Secondary electro-spray ionization (SESI) is an ambient ionization technique for the analysis of trace concentrations of vapors, where a nano-electrospray produces charging agents that collide with the analyte molecules directly in gas-phase. In the subsequent reaction, the charge is transferred and vapors get ionized, most molecules get protonated (in positive mode) and deprotonated (in negative mode). SESI works in combination with mass spectrometry or ion-mobility spectrometry.
The fact that trace concentrations of gases in contact with an electrospray plume were efficiently ionized was first observed by Fenn and colleagues when they noted that tiny concentrations of plasticizers produced intense peaks in their mass spectra. [1] However, it was not until 2000 when this problem was reframed as a solution, when Hill and coworkers used an electrospray to ionize molecules in the gas phase, [2] and named the technique Secondary Electrospray Ionization. In 2007, the almost simultaneous works of Zenobi [3] and Pablo Sinues [4] applied SESI to breath analysis for the first time, marking the beginning of a fruitful field or research. [5] With sensitivities in the low pptv range (10−12), SESI has been used in other applications, where the detection of low volatility vapors is important.
Detecting low volatility species in the gas phase is important because larger molecules tend to have higher biological significance. Low volatility species have been overlooked because it is technically difficult to detect them, as they are in very low concentration, and they tend to condensate in the inner piping of instruments. However, as this problem is solved, and new instruments are able to handle larger and more specific molecules, the ability to perform on-line, real time analysis of molecules naturally released in the air, even at minute concentrations, is attracting attention to this ionization technique.
In the early days of SESI, two ionization mechanisms were under debate.: the droplet-vapor interaction model postulates that vapors are adsorbed in the electrospray ionization (ESI) droplets, and then reemitted as the droplet shrinks, just as regular liquid phase analytes are produced in electrospray ionization; on the other hand, the ion-vapor interaction model postulates that molecules and ions or small clusters collide, and the charge is transferred in this collision. Currently available commercial SESI sources operate at high temperature so as to better handle low volatility species. [6] In this regime, nanodroplets from the electrospray evaporate very quickly to form ion clusters in equilibrium. This results in ion-vapor reactions dominating the majority of the ionization region. As charging ions originate from nano-droplets, and no high energy ions are involved at any point of the ionization process nor the creation of ionizing agents, fragmentation in SESI is remarkably low, and the resulting spectra are very clean. This allows for a very high dynamic range, where low intensity peaks are not affected by more abundant species. [7]
Some related techniques are laser ablation electrospray ionization, proton-transfer-reaction mass spectrometry and selected-ion flow-tube mass spectrometry.
The main feature of SESI is that it can detect minuscule concentrations of low volatility species in real time, with molecular masses as high as 700 Da, falling in the realm of metabolomics. These molecules are naturally released by living organisms, and are commonly detected as odors, which means that they can be analyzed non-invasively. SESI, combined with High Resolution Mass Spectrometry, provides time-resolved, biologically relevant information of living systems, where the system does not need to be interfered with. This allows to seamlessly capture the time evolution of their metabolism and their response to controlled stimuli.
SESI has been widely used for breath gas analysis for biomarker discovery, and in vivo pharmacokinetic studies:
It has been widely reported the identification of bacteria by their volatile organic compound fingerprint. SESI-MS has proven to be a robust technique for the identification of bacteria from cell cultures and infections in vivo from breath samples, after the development of libraries of vapor profiles. [8] [9] [10] [11] Other studies include: In vivo differentiation between critical pathogens Staphylococcus aureus and Pseudomonas aeruginosa. [12] or differential detection among antibiotic resistant S. aureus and its non-resistant strains. [13] Bacterial infection detection from other fluids such as saliva have also been reported. [14]
Many chronic respiratory diseases lack of an appropriate method of monitoring and differentiation among disease stages. SESI-MS has been used to diagnose and distinguish exacerbations from breath samples in chronic obstructive pulmonary disease. [15] [16] Metabolic profiling of breath samples has accurately differentiated healthy individuals from idiopathic pulmonary fibrosis [17] or obstructive sleep apnea patients. [18]
SESI-MS is being studied as a non-invasive detection system of cancer biomarkers in breath. A preliminary study differentiates patients suffering from breast neoplasia. [19]
Volatiles released from the skin can be detected by sampling the ambient gas surrounding it, providing a fast method for detecting metabolic changes in fatty acids composition patterns. [20] [21]
To study pharmacokinetics, it is necessary a robust technique because of the complex nature of the samples' matrix, be it plasma, urine, or breath. [22] Recent studies show that secondary electrospray ionization (SESI) is a powerful technique to monitor drug kinetics via breath analysis. [23] [24] Because breath is naturally produced, several datapoints can be readily collected. This allows for the number of collected data-points to be greatly increased. [25] In animal studies, this approach SESI can reduce animal sacrifice while yielding pharmacokinetic curves with unmatched time resolutions. [24] [25] In humans, SESI-MS non-invasive analysis of breath can help study the kinetics of drugs at a personalized level. [23] [26] [27] Monitoring exogenously introduced species allows tracking their specific metabolic pathway, which reduces the risk of picking confounding factors.
