Assessment of gas chromatography methodology approach for the trace evaluation of carcinogenic impurity, methyl chloride, in trimetazidine dihydrochloride.

OBJECTIVE: A simple and robust head space/gas chromatography with flame ionisation sensor (HS/GC/FIS) approach for the trace evaluation of carcinogenic impurity, methyl chloride, in trimetazidine dihydrochloride (TRD) drug ingredient and its formulation is described. METHOD: This HS/GC/FIS approach was based on separation and analysis of CH3Cl content on DB-624 [75.0m - length, 0.53mm - internal diameter, 3.0µm - film thickness] column using nitrogen as carrier gas flowing through the column at 3mL/min stream rate. Detection of eluted CH3Cl was accomplished with flame ionization sensor at a set temperature of 260̊C. RESULTS: The optimised HS/GC/FIS methodological approach was thoroughly validated, demonstrating that it was linear with range of 5.0ppm to 1508.4ppm, sensitive with detection limit of 1.65ppm and quantification limit of 5.01ppm, reproducible with RSD values of 2.10-2.35%, accurate with recoveries of 81.9-99.0%, robust with percent variation of 7.5-12.22% with respect to changes in oven temperature, injector temperature, detector temperature and practical for regular TRD quality control. CONCLUSION: The findings revealed that with this optimised HS/GC/FIS methodological approach, the trace amounts of carcinogenic impurity (methyl chloride) in TRD drug ingredient and formulation could be successfully measured.

Design and fabrication of new anticancer sensor for monitoring of daunorubicin using 1-methyl-3-octylimidazolinium chloride and tin oxide/nitrogen-doped graphene quantum dot nanocomposite electrochemical sensor.

In this study, a novel tin oxide/nitrogen-doped graphene quantum dot nanocomposite (SnO2-NDGQD) and 1-methyl-3-octylimidazolinium chloride (1M3OICl) ionic liquid amplified carbon paste electrode (CPE) was fabricated as an efficient and fast-response sensor to determine daunorubicin, an anticancer drug. The electrochemical characteristics of daunorubicin at the surface of the 1M3OICl/SnO2-NDGQD/CPE was explored via various voltammetric methods. The high-resolution transmission electron microscope (HR-TEM) images were recorded to examine the morphological structure of the as-synthesized nanocomposites. The 1M3OICl/SnO2-NDGQD/CPE offered a wide linear concentration of 0.001-280.0 µM with a low detection limit of 0.40 nM at the optimized experimental conditions using square wave voltammetric (SWV) method. In a nutshell, the developed electrode illustrated outstanding selectivity in the presence of interfering agents and long-term stability. The1M3OICl/SnO2-NDGQD/CPE was used as new and powerful analytical tool for determination of daunorubicin in real samples with recovery range 98.75%-104.8%.

13 C-chloromethane incubations provide evidence for novel bacterial chloromethane degraders in a living tree fern.

Chloromethane (CH3 Cl) is the most abundant halogenated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3 Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3 Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. We identified and characterized CH3 Cl-degrading bacteria associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13 C-labelled CH3 Cl combined with metagenomics. Metagenome-assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3 Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3 Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3 Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterized methylotrophic cmuA-independent pathway may drive CH3 Cl degradation in the investigated tree ferns.

Chlorine Isotope Fractionation of the Major Chloromethane Degradation Processes in the Environment.

