Determination of pharmacologically active chiral compounds (cPACs) at the enantiomeric level in environmental matrices is essential. Such an approach yields vital information for improved wastewater-based epidemiology, development of more accurate environmental risk assessment, and improved understanding of cPAC fate in wastewaters and the environment. This article gives an up-to-date commentary on chiral liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) to determine cPACs, including illicit drugs, in environmental matrices. Several applications are presented to demonstrate the benefits of performing environmental analysis of cPACs at the enantiomeric level. Finally, future perspectives in this rapidly developing field of research are outlined.

	Stereochemistry plays an important role in the life of plants and animals, but it is also vital in the agricultural, pharmaceutical, and chemical industries. Stereoisomers are compounds that differ only in the spatial arrangement of their constituent atoms or groups. They can be classified into two groups: enantiomers and diastereomers. Enantiomers are stereoisomers which are non-superimposable mirror images of one another and are described as chiral isomers (Figure 1). The fundamental difference between enantiomers and diastereomers is that enantiomers of the same chiral molecule, as opposed to diastereomers, have identical intramolecular distances between non-bonded atoms, the same energy content, and therefore the same physico-chemical properties. As a result they are very difficult to separate. Because of this difficulty, chiral pharmacologically active compounds (cPACs) are often sold as a 50:50 mixture of the two possible enantiomers (a so-called "

"). However, it is well known that biological activity is often associated with only one enantiomer.


	(PHOTO CREDIT: GETTYPHOTO/GETTY IMAGES)

	Chiral analysis relies on the separation of the analyte of interest in a chiral environment (for example, a chiral derivatizing agent or a chiral stationary phase), which enables differentiation of enantiomers through the formation of distinguishable diastereomers via multipoint interactions (1). The chiral recognition mechanism is usually explained by the "

" (Figure 2). In this a minimum of three simultaneous interactions between the chiral selector and at least one of the enantiomers, with at least one of these interactions being stereochemically dependent, is required (3). The physico-chemical properties of these diastereomers differ, enabling their separation using indirect or direct methods. In the indirect approach, the enantiomers are derivatized with an enantiomerically pure reagent followed by their separation in an achiral environment. In the direct approach, the enantiomer interacts with a chiral compound that can be either bound to an immobile support or as a mobile-phase additive.

Applications of Chiral Liquid Chromatography in Environmental Analysis 

	Despite the importance of chiral analysis in the environment, most studies do not explicitly account for individual stereoisomers. This leads to inaccurate conclusions because they incorrectly assume that enantiomers have identical environmental behaviour (4). For example, toxic effects of fluoxetine and propranolol to aquatic biota are enantiomer dependent (5,6).


	Figure 1: Structure of ketoprofen enantiomers.

	The enantiomeric fraction (EF) of a cPAC is the relative concentration of each enantiomer and a value of 0.5 denotes a racemic mixture. The EF can change significantly following intake by human metabolism and excretion, during biological wastewater treatment, and when present in the aquatic environment. Biological processes may discriminate between enantiomers of cPACs, which can lead to stereoselective enrichment or depletion of one enantiomer. Enantiomers can also undergo chiral inversion to form an enantiomer of potentially higher toxicity (7).


	Figure 2: The "three point interaction model". Adapted and reproduced with permission from Trends in Environmental Analytical Chemistry 1, Sian E. Evans and Barbara Kasprzyk-Hordern, Applications of Chiral Chromatography Coupled with Mass Spectrometry in the Analysis of Chiral Pharmaceuticals in the Environment, e34–e51 (2014) © Elsevier.

	Currently, the direct analysis approach is the method of choice for enantioseparations. The most important selectors are chiral stationary phases (CSPs) based on cyclodextrins, polysaccharides, macrocyclic antibiotics, synthetic chiral macrocycles (crown ethers, other synthetic macrocycles), chiral synthetic polymers, chiral imprinted polymers, protein-based CSPs, and ligand- or ion-exchange CSPs (8,9). CSPs based on polysaccharides, proteins, and macrocyclic antibiotics are the most used in enantioselective liquid chromatography tandem mass spectrometry (LC–MS–MS) separation methods for environmental analysis. Despite the large number of available CSPs, it is not possible to find a universal stationary phase for a range of different cPACs. This is a result of the different mechanisms involved and the high selectivity of chiral selectors required to achieve chiral recognition. For polysaccharide-based CSPs (made by various derivatives of cellulose and amylose molecules), H-bonding, π-π, dipole, and steric interactions are the main mechanisms involved, while for protein-based (α-acid glycoprotein, cellobiohydrolase, or albumin) and antibiotic-based (vancomycin, teicoplanin, or ristotecin A) CSPs, H-bonding, hydrophobic and ionic interactions, and inclusion complexes are the main interactions that determine chiral separation (Figure 3).


	Figure 3: (a) Potential binding sites and mechanisms in teicoplanin and (b) cellulose tris(3,5-dimethylphenylcarbamate). Adapted and reproduced with permission from Trends in Environmental Analytical Chemistry 1, Sian E. Evans and Barbara Kasprzyk-Hordern, Applications of Chiral Chromatography Coupled with Mass Spectrometry in the Analysis of Chiral Pharmaceuticals in the Environment, e34–e51 (2014) © Elsevier.

	Chiral analysis can provide useful information about the occurrence, fate, and transformation of cPACs and their metabolites in the environment because enantiomers of the same cPAC can interact in different ways when exposed to a chiral environment, such as in biological systems (enzymes, proteins). There are four main areas where chiral analysis of PACs in the environment is studied:

Fate of cPACs during Wastewater Treatment: 

Following consumption by the human population, cPACs reach wastewater treatment plants (WWTPs) in a modified form (for example, as metabolites) or with their EF altered. During wastewater treatment they are subjected to biotic processes that lead to further changes in enantiomeric composition. This stereoselective degradation needs attention because the discharged effluent might be enriched with one of the enantiomers and current risk assessment will not provide a realistic view. To date, there are relatively few studies that have reported enantiomeric concentrations of cPACs in influent and effluent wastewaters and in sewage sludge (7,12–18).

Wastewater-based epidemiology (WBE) is a new approach to estimate the consumption of illicit drugs by a community by determining their concentration in influent wastewater (19). The incorporation of enantiomer-specific analysis in this approach can help address current difficulties such as: distinguishing between legal and non-legal use of drugs; verification of the method of synthesis of illicit drugs; route of administration; identification of whether drug residue results from consumption of an illicit drug or metabolism of other drugs; and monitoring trends in drug abuse (20).

cPACs as Chemical Markers of Water Contamination with Wastewater: 

A small number of studies have revealed the potential for cPACs to be used as effective indicators of human sewage contamination in water courses, differentiating between sources of untreated effluents and treated effluents discharged from WWTPs. As Evans 

 (21) stated, such a marker must be consistent in samples where wastewater is found and must consistently and significantly change its EF during its residency in WWTPs. This must be in a way that is not characteristic with attenuation in the environment and propranolol was identified as a possible marker (12).

Environmental Risk Assessment and Fate in the Environment: 

Stereoselectivity requires attention because current environmental risk assessment does not account for individual enantiomers and, considering that enantiomeric specific toxicity can occur, the risk posed may be underestimated. Once present in the environment, changes in the EF could be used to differentiate biotransformation from other removal processes. Unlike abiotic mechanisms (that is, sorption, photochemical transformation, air-water, and soil-water exchange), which are believed to be the same for both enantiomers, biotic mechanisms can be enantioselective. However, the absence of enantioselectivity does not explicitly mean achiral degradation because not all biotic processes show preference for the transformation of one enantiomer over the other.

