Ionisation techniques that remove the need for sample preparation are taking mass spectrometry into new and exciting research areas, reports Steve Down
Ionisation techniques that remove the need for sample preparation are taking mass spectrometry into new and exciting research areas, reports Steve Down
Mass spectrometry is undergoing a revolution. One that is making the life of the mass spectrometrist far easier by removing the need for sample preparation, speeding up analysis and allowing ionisation to take place in the open air. Judging by the interest at the 2006 annual meeting of the American Society of Mass Spectrometry (ASMS), many of the delegates are already caught up in it. The excitement has been generated by two related but independently developed techniques for ionising compounds: direct analysis in real time (Dart) and desorption electrospray ionisation (Desi).
Ionisation is a basic requirement for mass spectrometry, which functions by measuring the mass-to-charge ratios (m/z) of ions. In conventional mass spectrometry, ionisation takes place inside the mass spectrometer. For many years, it occurred in a vacuum to preserve the ions formed by methods such as electron ionisation and, later, matrix-assisted laser desorption/ionisation (Maldi).
Atmospheric pressure ionisation techniques such as electrospray and atmospheric-pressure Maldi enabled solutions and analytes embedded in a chemical matrix to be analysed but still took place inside the mass spectrometer.
Most of this type of analysis requires some degree of extraction, sample preparation and manipulation to remove the analytes from their matrices (soil, food, tissue). In many cases, this is a time-consuming step even with automated procedures and is the principal bottleneck in the quest for high-throughput analysis. In short, sample preparation is the bane of mass spectrometrists.
Out in the open
In Dart and Desi, ionisation takes place in the open air, close to the mass spectrometer. No vacuum is required and neither is sample preparation. The sample is simply held within a gas or liquid stream at room temperature and the impact induces ionisation of analytes on the surface. The ions are drawn into the vacuum interface of the mass spectrometer for analysis and mass spectra are acquired within a few seconds of the analyst picking up the sample.
Desi and Dart were announced within months of each other but were developed independently.
Graham Cooks’ team at Purdue University, US, developed the Desi technique and publicly unveiled the device in October 2004. It produces a liquid stream of charged droplets by pneumatically assisted electrospray ionisation operating at high potential, typically 2-4kV. The analytes are ionised by heterogeneous charge transfer or a droplet charge pick-up mechanism.
Cooks has likened the technique to an atmospheric pressure version of secondary ion mass spectrometry. The Desi source has since been commercialised by Prosolia, US, but continues to be developed in Cooks’ lab.
The gas stream-based Dart ion source is a product of Japanese firm Jeol and was developed in the US by Robert ’Chip’ Cody from Jeol and James Laramee from EAI Corporation. The pair had previously designed a tuneable energy electron monochromator, which led them to investigate an atmospheric pressure version using a corona discharge as a source of electrons.
In Dart, applying a high potential, typically 2-4kV, to a stream of gas produces ions, electrons and excited-state atoms and molecules. A system of lenses or grids removes the charged species and the remaining metastable gas species are directed towards the target surface.
Dart’s launch prompted well-known mass spectrometry commentator David Sparkman of the University of the Pacific, US, to declare: ’Dart is the most significant event in mass spectrometry since the development of Maldi and electrospray and may be worthy of mass spectrometry’s next Nobel prize’.
In the short time since their introduction, it has transpired that very few combinations of surfaces and compounds are not amenable to Dart and Desi. Surfaces can be firm like tablets, hard like concrete, soft like paper or biological tissue, or liquid. So far, illegal drugs on paper currency, explosives on clothing, pesticides on foods, chemical warfare agents on concrete and biomolecules on human tissue have all been analysed with ease. Liquid samples are analysed as droplets on a glass rod or similar tool positioned by the source.
The pharmaceutical industry has been quick to examine the potential of Dart and Desi. Early demonstrations of the techniques showed that the active ingredient in a tablet could be determined simply by placing the pill in front of the source or, for coated pills, breaking them in half and exposing the internal surface. The speed of analysis was illustrated by early Desi mass spectrometry studies in which a 16-tablet strip was passed across the source in 16 seconds. Both normal and tandem mass spectra confirmed the presence of the active ingredient.
