Same outcome was seen in the KeratinoSens assay, investigating the activation of epidermal keratinocytes. The h-CLAT assay performed to determine the activation of dendritic cells provided positive results for SM and CEES but not for CHL and HN3. Altogether, following OECD requirements, our results suggest the classification of all investigated substances as skin sensitizers. Finally, a tentative AOP for SM-induced skin sensitization is suggested.

Determination of the Synthetic Antioxidant Ethoxyquin and Its Metabolites in Fish and Fishery Products Using Liquid Chromatography-Fluorescence Detection and Stable-Isotope Dilution Analysis-Liquid Chromatography-Tandem Mass Spectrometry.

The use of the synthetic antioxidant ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ) as a flame retardant in fish meal transported by sea is required by international authorities to prevent self-ignition. Because of extensive carry-over within the food chain, selective and sensitive analytical methods are required for investigations on human exposure and the safety of EQ and its metabolites. Therefore, a simple, fast, and rugged liquid-chromatography (LC) method was developed for the detection of EQ and its metabolites in fish and fishery products after liquid-liquid extraction using QuEChERS. For screening purposes, a fluorescence detector was used (LC-FLD) with the EQ-analogue methoxyquin serving as an internal standard. For stable-isotope dilution analysis by liquid chromatography-tandem mass spectrometry (SIDA-LC-MS/MS), deuterated analogues of EQ and its metabolites were synthesized for the first time and allowed for sensitive quantification and thus confirmation of screening results. Both methods were validated and successfully applied to commercially available fish samples.

Methionine329 in human serum albumin: A novel target for alkylation by sulfur mustard.

Exposure to the vesicant sulfur mustard (SM) may lead to erythema and blistering. Toxicity of SM is hypothesized due to the alkylation of DNA bases and nucleophilic amino acid side chains in proteins (adducts) by forming the hydroxyethylthioethyl (HETE) moiety. Despite its prohibition by the chemical weapons convention, SM still represents a serious threat to military personnel and civilians. Therefore, development and improvement of forensic analytical methods for the verification of SM exposure is of high interest. Protein adducts have been shown to be highly suitable and beneficial biomarkers of poisoning. Herein we present methionine329 in human serum albumin (HSA) as a novel target of SM forming a HETE-methionyl sulfonium ion. The alkylated tetrapeptide LeuGlyMet329 (-HETE)Phe, LGM(-HETE)F, was detected after pepsin-mediated proteolysis and subsequent analysis by microbore liquid chromatography-electrospray ionization-high-resolution tandem-mass spectrometry. Compound identity was confirmed by a synthetic reference. Proteolysis conditions for HSA were optimized towards maximum yield of LGM(-HETE)F and its limit of identification (32.3 nM SM in serum) was similar to those of the established HSA-derived biomarkers HETE-CysPro and HETE-CysProPhe (15.6 nM SM in serum). Stability of the alkylated Met329 in vitro and in vivo was limited to 5 days making this modification a beneficial short-time biomarker. Furthermore, it was found that the HETE-methionyl sulfonium ion can transfer its HETE moiety to the side chain of cysteine and glutamic acid as well as to the N-terminus of peptides and proteins in vitro thus revealing novel insights into the molecular toxicity of SM.

Collision-induced mass spectrometric fragmentation of protonated dimethoate and omethoate generated by electrospray ionization.

RATIONALE: Dimethoate (DIM, S=P(OMe)2 -S-CH2 -C(O)-NH-CH3 ) is a dimethyl phosphorodithioate pesticide widely used in agri- and horticulture that undergoes biotransformation in vivo by desulfuration into its more toxic oxono-derivative omethoate (OM, O=P(OMe)2 -S-CH2 -C(O)-NH-CH3 ). OM inhibits acetylcholinesterase thus provoking cholinergic crisis in vivo, ultimately leading to death. Quantitative approaches for the determination of DIM and OM in environmental and toxicological samples make use of tandem mass spectrometry (MS2 ). Nevertheless, so far interpretation of resulting product ions is incomplete and sometimes contradictory. METHODS: DIM and OM as well as their deuterated analogues (fully deuterated at both methoxy groups bound to the phosphorus atom) were analyzed by MS2 and MS3 after positive electrospray ionization and collision-induced dissociation (CID) in a linear ion trap to characterize fragmentations. The accurate masses of product ions were determined in a time-of-flight mass analyzer. Hydrogen/deuterium (H/D)-exchange experiments were carried out for further support of product ion identification. In addition, density functional theory (DFT) computations were used to calculate both the most stable protonation sites of DIM and OM and the changes in the diverse bond lengths after protonation. RESULTS: Some identical and some related product ions of DIM and OM were found but also striking individual differences. Fragmentation pathways were proposed and product ions identified. Most fragmentations followed the common rules of charge migration fragmentation. DFT calculations supported experimental findings. CONCLUSIONS: Discrepancies present in the literature so far are clarified and a deeper insight is provided into the fragmentation processes of organophosphorus pesticides. The combination of diverse experimental and theoretical approaches yielded consistent results, thus demonstrating continuous progress in understanding gas-phase reactions in MS experiments.