Chloromethane (CH3Cl) is an important source of chlorine in the stratosphere, but detailed knowledge of the magnitude of its sources and sinks is missing. Here, we measured the stable chlorine isotope fractionation (εCl) associated with the major abiotic and biotic CH3Cl sinks in the environment, namely, CH3Cl degradation by hydroxyl (·OH) and chlorine (·Cl) radicals in the troposphere and by reference bacteria Methylorubrum extorquens CM4 and Leisingera methylohalidivorans MB2 from terrestrial and marine environments, respectively. No chlorine isotope fractionation was detected for reaction of CH3Cl with ·OH and ·Cl radicals, whereas a large chlorine isotope fractionation (εCl) of -10.9 ± 0.7‰ (n = 3) and -9.4 ± 0.9 (n = 3) was found for CH3Cl degradation by M. extorquens CM4 and L. methylohalidivorans MB2, respectively. The large difference in chlorine isotope fractionation observed between tropospheric and bacterial degradation of CH3Cl provides an effective isotopic tool to characterize and distinguish between major abiotic and biotic processes contributing to the CH3Cl sink in the environment. Our findings demonstrate the potential of emerging triple-element isotopic approaches including chlorine to carbon and hydrogen analysis for the assessment of global cycling of organochlorines.

Methyl Chloride and Methyl Bromide Production and Consumption in Coastal Antarctic Tundra Soils Subject to Sea Animal Activities.

Methyl chloride (CH3Cl) and methyl bromide (CH3Br) are the predominant carriers of natural chlorine and bromine from the troposphere to the stratosphere, which can catalyze the destruction of stratospheric ozone. Here, penguin colony soils (PCS) and the adjacent tundra soils (i.e., penguin-lacking colony soils, PLS), seal colony soils (SCS), tundra marsh soils (TMS), and normal upland tundra soils (UTS) in coastal Antarctica were collected and incubated for the first time to confirm that these soils were CH3Cl and CH3Br sources or sinks. Overall, tundra soil acted as a net sink for CH3Cl and CH3Br with potential flux ranges from -18.1 to -2.8 pmol g-1 d-1 and -1.32 to -0.24 pmol g-1 d-1, respectively. The deposition of penguin guano or seal excrement into tundra soils facilitated the simultaneous production of CH3Cl and CH3Br and resulted in a smaller sink in PCS, SCS, and PLS. Laboratory-based thermal treatments and anaerobic incubation experiments suggested that the consumption of CH3Cl and CH3Br was predominantly mediated by microbes while the production was abiotic and O2 independent. Temperature gradient incubations revealed that increasing soil temperature promoted the consumption of CH3Cl and CH3Br in UTS, suggesting that the regional sink may increase with Antarctic warming, depending on changes in soil moisture and abiotic production rates.

A bioinspired strategy for designing well-ordered nanotubular structures by templateless electropolymerization of thieno[3,4-b]thiophene-based monomers.

Here, a bioinspired strategy is used to prepare well-ordered nanotubular structures, as observed in animals and plants, such as gecko toe pads or corals. The nanotubes are obtained by templateless electropolymerization of thieno[3,4-b]thiophene-based monomers with various aromatic groups in an organic solvent (dichloromethane). The most interesting and robust structures were obtained with carbazole and pyrene substituents to the base monomer structure, since these groups participate significantly in the polymerization and also have strong π-stacking interactions. The addition of water to electropolymerization solvent significantly impacted the formation of nanotubes, as it caused the release of a significant amount of H2 and O2 bubbles, depending on the electropolymerization method. Identifying templateless approaches to vary nanotubular structures is very interesting, as these materials are sought-after for applications in water harvesting systems. This article is part of the theme issue 'Bioinspired materials and surfaces for green science and technology (part 3)'.

Methylotrophs and Methylotroph Populations for Chloromethane Degradation.

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle.

Isotopic Characterization (2H, 13C, 37Cl, 81Br) of Abiotic Degradation of Methyl Bromide and Methyl Chloride in Water and Implications for Future Studies.