Critical Aspects of Chiral Environmental Analysis

Although often overlooked, sampling is a critical aspect of any environmental monitoring procedure. An excellent critical evaluation of different sampling modes has been conducted by Ort

 (22). The collection of grab/spot samples has obvious limitations because it can only give information for a specific point in time. Findings therefore may not be representative as cPAC concentration and wastewater or river flow can vary throughout the day. Alternatively, automated samplers are used to collect a composite sample that can be either time or volume proportional over a 24 h period. Volume proportional sampling is considered to be the most representative mode of sampling because it accounts for variations in flow. However, the stability of cPACs in composite samplers is unknown for a variety of matrices. Significant degradation (>15%) has been observed for some PACs in wastewater over a 12 h period despite storage at 4 °C (23). This is even more important for chiral analysis because stereoselective degradation occurs for several cPACs (24). Consequently, EF may change substantially from the time of sample collection to sample processing. To overcome this, biological activity can be inhibited by acidification or the addition of sodium azide to the collection bottles within the samplers. This approach has yet to be validated for chiral analysis.

Sample preparation procedures usually applied in the analysis of cPACs are achiral (that is, they should not show any stereoselectivity towards chiral analytes). The preparation of environmental matrices (typically 50 mL to 500 mL) for aqueous phase analysis is often conducted offline and involves centrifugation and/or vacuum filtration through glass fibre filters, solid-phase extraction (SPE), elution and evaporation in a suitable solvent, reconstitution in mobile phase, and filtration through pre-LC–MS filter membranes. Such a large number of steps during sample preparation increase the likelihood of incorporating error into the final measurement (25). Therefore the use of deuterated surrogates is essential to correct for such errors and ensure accuracy of the final measurement. Despite the use of deuterated surrogates, recovery of target PACs must be maximized because very low method quantitation limits are required for environmental monitoring. The loss of PACs to glassware surfaces (specifically during evaporation of SPE extracts) can impact analyte recovery significantly. For example, using silanized SPE vials gave recoveries six times higher than non-silanized vials for some PACs (25). Even with this knowledge, silanized glassware is often not used in reported analytical methods.

Coupling Chiral Chromatography with Mass Spectrometry: 

Mobile phase composition is essential for chiral chromatography, not only for achieving enantiomeric separation while using chiral stationary phases, but also to enable compatibility with mass spectrometry (MS) (21). Coupling chiral chromatography to MS is crucial for environmental analysis where complex heterogeneous matrices are encountered. The high selectivity and sensitivity of MS detection enables the quantification of cPACs present at low levels (ng to µg/L) in environmental matrices. However, the majority of established analytical methods for chiral analysis use UV detection, often for quality control purposes within the pharmaceutical industry. These mobile phases often contain non-volatile buffers or additives not compatible with MS. There is therefore a need to develop more chiral methods that can be coupled to MS for environmental analysis. Furthermore, there is a lack of chiral ultrahigh-performance liquid chromatography (UHPLC) columns available. Most chiral columns used in environmental analysis are only capable of high performance liquid chromatography (HPLC) performance and are limited to maximum operating pressures of 2000 psi. The introduction of chiral UHPLC columns would enable improved separations in shorter analysis times; this is currently the rate limiting step of current analytical methods.

	A further issue of coupling chiral chromatography or any LC system with MS for environmental analysis is matrix suppression, particularly with electrospray ionization (ESI) (26). The majority of methods in the literature report the use of polymeric-based SPE cartridges for sample preparation because of their suitability for PACs which possess a wide range of chemistries (21). However, this type of sorbent is non-selective and unwanted materials are co-extracted with target chemicals. This can result in >50% signal suppression of analyte signal strength in some cases (7). Furthermore, matrix suppression is known to vary between enantiomers of the same cPAC, reinforcing the importance of using racemic mixtures of deuterated surrogates in analytical methods. López-Serna 

 (27) reported that several compounds (propranolol, metoprolol, and timolol) are subject to stereoselective matrix effects. To demonstrate, EF for propranolol-d7 was 0.50 in a standard solution, 0.58 in surface water, and 0.66 in wastewater influent.

Chiral Liquid Chromatography Coupled with Mass Spectrometry for the Determination of cPACs in Environmental Matrices

	Most enantiomeric profiling of cPACs in environmental matrices has focused on beta-blockers, antidepressants and ephedrines. Such methods tend to use columns containing vancomycin or cellobiohydrolase stationary phases as chiral selectors. Lopez-Serna 

 (27) successfully separated 14 cPACs including metabolites at the enantiomer level. These belonged to several therapeutic classes including beta-blockers, antidepressants and anti-inflammatories. This was achieved using a column containing a vancomycin-based stationary phase (250 mm × 2.1 mm, 5-µm) operated under isocratic conditions at a flow rate of 0.1 mL/min. The mobile phase consisted of 4 mM ammonium acetate in methanol containing 0.005% formic acid. The total run time was 66 min. The addition of ammonium acetate as a mobile phase buffer was essential because it improves ionization but, more importantly, it increased the resolution of enantiomers during LC separation (27). A column containing a vancomycin-based stationary phase is versatile as it can also be used in either normal- or reversed-phase modes. On the other hand, a column with a cellobiohydrolase stationary phase is more restrictive, only enabling operation in reversed-phase mode. Nevertheless, it has successfully resolved enantiomers of amphetamines, ephedrines, some antidepressants, and beta-blockers in environmental matrices.

	Beta-blockers are ubiquitous in wastewaters and they are the best studied of the cPACs at the enantiomeric level. Many studies have found atenolol to be enriched with the 

(–)-enantiomer in raw wastewater (18,28,32,34). However, a recent study by Vazquez-Roig 

 (32) found a racemic EF that coincided with a higher estimated weekly population usage (1.3 g d

 inhabitants), suggesting the higher concentration may be from disposal of the drug, rather than human usage and metabolism. Stereoselective biodegradation of atenolol has been observed in some WWTPs (13,18,32). It is hypothesized that the degree of atenolol stereoselectivity during wastewater treatment is dependent on the season (34) and WWTP technology (32,34).

	Other beta-blockers including metoprolol (12,13,34), propranolol (13,27,29,34), and albuterol (27) behave similarly and undergo enantioselective biodegradation. The antidepressant fluoxetine is also found to exhibit steroeselectivity. In wastewater effluents fluoxetine has been found to be enriched with S(+)-fluoxetine (29). This is a significant observation as 

(+)-fluoxetine is more toxic than the corresponding enantiomer to 

 (5,6). Interestingly, the stimulant ephedrine has two chiral centres and therefore occurs as natural, 

(–)-pseudoephedrine. Only the natural diastereomers are usually found in raw wastewater (18,32). During the winter months concentrations of the whole drug were higher and enriched with 

(+)-pseudoephedrine. This is likely a result of higher usage of over-the-counter medications that contain this enantiomer for the treatment of colds.

Enantiomeric Profiling of Illicit Drugs in Environmental Matrices

	Many illicit drugs are chiral. Since human pharmacokinetics show stereoselectivity in many chiral xenobiotics, differences in potency and activity are also found in illicit drugs. For example, 

-(–)-MDMA has a more hallucinogenic effect than the 

-(+)-form (21), while (+)-LSD is 20 times more psychoactive than (–)-LSD (20). Despite this knowledge and possible differences in ecological impact between enantiomers of the same drug, there are a very limited number of illicit drugs analyzed at the enantiomeric level in environmental matrices.


	Figure 4: Extracted influent wastewater showing cPACs separated using a chiral column (METH: methamphetamine; AMPH: amphetamine; MDMA: 3,4-methylenedioxymethamphetamine; MDA: 3,4-Methylenedioxyamphetamine). Adapted and reproduced with permission from Environmental Science & Technology 46, Barbara Kasprzyk-Hordern and David S. Baker, Enantiomeric Profiling of Chiral Drugs in Wastewater and Receiving Waters, 1681–1691 (2012) © American Chemical Society.

	The enantiomeric analysis of amphetamine-like compounds in wastewater has been performed previously using a column with a cellobiohydrolase stationary phase (100 mm × 2 mm, 5-µm) operated under isocratic conditions with a mobile phase consisting of 1 mM ammonium acetate/2-propanol 9:1 (Figure 4) (7). Enantioselective separation was achieved by using a pH of 5. This enables ionic bindings between positively charged analytes and the chiral selector (isoelectric point = 3.9).