Verification of organophosphorus pesticide poisoning: Detection of phosphorylated tyrosines and a cysteine-proline disulfide-adduct from human serum albumin after intoxication with dimethoate/omethoate.

A method is described allowing forensic analysis of plasma samples to prove human poisoning with the organophosphorus pesticides omethoate (OM) and dimethoate (DIM). Upon incubation of human serum albumin (HSA) with both pesticides tyrosine residues were phosphorylated. In addition, a novel disulfide-adduct between the identical thiol-containing leaving group of OM and DIM (2-mercapto-N-methylacetamide, MNMA) and the only free cysteine residue in HSA (Cys34) was formed. Following pronase-catalyzed proteolysis either O,O-dimethyl phosphotyrosine (Tyr-dmp) or O,O-dimethyl thiophosphotyrosine (Tyr-dmsp) as well as the cysteine-proline dipeptide disulfide-adduct (MNMA-CysPro) were produced. All biomarkers were simultaneously detected using modern microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS). Corresponding limits of identification (LOI) for tyrosine-adducts (LOIOM: 30 µM, LOIDIM: 120 µM) and disulfide-adducts (LOIOM: 1.2 µM, LOIDIM: 30 µM) demonstrated that MNMA-CysPro allowed a considerably more sensitive detection. Finally, this novel method was applied to a plasma sample of an 87-year-old man, who had unintentionally ingested the pesticide Roxion® containing DIM as active ingredient. Unambiguous proof of poisoning demonstrated suitability of the novel biomarkers for sensitive verification analysis.

Novel cysteine- and albumin-adduct biomarkers to prove human poisoning with the pesticide oxydemeton-S-methyl.

We herein report on the forensic analysis of plasma samples to prove human poisoning with oxydemeton-S-methyl (ODM), S-(2-(ethylsulfinyl)ethyl)-O,O-dimethyl phosphorothioate. This organophosphorus pesticide is the active ingredient of Metasystox®, that was swallowed by a 77-year-old woman to commit suicide. ODM belongs to the class of dimethyl phosphoryl (DMP) pesticides, contains a 2-(ethylsulfinyl)ethanethiol (ESOET) leaving group and undergoes adduct formation with endogenous molecules as elaborated herein with human serum exposed to pesticides in vitro. A novel bioanalytical micro liquid-chromatography-electrospray ionization tandem high-resolution mass spectrometry method (µLC-ESI MS/HR MS) was developed to target multiple biomarkers of exposure. Following pronase-catalyzed proteolysis of patient plasma and subsequent ultrafiltration, the filtrate was analyzed. Diverse reaction products of ODM as well as of its oxidized biotransformation product demeton-S-methyl sulfone (DSMS), that possesses a 2-(ethylsulfonyl)ethanethiol (ESO2ET) leaving group, were simultaneously detected. Phosphorylated tyrosine residues (Tyr-DMP) derived from human serum albumin (HSA) as well as novel dipeptide-adducts containing the Cys34 residue of HSA coupled to ESOET and ESO2ET via a disulfide bond (ESOET-CysPro and ESO2ET-CysPro) were found. In addition, a related disulfide-product was detected comprising the single amino acid cysteine and ESOET (ESOET-Cys). Whereas Tyr-DMP only proved the intake of any DMP pesticide in general, its simultaneous detection with ESOET-CysPro, ESO2ET-CysPro and ESOET-Cys allowed unambiguous identification of the ingested pesticide. Therefore, the novel biomarkers and the method developed expand the possibilities of forensic investigations of ODM poisoning.

Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin.

Nerve agents still represent a serious threat to civilian and military personnel as demonstrated by the violent conflict in the Middle East. For verification of poisoning, covalent adducts with endogenous proteins (e.g., human serum albumin, HSA) are valuable long-term biomarkers. Accordingly, we developed a microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (µLC-ESI MS/HR MS) method for simultaneous detection of HSA-adducts with the V-type nerve agents VX, Chinese VX (CVX), and Russian VX (RVX). Following Pronase-catalyzed proteolysis, novel disulfide-adducts were detected in addition to phosphonylated tyrosine residues. Dipeptide disulfide-adducts were formed between the thiol-containing leaving group of the V-type nerve agents (2-(diisopropylamino)ethanethiol, DPAET, for VX and 2-(diethylamino)ethanethiol, DEAET, for CVX and RVX) and the free thiol group of Cys34 in HSA (DPAET-CysPro, DEAET-CysPro). We also identified tripeptide disulfide-adducts containing Cys448 (MetProCys-DPAET, MetProCys-DEAET) and to a lesser extent Cys514 (AspIleCys-DPAET, AspIleCys-DEAET). Synthetic tripeptide references were used for confirmation of the postulated structures by µLC-ESI MS/HR MS. Lower limits of detection were determined in human plasma, being nearly identical for the three V-type nerve agents, and corresponded to 1-6 µM nerve agent for tyrosine-adducts, 1-3 µM nerve agent for CysPro-adducts, and 6 µM nerve agent for MetProCys-adducts, thus covering concentrations of toxicological relevance. Characterization of proteolysis kinetics revealed stable plateaus for all adducts being reached between 60 and 90 min at 37 °C. Adduct formation kinetics were characterized by simultaneously monitoring the V-type nerve agent, its leaving group, and the corresponding disulfide dimer. Furthermore, adduct formation patterns were investigated as a function of the molar ratio of HSA to V-type nerve agent. Graphical abstract Modification of human serum albumin (HSA) by V-type nerve agents Chinese VX (CVX) and RussianVX (RVX). Various tyrosine residues (Tyr???)n (e.g. most reactive Tyr411) were phosphonylated and disulfide-adducts were formed between the thiol-containing leaving group 2-(diethylamino)ethanethiol (DEAET) and at least three cysteine residues (Cys34, Cys448 and Cys514). Pronase-mediated proteolysis produced low-molecular cleavage products including phosphonylated tyrosines, dipeptide (Cys34Pro) and tripeptide (MetProCys448, AspIleCys514) disulfide-adducts that were detected by microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (µLC-ESI MS/HR MS).

N-Acetylcysteine as a chemical scavenger for sulfur mustard: New insights by mass spectrometry.

The vesicant sulfur mustard (SM) is a banned chemical warfare agent. Although, SM has been used in combat since WWI, there is no causal therapy currently available. Accordingly, development and investigation of antidotes and scavengers targeting SM are of high clinical relevance. N-acetylcysteine (NAC) was shown to mitigate symptoms of SM intoxications in vitro and in vivo. However, it is still unclear whether the beneficial effects of NAC are only due to physiological processes or also due to chemical scavenging of SM. Therefore, in this study, we examined the scavenging potential of NAC toward SM. Co-incubations of SM and different NAC concentrations in human serum were performed to monitor diverse adducts (covalent reaction products) of human serum albumin (HSA), NAC, and SM. After proteolytic cleavage of HSA with proteinase K the alkylated tripeptide hydroxyethylthioethyl-CysProPhe (HETE-CPF) and the disulfide bridged tripeptide NAC-CPF were detected. Samples were analyzed by microbore liquid chromatography-electrospray ionization-high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS). Furthermore, degradation kinetics of SM in phosphate buffered saline were measured in the presence and absence of NAC. Although NAC-CPF was identified and characterized for the first time by mass spectrometry and reaction products of NAC and SM were detected and identified by MS/HR MS, analyses clearly documented minor reactivity not significantly contributing to reduction of SM concentrations. Therefore, we conclude that chemical scavenging of SM by NAC does not play the key role in NAC therapy of SM poisoning.

Glutathione as an antidote for sulfur mustard poisoning: Mass spectrometric investigations of its potency as a chemical scavenger.