Methyl bromide (CH3Br) and methyl chloride (CH3Cl) significantly contribute to stratospheric ozone depletion. The atmospheric budgets of both compounds are unbalanced with known degradation processes outweighing known emissions. Stable isotope analysis may be capable to identify and quantify emissions and to achieve a balanced budget. Degradation processes do, however, cause isotope fractionation in methyl halides after emission and hence knowledge about these processes is a crucial prerequisite for any isotopic mass balance approach. In the current study, triple-element isotope analysis (2H, 13C, 37Cl/81Br) was applied to investigate the two main abiotic degradation processes of methyl halides (CH3X) in fresh and seawater: hydrolysis and halide exchange. For CH3Br, nucleophilic attack by both H2O and Cl- caused significant primary carbon and bromine isotope effects accompanied by a secondary inverse hydrogen isotope effect. For CH3Cl only nucleophilic substitution by H2O was observed at significant rates causing large primary carbon and chlorine isotope effects and a secondary inverse hydrogen isotope effect. Observed dual-element isotope ratios differed slightly from literature values for microbial degradation in water and hugely from radical reactions in the troposphere. This bodes well for successfully distinguishing and quantifying degradation processes in atmospheric methyl halides using triple-element isotope analysis.

Evaluation of developmental toxicity of Methyl Chloride (Chloromethane) in rats, mice, and rabbits.

Methyl Chloride (MeCl; Chloromethane) is a high production volume chemical (>1000 t/a) and is used as an industrial solvent. Based on cardiac lesions reported in developmental toxicity studies in mice, but not in rats, manufacturers decided to classify MeCl as a developmental toxicant, cat. 2. Recently, the European Chemical Agency required a developmental toxicity study in a non-rodent species. No developmental toxicity was observed in rabbits in the recently completed, GLP, OECD 414 guideline study. In view of the absence of cardiac effects in rats and rabbits, the purpose of this review is to consider whether the cardiac effects reported in mice should be considered real effects and, if so, their potential for relevance to humans. This paper provides substantive new evidence with data from a third species and shows that an evaluation of the integrated scientific evidence indicates the reported developmental cardiac effects in mice, if not an artifact, are unlikely to be relevant to humans. As such the classification of MeCl for developmental toxicity was reconsidered.

Modelling of the methyl halide biodegradation in bacteria and its effect on environmental systems.

Methyl halide group of pesticides are being used widely in past decades as fumigant but due to their hazardous effect, these pesticides are not sold directly. They are volatile and gaseous in nature and may easily come in the contact of trophosphere and stratosphere. In troposphere, they are harmful to the living beings; nevertheless, in stratosphere they react with ozone and degrade the ozone layers. In this study, we have investigated the in-silico pathways of methyl halide and its toxic effect on living systems like pest, humans and environment. Till date, limited studies provide the understanding of degradation of methyl halide and its effect on the environment. This leads to availability of scanty information for overall bio-magnifications of methyl halides at molecular and cellular level. The model developed in the present study explains how a volatile toxic compound not only affects living systems on earth but also on environmental layers. Hub nodes were also evaluated by investigating the developed model topologically. Methyl transferase system is identified as promising enzyme in response to degradation of methyl halides.

Chloromethane Degradation in Soils: A Combined Microbial and Two-Dimensional Stable Isotope Approach.

Chloromethane (CHCl, methyl chloride) is the most abundant volatile halocarbon in the atmosphere and involved in stratospheric ozone depletion. The global CHCl budget, and especially the CHCl sink from microbial degradation in soil, still involves large uncertainties. These may potentially be resolved by a combination of stable isotope analysis and bacterial diversity studies. We determined the stable isotope fractionation of CHCl hydrogen and carbon and investigated bacterial diversity during CHCl degradation in three soils with different properties (forest, grassland, and agricultural soils) and at different temperatures and headspace mixing ratios of CHCl. The extent of chloromethane degradation decreased in the order forest > grassland > agricultural soil. Rates ranged from 0.7 to 2.5 µg g dry wt. d for forest soil, from 0.1 to 0.9 µg g dry wt. d for grassland soil, and from 0.1 to 0.4 µg g dry wt. d for agricultural soil and increased with increasing temperature and CHCl supplementation. The measured mean stable hydrogen enrichment factor of CHCl of -50 ± 13‰ was unaffected by temperature, mixing ratio, or soil type. In contrast, the stable carbon enrichment factor depended on CHCl degradation rates and ranged from -38 to -11‰. Bacterial community composition correlated with soil properties was independent from CHCl degradation or isotope enrichment. Nevertheless, increased abundance after CHCl incubation was observed in 21 bacterial operational taxonomical units (OTUs at the 97% 16S RNA sequence identity level). This suggests that some of these bacterial taxa, although not previously associated with CHCl degradation, may play a role in the microbial CHCl sink in soil.

