Advanced MS techniques and tools have revolutionized the pharma lab
Editor’s Note: This article on the history and impact of mass spectrometry in the pharmaceutical industry is the second in a new series for Pharmaceutical Formulation & Quality. In “PharmaTools: Technologies That Changed Pharma and Biotech,” we look at various technologies such as mass spectrometry that have played a key role and had an indelible impact on the pharma and biotech industries. In our next two issues, we will examine the evolution of liquid chromatography and gas chromatography. To view other mass spectrometry materials, please see below.
Even in the midst of a world economic downturn, some products continue to attract buyers. Within the pharmaceutical industry, one standout is mass spectrometry (MS) systems. According to Strategic Directions International, a market research firm that tracks instrument business trends, the mass spectrometry market, already a $2 billion annual concern, is expected to grow at a 9% annual rate through 2012.
“The market will be led for the foreseeable future by the more advanced methods, including Fourier transform (FT)-MS, tandem LC-MS, and quadrupole time-of-flight QTOF LC-MS (9),” the report states.
In the last issue of Pharmaceutical Formulation & Quality, we looked at the overall history of mass spectrometry in the pharmaceutical industry and how its evolution from the “expert-from-Switzerland” mode to easy-to-operate, benchtop tandem LC-MS systems has made it a ubiquitous tool in virtually every pharma lab (“Bringing Mass Spec to the Masses,” September 2009, pgs. 16-21). In this issue, we’ll explore the history of some of the more advanced methods and adaptations of mass spectrometry that are now helping to drive the field’s growth.
One technology that waited decades for its time to come is time-of-flight (TOF). W.E. Stephens, at the University of Pennsylvania, developed the concept in 1946. In 1948, Cameron and Eggers, at Oak Ridge Laboratories, built the first TOF instrument with very low mass resolution. But it took more than five decades for improvements in electronics, software, and engineering design to make TOF mass spectrometry the indispensable industry tool it is today.
“The issue was fundamentally low performance in terms of analytical service, resolution, sensitivity, and mass accuracy,” says Iain Mylchreest, PhD, vice president and general manager of life sciences mass spectrometry for Thermo Fisher Scientific. “Quadrupoles were much easier to interface with, so TOF took a back burner until the supporting technology and the means to interface with that technology caught up.”
Quad TOF Vital for Proteomics
TOF made a key leap forward in 1984 when Gary Glish, PhD, now the president of the American Society for Mass Spectrometry, published the first paper (in Analytical Chemistry) on quadrupole TOF mass spectrometry. “The evolution of the quad TOF over the past two decades has been very important, particularly for proteomics and metabolomics,” said Gary Siuzdak, PhD, senior director of the Scripps Center for Mass Spectrometry in La Jolla, Calif. “Having quad in the front and TOF in the back gives you accurate mass measurements.”
Previous quad-TOF instruments notoriously lacked robustness and accuracy, but that has changed in recent years, according to Dr. Siuzdak. “Previously, they tried hard to achieve five PPM [parts per million] accuracy, but more often than not the range was more like 20 or 30. Now, with new techniques and improved detectors, they’re routinely getting sub-five PPM accuracy, and in some reports I’ve even heard of sub-part-per-million accuracy.”
Engineering improvements, such as changing the composition of the flight tubes, have made this possible. “They’re now making them out of a ceramic-like material that has a very low coefficient of expansion, so even if the temperature changes in the room, the flight tube won’t change,” Dr. Siuzdak said. “When you’re talking about PPM accuracy, having a flight tube change in size can make a pretty big difference.”
Quad-TOF instruments are particularly useful for proteomic and metabolomic accuracy. “Now, people can create a profile of the sample using quad TOF without generating MS-MS data and then do comparative analysis between the profiles and look at which peaks are changing significantly between different samples, such as with a benign versus malignant cancer sample,” Dr. Siuzdak explains. “Instead of getting fragmentation data on everything, you can go after what’s significantly different.
“It gives you a more manageable quantity of information, and the fragmentation you get is of higher quality, only focusing on the molecules that matter. This all means that we can do much more direct lead analysis.”
The coupling of TOF with MALDI (matrix-assisted laser desorption/ionization), a soft ionization technique that allows the analysis of biomolecules and large organic molecules that are vulnerable to fragmentation with conventional ionization, has advanced both tools, Dr. Mylchreest said. “It’s a natural marriage, since MALDI is a pulse technology and TOF deals with pulses of ions.”
Introduced before electrospray was in wide use, MALDI allowed the direct analysis of big proteins and peptides, something very difficult to do with the technology of the time. “It was attractive to the biologists, because it had gel spots you could directly analyze in the mass spectrometer and get some idea of molecular weights, which you could never do before. You had to use chromatographic methodologies, which were very inaccurate,” Dr. Mylchreest said.
Pioneered in the early 1980s, the first MALDI instruments were linear, single-stage instruments. “With those, the primary application within proteomics was measuring the mass of intact proteins,” says Ronan O’Malley, PhD, MALDI product manager for the Waters Corporation. “The next development in MALDI, in the late 1980s and early 1990s, was the introduction of a reflectron into the instrument, an ion mirror that has the advantage of lengthening the flight tube, thereby increasing TOF and improving the mass accuracy that can be achieved.”