	Consequently, a higher retention for these analytes was observed in response to an increased degree of net charge of the chiral selector. A comparison between a column with a cellobiohydrolase stationary phase and a column containing a vancomycin-based stationary phase (the two most commonly used chiral columns for environmental analysis) for the analysis of several illicit drugs was performed by Bagnall 

 (29). Greater resolution was achieved with a column with a cellobiohydrolase stationary phase than a column containing a vancomycin-based stationary phase (i.e., R

 1.0) because of combinations of ion exchange, hydrogen bonding, and hydrophobic interactions. These methods operate at flow rates of ≤0.1 mL/min and run times of ≥1 h to achieve adequate enantiomeric resolution.

	Studies have shown enantioselective degradation of MDMA leading to the enrichment with the 

-(–)-enantiomer during wastewater treatment in the UK (23) and Spain (32). The extent of enantioselective degradation was found to vary between different types of chiral drugs and was dependant on the wastewater treatment technology used and the time of year (18). Enantiomer profiling of some illicit drugs and metabolites (MDMA, MDA, amphetamine and methamphetamine) has also been applied in WBE. All illicit synthesis methods produce racemic MDMA (EF = 0.5). However, 

-(+)-MDMA undergoes preferential metabolism over 

	This leads to the enrichment of residual MDMA excreted in urine (and then found in wastewater) with the 

-(–)-enantiomer (EF>0.5). This characteristic change in EF of MDMA allows the verification of whether drug residues present in wastewater results from its consumption (EF>0.5) or direct disposal of unused drug (EF = 0.5). For example, the results of several sampling campaigns in England (35) and in the Netherlands (37) showed that MDMA is usually present in wastewater as a result of direct consumption (EF>0.5; MDMA enriched with 

-(–)-enantiomer). However, excessively high mass loads of MDMA during one sampling campaign in a WWTP in Utrecht (the Netherlands) proved to be racemic, indicating direct disposal of unused MDMA. This coincided with a police raid earlier at a nearby illegal production facility within the catchment area (37).

	Chiral chromatography coupled with MS is an emerging tool in environmental analysis of cPACs. Enantiomeric profiling of cPACs in environmental matrices could aid risk assessment to help determine the fate and toxicity of these chiral pollutants. A selection of contemporary applications to detect a range of important cPACs in environmental matrices can be obtained from reference (21).

	Unfortunately, because of gaps in knowledge and the lack of robust analytical protocols for the analysis of chiral compounds at the enantiomeric level, false conclusions might be drawn from environmental data. One example concerns enantiomer-dependent signal suppression in the ESI–MS interface, which can lead to false measurements of individual enantiomers if undertaken in the absence of labelled analogues of target analytes.

	There are several aspects of stereoselective environmental analysis using chiral LC coupled with MS that require immediate attention. There is an urgent need to: (i) introduce robust protocols for sampling and adequate sample preparation approaches limiting stereoselective microbial degradation and enantiomer-dependent enrichment of analytes; (ii) introduce new chiral stationary materials, which could be applied in UHPLC applications with the aim of increasing the speed of analysis and column efficiency while maintaining high enantioselectivity, as well as increasing the capability to resolve as wide a spectrum of analytes as possible; and (iii) ensure high precision and accuracy of measurements at the enantiomeric level by using labelled analogues of target analytes to limit analytical errors leading to changes in chiral recognition (for example, stereoselective signal suppression in the ESI interface). In the future we will see significant advances in environmental MS in terms of both sensitivity and selectivity. It is important to realize that without tackling the issue of stereochemistry and the effects of chiral pollutants, false, inaccurate, or misleading conclusions will still be drawn from the environmental data.

	This work was supported by the UK Engineering and Physical Sciences Research Council [grant number EP/I038608/1 and EP/K503897/1/], the UK Natural Environment Research Council [grant number NE/I000534/1], and the European Union's Seventh Framework Programme for research, technological development, and demonstration [grant agreement 317205, the SEWPROF MC ITN project, 'A new paradigm in drug use and human health risk assessment: Sewage profiling at the community level'] and [grant agreement 629015, the MC IEF project 'Chiral veterinary medicines in the environment']. The support from Wessex Water is also greatly appreciated.

 is a research officer at the University of Bath. He was awarded his PhD in water engineering at Cranfield University. Dr Petrie moved to Bath in 2013 to work on the major interdisciplinary programme co-funded by Wessex Water and the University of Bath's EPSRC Impact Acceleration Account, titled Holistic Approaches to Sustainable Water Supply.

 has been a Marie Curie Postdoctoral IEF Fellow at the University of Bath since 2014. Originally from Spain she completed her PhD in chemistry at the University of Seville (Spain) in 2013. She also obtained a MSc in advanced studies in chemistry (University of Seville) and an MA in quality management systems (IMF Business School, Spain).

 is a Marie Curie Early Stage Researcher (Department of Chemistry, University of Bath, UK). She has a MSc in pharmaceutical chemistry (La Sapienza, Rome, Italy). She has also graduated from the National School on Addiction and the National School of Analytical Methodologies in Mass Spectrometry.

 is an EPSRC and University of Bath funded PhD student (Department of Chemistry, University of Bath, UK). She has a MSc in toxicology (Birmingham University, UK). She has also graduated from Plymouth University with a BSc (Hons) in environmental Sciences.

 is a reader in environmental and analytical chemistry in the Department of Chemistry at the University of Bath. She received her PhD in chemistry from Adam Mickiewicz University in 2004. Having completed her PhD she has worked as a lecturer at the Faculty of Chemistry at Adam Mickiewicz University, a Marie Curie Transfer of Knowledge Research Fellow in analytical environmental toxicology at the University of Glamorgan, and a lecturer/senior lecturer in analytical science at the University of Huddersfield and at the University of Bath.

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Articles

Features

This article gives an up-to-date commentary on chiral liquid chromatography coupled with mass spectrometry for the determination of pharmacologically active chiral compounds (cPACs) (including illicit drugs) in environmental matrices. Several applications are discussed to demonstrate the benefits of performing environmental analysis of cPACs at the enantiomeric level. Finally, future perspectives in this rapidly developing field of research are outlined.

Chiral Liquid Chromatography Coupled with Tandem Mass Spectrometry for Environmental Analysis of Pharmacologically Active Compounds

Redox reactions are integral parts of many cellular processes. They are therefore extensively studied

. Electrochemistry (EC) represents a purely instrumental approach to characterize direct and indirect effects of redox reactions on bioorganic molecules, such as peptides, proteins, and endogenous and exogenous small molecules as well as nucleic acids. In addition to direct infusion electrospray ionization (ESI)–mass spectrometry (MS), hyphenated techniques such as liquid chromatography–mass spectrometry (LC–MS) are often applied for comprehensive characterization of reaction mixtures generated by EC experiments. EC–LC–MS represents a fast, automatable, and "green" approach that can be used in a variety of "omics" disciplines. This review highlights important trends and recent developments. 

	Redox reactions are essential for life. There are two important groups of biologically important redox reactions: enzymatic and non-enzymatic reactions. Enzymatic redox reactions often involve complex mechanisms of several enzymes. The electrons are transported by flavin- or heme-containing coenzymes from one reaction to another. These reactions represent an integral part of many metabolic pathways. Biological energy, for instance, is stored and released by means of redox reactions. Cellular respiration is the oxidation of glucose to carbon dioxide and the reduction of oxygen to water; photosynthesis involves the reduction of carbon dioxide into sugars and the oxidation of water into molecular oxygen. Furthermore, oxidoreductases such as the members of the cytochrome P450 superfamily generate structural complexity during natural product biosynthesis, and they play a central role in the biotransformation of drugs and toxins ("phase I metabolisms"). Biotransformation reactions involving xenobiotics are usually intended to make them more polar and, thus, enable a more rapid excretion. Biotranformation can lead to loss or gain of activity. Furthermore, in some cases reactive intermediates are produced that can bind to proteins, lipids, and nucleic acids giving rise to cellular damage.