Empirical data in support of a skin notation for methyl chloride.

This article presents the first empirical experimental data on the skin absorption of methyl chloride gas using an in vitro technique and human skin. Methyl chloride is a commonly used industrial agent that is known to be an inhalational hazard but is also reported to be absorbed through human skin in amounts that contribute substantially to systemic intoxication. As a result, is has been assigned a skin notation by the ACGIH. Other than predictive models, there is a general paucity of experimental data on the skin absorption of methyl chloride and therefore a distinct lack of empirical evidence in the open literature to support the assignment of a skin notation for this chemical. This study found that methyl chloride permeates through human epidermis when exposed at high atmospheric concentrations within relatively short timeframes. Therefore, providing important initial empirical evidence in support of the assignment of a skin notation.

Mutualistic interaction between dichloromethane- and chloromethane-degrading bacteria in an anaerobic mixed culture.

The microbial mixed culture RM grows with dichloromethane (DCM) as the sole energy source generating acetate, methane, chloride and biomass as products. Chloromethane (CM) was not an intermediate during DCM utilization consistent with the observation that CM could not replace DCM as a growth substrate. Interestingly, cultures that received DCM and CM together degraded both compounds concomitantly. Transient hydrogen (H2 ) formation reaching a maximum concentration of 205 ± 13 ppmv was observed in cultures growing with DCM, and the addition of exogenous H2 at concentrations exceeding 3000 ppmv impeded DCM degradation. In contrast, CM degradation in culture RM had a strict requirement for H2 . Following five consecutive transfers on CM and H2 , Acetobacterium 16S rRNA gene sequences dominated the culture and the DCM-degrader Candidatus Dichloromethanomonas elyunquensis was eliminated, consistent with the observation that the culture lost the ability to degrade DCM. These findings demonstrate that culture RM harbours different populations responsible for anaerobic DCM and CM metabolism, and further imply that the DCM and CM degradation pathways are mechanistically distinct. H2 generated during DCM degradation is consumed by the hydrogenotrophic CM degrader, or may fuel other hydrogenotrophic processes, including organohalide respiration, methanogenesis and H2 /CO2 reductive acetogenesis.

Recent advances in QM/MM free energy calculations using reference potentials.

BACKGROUND: Recent years have seen enormous progress in the development of methods for modeling (bio)molecular systems. This has allowed for the simulation of ever larger and more complex systems. However, as such complexity increases, the requirements needed for these models to be accurate and physically meaningful become more and more difficult to fulfill. The use of simplified models to describe complex biological systems has long been shown to be an effective way to overcome some of the limitations associated with this computational cost in a rational way. SCOPE OF REVIEW: Hybrid QM/MM approaches have rapidly become one of the most popular computational tools for studying chemical reactivity in biomolecular systems. However, the high cost involved in performing high-level QM calculations has limited the applicability of these approaches when calculating free energies of chemical processes. In this review, we present some of the advances in using reference potentials and mean field approximations to accelerate high-level QM/MM calculations. We present illustrative applications of these approaches and discuss challenges and future perspectives for the field. MAJOR CONCLUSIONS: The use of physically-based simplifications has shown to effectively reduce the cost of high-level QM/MM calculations. In particular, lower-level reference potentials enable one to reduce the cost of expensive free energy calculations, thus expanding the scope of problems that can be addressed. GENERAL SIGNIFICANCE: As was already demonstrated 40 years ago, the usage of simplified models still allows one to obtain cutting edge results with substantially reduced computational cost. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

Development and validation of an universal interface for compound-specific stable isotope analysis of chlorine (37Cl/35Cl) by GC-high-temperature conversion (HTC)-MS/IRMS.