The techniques are being extended by pharma scientists. Melissa Gomez and colleagues at GlaxoSmithKline in North Carolina, US, have used Dart to monitor the progress of chemical reactions by analysing the reaction mixtures. They sampled these regularly by dipping the closed end of a glass capillary in the mixture and placing it in the gas stream. The technique is promising but is still in its early stages.
’There are many factors to consider when comparing it with other sources, such as finding the ’’sweet spot’’ [ensuring the gas jet hits the sample], ion suppression, and effects of temperature, substrate and the solvent in which the compound is dissolved,’ says Gomez. The principal advantage is time saving and Gomez envisages such instrumentation eventually being located within synthetic chemists’ laboratories to support reaction monitoring and purification efforts. ’Analysis time could be decreased as much as 10-fold,’ she predicts.
Novel ways to trap ions
Mass spectrometry is going through a phase of rapid development, partly driven by proteomics and metabolomics research, which demands high performance at low cost.
One piece of equipment to emerge on the market recently is the Orbitrap, developed by Alexander Makarov at Thermo Electron in Bremen, Germany. This traps ions radially around a central spindle electrode within a barrel-shaped electrode, without the use of a magnet. Mass/charge values are measured from the frequency of harmonic ion oscillations along the axis of the spindle. The size of a walnut, the mass analyser provides high mass accuracy, high mass resolution and high sensitivity.
In its LTQ orbitrap mass spectrometer, Thermo Electron has coupled the analyser to a linear ion trap (LIT). LITs confine ions in two dimensions along the axis of the analyser, rather than in three dimensions as in conventional ion traps, to provide improved capacity, trapping efficiency and scan speed.
LITs are currently available as stand-alone systems or as part of hybrid instruments in combination with triple quadrupoles, the Orbitrap analyser, or Fourier transform ion cyclotron resonance cells.
Real-life applications of Desi and Dart are springing up around the world. For example, Facundo Fernandez from Georgia Institute of Technology, US, and colleagues in the US, UK, Thailand and Lao PDR have investigated intact counterfeit antimalarial tablets seized in southeast Asia. Fake tablets containing replacement ingredients in place of the active component artesunate have led to at least one documented death. Reduced amounts of artesunate in the tablets render them ineffective but some may still give positive results in the standard colorimetric test.
In normal mode, Desi gave [M+Na]+and [M+NH4]+adducts (M is the molecular ion) and much fragmentation, but adding dodecylamine (DDA) to the Desi solution, in a mode dubbed reactive Desi, produced strong [M+DDA+H]+adducts of artesunate with high ion yields and no fragmentation. The signals depend on artesunate concentration, allowing tablet contents to be determined once matrix effects and other factors have been studied.
Adding ammonia to the gas stream in reactive Dart gave strong [M+NH4]+adducts for artesunate. Using high resolution time of flight mass spectrometry, the researchers detected adulterants in fake tablets without sample preparation in a few seconds, an improvement in speed of two orders of magnitude compared with liquid chromatography-mass spectrometry. The additives were found to include analgesics such as acetaminophen and the first-generation antimalarials chloroquine and sulfadoxine/pyrimethamine that are now ineffective against malaria.
The US Drug Enforcement Administration (DEA) is using Desi mass spectrometry to analyse rapidly counterfeit tablets, cannabinoid-containing foods and drug-related material from cannabis and khat plants. Sandra Rodriguez-Cruz from the DEA agrees that the principal advantage is time saving compared with traditional techniques such as gas chromatography-mass spectrometry (GC-MS) and IR spectroscopy. ’For use as evidence in a court case, accurate mass data is not necessary but it should be demonstrated that Desi MS data matches that from an authenticated standard, taken under identical instrumental conditions,’ she says. ’It takes some practise for new users to get used to the technique, but once they have tried it they love it. The hardest part has been to explain the principles of Desi, and electrospray for that matter, to scientists that have been doing GC-MS all their lives.’
Jeffrey Leibowitz and colleagues at the FBI at Quantico, US, have tested Dart on many types of chemicals and physical evidence representing different types of analyte and surface, including banknote dye on clothing, pesticide pellets, controlled drugs and nitro explosives. For trace analysis of complex matrices such as blood plasma, they have used simple sample preparation to concentrate the target analytes. With microextraction in a packed syringe, detection limits reached parts-per-billion levels.