	Biologically important redox reactions can also involve nonenzymatic processes. Reactive oxygen species (ROS) are natural by-products of the normal metabolism of oxygen. However, during times of environmental stress (for example, UV or heat exposure), ROS levels can increase dramatically. The availability of ROS can be of importance for an organism, because they are used by the immune system as a way to attack and kill pathogens. Furthermore, some ROS function as physiological regulators of intracellular signalling pathways. In the majority of cases, however, ROS production is considered to be harmful. ROS can react with proteins, lipids, and nucleic acids, which usually gives rise to cell damage called oxidative stress. In humans oxidative stress is thought to be involved in the pathogenesis of different kinds of disease, including neurodegenerative disease, cardiovascular disease, and cancer.

	Because of their importance, biological redox reactions are extensively studied in 

 models. Electrochemistry (EC) was found to be a powerful approach to complement existing assays in characterizing direct and indirect effects of redox reactions on bioorganic molecules. EC was particularly useful in generating oxidation products as well as reactive intermediates, which can be trapped by different kinds of electrophiles and nucleophiles. Moreover, EC is a purely instrumental approach. Experimental conditions, including the electrochemical potential, the electrode material, pH, as well as the kind and concentration of reactants, can be precisely controlled. In addition, the use of EC means costly and often non-specific enzymes and the use of harsh chemicals can be made obsolete (1–17). Thus, EC can be regarded as a 

	Redox reactions may give rise to the formation of complex mixtures of intermediates, products, and by-products. Different kinds of analytical techniques can be used for comprehensive analysis. Mass spectrometry (MS) in particular has found widespread application for that purpose. Mass spectrometric techniques enable qualitative (that is, identification and structure elucidation) and quantitative analysis. Among the different ionization techniques available, electrospray ionization (ESI) is the most commonly applied technique. ESI allows the MS characterization of a large variety of compounds ranging from small molecules to large biopolymers. The importance of EC–ESI–MS techniques is emphasized by the large number of reviews that have been published in recent years (1–17). Particularly in life sciences, EC–ESI–MS has found widespread application. This review will give a short overview on recent advances in that important field of research (Figure 1).


	Figure 1: Sketch summarizing the different kinds of applications that are realizable with EC–(LC)–ESI–MS in metabolomics, proteomics, and nucleic acids research.

	Different experimental setups have been developed that enable EC–ESI–MS experiments. In the simplest setup, the inherent EC of ESI is used (14). From the electrochemical point of view (18–20), the ESI source represents a controlled-current cell consisting of two electrodes. One electrode is the capillary emitter; the mass spectrometer acts as the counter electrode. The two electrodes are connected on the one hand by the power supply and on the other by a series of resistors consisting of the electrochemical contact to the solution, the solution resistance, the resistance at the solution-air interface and in the gas-phase, and the charge neutralization at the counter electrode. During operation, charges are transported from the emitter via the gas phase to the mass spectrometer. The loss of charges needs to be balanced in solution, and this is accomplished by electrochemical processes at the emitter electrode. These redox reactions may involve analytes (21–23), the solvent (24,25), or the electrode material (18,26). Practitioners have learned to control the electrochemical part of ESI in a way that the impact of EC on the mass spectra observed can be very much tuned. In the majority of cases, experimental conditions are chosen that prevent the mass spectrometric detection of ESI-inherent electrochemical reactions. However, in some situations EC can be used as an analytical advantage. A clear limitation of this setup is its inability to precisely control the electrochemical potential at the emitter electrode. Thus, experimental setups allowing separation of the electrochemical processes studied from the ESI-inherent EC are more commonly used.

	Discrete electrochemical cells can be on-line hyphenated to ESI–MS (Figure 2[a]). The electrochemical cells used are typically controlled-potential cells consisting of three electrodes (that is, working electrode, reference electrode, and auxiliary electrode). The cells contain either porous flow-through or planar, thin-layer, flow-by working electrodes. Cells with porous electrodes are considered to provide good conversion rates even at high flow rates because of the large surface area provided. Typically, glassy carbon is used as electrode material. A clear disadvantage of glassy carbon is the occurrence of analyte adsorption on the electrode surface. Thus, the cells are usually operated with solvents containing high organic contents. Other problems of this cell type are limited robustness and reproducibility. Life history or age of the electrochemical cell can sometimes have an impact on the oxidation reactions observed (27). For the planar thin-layer cells, adsorption on the working electrode is usually a less common problem (28). Another advantage of this cell type is the possibility to use different kinds of electrode materials, such as glassy carbon, boron-doped diamond, platinum, and titanium. For obtaining high conversion rates, thin-layer cells are usually operated at very low flow rates (<10 μL/min) (29).


	Figure 2: Commonly used setups for combining EC with LC and ESI–MS: (a) EC–MS, (b) LC–EC–MS, and (c) EC–LC–MS.

	Electrochemical cells integrated in microfluidic devices were presented (30–35). With this design, high electrochemical conversion efficiency can be obtained with only small amounts of reagent being used. Another advantage of such a setup is the short transit time between EC and MS, which enables the detection of short-lived oxidation products (35).

	Configurations with electrochemical cells integrated into the ESI emitter are available (36–39). Very recently, we demonstrated that such devices can characterize the reducing potencies of natural antioxidants such as polyphenols in wine (39). The ESI source represents a controlled-current electrochemical cell. The interfacial potential at the emitter electrode is at or near the electrochemical potential of those reactions that sufficiently supply all the required current for the ESI circuit. Indicator molecules prone to oxidation in ESI, such as amodiaquine, can be used to visualize the impact of reducing compounds on the interfacial potential. The extent of inhibition of the oxidation of the indicator molecule is dependent on the kind and amount of antioxidant added.

	The coupling of a discrete electrochemical cell with the ESI–MS system is complicated by the need to decouple the cell voltage from the ESI high voltage (40). Decoupling is necessary for proper control of the electrochemical potential at the working electrode, and is usually accomplished either by using sufficiently long transfer lines (~30 cm) between the electrochemical cell and the ESI source (19,36), the insertion of a ground point between the electrochemical cell and the ESI source (36), or by allowing the whole system to float on the potential induced by the ESI high voltage (36).

	For proper setup of an EC–ESI–MS system, sites for electrochemical reactions with limited user control should be kept to a minimum. Unwanted secondary reactions can take place at ground points, the ESI source, and auxiliary and reference electrodes (40–42). The mass transport to these sites is usually limited by using high solution flow rates, minimizing their accessible surface area, or avoiding any direct contact with the analyte in the flow stream (for example, separate electrode compartments).

	The analytical power of EC–ESI–MS can be increased by integrating liquid chromatography (LC) as an additional dimension of separation. Chromatographic separation is particularly useful to reduce the complexity of the sample submitted either to the EC cell or to ESI–MS. Because of its compatibility with EC and ESI, reversed-phase chromatography is the preferred chromatographic mode of operation. The electrochemical cell can be integrated at different positions into the LC–MS system.

	Post-column EC (Figure 2[b]) can be used to study the redox chemistry of selected compounds within complex mixtures (43,44). Pre-column EC (Figure 2[c]) enables the comprehensive characterization of redox reaction products via LC fractionation and subsequent MS detection (45,46). As the commonly applied chromatographic systems are operated at flow rates of several hundred microlitres per minute, electrochemical cells with porous flow-through working electrodes with very high conversion efficiencies are exclusively used for both types of experimental setups. To enable the use of thin-layer cells, the EC cell can be integrated into the injection device (29,47). In such a setup, the sample solution is delivered through the electrochemical cell into the injection loop and subsequently transferred into the LC flow path.