The banned chemical warfare agent sulfur mustard (SM) still represents a serious threat to civilians and military personnel. Therefore, identification of antidotes and scavengers is of high concern. One promising substance is glutathione (GSH). GSH is known to mitigate symptoms of SM poisoning in vitro and in vivo. However, the mechanism of action remains unclear with respect to physiological impact as well as chemical scavenging by reaction between GSH and SM. Therefore, a novel in vitro method was used to characterize the scavenging potential of GSH. Accordingly, alkylation of human serum albumin (HSA), which represents an established biomarker for SM intoxication, was used as a measure for remaining SM. Coincubation of GSH and SM in human serum was performed, and time-dependent degradation of SM was monitored in the presence and absence of GSH. Protein-derived and small molecular reaction products between GSH, HSA, and SM were analyzed using microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Although covalent modification of GSH by SM was observed, measurements clearly documented no significant reduction of SM concentration in the presence of GSH. Accordingly, beneficial therapeutic mechanisms of GSH in the case of SM poisoning would appear to be based on physiological effects than on chemical scavenging.

Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin.

Chemical warfare agents represent a continuous and considerable threat to military personnel and the civilian population. Such compounds are prohibited by the Chemical Weapons Convention, to which adherence by the member states is strictly controlled. Therefore, reliable analytical methods for verification of an alleged use of banned substances are required. Accordingly, current research focuses on long-term biomarkers derived from covalent adducts with biomolecules such as proteins. Recently, we have introduced a microbore liquid chromatography/electrospray ionization high-resolution tandem mass spectrometry method allowing for the investigation of two different classes of adducts of the nerve agent VX with human serum albumin (HSA). Phosphonylated tyrosine residues and novel disulfide adducts at cysteine residues of HSA were produced by enzymatic cleavage with pronase and detected simultaneously. Notably, the thiol containing leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) formed disulfide adducts that were released as cysteine and proline containing dipeptides originating from at least two different sites of HSA. Aim of this study was to identify assumed and novel adducts of DPAET with HSA using synthetic peptide reference compounds. Two novel tripeptides were identified representing disulfide adducts with DPAET (Met-Pro-Cys-DPAET, MPC-DPAET and Asp-Ile-Cys-DPAET, DIC-DPAET). MPC-DPAET was shown to undergo partial in-source decay during electrospray ionization for MS detection thereby losing the N-terminal Met residue. This results in the more stable Pro-Cys-DPAET (PC-DPAET) dipeptide detectable as protonated ion. The limit of detection for MPC-DPAET was evaluated, revealing toxicologically relevant VX plasma concentrations. The results provide novel insights into the reactivity of VX and its endogenous targets. Copyright © 2016 John Wiley & Sons, Ltd.

Modification of human serum albumin by the nerve agent VX: microbore liquid chromatography/electrospray ionization high-resolution time-of-flight tandem mass spectrometry method for detection of phosphonylated tyrosine and novel cysteine containing disulfide adducts.

RATIONALE: Organophosphorus nerve agents still constitute a considerable threat to the health of military personnel and the civilian population. Long-term biomarkers are crucial for reliable verification of exposure to banned substances. Therefore, current research focuses on identification of endogenous protein targets showing covalent modifications by organophosphorus nerve agents (adducts). METHODS: Purified human serum albumin and human plasma were incubated with the nerve agent VX followed by enzymatic proteolysis with pronase. Resulting peptide cleavage products were separated by microbore liquid chromatography (µLC) online coupled to positive electrospray ionization (ESI) with subsequent high-resolution time-of-flight tandem mass spectrometry (HR MS/MS) allowing identification of known and novel adducts. RESULTS: In addition to known phosphonylation of various tyrosine residues, albumin was found to be modified at diverse cysteine residues by covalent attachment of the leaving group of VX. These novel disulfide adducts were cleaved from at least two regions of the intact protein as dipeptides containing cysteine and proline either as CP or PC. A rapid and sensitive method was developed for simultaneous detection of the diverse covalent modifications of human albumin by VX. CONCLUSIONS: Identification of the novel leaving group adducts with human albumin expands the basic knowledge on molecular toxicology of the nerve agent VX. Furthermore, the presented µLC/ESI HR MS/MS method might be of relevance for verification of VX poisoning. Copyright © 2016 John Wiley & Sons, Ltd.