A universal application of compound-specific isotope analysis of chlorine was thus far limited by the availability of suitable analysis techniques. In this study, gas chromatography in combination with a high-temperature conversion interface (GC-HTC), converting organic chlorine in the presence of H2 to gaseous HCl, was coupled to a dual-detection system, combining an ion trap mass spectrometer (MS) and isotope-ratio mass spectrometer (IRMS). The combination of the MS/IRMS detection enabled a detailed characterization, optimization, and online monitoring of the high-temperature conversion process via ion trap MS as well as a simultaneous chlorine isotope analysis by the IRMS. Using GC-HTC-MS/IRMS, chlorine isotope analysis at optimized conversion conditions resulted in very accurate isotope values (δ(37)Cl(SMOC)) for measured reference material with known isotope composition, including chlorinated ethylene, chloromethane, hexachlorocyclohexane, and trichloroacetic acids methyl ester. Respective detection limits were determined to be <15 nmol Cl on column with achieved precision of <0.3‰.

Accurate ab initio vibrational energies of methyl chloride.

Two new nine-dimensional potential energy surfaces (PESs) have been generated using high-level ab initio theory for the two main isotopologues of methyl chloride, CH3 (35)Cl and CH3 (37)Cl. The respective PESs, CBS-35( HL), and CBS-37( HL), are based on explicitly correlated coupled cluster calculations with extrapolation to the complete basis set (CBS) limit, and incorporate a range of higher-level (HL) additive energy corrections to account for core-valence electron correlation, higher-order coupled cluster terms, scalar relativistic effects, and diagonal Born-Oppenheimer corrections. Variational calculations of the vibrational energy levels were performed using the computer program TROVE, whose functionality has been extended to handle molecules of the form XY 3Z. Fully converged energies were obtained by means of a complete vibrational basis set extrapolation. The CBS-35( HL) and CBS-37( HL) PESs reproduce the fundamental term values with root-mean-square errors of 0.75 and 1.00 cm(-1), respectively. An analysis of the combined effect of the HL corrections and CBS extrapolation on the vibrational wavenumbers indicates that both are needed to compute accurate theoretical results for methyl chloride. We believe that it would be extremely challenging to go beyond the accuracy currently achieved for CH3Cl without empirical refinement of the respective PESs.

Investigation of the CH3Cl + CN(-) reaction in water: Multilevel quantum mechanics/molecular mechanics study.

The CH3Cl + CN(-) reaction in water was studied using a multilevel quantum mechanics/molecular mechanics (MM) method with the multilevels, electrostatic potential, density functional theory (DFT) and coupled-cluster single double triple (CCSD(T)), for the solute region. The detailed, back-side attack SN2 reaction mechanism was mapped along the reaction pathway. The potentials of mean force were calculated under both the DFT and CCSD(T) levels for the reaction region. The CCSD(T)/MM level of theory presents a free energy activation barrier height at 20.3 kcal/mol, which agrees very well with the experiment value at 21.6 kcal/mol. The results show that the aqueous solution has a dominant role in shaping the potential of mean force. The solvation effect and the polarization effect together increase the activation barrier height by ∼11.4 kcal/mol: the solvation effect plays a major role by providing about 75% of the contribution, while polarization effect only contributes 25% to the activation barrier height. Our calculated potential of mean force under the CCSD(T)/MM also has a good agreement with the one estimated using data from previous gas-phase studies.

Charge-dependent many-body exchange and dispersion interactions in combined QM/MM simulations.