	Despite considerable success of the currently available EC instrumentation in converting bioorganic molecules, there is still a need for improved experimental conditions. A lot of research is focused on increasing predictability, yield, and reproducibility of electrochemical reactions. The yield can be improved by optimization of reaction parameters including solvent composition, pH, and electrode material (29). Another strategy to increase the rate of conversion is based on the application of square-wave potential pulses (48,49). The superior performance with pulsing was attributed to an increased desorption of reaction products and continuous renewal of the electrode surface. The potential pulses were also found to give rise to the formation of products that were not seen in direct EC alone. Other approaches developed to expand the reactivity of EC include enzyme-modified systems (50) and hydroxyl radical-producing systems (51–54).

Metabolomics, Lipidomics, and Related Applications

	Based on the number of published articles, one of the most important fields of application of EC–ESI–MS lies in studying the redox chemistry of small bioorganic molecules. In this context, EC–ESI–MS techniques are particularly useful to mimic biotransformation reactions.

	The first attempts at using EC–MS to characterize oxidation processes of organic molecules date back to the pioneering work of Hambitzer and Heitbaum as well as Yost, Brajter-Toth, and colleagues in the late 1980s (55–59). However, Bruins and colleagues were the first who systematically studied the redox reactivity of pharmaceutical compounds with EC–ESI–MS (60,61). Their work laid the foundation for the use of EC–ESI–MS techniques to mimic phase I oxidative reactions in drug metabolism studies. Because valuable information concerning the sensitivity of a substrate towards oxidation can be obtained from EC–ESI–MS experiments, the technique is regarded as an efficient tool in the drug development process that complements existing 

 screening techniques. EC was found to be particularly useful in cases where P450 enzyme catalyzed reactions are supposed to proceed via a mechanism initiated by a one-electron oxidation. Typically, direct EC oxidation successfully mimics benzylic hydroxylation, hydroxylation of aromatic rings containing electron-donating groups, 

-dealkylation, S-oxidation, dehydrogenation, and, less efficiently, 

-dealkylation. The range of oxidizable moieties was extended by the application of hydroxyl radical-producing systems (51–54). For instance, with electrochemically assisted Fenton chemistry aliphatic hydroxylation, aromatic hydroxylation, 

-dealkylation were also efficiently mimicked. Furthermore, combining EC with enzyme-modified systems holds the promise to enable simulation of the full range of biotransformation reactions occurring 

	Although EC–MS has been used extensively to simulate oxidation reactions, it can also be applied to mimic reductive metabolism reactions, and this has been demonstrated for nitro aromatics (62).

	The applicability of EC–ESI–MS in drug metabolism studies has been demonstrated for a number of important pharmaceutical compounds, including amodiaquine (50, 63–65), diclofenac (66,67), lidocaine (48,53,54), acetaminophen (63), haloperidol (68), troglitazone (69–71), clozapine (63), trimethoprim (63), flunitrazepam (72), clonazepam (72), chlorpromazine (72), zotepine (73,74), acebutolol (75,76), alprenolol (75), albendazole (77), tetrazepam (47), verapamil (78), and galantamin (79).

	Phase I metabolism of drug compounds often gives rise to the formation of reactive species that are generally short-lived and unstable. These species can react with lipids, proteins, and nucleic acids giving rise to substantial damage. Within living organisms reactive intermediates are usually detoxified by binding to glutathione (phase II reaction). Reactive species are also produced in EC experiments. For the detection of these compounds, and to mimic phase II metabolism, trapping agents are commonly applied. Appropriate nucleophiles include glutathione (65,80), glutathione derivatives (81), cysteine (82), and proteins (83). These compounds can be added to the sample solution after or before EC. The study of the skin-sensitizing potential of chemicals is a very interesting application of the ability of EC to produce electrophiles that subsequently react with proteins (84–86). Allergic reactions are most often caused by haptenation of proteins. A presupposition for this reaction is the ability of chemical allergens (small organic molecules and their metabolites) to form covalent bonds to nucleophilic sites in proteins (for example, cysteine and lysine). These activation and conjugation reactions can be studied in a well defined environment of selected nucleophiles with EC–ESI–MS, and this has been demonstrated for p-phenylendiamine, eugenol, and isoeugenol.

	The study of biotransformation reactions involving pharmaceutical compounds is also gaining importance in environmental research. Xenobiotics are emitted from living organisms after biotransformation. Furthermore, biotransformation reactions may occur in the environment after exposure to terrestrial and aquatic microbial systems. Accordingly, comprehensive risk assessment of any chemical requires a detailed understanding of its different manifestations. Here, EC–ESI–MS can be used to produce putative metabolites as well as their conjugates with natural organic matter (87–91).

	Other emerging fields of application for EC–MS-assisted drug metabolism studies are doping analysis (92,93) and forensic toxicology (94–96). There, EC–MS can be used to predict potential human metabolites of illicit drugs that can be used as targets to verify consumption.

	In comparison to drug metabolism studies, the application of EC–ESI–MS to study transformation reactions of endogenous compounds is less common. So far, there are only a few publications demonstrating the potential of this approach (45,46,97–100). Nevertheless, we believe that in the near future EC–ESI–MS will gain in importance in this field of research. EC reactions could become particularly useful to produce sufficient quantities of likely metabolites using known metabolites as reactants to enable their structural elucidation and subsequent confirmation in authentic samples by making use of spectral data stored in libraries or similar systems (101,102).

	An example of the successful application of EC–ESI–MS to the study of transformation reactions of an endogenous compound is shown in Figure 3. Cholesterol was oxidized in an electrochemical cell, and the produced oxysterols were characterized by LC–MS–MS. Nine different species were unequivocally identified by comparing the mass spectrometric information to data obtained from reference compounds. The identified products were mostly oxidized near the double-bound at the B-ring (and to a smaller extent at the tertiary carbon in position 25), which is in agreement with susceptibility of cholesterol to free radical driven oxidation.


	Figure 3: Putative products of electrochemical cholesterol oxidation identified by LC–MS–MS. The carbon numbering at the cholesterol backbone is shown in blue. The sites of oxidation are highlighted by red circles. Indicated are the free radical driven and possible consecutive reactions leading to the formation of the identified compounds, as well as enzymes (in brackets) involved in the generation of oxysterols in vivo. Courtesy of Maria Fedorova and Dieter Weber, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Leipzig, Germany. 

	In proteomics, there is a continuously growing interest in EC–ESI–MS. The technique can be used to study the oxidation of peptides and proteins as well as to selectively tag, label, or cleave these species.

	Direct oxidation of peptides and proteins is widely studied, because of the role of oxidative damage in disease. The amino acids that most easily undergo oxidation are tyrosine, tryptophan, cysteine, methionine, and histidine (103). Tyrosine and tryptophan are hydroxylated. Cysteine and methionine are converted to their sulphonic acid and sulphone derivatives, respectively. In addition, cysteines can form disulphide bonds, which may be oxidatively cleaved.

	The direct oxidation of proteins can be used for analytical advantage to probe their higher order structure by oxidizing solvent-exposed amino acid residues (104,105). Oxidization can be accomplished at physiologically relevant conditions and monitored on-line by MS. A clear drawback of the technique is that, even when only one protein is involved, complications such as low abundances across a range of different oxidized peptides, a wide variety of possible mass shifts, and fragmentation of coeluted oxidized peptide isoforms pose challenges to data interpretation, which currently renders EC more laborious and time-consuming than chemical approaches.

	In 2003, Bruins and colleagues realized that direct oxidation of proteins and peptides can induce cleavage of the amide bond at the C-terminal side of tyrosine and tryptophan residues (103). These cleavage reactions were observed in most analyzed peptides and proteins, which clearly suggested that EC could represent an instrumental alternative to chemical and enzymatic cleavage strategies. The main advantage of EC is the direct hyphenation to MS without the need to remove reagents, enzymes, or buffers. Thus, EC–ESI–MS enables direct cleavage and sequencing of proteins. The main problems encountered are related to yield, reproducibility, and adsorption phenomena. The cleavage rate can vary between different species, and a considerable amount of noncleavage oxidation products are often observed (27,106). Nevertheless, the yield of cleavage products can be improved by proper control of the oxidation potential and the application of strongly acidic conditions (pH 2–3) (106). Furthermore, adsorption phenomena and electrode fouling can be reduced by changing from glassy carbon to boron-doped diamond electrodes and by applying regeneration procedures (107).

