Recent Developments in Analytical Science - Mass Spectrometry

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  • Published: Jul 18, 2016
  • Channels: Ion Chromatography / Gas Chromatography / HPLC / Electrophoresis / Proteomics & Genomics / X-ray Spectrometry / Base Peak / Raman / NMR Knowledge Base / MRI Spectroscopy / Infrared Spectroscopy / Atomic / Proteomics

Mass spectrometry

Mass spectrometry has been described as “the Swiss Army knife of the analytical field” by Karl Burgess, Head of Metabolomics at the University of Glasgow, UK. He expanded upon this by explaining that it has many different capabilities and can be used for many different applications. It is this flexibility that has made mass spectrometry so popular within the analytical world and this is reflected in the global market value which is expected to rise by USD 2,360 million between 2015 and 2020.

Most scientists will be familiar with gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry as they are well established but both of these so-called hyphenated techniques continue to flourish, driven by regular new developments. Their capabilities have been boosted by improvements in the mass resolution and data acquisition rates of mass spectrometers, exemplified by the OrbitrapTM. This unique mass analyser traps ions that oscillate axially across the cell and the resultant residence times enable higher resolving powers than conventional ion traps and quadrupoles, with values up to 500,000 in hybrid instruments.

When the mass resolution is linked to improved chromatographic resolution in a GC-orbitrap system, Burgess expects to be able to run metabolomics studies in a proteomics-style way. In human proteomics experiments, there is a very wide dynamic range of protein concentrations but you know which ones should be there, due to their prediction from the genome. This is not the case for metabolomics. So, the enhanced resolving power and large mass accuracy will give better identifications, allowing scientists to look at the chemical formulas and structures of many small molecules in one run.

While the GC/MS Orbitrap system was developed more recently, the LC/MS equivalent has been available for around a decade. The most recent version consists of a quadrupole linked to an ultra-high-field Orbitrap to give resolving powers up to 240,000 and faster scan speeds. It can scan at twice the rate of its predecessor at the same resolution, allowing more samples to be analysed in the same time, or more data points to be collected across a chromatographic peak. Alternatively, it can be operated at twice the resolving power at the same scan rate.

For proteomics studies, in which multiple samples are studied and compared, the increased resolving power of this and other high resolution mass spectrometers will help to identify closely eluting peptides, leading to the identification of more proteins, or of existing proteins with more certainty. The faster scan rates will give time to run more experiments under different conditions and will encourage the analysis of more fractions and replicates to give in-depth coverage.

The power of these two instruments is indisputable but a recent publication has demonstrated that different instrument platforms give the same performance in proteomics studies. Working with a yeast extract, the results were comparable across quadrupole-time-of-flight, quadrupole-Orbitrap, triple quadrupole, triple quadrupole-linear ion trap and time-of-flight systems. Moreover, it was found that selected reaction monitoring and parallel reaction monitoring, which are targeted data acquisition methods, were more accurate and precise than data-independent acquisition.1

Mass spectrometry has been one of the powerhouses behind the rise of proteomics, with its ability to identify thousands of peptides and proteins in one run. The contrasting top-down and bottom-up approaches, which target intact proteins or their constituent peptides, respectively, have been the major analytical technique in the study of biological systems, be they human, plant, animal of microbial.

The principal aim of most human proteomics studies is the identification of proteins and peptides which can act as indicators of the onset or progression of diseases. This must be accomplished by quantitative studies since the amounts of these so-called biomarkers differ from those in healthy people. The multiple reaction monitoring mode in mass spectrometry is often used in conjunction with isotope-labelled peptide standards. Alternatively, specific labelling agents such as the iTRAQ series are used to compare the abundances of peptides in subsets of the same biological system that have been subjected to different conditions. Label-free methods such as spectral counting are also available, being quicker and less expensive than labelling methods. However, these are more highly dependent on instrument reliability and typically have low throughput.2 All of these experiments are most typically carried out on triple quadrupole instruments but others also produce comparable data.

