Light work provides many hands

Light work provides many hands

Mass spectrometry software plays a major role in scientific detective work, writes Sian Harris.

Scientific Computing World: June/July 2007

Pet owners in North America have had a worrying few months. Cats and dogs have been mysteriously falling ill and dying after eating hearty meals of their cat or dog food. The sudden deaths, mostly from kidney failure, of these pets have resulted in millions of cans and pouches of pet food being recalled.

But that is just the start: the big challenge has been in identifying what was in the food that caused all these deaths. The culprit is thought to be some of the wheat gluten from China that is used to bulk up the gravy in the pet food. Several labs across North America have analysed the food and the wheat gluten and found traces of melamine and cyanuric acid. At certain pH levels in the pets' kidneys these compounds are thought to react to produce crystals that are very harmful to the animals. The current theory in the USA is that these compounds might have been added to the gluten to increase its nitrogen percentage so that it would appear to contain higher levels of protein and hence fetch more money.

This is not an isolated incident of contamination, of course: in the UK last year, for example, many curry powders and other products were recalled from supermarket shelves after they were found to be contaminated with Sudan dyes, the dyes used in shoe polish. And US shoppers have recently been concerned about the threat of ethylene glycol contamination in some toothpastes.

With such scares, government bodies, manufacturers and consumers want answers quickly. The scientific detective work to give these answers can mean research teams working through their weekends to try to work out what is going on. But such challenges also have their unsung heroes and one of these is the mass spectrometer.

According to Mark Kuracina, applied markets field marketing manager of Applied Biosystems/MDS SCIEX, mass spectrometers have been instrumental in identifying the melamine and cyanuric acid in the wheat gluten used in pet food and also in investigating other elements of the food chain (the same wheat gluten was fed to pigs and poultry, for example) and whether food for humans is therefore also at risk. ‘The FDA used mass spec in its analysis because of its incredible sensitivity and selectivity,’ Kuracina points out. ‘There are other lower-cost techniques out there, but mass spectrometry gives very valuable information.’

LC/MS/MS chromatograms of a blank and contaminated cat food sample (Applied Biosystems).

Hardware improvements

Obviously the instrument hardware plays a large part in the effectiveness of mass spectrometry in such tasks, and great strides have been made in recent years in improving this equipment.

‘In the early stages of mass spectrometry, companies were more concerned to make it work and a lot of work was done on sheer processing power, but now faster computers are expanding the possibilities of analysis,’ comments Margaret Antler, chromatography technical specialist at ACD/Labs.

One of the hardware improvements that is boosting the analysis capabilities of mass spectrometers, according to Antler, is the increase in mass accuracy. ‘Spectrometers such as Thermo Fisher Scientific’s Orbitrap can give mass to five decimal places. With such accuracy and knowledge of natural distributions of isotopes, you can narrow down the molecular formula to perhaps just one option,’ she explains.

Another hardware improvement that is helping customers gain more from the technique is increased sensitivity and selectivity, giving the ability to detect analytes in samples at lower levels, according to Jim Edwards, who is software product manager for gas chromatography-mass spectrometry (GC-MS) applications for Thermo Scientific products at Thermo Fisher Scientific. ‘This helps to reduce other labour-consuming tasks in the laboratory such as the number of purification or separation steps.’

But the instrument hardware is only part of the story in situations such as solving the pet food mystery. ‘We are now finding with increased sensitivity from hardware (better, stronger, faster) there are many very high-quality data files and customers are now looking to software to reduce all this,’ points out Edwards. This is why, over the past few years, much more emphasis has been placed on developing the software to control the spectrometers and analyse the resulting spectra.

Antler, of ACD/Labs, agrees about the valuable role of software in helping make sense of what can sometimes end up being gigabytes of data generated by mass spectrometers. ACD/Labs is a third-party software company that specialises in software to analyse chemical data from many types of analytical instruments. One such product is ACD/IntelliXtract, which looks at the mass spectra for all the chromatographic components from a sample. It, for example, looks at the different isotopes of the elements involved and analyses whether the isotope patterns correspond with what is expected. It can also work out the molecular weight and empirical formulae of compounds.

One example of how this is used is in metabolism experiments. ‘In the body, drugs are modified to make them more polar so we are generally looking for something in the body that is related to the parent drug but more polar. From this you can find out what part of drug has been modified and build up structures of the metabolites,’ explains Antler.

Spectral libraries

Another area of software that has developed recently is the building up of libraries of mass spectral data on known compounds. Applied Biosystems, for example, works with partners around the world to develop libraries based on its Cliquid software for liquid chromatography-mass spectrometry (LC-MS) that provide the ability to test according to regulatory requirements. Some of these libraries are in the public domain and some are being incorporated into Applied Biosystems’ products.

‘We’re trying to make screening with libraries easier and faster,’ says Applied Biosystems’ Kuracina. ‘For example, there are 800 to 1000 pesticides in circulation. They each give different fragments under different energies so it is like a library of fingerprints.’ The company is also working on a new product to screen for drug abuse that will screen a sample for 12,000 drugs in less than 20 minutes and is working with a customer in Singapore to develop a library of spectra for banned substances in race horses.