But the earliest MALDI machines had significant disadvantages. “The M in MALDI is for matrix. In the early days, you never knew why you’d get a good signal with a matrix in one case and not in another,” said Richard Caprioli, PhD, director of the Mass Spectrometry Research Center at Vanderbilt University in Tennessee and a developer of MALDI MS imaging.
“We might play all day with sample preparation to get our signal and try to understand these things, which is fine in academia, but not what they’re getting paid for in pharma. Today, there is a much better functional and chemical understanding of how to get a better signal,” Dr. Caprioli said.
Advantages of MALDI MS
Dual-stage MALDI, which came on the scene around the turn of the 21st century, allowed people to select ions in the first stage of mass spectrometry and fragment in the second. “This gave much more specificity, allowing you to add fragmentation to the molecular mass experiments,” said Dr. O’Malley. “Proteomics was the mainstream market for this capability, but there were also applications in quality control for formulated compounds and for analyzing oligonucleotides.”
Not long after, MALDI sources were combined with orthogonal instrumentation. “With an orthogonal system measuring TOF from the pusher to detector, you don’t have to take into account the TOF from source to detector,” said Dr. Mylchreest. “That’s important in MALDI, because there can be variations in the uniformity of the analyte across the target plate. With an orthogonal system, that doesn’t matter anymore. This brought the advantages of high resolution and exact mass to MALDI.”
Another advantage that MALDI MS brought to the table is speed. “The ability to do 2-5K laser shots per second—and you only need 10 or even less to give you a good analysis—enables a really rapid screening process,” said Dr. Caprioli. “Other MS techniques, although very valuable, are much slower. LC-MS might take one to three hours, whereas MALDI would take you one second to acquire the same data.”
Like TOF, Fourier transform mass spectrometry (FTMS) also took decades to reach its full potential. First developed in the mid-1970s, Fourier transform to ion cyclotron resonance (FTICR) mass analysis made FTMS applicable to the study of biomolecules. But FTMS and FTICR have only taken off within the last decade.
“You’re dealing with big magnet technology, which wasn’t that advanced back then,” said Dr. Mylchreest. “The magnets were huge, expensive, and weren’t shielded, and it took a long time to adapt that more academic technology for commercial usage.”
Today, FTICR offers ultra-high resolution along with impressive stability and accuracy. “It’s possible, with a skilled user, to get on the order of 500 parts per billion accuracy,” said Dr. Siuzdak. “This can really allow you to nail down elemental composition with relatively low ambiguity.”
FT has one significant downside: It is much more complex than TOF and quadrupole instrumentation. “FT instruments require a more advanced user,” said Dr. Siuzdak. “Within a day or so you can get reasonably familiar with [a] TOF or quadrupole instrument. With an FT system, especially the ICR instruments, it can take longer to learn all their aspects.” He added that some instruments on the market have made this easier, but they don’t offer resolution and accuracy as high as that attained from FTICR.
Ion Mobility Plays Important Role
Driving the utility of many of these advanced instruments is ion mobility. As a technique, it’s been around for decades. Some of the first measurements were reported by researchers as early as the 1930s. Researchers at Bell Labs developed an instrument in 1967 that was “essentially an ion mobility drift tube combined with an orthogonal time-of-flight type analyzer,” said Alistair Wallace, PhD, Synapt product manager for Waters. But, just as with TOF, MALDI, and Fourier transform, ion mobility’s time had not yet arrived. “Electronics then were far less evolved, and the analyzers in use at that time were only capable of analyzing a single ion arrival event.”
It took another 25 years for pioneers like Michael Bowers’ group at the University of California, Santa Barbara, and David Clemmer’s at the University of Indiana, to move ion mobility mass spectrometry into the modern age. “Today, ion mobility is consistently increasing and driving the performance one can get from things like a TOF analyzer,” said Dr. Wallace.
He compares the TOF analyzer to a big molecular dustbin. “You throw thousands of ions in there, and at the end of the day the limiting factor is the speed with which you can acquire the data. The faster and more powerful the electronics are, the more you can get out,” Dr. Wallace said.
Introduced in 2006, Waters’ Synapt instrument takes advantage of tri-wave technology to perform ion mobility at the limits of MS detection as it is currently known. “It can trap and accumulate ions prior to ion mobility separation, and the tight radial confinement of the T-wave enables you to get very high transmission of ions—nearly 100%—through the entire device,” said Dr. Wallace.
The impact of all of these advanced MS technologies on the pharmaceutical industry has been nothing short of revolutionary. “They’ve opened up new areas in terms of very high resolution mass analysis and accurate mass, something that has always been a big challenge in pharma. A lot of us have big fish stories on accurate mass,” said Dr. Mylchreest.
“They also made these experiments available to every lab. Today, a technician or a grad student can come along, put in samples, and get accurate measurements from one to five PPM. That’s been a massive change for industry. Although they all have different characteristics as to what they can do in terms of resolution, capabilities, and performance, they all address the same market space: opening up drug metabolism and structural analysis. They’ve opened up areas in proteomics and peptide sequencing and characterization that simply couldn’t be done before,” Dr. Mylchreest said.