Hundreds of these biomarkers have been identified to date but very few have been converted into diagnostic tests. In the US, only an average of 1.5 biomarkers per annum have been approved by the FDA since 1990.3 The reasons for this poor conversion rate have been attributed to high false discovery rates and the inability to produce the corresponding antibodies for ELISA tests. ELISA is the conventional next step after candidates have been identified but is time consuming, creating a bottleneck in biomarker discovery. However, multiple reaction monitoring is emerging as an alternative method and will speed up the overall process while reducing the cost.4

The most common mass spectrometry systems are conventional GC/MS and LC/MS, which are workhorses in a variety of practical areas, such as the authentication of a range of foodstuffs. Complex mixtures of volatiles can produce coeluting peaks which are difficult to measure accurately but the introduction of 2D GC, in which the compounds are separated on two columns in series, affords much improved chromatographic resolution. However, in a recent review on 2D GC, the current and future evolution of mass spectrometry, rather than GC, was seen as far more important.5 “The number of GCxGC/MS experiments with novel MS instrumentation (i.e. triple quadrupole, high-resolution time-of-flight and hybrid devices) will certainly increase”, the authors concluded.

High-throughput, automated analyses with low detection limits and high accuracy maintain the popularity of GC/MS but problems remain, particularly the potential for long run times. These can be reduced by the use of columns on a chip although faster elution times tend to reduce the resolution of peaks.6 Other advantages of chip-based systems are the ability to test smaller samples using less carrier gas with reduced energy costs.

Also of increasing use is the application of GC coupled to inductively coupled plasma mass spectrometry to measure organo-metallic compounds such as methylmercury in foods, especially fish. In addition, GC with combustion isotope ratio mass spectrometry can be exploited to measure isotope compositions to confirm the geographic origins of food and any adulterants present.

GC/MS and LC/MS are also popular techniques for environmental monitoring and have been employed to measure a host of pollutants in air, soil, water and other matrices. GC/MS is generally preferred for many of the so-called emerging contaminants, like pharmaceuticals and personal care products but for their transformation products, which are usually polar, LC/MS is the method of choice.7 The lack of available reference standards and mass spectral libraries enhances the importance of high-resolution mass spectrometry for proposing identities from exact masses. A number of detectors such as ion traps and time-of-flight instruments, as well as some hybrid instruments are capable of this improved performance.

Many of these GC/MS and LCMS illustrations take place in the lab but a drive is underway to develop small mass spectrometers that can be used in other environments such as airports, illicit drug labs and disaster scenarios. Several organisations have successfully brought instruments to market based on different types of mass spectrometer. The successful systems will be those that are plug-and-play (or switch-on-and-play for battery operated ones) that function like a black box to give a yes/no answer, so that they can be can be operated by non-scientists in the field.

So, different technologies are being developed and adapted for mobile and miniature mass spectrometers. The successful systems will be those that are plug-and-play (or switch-on-and-play for battery operated ones) that function like a black box to give a yes/no answer, so that they can be can be operated by non-scientists in the field.


One of the biggest innovations in recent years was ambient mass spectrometry. This surprisingly simple technique turned mass spectrometry on its head, showing that detectable ions can be produced in the open air and drawn into an ionisation source for analysis. The two principle techniques were announced within months of each other in 2004-2005.

Desorption electrospray ionisation8 produces ions by spraying a surface with droplets of an electrosprayed liquid that is sheathed in a gas such as nitrogen. In Direct Analysis in Real Time,9 ionisation is induced by directing a stream of metastable atoms and molecules, typically of helium and/or nitrogen, towards the target. In both cases, the sample is positioned a few millimetres from the source on one side and the inlet to the mass spectrometer one the other, so, in theory, the surface of any item can be placed in that location and analysed.