Accurate modeling of the molecular environment is critical in condensed phase simulations of chemical reactions. Conventional quantum mechanical/molecular mechanical (QM/MM) simulations traditionally model non-electrostatic non-bonded interactions through an empirical Lennard-Jones (LJ) potential which, in violation of intuitive chemical principles, is bereft of any explicit coupling to an atom's local electronic structure. This oversight results in a model whereby short-ranged exchange-repulsion and long-ranged dispersion interactions are invariant to changes in the local atomic charge, leading to accuracy limitations for chemical reactions where significant atomic charge transfer can occur along the reaction coordinate. The present work presents a variational, charge-dependent exchange-repulsion and dispersion model, referred to as the charge-dependent exchange and dispersion (QXD) model, for hybrid QM/MM simulations. Analytic expressions for the energy and gradients are provided, as well as a description of the integration of the model into existing QM/MM frameworks, allowing QXD to replace traditional LJ interactions in simulations of reactive condensed phase systems. After initial validation against QM data, the method is demonstrated by capturing the solvation free energies of a series of small, chlorine-containing compounds that have varying charge on the chlorine atom. The model is further tested on the SN2 attack of a chloride anion on methylchloride. Results suggest that the QXD model, unlike the traditional LJ model, is able to simultaneously obtain accurate solvation free energies for a range of compounds while at the same time closely reproducing the experimental reaction free energy barrier. The QXD interaction model allows explicit coupling of atomic charge with many-body exchange and dispersion interactions that are related to atomic size and provides a more accurate and robust representation of non-electrostatic non-bonded QM/MM interactions.

Theoretical foundation for the presence of oxacarbenium ions in chemical glycoside synthesis.

Glycoside formation in organic synthesis is believed to occur along a reaction path involving an activated glycosyl donor with a covalent bond between the glycosyl moiety and the leaving group, followed by formation of contact ion pairs with the glycosyl moiety loosely bound to the leaving group, and eventually solvent-separated ion pairs with the glycosyl moiety and the leaving group being separated by solvent molecules. However, these ion pairs have never been experimentally observed. This study investigates the formation of the ion pairs from a covalent intermediate, 2,3,4,6-tetra-O-methyl-α-d-glucopyranosyl triflate, by means of computational chemistry. Geometry optimization of the ion pairs without solvent molecules resulted in re-formation of the covalent α- and ß-triflates but was successful when four solvent (dichloromethane) molecules were taken into account. The DFT(M06-2X) computations indicated interconversion between the α- and ß-covalent intermediates via the α- and ß-contact ion pairs and the solvent-separated ion pairs. The calculated activation Gibbs energy of this interconversion was quite small (10.4-13.5 kcal/mol). Conformational analyses of the ion pairs indicated that the oxacarbenium ion adopts (4)H3, (2)H3/E3, (2)H3/(2)S0, E3, (2,5)B, and B2,5 pyranosyl ring conformations, with the stability of the conformers being strongly dependent on the relative location of the counteranion.

Solvent effects on the intramolecular conversion of trimethylsulfonium chloride to dimethyl sulfide and methyl chloride.

The formation of CH3Cl from (CH3)3SCl in various solvents has been studied based on M05/6-311+G(2d,p) DFT calculations to quantify the influence of the solvent on the stability of sulfonium cations. Four different pathways (one SN1, one backside and two frontside attacks for SN2) as well as the formation of different ion pairs (tripod, seesaw, and linear) are discussed to investigate the origin of the kinetic solvent effect (KSE) and the contribution of ion pairs to the overall reaction. Ion pairs are formed only in solvents with a permittivity ε lower than 28, but the reaction proceeds via a standard SN2 mechanism with a backside attack in all solvents. The formation of ion pairs does not change the order of the rate law, but it strongly influences the KSE, which can distinguish between reactions starting from free ions and those starting from ion pairs, in contrast to standard kinetic analysis.