Leibowitz has also quantified the date rape drug gamma-hydroxybutyrate (GHB) in urine using Dart, by adding a deuterated GHB internal standard and dipping with a glass rod. He has obtained linear calibration curves from five to 800 ppm and suspect samples can be analysed and confirmed within a matter of seconds.
Dart and Desi may even spread to the operating theatre. Richard Caprioli from Vanderbilt University, US, has been collaborating with Cooks on applying Desi as a surgical tool to differentiate between cancerous and healthy tissue. He has analysed tumour biopsy tissue and found that lipids are readily detectable, yielding distinct molecular distributions in adjacent sections of healthy and diseased tissue. These could eventually be used to help surgeons identify the boundaries of a tumour.
’Currently, development is at an early but interesting stage but the technique is a long way from the operating room,’ says Caprioli. ’The data have not yet been correlated with patient treatment and prognosis.’
He envisages it developing into a near-surgical, rather than a surgical procedure, not least because the liquid spray used for ionisation is generally acidic and should not be sprayed into the patient. Swabs taken from a patient on the operating table, possibly from the region of a glioma on the brain, would be analysed by Desi MS in the vicinity of the surgeon to give immediate feedback on the boundaries of the tumour.
’The ultimate goal is to use the technique in the operating room to detect signatures of disease but there are many issues to resolve, including ethical, surgical, and medical implications,’ says Caprioli.
The heart of the matter
Desi and Dart are essentially surface ionisation techniques, desorbing molecules from the upper layers of the sample. There are some tricks to get around this. Troy Lowe and colleagues from the University of Wollongong, Australia, wanted to analyse hindered amine light stabilisers (Hals) in polymer coatings using Desi but the target molecules were distributed throughout the coating. However, wetting the polymer surface with dichloromethane encouraged migration of the Hals molecules and enabled detection.
As with all new techniques, developments and applications continue apace. At ASMS 2006, Cody reported on the expansion of Dart to materials analysis.
The mass spectra of polymers are similar to those from pyrolysis mass spectrometry but are generally simpler and more reproducible. Dart also has the advantage of being a non-destructive technique. Searchable libraries of polymer mass spectra have been compiled and used to identify unknown materials.
Because of its ability to produce multiple-charged ions, Desi mass spectrometry can also detect intact proteins and other large biomolecules, illustrating its potential for proteomics studies.
Christopher Mulligan in the Cooks lab has developed a portable Desi mass spectrometer. ’Desi was rather quick and painless to implement on the portable instrument. Currently, the sample distance is 1-2cm from the end of the inlet capillary, but this capillary could be extended up to 15cm without too much effect on signal intensity,’ says Mulligan. Mobile instruments could find many applications in homeland security.
Desi is already spawning offspring. Jet desorption ionisation overcomes one of the main limitations of Dart and Desi by permitting depth profile analysis of biological tissue. Described at ASMS 2006 by Zoltan Takats from the Hungarian Academy of Sciences, it employs a high velocity aqueous jet with a high voltage (2-6kV) applied. For proteins, peptides and drugs, detection limits of 100 femtogram to 100 picogram have been achieved. The technique has been applied safely to the in vivo analysis of heart tissue.
The simplicity of Desi and Dart is bringing mass spectrometry to a broader range of scientists in different areas and, some say, will herald the advent of personal mass spectrometers.
Steve Down in a science writer based in Nottingham, UK
- Z Takats et al, Science, 2004, 306, 471
- R B Cody et al, Anal. Chem., 2005, 77, 2297
An invisibility shield
A chemical cloak that conceals people from hungry mosquitoes has recently been developed thanks to mass spectrometry.
In June this year, Ulrich Bernier, a chemist at the US department of agriculture’s mosquito and fly research unit in Gainesville, Florida, US, completed the final set of mass spectrometry experiments he needed for a patent application.
’I have about 17 really good chemicals that, if we supply them in sufficient dosage, make us essentially invisible,’ he says.
Unlike most conventional insect sprays, these chemicals are not primarily mosquito repellents, explains Bernier. ’The compounds that I work with don’t necessarily deter the feeding once the mosquito is on the skin, but they make the mosquito less sensitive to finding host odours,’ he says.
