Ruth Barak

Direct determination of the chemical composition of acetylcholinesterase phosphonylation products utilizing electrospray-ionization mass spectrometry

While non‐reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post‐inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methyl‐phosphonofluoridates, were measured by electrospray‐ionization mass spectrometry (ESI‐MS). The measured mass of the free enzyme was 64 700 Da (calculated 64 695 Da) and those of the methylphosphono‐HuAChE adducts, bearing isopropyl, isobutyl, 1,2‐dimethylpropyl and 1,2,2‐trimethylpropyl substituents, were 64 820, 64 840, 64 852 and 64 860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64 780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2‐dimethylpropyl methylphosphono‐HuAChE, the change in the molecular mass and the kinetics of non‐reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to ‘aging’ of phosphonyl‐AChE adducts.

N1-(2-hydroxyethylthioethyl)-4-methyl imidazole (4-met-1-imid-thiodiglycol) in plasma and urine: a novel metabolite following dermal exposure to sulphur mustard

There is increasing interest in the mode of action and metabolism of alkylating agents and specifically sulphur mustard (HD). We report the detection of a new metabolite, 4-met-l-imid-thiodiglycol, formed following dermal exposure of pigs to HD. Alkylating agents are electrophilic in nature and react in vivo with N7 of guanine moieties in nucleic acids (Brookes et al. 1960). Proteins are predominantly alkylated on the cysteine, histidine and N-terminal amino acids (Bailey et al. 1987). Several alkylated metabolites of HD have been previously reported in the literature (Davison et al. 1960). The principal reaction product of DNA with HD, isolated from mice bearing Ehrlich ascites tumor, is 7-(2-hydroxyethylthioethyl) guanine (Brookes et al. 1960). The major metabolites in urine after intraperitoneal injection of (S35)-HD to rats are conjugates of 2,2-dicysteinyl ethyl sulphone (Roberts et al. 1963). There have been no case reports or pharmacological studies reporting the formation of alkylated histidine by HD. However, it is known that histidine in hemoglobin may undergo alkylation processes like 2-hydroxyalkylation following exposure to propylene oxide and ethylene oxide (Bailey et al. 1987). In the search of metabolites following dermal exposure of HD in pigs we have isolated a previously unknown compound. It was isolated from plasma and urine samples of pigs by extraction and purification on a C18 Sep-Pak column. Various modes of FAB/MS (fast atom bombardment/mass spectrometry) methods were used for identification. Low resolution measurements and linked scans were obtained on a VG 7035 MS instrument equipped with a Xenon gun. For high resolution measurement the VG 70 VSEQ with a Cesium gun was used.

Mass spectrometric investigation of the presence of 7-methyl ring-opened guanine derivatives in urine

The involvement of chemical alkylating agents in tumorigenesis and chemotherapy is well established and it was shown that one of the main sites of alkylation is the N-7 of guanine in DNA. Though excision of damaged bases is regarded as one of the repair mechanisms in damaged DNA there is a scarcity of information concerning the excised final metabolites in body fluids. This study attempts to demonstrate the usefulness of CAD MS/MS for the detection of the final metabolite-deformylated ring-opened 7 alkylguanine in urine. Such mass spectrometric methods can be used in biomedical studies.

GC columns as micro-air samplers for the quantitative analysis of naphthalene vapours

Although linked to a number of adverse health effects, studies of naphthalene are lacking in the exposure-relevant literature. This compound, a major by-product of the incomplete combustion of most organic materials (traffic vehicle exhaust, power generation, tobacco smoke, etc.), has characteristics that are in between those of volatile and semivolatile organic compounds (VOCs and SVOCs) and has consequently often been excluded from studies of both compound classes. A new, highly quantitative, rapid and spatially well-resolved micro-sampling method was developed for the sampling of naphthalene vapours in indoor, outdoor and personal air. The method is based on GC capillary columns as air samplers, solvent extraction and GC-MS analysis. For the first time, naphthalene concentrations were quantitatively determined at the surface level (around the boundary layer) and at 1 mm height intervals above an emitting surface. Minimal sampling flow rates of 5 mL min−1 were applied to reduce any geometrical or dynamical disturbance to the environmental air flow regime to a minimum. Toluene was highly efficient, precise and easy to apply in the extraction of the target compound from the GC column sampler. Method quantification was confirmed using a unique calibrated vapour generator based on an inert GC inlet. Using this source, a 13 ng L−1 limit of quantitation (LOQ) with 90% mass recovery over a linear dynamic range of five orders of magnitude in high precision and accuracy of ±13% and 35%, respectively, were determined and validated. The applicability and flexibility of the method was demonstrated by investigating the vapour concentration profiles generated “0”–20 mm above a naphthalene-emitting surface in outdoor air. The concept introduced in this research could be easily adopted for the reliable, sensitive and quantitative monitoring of other VOCs and SVOCs in different micro-spaces for various applications.

ESMS as a Unique Tool for the Molecular Monitoring of Reactions between HuAChE and Various OP-Agents

The molecular masses of the bacterially expressed recombinant HuAChE and its conjugates with series of alkyl methylphosphonofluoridates and with diisopropyl fluorophosphate (DFP), were measured by electrospray - ionization mass spectrometry (ESMS). The mass of HuAChE (measured as 64700 Da, calc. 64695 Da) increased, following reactions with sarin, isobutyl methyl phosphorofluoridate (IBMPF), 1, 2-dimethylpropyl methyl phosphorofluoridate (DMPF), soman and DFP, by 120, 140, 150, 160 and 160 Da respectively. These values were in excellent agreement with the calculated masses of the adducts and reflected both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of the phosphyl adducts change with time to yield a common product with molecular mass of 64780 Da, which is consistent with dealkylation of the phosphonyl moieties. By sequential ESMS measurements we were able to estimate the kinetics of evolution of the aged product of the HuAChE-soman adduct (t1/2 ≈ 50 sec, at pH 6.0). This rate is in good agreement with that determined by kinetic measurement of the development of non-reactivability under similar conditions. It is important to note that in agreement with the accepted mechanism of dealkylation, only two molecular species are evident in the sequential ESMS measurements during the aging process.