Introducing known stimuli, such as specific metabolites isotopically labeled compounds, or other sources of stress triggers metabolic changes which can be easily monitored with SESI-MS. Some examples if this include: cell culture volatile compounds profiling; [28] and metabolic studies for plant [29] or trace human metabolic pathways. [30] [31] [32]
Other applications developed with SESI-MS include:
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.
An ion source is a device that creates atomic and molecular ions. Ion sources are used to form ions for mass spectrometers, optical emission spectrometers, particle accelerators, ion implanters and ion engines.
Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. ESI is different from other ionization processes since it may produce multiple-charged ions, effectively extending the mass range of the analyser to accommodate the kDa-MDa orders of magnitude observed in proteins and their associated polypeptide fragments.
Lipidomics is the large-scale study of pathways and networks of cellular lipids in biological systems. The word "lipidome" is used to describe the complete lipid profile within a cell, tissue, organism, or ecosystem and is a subset of the "metabolome" which also includes other major classes of biological molecules. Lipidomics is a relatively recent research field that has been driven by rapid advances in technologies such as mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarisation interferometry and computational methods, coupled with the recognition of the role of lipids in many metabolic diseases such as obesity, atherosclerosis, stroke, hypertension and diabetes. This rapidly expanding field complements the huge progress made in genomics and proteomics, all of which constitute the family of systems biology.
Selected-ion flow-tube mass spectrometry (SIFT-MS) is a quantitative mass spectrometry technique for trace gas analysis which involves the chemical ionization of trace volatile compounds by selected positive precursor ions during a well-defined time period along a flow tube. Absolute concentrations of trace compounds present in air, breath or the headspace of bottled liquid samples can be calculated in real time from the ratio of the precursor and product ion signal ratios, without the need for sample preparation or calibration with standard mixtures. The detection limit of commercially available SIFT-MS instruments extends to the single digit pptv range.
Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography – MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify each separated component. MS is not only sensitive, but provides selective detection, relieving the need for complete chromatographic separation. LC–MS is also appropriate for metabolomics because of its good coverage of a wide range of chemicals. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC–MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. Since the early 2000s, LC–MS has also begun to be used in clinical applications.
Explosives trace detectors (ETD) are explosive detection equipment able to detect explosives of small magnitude. The detection is accomplished by sampling non-visible "trace" amounts of particulates. Devices similar to ETDs are also used to detect narcotics. The equipment is used mainly in airports and other vulnerable areas considered susceptible to acts of unlawful interference.
Desorption electrospray ionization (DESI) is an ambient ionization technique that can be coupled to mass spectrometry (MS) for chemical analysis of samples at atmospheric conditions. Coupled ionization sources-MS systems are popular in chemical analysis because the individual capabilities of various sources combined with different MS systems allow for chemical determinations of samples. DESI employs a fast-moving charged solvent stream, at an angle relative to the sample surface, to extract analytes from the surfaces and propel the secondary ions toward the mass analyzer. This tandem technique can be used to analyze forensics analyses, pharmaceuticals, plant tissues, fruits, intact biological tissues, enzyme-substrate complexes, metabolites and polymers. Therefore, DESI-MS may be applied in a wide variety of sectors including food and drug administration, pharmaceuticals, environmental monitoring, and biotechnology.
Sample preparation for mass spectrometry is used for the optimization of a sample for analysis in a mass spectrometer (MS). Each ionization method has certain factors that must be considered for that method to be successful, such as volume, concentration, sample phase, and composition of the analyte solution. Quite possibly the most important consideration in sample preparation is knowing what phase the sample must be in for analysis to be successful. In some cases the analyte itself must be purified before entering the ion source. In other situations, the matrix, or everything in the solution surrounding the analyte, is the most important factor to consider and adjust. Often, sample preparation itself for mass spectrometry can be avoided by coupling mass spectrometry to a chromatography method, or some other form of separation before entering the mass spectrometer. In some cases, the analyte itself must be adjusted so that analysis is possible, such as in protein mass spectrometry, where usually the protein of interest is cleaved into peptides before analysis, either by in-gel digestion or by proteolysis in solution.
Matrix-assisted laser desorption electrospray ionization (MALDESI) was first introduced in 2006 as a novel ambient ionization technique which combines the benefits of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). An infrared (IR) or ultraviolet (UV) laser can be utilized in MALDESI to resonantly excite an endogenous or exogenous matrix. The term 'matrix' refers to any molecule that is present in large excess and absorbs the energy of the laser, thus facilitating desorption of analyte molecules. The original MALDESI design was implemented using common organic matrices, similar to those used in MALDI, along with a UV laser. The current MALDESI source employs endogenous water or a thin layer of exogenously deposited ice as the energy-absorbing matrix where O-H symmetric and asymmetric stretching bonds are resonantly excited by a mid-IR laser.