	Direct reduction of peptides mainly targets disulphide bond bridges. Disulphide bonds are one of the most common post-translational modifications and provide covalent cross-linkages in native proteins for maintaining the three-dimensional structures of proteins and their biological activities. However, the presence of disulphide linkages increases the complexity for the protein structure elucidation by MS. Accordingly, they need to be reduced, and this is usually accomplished by chemical reduction. Recently, electrochemical reduction has been shown to represent a fast and efficient alternative for disulphide bond cleavage with immediate application in bottom-up and top-down proteomics (72,108–114). A clear advantage of EC is the possibility to analyze the reaction products directly by MS. EC is a reagent-free cleavage approach. There is no need for any kind of sample preparation between cleavage and MS analysis. High conversion yields can be obtained with proper selection of experimental conditions (that is, Ti-based electrode, 1% formic acid in the solvent). In peptide mapping, EC enables the identification of disulphide-bridged peptides within enzymatic digest mixtures by inducing changes in ion abundance. Furthermore, in combination with tandem MS analysis, disulphide linkage pattern and sequence information for the examined peptides can be determined. At the protein level, electrochemical reduction of disulphide bonds enables tandem MS sequencing with high sequence coverage. For instance, the van der Burgt group reported sequence coverage of over 80% for oxytocin and hepcidin after electrochemical reduction (111). In the case of hepcidin, 21 of the 24 peptide bonds were cleaved after full EC reduction of the four disulphide bonds, while only seven peptide bonds were fragmented in the native hepcidin. Recently, EC has also been applied for the reduction of large proteins such as monoclonal antibodies (mAbs) (113), thereby generating light and heavy chains with high selectivity (Figure 4). Besides the reduction of the four inter-chain disulphide bonds, it was also possible to reduce most of the intra-chain disulphide bonds.


	Figure 4: EC reduction of the disulphide bonds of a commercially available monoclonal antibody (mAb) monitored by on-line ESI–FTICR–MS. Cleavage of the four inter-disulphide bonds yields two light and two heavy chains. Courtesy of Simone Nicolardi and Yuri E. M. van der Burgt, Leiden University Medical Center (LUMC), Center for Proteomics and Metabolomics, Leiden, The Netherlands. 

	Another promising field of EC-based cleavage of disulphide bonds is hydrogen/deuterium exchange monitored mass spectrometry (HDXMS) (114). HDXMS is increasingly being used to characterize the dynamic properties of proteins. When a protein is incubated in D

O, the backbone amide hydrogen/deuterium exchange kinetics directly reflect the conformational dynamics of the polypeptide backbone. HDXMS experiments usually involve digestion of proteins. A critical step in sample preparation is the reduction of disulphide bonds. Cleavage has to be performed under cold and acidic conditions where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). In addition, the reduction must be performed as quickly as possible (within a few minutes) to minimize artifactual deuterium loss or gain in the backbone amides. Here, EC is used for the controlled reduction of the disulphide bonds, replacing the chemical reducing agent Tris(2-carboxyethyl)-phosphine (TCEP), which often causes serious adverse effects on H/D exchange, chromatography, and MS.

	EC can also indirectly be used for peptide and protein characterization. Mass tags or labels for specific amino acid residues can be generated electrochemically (9,115–119). Usually, the inherent EC of ESI is applied for activation. Metal ions are used for non-covalent labelling, copper ions for targeting cysteine residues, and zinc ions for targeting histidine residues and phosphorylation sites. Covalent labelling usually involves activated hydroquinone species, which selectively react with cysteine residues. Electrochemically-assisted labelling was found to be useful to determine the number of cysteine residues, which improves protein identification by database searching. Furthermore, based on the labelling extent of different residues within peptide and proteins, the accessibility, reactivity, or affinity of these sites can be tested. The usefulness of this approach has, for instance, been demonstrated by identifying the preferred binding sites of copper ions to β-amyloid 16, which is a protein that is involved in the development of Alzheimer's disease (119).

	The conical nucleosides - adenosine, cytidine, guanosine, uridine, and thymidine - are the main building blocks of all naturally- occurring nucleic acid polymers (DNA and RNA). Nevertheless, a large number of nucleoside derivatives have been identified in DNA and RNA of living organisms, as well as of viruses, mitochondria, and chloroplasts. The variety of modification reactions is enormous; often oxidation reactions are involved either as part of enzymatic or non-enzymatic processes.

	Non-enzymatic oxidation reactions are typically involved in processes leading to DNA damage. Oxidative stress can lead to the production of ROS, which react with nucleic acids. Furthermore, exogenous chemical agents, such as toxins, pharmaceuticals, or pollutants, are often activated by oxidation to react with the genetic material. Produced lesions include base and sugar damages, strand breaks, and crosslinks with proteins as well as the formation of bulky adducts. The cellular response to damage involves several processes, such as DNA repair, cell cycle arrest, and apoptosis, while irreversible mutations contribute to oncogenesis.

	To enable the development of strategies to protect the genetic material from oxidative stress, mechanistic aspects of nucleic acids oxidation have been extensively studied. The electroactivity of nucleic acids was discovered by Palecek in the 1960s (120). In the following years, a number of oxidation products of nucleobases, nucleosides, and nucleotides were identified using EC combined off-line with analytical techniques, such as UV–vis spectroscopy, gas chromatography (GC)–MS after derivatization, or LC–MS (121–123). In this context, EC–ESI–MS holds the promise to facilitate and accelerate the identification process by eliminating laborious and time-consuming isolation and derivatization steps (29,99,124–129).

	EC–ESI–MS was applied to study oxidation of guanine-containing species. Guanine exhibits the lowest oxidation potential among nucleic acids components and is, therefore, the preferential target of oxidation within nucleic acids. The primary products of guanine oxidation were identified as 8-hydroxyguanine and cross-linked guanines. The product 8-hydroxyguanine represents the most important biomarker to indicate oxidative damage of genetic material, and the formation of this compound clearly demonstrates that EC is able to mimic 

 oxidation of nucleic acids induced by oxidative stress.

	The formation of bulky adducts is an alternative mode of nucleic acids alteration (130). Here, oxidation reactions are involved in activation processes; the produced electrophiles react with nucleophilic sites within the genetic material. EC–MS represented a useful tool to study the formation of adducts between nucleic acids and environmental pollutants (131), amino acids (132), and pharmaceutical compounds (124,125).

	The most important enzymatic modification in genomic DNA is methylation of the C5 atom of cytosine (C). Production of 5-methylcytosine (5mC) is catalyzed by DNA methyltransferase enzymes. Methylation of C is an important epigenetic DNA modification that is essential for development and normal function in all mammals (133). In 2009 it was demonstrated that genomic 5mC is transformed to 5-(hydroxymethyl)cytosine (5hmC) (134,135). Oxidation was accomplished by the ten-eleven translocation enzymes. These iron and 2-oxoglutarate-dependent enzymes were furthermore able to convert 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (136). The later oxidation products are considered to represent important intermediates in active demethylation of 5mC. They are recognized and excized by the mammalian thymine-DNA glycosylase, and subsequently converted to C through base excision repair (137,138). There is increasing evidence that 5hmC may also serve as a stable epigenetic mark that possesses unique regulatory functions (139,140). It binds to specific regulatory proteins and is mainly present at actively transcribed genes (141,142).

 experiments employing different sources of radicals demonstrated that 5hmC and 5fC can also be formed by one-electron oxidation of 5mC (143,144). However, both species were considered to represent only minor products of non-enzymatic 5mC oxidation (145). The 5,6-double bond was considered to be more reactive than the methyl group. Thus, oxidation should mainly lead to 5,6-dihydroxy-5,6-dihydro-5-methylcytosine (glycolmC).