Optimized verification method for detection of an albumin-sulfur mustard adduct at Cys(34) using a hybrid quadrupole time-of-flight tandem mass spectrometer after direct plasma proteolysis.

The vesicant sulfur mustard (SM) is a banned chemical warfare agent that is controlled by the Organisation for the Prohibition of Chemical Weapons (OPCW). Bioanalytical procedures are mandatory for proving an alleged use and incorporation of SM into the body. We herein present the development and application of a novel optimized procedure suitable for qualitative verification analysis of plasma targeting the SM-adduct of human serum albumin (HSA) alkylated at the cysteine(34) residue. Diluted human plasma is directly mixed with pronase in an ultrafiltration device (10kDa cut-off) for proteolysis (4h, 37°C). Following ultrafiltration the filtrate is diluted and analyzed by microbore liquid chromatography-electrospray ionization high resolution tandem-mass spectrometry (µLC-ESI HR MS/MS) targeting the alkylated dipeptide hydroxyethylthioethyl-CysPro (HETE-CP). A hybrid quadrupole time-of-flight mass spectrometer provided high mass spectrometric resolution in the MS/MS mode enabling highest selectivity and sensitivity (lower limit of detection corresponding to 9.8nM SM in plasma). Kinetics of HETE-CP formation from heparin-, citrate-, and EDTA-plasma as well as serum are presented and the influence of different EDTA and pronase concentrations was characterized. The novel procedure was applied to plasma samples provided by the OPCW as well as to patients plasma derived from real cases of SM-poisoning.

Elimination kinetics and molecular reaction mechanisms of cyclosarin (GF) by an oxime substituted ß-cyclodextrin derivative in vitro.

Detoxification mechanisms of the chemical warfare agent cyclosarin (GF) in presence of 6-OxP-CD were investigated in detail in in vitro model systems. Most important finding was the preference of 6-Ox-P-CD to eliminate the more toxic (-)-GF. However, elimination of GF enantiomers was dependent on the 6-OxP-CD:GF ratios showing decreasing stereoselectivity and speed of the reaction with increasing GF concentrations. Formation of covalent mono, bis, tris and tetrakis conjugates ((CHMP)n-6-OxP-CD) and appearance of small molecular fragments (SMF) as possible decomposition products after consumption of 6-OxP-CD could be observed.. Interestingly, the non-toxic metabolite O-cyclohexyl methylphosphonic acid (CHMPA) and covalent mono and bis conjugates of 6-OxP-CD and GF were immediately formed, indicating that GF elimination proceeds along different pathways. These important new insights provide information about the mode of action of 6-Ox-P-CD including the role of the pyridinium aldoxime group on the cyclodextrin ring. They are the basis for further investigations in biological media, which could eventually lead to approval of 6-OxP-CD as a new antidote against nerve agent toxicity.

Elimination pathways of cyclosarin (GF) mediated by ß-cyclodextrin in vitro: pharmacokinetic and toxicokinetic aspects.

Cyclodextrins (CD) are promising small molecular scavengers showing favourable degradation of extremely toxic organophosphorus compounds (OP) such as tabun (GA), soman (GD) or cyclosarin (GF). For ß-CD derivatives as potential OP antidotes with low intrinsic toxicity it is of great interest to completely understand the modes of interaction of both compounds in terms of OP detoxification. The mechanisms of CD action are not completely understood which prompted us to investigate the interactions of GF and ß-cyclodextrin (ß-CD) as model compounds. Using positive electrospray ionization mass spectrometry (ESI/MS), the formation of covalent conjugates of ß-CD with O-cyclohexylmethylphosphonate (CHMP) residue was detected for the first time and was examined in vitro. With a newly developed LC-MS method the formation of O-cyclohexylmethylphosphonic acid (CHMPA) (i.e. GF hydrolysis) and covalent CHMP-ß-CD conjugates was analyzed. Compared to water, tris(hydroxymethyl)aminomethane (TRIS) reduced the formation of covalent conjugates but amplified formation of CHMPA. Depending on experimental conditions the degradation of GF by ß-CD may be preferably catalytic or stoichiometric. For illustrating different possible reaction pathways a scheme was established that could support the idea of ß-CD acting as an artificial enzyme. These results provide an important insight into the ß-CD mediated detoxification pathways of GF.