Applied Biosystems also has software to screen for differences, for example to compare normal water with potentially contaminated water or tobacco from one brand with that from another. This software is being used to identify drug impurities or counterfeit drugs, an important issue in China where around 60 to 70 per cent of drugs are thought to be counterfeit. The software is also helping to determine where olive oils come from and which ones really are entitled to be labelled as ‘extra virgin’. Another potential application, according to Kuracina, is in comparing traditional Chinese medicines. ‘When plants are grown in different places they can have different ratios of the active compounds in them, which impacts their effectiveness,’ he explains.

In addition, the company has methods for testing for known compounds. For example, it has collaborated with Campden and Chorleywood Food Research Association (CCFRA) in the UK, which developed a method for identifying Sudan dyes, the dyes that contaminated some food in the UK last year. Similarly, the company has recently released a method for melamine and cyanuric acid in response to the latest pet food scare in North America. ‘Users can download methods just as they would download music for their iPod,’ says Kuracina.

Not just for experts

Such activities are part of Applied Biosystems’ vision to make mass spectrometry into a tool that the average person could use to process thousands of samples per day for regulatory authorities or quality control. ‘Our Cliquid software has four simple steps and all the tests are pre-configured. Our design goal was that people who’ve never used mass spec can get it up and running with samples within half a day,’ explains Kuracina. He adds that this approach also appears to be popular with the traditional ‘expert’ customer base. ‘We thought we were making this for non-experts, but pharmaceutical customers are also interested in it, because it enables such companies to employ less specialised people to do the routine work and so save money,’ he points out.

Edwards, of Thermo Fisher Scientific, has observed a similar trend. ‘For certain mass spectrometry types there are high-level users, but more and more often there is routine use of instruments to answer immediate questions,’ he says. The labs are probably still staffed by experienced mass spectrometrists, but the bench chemists are now much more part of the process. This means that the expert doesn’t have to spend time on routine sample processing, but can set up the methods and then concentrate on specialised instruments and tasks.’

This observation is shaping Thermo Scientific’s software plans. ‘We want to take advantage of increasing hardware and software and look at, for example, what the new Windows Vista will allow and how this can be pulled into scientific packages so that they will be intuitive to users without the large burden of customer training and development,’ he comments. ‘For word processors, spreadsheets and scientific applications, the common element is the human. Scientific software needs to have a huge knowledge of the science internally, but the user interface can build on what has been learnt in other areas.’

The trend towards non-expert users is also reflected in customers’ desires to have software more tailored to their application area, irrespective of the underlying mass spectrometry technique or instrument, according to Edwards. ‘Increasingly, people are asking for workflow-specific tools with whatever technology is most applicable to the samples and analytes,’ he says. A portion of Thermo Fisher Scientific’s growth has been through acquisitions and this inevitably led to a wide range of mass spectrometry techniques and instruments. It dealt with this by deciding, a decade ago, to combine all its data systems onto one platform, using virtual instrumentation technology, called Xcalibur, which is provided with all GC-MS and LC-MS instruments. ‘All our instruments output Xcalibur raw files. By the time users are ready to do processing and analysis, the data is all in the same format,’ says Edwards.

‘This makes it easier to put workflow-orientated applications on top,’ he continues. These applications include specialist tools such as EnviroLab Forms and ToxLab Forms, which cater to environmental and toxicology applications respectively. And the tools can be further specialised too: for example, the regulatory requirements for environmental monitoring in the USA may not be the same as those in Europe or Asia so systems for each market need to have different methods and libraries.

Speeding up the process

Automation is also an important requirement for today’s mass spectrometry software, especially for laboratories where high throughput is required. ‘Customers have described to us that they want as much automation as possible in areas that don’t really require much user involvement, such as loading samples and getting information about the samples from the LIMS, but at the analysis stage they want a higher level of user involvement,’ comments Thermo Fisher Scientific’s Edwards.

An example of this is the Intelligent Sequencing capability of the company’s ToxLab application. This is a data review step that is done by the computer automatically to, for example, change the sample order. For example, in testing drugs of abuse, high concentrations of a drug like cocaine might carry over to the next sample. Traditionally, the mass spectrometrists would either have to watch all the samples and run blanks after a highly positive sample or come back and rerun any samples that showed as positive soon after the highly-positive sample, according to Edwards. However, the Intelligent Sequencing software automatically detects positive samples and inserts blank solvent samples into the sequence after the positive sample until the samples no longer show any traces of the cocaine or whatever was detected.

‘This frees up the users’ time to do other things and we’ve seen great benefits of this. In one study we’ve calculated that this capability could save our customers up to $25,000 per year by freeing up the spectrometrists time to do other, less-routine tasks and not needing to repeat samples,’ comments Edwards.

With the increasing power and sensitivity of the instrument hardware, and the evolving usability and capabilities provided by the software, mass spectrometry looks set to continue making its mark. What’s more, it should carry on helping to check that we and our pets are eating safely.

Top image courtesy of Applied Biosystems.