	However, there is convincing evidence from studying the electrochemical oxidation of cytidine and 5-methylcytidine that 5hmC and 5fC are major products of 5mC oxidation (Figure 5)(126). Simulation experiments have further revealed that C5-methylation reduces stability and increases reactivity of pyrimidine bases. Based on these results, it can be speculated that oxidative stress may induce epigenetic alterations by influencing the previoualy described equilibrium of the oxidized forms of 5mC.


	Figure 5: Products of the electrochemical oxidation of C and 5mC.

	EC–ESI–MS represents a versatile tool for studying redox processes involving different kinds of bioorganic molecules, such as peptides, proteins, endogenous, and exogenous small molecules as well as nucleic acids. EC–ESI–MS is a purely instrumental approach. EC enables the fast, automated, and cost-effective generation of redox reaction products, and is a "green chemistry" technique. EC–ESI–MS, often in combination with LC separation, allows the comprehensive characterization of the reaction mixture obtained. EC–ESI–MS represents a very useful complement to 

 techniques in metabolomics, proteomics, genomics, and related areas of application. Despite the applicability of EC in general and EC–MS in particular to "omics" this approach is relatively new and therefore unknown to analytical chemists. Nevertheless, the fast growing number of EC–MS publications illustrates the power of this technology and will help to create a broader acceptance and use in life science applications.

	This work was funded by the Austrian Science Fund (FWF): P 22526-B11. Furthermore, the authors want to thank Maria Fedorova and Dieter Weber (Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Leipzig, Germany) as well as Simone Nicolardi and Yuri E. M. van der Burgt (Leiden University Medical Center (LUMC), Center for Proteomics and Metabolomics, Leiden, The Netherlands) for providing Figures 3 and 4.

is associate professor for bioanalytical chemistry at the Medical University of Innsbruck (Innsbruck, Austria). His research focuses on the development and application of analytical techniques for the analysis of bioorganic molecules with special emphasis on nucleic acids and small molecules. Please direct any correspondence to: 

 is research associate at the Institute of Legal Medicine of the Medical University of Innsbruck (Innsbruck, Austria). His main research interests are related to the development and application of LC–MS methods for the qualitative and quantitative analysis of small molecules.

 is President and CEO of Antec BV, headquartered in Zoeterwoude, The Netherlands, and with a subsidiary in Boston, USA. His main research interest is the development of electrochemistry–mass spectrometry as a new, sustainable analytical approach.

	1. H. Oberacher, F. Pitterl, R. Erb, and S. Plattner, 

	6. H. Faber, S. Jahn, J. Kunnemeyer, H. Simon, D. Melles, M. Vogel, and U. Karst, 

	9. C. Roussel, L. Dayon, N. Lion, T.C. Rohner, J. Josserand, J.S. Rossier, H. Jensen, and H.H. Girault, 

	10. H.P. Permentier, A.P. Bruins, and R. Bischoff, 

	11. E. Nouri-Nigjeh, R. Bischoff, A.P. Bruins, and H.P. Permentier, 

	12. J. Roeser, R. Bischoff, A.P. Bruins, and H.P. Permentier, 

	13. J.F. de la Mora, G.J. Van Berkel, C.G. Enke, R.B. Cole, M. Martinez-Sanchez, and J.B. Fenn, 

	15. J. Gun, S. Bharathi, V. Gutkin, D. Rizkov, A. Voloshenko, R. Shelkov, S. Sladkevich, N. Kyi, M. Rona, Y. Wolanov, D. Rizkov, M. Koch, S. Mizrahi, P.V. Pridkhochenko, A. Modestov, and O. Lev, 

	16. P.Y. Liu, M. Lu, Q.L. Zheng, Y. Zhang, H.D. Dewald, and H. Chen, 

	18. A.T. Blades, M.G. Ikonomou, and P. Kebarle, 

	21. G.J. Van Berkel, S.A. Mcluckey, and G.L. Glish, 

	22. X.M. Xu, S.P. Nolan, and R.B. Cole, 

	23. R. Vessecchi, A.E.M. Crotti, T. Guaratini, P. Colepicolo, S.E. Galembeck, and N.P. Lopes, 

	24. G.J. Van Berkel, F.M. Zhou, and J.T. Aronson, 

	25. S. Plattner, R. Erb, J.P. Chervet, and H. Oberacher, 

	26. G.J. Van Berkel, K.G. Asano, and P.D. Schnier, 

	28. A. Baumann, W. Lohmann, T. Rose, K.C. Ahn, B.D. Hammock, U. Karst, and N.H. Schebb, 

	29. R. Erb, S. Plattner, F. Pitterl, H.J. Brouwer, and H. Oberacher, 

	30. V. Mengeaud, O. Bagel, R. Ferrigno, H.H. Girault, and A. Haider, 

	31. G. Liljegren, A. Dahlin, C. Zettersten, J. Bergquist, and L. Nyholm, 

Cover Story

Redox reactions are integral parts of many cellular processes. They are therefore extensively studied in vitro and in vivo. Electrochemistry (EC) represents a purely instrumental approach to characterize direct and indirect effects of redox reactions on bioorganic molecules. This review highlights important trends and recent developments.

“Omics” Applications of Electrochemistry Coupled to Mass Spectrometry — A Review

The transit of peaks through a gas chromatography (GC) column depends strongly on the thermal profile they encounter on the way. Gas chromatographers primarily rely on the classic hot-air-bath type of oven, but several alternatives are also in use. This instalment examines ovens for GC in several forms plus how oven thermals affect peak retention behaviour.

	Gas chromatography (GC) ovens were not developed for cooking purposes, but rather to provide a suitable thermal environment for the elution of chromatographic peaks from GC columns. That being said, I have seen a number of inappropriate applications for GC ovens including reheating of cold pizza for lunch and the warming of casseroles for company potluck dinners. Some of us have even been known to trick out the oven door switch and use an idle GC system as a room heater on cold days.

	GC ovens have gone through an evolutionary development just like other GC components have. Today, the majority of GC ovens use rapidly circulating air inside an insulated enclosure to transfer heat to and from the column, much like the convection ovens found in many high-end kitchen ranges. Some readers may be familiar with other means that chromatographers use to control the thermal environment surrounding a column; some of these are quite ingenious. First, though, let's take a look at what kind of thermal conditions a column needs to deliver excellent separation performance.

	In gas chromatography, the relationship between peak retention and column temperature is fundamental. Changes in temperature cause significant exponential retention shifts, so GC instruments control solute retention times by carefully managing the column temperature along with the column pressure drop. In general, retention times decrease by a factor of two for every 15–20 °C increase in column temperature (

). Peak retention depends differently on column temperature with isothermal versus temperature-programmed elution, but in both cases tight control of the temperature is paramount. This same temperature dependency also causes minor temperature shifts to randomize peak retention times by a small amount from one run to the next. Slightly higher overall oven temperatures cause peaks to be eluted a little earlier, and lower temperatures shift retentions to longer times. In addition to changes in the average oven temperature from one run to the next, other important temperature variabilities include spatial fluctuations along the column because of temperature gradients in the GC oven and dynamic short-term oven temperature fluctuations because of electronic and software temperature-control processes. If large enough, such spatial and short-term changes will affect peak shapes and resolution.