A key driving force behind ambient sources was the need to design portable mass spectrometers, open-air ionisation being compatible with taking measurements in the field. The mass spectra from ambient sources tend to be simple, featuring only protonated or deprotonated molecules. However, in a screening context, that means that they can be checked rapidly against a targeted database to register positive or negative findings. It was envisaged that the technique would allow security screening at airports for drugs and explosives which has proved to be possible. A host of other applications feature the analysis of pesticides on fruit, inks on documents, active components and impurities in pharmaceutical tablets, melamine in foods, and lipids and hormones in tissue.

In the wake of DESI and DART, many related techniques were spawned, some major developments, others minor tweaks that have led to disingenuous claims of new techniques. Easy ambient sonic spray ionisation,10 flowing atmospheric pressure afterglow,11 dielectric barrier discharge ionisation12 and electrospray-assisted laser desorption/ionisation13 are just a few of the many that are now being practised.

A notable real-life application of ambient mass spectrometry is the development of the iKnife for use in surgery.14 Based on rapid evaporative ionization mass spectrometry, it is designed to sample tissue in real time in the operating theatre to distinguish between tumours and normal tissue, giving results in seconds. The smart device is connected to an electrosurgical knife and the vapours are drawn up as the surgeon operates and analysed for their phospholipid content. Tumours have different phospholipid profiles to healthy tissue, so, the surgeons will be guided to remove the entire tumour accurately. Currently, the iKnife is available as a research tool only, fitted to quadrupole-time-of-flight mass spectrometers.

All of the aforementioned ambient ionisation techniques were designed to analyse compounds on surfaces but not every compound can be ionised this way. This obstacle can be overcome by adding reagents to the surface to derivatise the target compounds and a number of research groups are investigating this concept. In reactive DESI, a reagent is added to the electrospray to react with the compounds after they are coerced into secondary droplets via a process of thin-film solution phase extraction. A recent review15 summarises the type of reactions that have been completed in this fashion. Most of the common functional groups apart from ethers can be derivatised and the complement of useful reactions will grow in the future. As a spin-off, the products can be collected easily in small-scale synthetic reactions.


The long-standing matrix-assisted desorption/ionisation mass spectrometry (MALDI MS) technique has developed a growing reputation for the analysis of microorganisms, with a number of commercial platforms designed for this one application and giving accurate identification of bacteria.16 However, MALDI also influences the imaging arena, where it is being used to visualise peptides, proteins, lipids, drugs and metabolites in biological tissue.

Such is the strength of the mass spectrometry imaging field that there are currently 13 commercial MALDI systems available, mounted on different instrument configurations.17 During operation, the laser beam is moved in a 2D pattern across the sample so that the distributions of the compounds in the tissue can be positioned accurately.

In a targeted or non-targeted mode of operation, MALDI imaging can decipher the sites of drugs and their metabolites to show their active sites. In studies of cancers or other diseases, the locations of proteins, lipids and other compounds that are potential biomarkers can be pinpointed and followed as treatments progress. The technique has also found to be valuable in neurochemistry and is regarded by some as a replacement for conventional histology studies. Metabolites in plant tissue can also be visualised.

The other two dominant mass spectrometry imaging techniques involve ambient mass spectrometry and secondary ion mass spectrometry. All of the reports to date are on extracted tissue samples that are analysed in the lab but the use of mass spectrometry imaging on living tissue is a clear objective for the future, building on the success of the iKnife. However, in the meantime, instruments can be installed in hospitals alongside the operating theatres to facilitate the rapid relay of results back to the surgeon.

The vast amounts of data that are collected from the 2D scanning of biological tissue are difficult for non-mass spectrometrists to interpret, highlighting the need for data processing software that simplifies the results and presents them in a clear manner. This dilemma led to the establishment of OpenMSI to make mass spectrometry imaging more accessible. The stated goal is “To make the most high-performance, advanced data management, model building, analysis and visualization resources for mass spectrometry imaging accessible to scientists via the web.”

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