This covers the breadcrumb trail of chemical attractants that humans release, preventing mosquitoes from homing in on their prey. ’We’re essentially making it so the mosquitoes don’t know that the attractants are there,’ says Bernier.
All in the blend
The research to find this invisibility shield began in the 1990s, when Bernier’s team tried to identify blends of compounds that would actually attract mosquitoes.
’We were failing miserably, because every time we had a 30-component blend we’d attract no mosquitoes,’ he says. ’But when we separated the blend and broke it down into smaller subsets of three or five components we started to see activity.’
With just lactic acid and acetone in the mix, the mosquitoes really started to bite. ’All of a sudden we had 90 per cent of mosquitoes coming upwind in the trap,’ he says.
Bernier realised that some of the chemicals in the more complex blends must have been masking these simple attractants. Using gas chromatography-mass spectrometry (GC-MS), the scientists isolated the best: ’we have some that bring it down from 100 per cent to 5 per cent or less attraction,’ he recalls.
Bernier uses standard GC-MS techniques to analyse his samples. ’I have always been a fan of pulsed positive ion/negative ion chemical ionisation,’ he says. ’I still work in the small molecule side of things, but flavours and fragrances are now such a small part: it’s amazing to see the number of large molecules that are now studied using MS.’
Eau de giraffe
Bernier has recently been working with Paul Weldon from the Smithsonian Institution in Virginia, US, to investigate volatile chemicals given off by giraffes that ward off mosquitoes and ticks.
Bernier and Weldon analysed giraffe hair and wax samples using GC-MS in a bid to develop chemical mixtures that change the human odour profile. ’So we’ll smell somewhat like a giraffe or somewhat like a water buck,’ says Bernier.
Holidaymakers concerned that these smells may also ward off their travelling companions need not worry: ’these are odours that we can’t detect but that the mosquitoes can detect,’ says Bernier.
The work builds on previous research by Weldon and Bill Wood at Humboldt State University, US, which identified the major volatile constituents. Bernier and Weldon have now also identified prasterone sulfate, an adrenal hormone metabolite in the hair. ’It’s one of the compounds that comprise the caramel-coloured, sticky, waxy, pungent, substance found in the hair,’ says Bernier.
They have also identified a large number of additional compounds in the hair, including many attractant aldehydes and ketones, and repellent carboxylic acids.
Despite this, Bernier admits he is surprised the team did not find more chemicals in their analysis. ’I have a sneaking suspicion that liquid chromatography-MS analysis may reveal a much larger number of compounds due to the low volatility of those in the wax,’ he says. ’We may try an examination of that nature sometime soon.’
Weldon is now testing the compounds’ effectiveness as mosquito deterrents.
- Thermo Electron, Waltham, Massachusetts, US, sells a wide range of mass spectrometry (MS) products, including high resolution MS and Fourier transform MS. Its LTQ Orbitrap combines a linear ion trap and an Orbitrap analyser that traps ions radially around a central electrode (see box).
- Waters, based in Mitford, Massachusetts, US, sells MS products, ranging from single quadrupole to hybrid quadrupole time of flight (Tof) MS.
- Shimadzu, Kyoto, Japan, sells a range of MS products. Through Kratos Analytical, a Shimadzu subsidiary based in Manchester, UK, the company sells matrix-assisted laser desorption/ionisation (Maldi) tof machines.
- Jeol, Tokyo, Japan, sells magnetic sector and tof mass spectrometers. Its AccuTofDart (Direct analysis in real time) can analyse solid, liquid and/or gaseous samples at atmospheric pressure when a sample is placed between the Dart source and the MS.
- Bruker, based in Billerica, Massachusetts, US, sells MS equipment through Bruker Daltronics. This includes Fourier-transform MS, and Maldi-Tof systems.
- Prosolia, Indiana, US, was set up to commercialise technology from Graham Cooks’ lab at Purdue University, US. It is working on a range of products including omni spray ion sources and desorption electrospray ionisation (Desi).
- Hiden Analytical, Warrington, UK, manufactures quadrupole MS equipment for applications in gas analysis, plasma diagnostics, surface science and vacuum science.
- M-Scan is a group of companies that provide analytical and training services based on MS and chromatography. M-Scan has analytical labs in Wokingham, UK, Geneva, Switzerland, and West Chester, US.