Run-to-Run Average Temperature Deviations

	In general, run-to-run retention time deviations should stay within a window of about ±10% of the peak width at base. That way there is little ambiguity concerning peak identification by absolute retention time, including for partially separated peaks. The narrowest practical peak widths will put the most demands on the oven temperature repeatability. Relatively short, 10-m long, 0.20-mm i.d., thin-film capillary columns elute significantly retained peaks - those with retention factors of two or greater - with base widths of about 1.5 s minimum. This figure assumes that the column operates at an optimum average linear helium carrier gas velocity of about 30 cm/s with the best theoretical efficiency. Chromatographers can obtain narrower peaks by operating columns at higher than optimum carrier-gas velocities, or by choosing narrower or shorter columns - in other words, by going to "fast" GC. But in general this is about as fast as peaks go for the vast majority of GC systems in operation today.

	The oven is not the only factor that contributes to run-to-run retention time variations. The pneumatic system also influences retention times significantly, and the data handling system introduces other uncertainties including its determination of the exact run start time and the peak apex or centroid. I assembled a statistical analysis of these factors and the GC oven using typical values for modern GC instrumentation and columns. Interested readers can refer to a website for this supplemental information (1). In this analysis, the room temperature and GC line voltage supply are assumed to be stable and to not influence the actual oven temperature and column pressure drop. This is not always the case, of course, and such problems fall into the category of instrument "environmental" difficulties that should be resolved before attempting to obtain the best retention time reproducibilities.

 According to this analysis, the average oven temperature needs to repeat from run to run within less than ±0.05 °C to hold retention times inside a window of ±10% of peak base widths, for commonly encountered solutes. Lee, Yang, and Bartle cite a similar requirement in their 1984 book (2). These figures are for typical test mixture solutes separated at moderate isothermal temperatures on short narrow-bore thin-film capillary columns, which represent the more difficult cases. Columns with thicker stationary films, columns that are longer, wide-bore columns, and, of course, packed columns generate broader peaks and may tolerate a less stringent temperature environment while still delivering retention time repeatability that is adequate for peak identification purposes. On the other hand, fast GC separations performed on microbore columns with inner diameters under 0.2 mm, on short columns under 10 m, or with elevated carrier-gas velocities over 100 cm/s, may require better temperature control to contain retention times within a ±10% of base-width window.

 With isothermal elution, peak base widths increase approximately in proportion to the retention time, but with single-ramp programmed temperature elution the base widths stay approximately constant across a GC run, for peaks that are eluted during the temperature ramp. Most peaks are essentially immobilized upon injection at the initial column temperature; as the oven temperature increases, solutes begin to move along the column. Each spends about the same time moving through the column (3), which results in the peaks having approximately the same widths as they are eluted. For temperature programming, oven temperature ramps need to repeat the time and temperature curve with the same precision as required for isothermal elution. The oven heating system must have sufficient power to maintain a linear programme ramp from the start temperature to the final temperature. The maximum rate at which the oven temperature can increase linearly is related to the thermal mass of the oven cavity, including the column, the power that the heater can dissipate into the oven, the efficiency of the oven boundary insulation, and the differential temperature between the inside of the oven and the external environment.

	One principal difference between isothermal and programmed elution is the slight time lag between the actual column temperature and the setpoint temperature of the temperature ramp. As the oven temperature controller increases the setpoint temperature according to the demands of the programme ramp, the actual oven temperature lags behind by a small amount and the temperature inside the column lags a little more. The temperature lag is larger at higher programme rates. This temperature differential is a necessary by-product of both the nature of temperature-control algorithms and the physics of heat transfer from the heater to the column. For our purposes, it is the temperature in the column itself that counts.

	The electronics and software must repeat not only the same time and temperature profile across the run, but also the thermal conditions in the oven must be consistent. This is one reason why temperature-programmed ovens require a temperature equilibration period after cooling down before a new run can commence. Shortly after the oven cools down, the oven temperature probe may indicate the initial oven temperature, but there is still a significant amount of heat remaining in the oven; the walls and the column can be significantly hotter than the indicated temperature. This is the reverse of the temperature lag encountered with oven heating, and good practice dictates that sufficient time be allowed to dissipate the residual oven heat and bring the column as close as possible to the initial oven temperature. An equilibration time of 2–4 min is typical for most air-bath GC ovens with temperature programming capability. With isothermal elution, no equilibration time is required after the oven reaches its temperature for the first time, because the temperature is not changed during the run.

 A high degree of run-to-run average temperature repeatability ensures the best retention time precision. GC ovens also must maintain a uniform and stable temperature profile across the column during elution. The process of solute partitioning and migration along the column averages out temperature gradients so that peaks are eluted as if they experienced the average column temperature along the length of the column. Minor temperature fluctuations of less than 1 °C along the column do not affect retention times seriously, as long as these gradients are stable and repeatable. Unstable gradients that change from run to run, however, will impose additional uncertainty on retention times because they affect the average temperature that solutes experience.

	Some temperature gradients are normal and necessary. The column inlet and outlet connect to the injector and detector, which are normally at elevated temperatures relative to the oven. Where GC column ends are positioned inside the inlet and detector, a negative temperature gradient exists from the inlet into the column and a positive gradient out into the detector. The net effect on retention is negligible, since solutes will be weakly retained, if at all, in the short sections of heated column ends, which act more like connecting tubing. Gradients also exist close to the oven walls because of heat losses or gains from cooler oven walls or warmer heated inlets and detectors. In a conventional air-bath oven, the column is normally positioned in the centre of the oven to immerse it in the most thermally uniform area. These stable temperature gradients do not seriously affect performance if they are stable both during a run and in the long term from run to run.

	Gradients that fluctuate from run to run will affect retention time stability, but gradients that fluctuate rapidly during a run will affect peak shapes. A poorly sealed air-bath oven can generate this type of gradient. A controlled amount of air normally circulates through an air-bath oven, to prevent the buildup of explosive hydrogen gas. This is done in such a way that significant temperature gradients are not generated. But if the oven door or vent isn't sealed properly, significant amounts of cold air can enter and blow directly on the column and create a rapidly fluctuating localized temperature gradient. For fused-silica columns in particular, with their thin walls and rapid thermal time constants, fluctuating gradients will alternately trap and release portions of solute bands as they pass by, which will distort the peak shapes and may even split peaks up into apparent multiples.

	GC operators play an important role in ensuring the minimum possible gradients, by installing columns correctly. As much as possible, columns should be centred within the column oven, with no length of the column closer than about 1 in. (2.5 cm) from the oven walls, except of course for the inlet and detector connections. Column mounting brackets provided by the manufacturer are the best way to provide secure and centred column mounting.

	Over the years, GC researchers have envisioned and built a wide variety of column heating schemes. Early workers were well aware of the requirements imposed by the sensitivity of GC retention times to temperature changes and by the problem of transient thermal gradients. They had it easier than present-day chromatographers, however, because they had to accommodate packed columns and metal or thick-walled glass capillary tubing, but not fused-silica tubing. Thin-walled fused-silica tubing responds more rapidly to oven temperature changes and is more susceptible to the effects of rapidly changing oven gradients. The newest generation of GC ovens includes features designed to maximize performance with all columns.

	Oven heating schemes fall into two broad categories: those that rely on a fluid to transfer heat to and from the column, and those that use solid-state materials for heating purposes. Today, of course, air is the heat-transfer fluid of choice for most of us, but that was not always the case. Chromatographers have experimented with various vapours and liquids, as well. Solid-state ovens have been applied to temperature programming metal capillary columns, and small, completely solid-state ovens are found primarily in portable instruments because of their low power requirements and good long-term stability. Columns installed inside resistively heated metal tubing or directly heated column tubing, suspended inside a conventional air-bath oven, deliver very fast programming rates for high-speed GC.

 The earliest report I could find of a fluid oven for GC involves the suspension of the column inside a vapour bath over a boiling pure substance (4). This technique of temperature regulation had been used in various related disciplines even before partition GC came into its prime in the 1950s, and it was a natural step to use it for the then-new technique. The operator could change the temperature of the vapour bath up or down by regulating the pressure inside a sealed oven container. For example, 

This instalment examines ovens for GC in several forms plus how oven thermals affect peak retention behaviour.

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Vol 28 No 3 LCGC Europe March 2015 Regular Issue PDF