Desorption atmospheric pressure photoionization (DAPPI) is an ambient ionization technique for mass spectrometry that uses hot solvent vapor for desorption in conjunction with photoionization. Ambient Ionization techniques allow for direct analysis of samples without pretreatment. The direct analysis technique, such as DAPPI, eliminates the extraction steps seen in most nontraditional samples. DAPPI can be used to analyze bulkier samples, such as, tablets, powders, resins, plants, and tissues. The first step of this technique utilizes a jet of hot solvent vapor. The hot jet thermally desorbs the sample from a surface. The vaporized sample is then ionized by the vacuum ultraviolet light and consequently sampled into a mass spectrometer. DAPPI can detect a range of both polar and non-polar compounds, but is most sensitive when analyzing neutral or non-polar compounds. This technique also offers a selective and soft ionization for highly conjugated compounds.
Ambient ionization is a form of ionization in which ions are formed in an ion source outside the mass spectrometer without sample preparation or separation. Ions can be formed by extraction into charged electrospray droplets, thermally desorbed and ionized by chemical ionization, or laser desorbed or ablated and post-ionized before they enter the mass spectrometer.
Laser ablation electrospray ionization (LAESI) is an ambient ionization method for mass spectrometry that combines laser ablation from a mid-infrared (mid-IR) laser with a secondary electrospray ionization (ESI) process. The mid-IR laser is used to generate gas phase particles which are then ionized through interactions with charged droplets from the ESI source. LAESI was developed in Professor Akos Vertes lab by Peter Nemes in 2007 and it was marketed commercially by Protea Biosciences, Inc until 2017. Fiber-LAESI for single-cell analysis approach was developed by Bindesh Shrestha in Professor Vertes lab in 2009. LAESI is a novel ionization source for mass spectrometry (MS) that has been used to perform MS imaging of plants, tissues, cell pellets, and even single cells. In addition, LAESI has been used to analyze historic documents and untreated biofluids such as urine and blood. The technique of LAESI is performed at atmospheric pressure and therefore overcomes many of the obstacles of traditional MS techniques, including extensive and invasive sample preparation steps and the use of high vacuum. Because molecules and aerosols are ionized by interacting with an electrospray plume, LAESI's ionization mechanism is similar to SESI and EESI techniques.
Breath gas analysis is a method for gaining information on the clinical state of an individual by monitoring volatile organic compounds (VOCs) present in the exhaled breath. Exhaled breath is naturally produced by the human body through expiration and therefore can be collected in non-invasively and in an unlimited way. VOCs in exhaled breath can represent biomarkers for certain pathologies. Breath gas concentration can then be related to blood concentrations via mathematical modeling as for example in blood alcohol testing. There are various techniques that can be employed to collect and analyze exhaled breath. Research on exhaled breath started many years ago, there is currently limited clinical application of it for disease diagnosis. However, this might change in the near future as currently large implementation studies are starting globally.
Renato Zenobi is a Swiss chemist. He is Professor of Chemistry at ETH Zurich. Throughout his career, Zenobi has contributed to the field of analytical chemistry.
Extractive electrospray ionization (EESI) is a spray-type, ambient ionization source in mass spectrometry that uses two colliding aerosols, one of which is generated by electrospray. In standard EESI, syringe pumps provide the liquids for both an electrospray and a sample spray. In neutral desorption EESI (ND-EESI), the liquid for the sample aerosol is provided by a flow of nitrogen.
In mass spectrometry, an ion funnel is a device used to focus a beam of ions using a series of stacked ring electrodes with decreasing inner diameter. A combined radio frequency and fixed electrical potential is applied to the grids. In electrospray ionization-mass spectrometry (ESI-MS), ions are created at atmospheric pressure, but are analyzed at subsequently lower pressures. Ions can be lost while they are shuttled from areas of higher to lower pressure due to the transmission process caused by a phenomenon called joule expansion or “free-jet expansion.” These ion clouds expand outward, which limits the amount of ions that reach the detector, so fewer ions are analyzed. The ion funnel refocuses and transmits ions efficiently from those areas of high to low pressure.
Probe electrospray ionization (PESI) is an electrospray-based ambient ionization technique which is coupled with mass spectrometry for sample analysis. Unlike traditional mass spectrometry ion sources which must be maintained in a vacuum, ambient ionization techniques permit sample ionization under ambient conditions, allowing for the high-throughput analysis of samples in their native state, often with minimal or no sample pre-treatment. The PESI ion source simply consists of a needle to which a high voltage is applied following sample pick-up, initiating electrospray directly from the solid needle.
Pablo Sinues is an associate professor at the Department of Biomedical Engineering at the University of Basel and lecturer at the Department of Chemistry and Applied Biosciences at ETH Zürich. He received his Ph.D. in Mechanical Engineering from the Charles III University of Madrid (Spain) and Habilitation in Analytical Chemistry at ETH Zürich. Sinues heads the Translational Breath Research group located at the University Children’s Hospital Basel
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