When it comes to characterizing the molecular composition of complex samples, mass spectrometry (MS) is tough to beat. If it’s in there and can be ionized, the mass spec can probably find it.
That’s a qualitative question, though—is a compound present, or isn’t it? Quantification is another matter. Despite their exquisite sensitivities, mass spectrometers are not necessarily adept at measuring abundance. One instrument, though, is synonymous with quantitative mass spectrometry: The triple-quadrupole mass spectrometer.
As its name suggests, a triple-quadrupole mass spectrometer consists of three single-quadrupole mass analyzers strung end to end. These are typically relatively inexpensive, low-resolution devices—in a different class altogether from high-resolution, high-mass-accuracy instruments such as Thermo Fisher Scientific’s popular Orbitraps, for instance. But according to Keith Waddell, director of application marketing for mass spectrometry products at Agilent Technologies, triple quads are unparalleled in terms of absolute sensitivity and dynamic range.
“Those are two pretty strong arguments for why one would not choose an Orbitrap for high-level, high-sensitivity quantitation,” he says.
Quantification on a triple quad is generally accomplished via a tandem MS/MS assay known as selected reaction monitoring (SRM; also called single reaction monitoring or alternatively, multiple reaction monitoring, or MRM). SRM assays enable researchers to both identify an ion and measure its abundance. That’s because mass spectrometers identify molecular species based on, well, their mass. Sometimes, though, weight is not sufficiently discriminating, especially in a complex sample. After all, many molecular species weigh the same, or very nearly so. One way to increase confidence in a putative identification is to break the ion apart and see what fragments it produces.
That, explains Shane Tichy, triple quadrupole LC-MS product manager at Thermo Fisher Scientific, is the equivalent of determining whether a Mercedes Benz E500 was involved in a multicar pileup by looking for its nameplate among the rubble.
That’s where SRM comes in, and it is an assay uniquely suited to the triple quad. In an SRM assay, the first quad acts like a mass filter, sending a specified ion—the one to be quantified—into the second mass analyzer. The second quad is a collision cell, where the selected ion collides with a neutral gas and fragments. Those fragment ions then pass into the third quad, where they are finally measured. If it is chosen well, the "transition," as it is called, from precursor ion to fragment ion can be diagnostic for a particular molecular species, and a quantitative one, as well.
“By doing this type of analysis you increase detection specificity of known molecules,” says Tichy. “So you’re quantitating on a pure ion signal without the interferences of chemical noise.”
Today, says Keith Worrall, senior product manager for tandem quadrupole mass spectrometers at Waters Corp., MRM “has become the gold-standard technique for quantitative applications, especially where you have a complex matrix.” Tichy estimates there could be 2,500 to 3,000 new systems sold this year worldwide, crunching ions in everything from proteomics to forensics, from biopharmaceuticals to environmental testing.
For scientists looking to incorporate this technology into their own research, there exists a wide array of commercial triple quads to choose from.
Among the companies offering triple-quad mass spectrometers are AB SCIEX, Agilent Technologies, Bruker Daltonics, Thermo Fisher Scientific and Waters. Each company offers several options, which tend to differ mainly in sensitivity.
AB SCIEX, for instance, offers six different flavors of triple quad in its portfolio: the API3200™, API4000™, Triple Quad™ 4500, API5000™, Triple Quad™ 5500 and Triple Quad™ 6500, the most sensitive of the bunch, which was launched at ASMS 2012.
Sensitivity depends on a number of variables, including the matrix and the ion itself, but according to Mauro Aiello, senior product manager at AB SCIEX, the 6500, with its new IonDrive ionization source, optics and detection systems, generally yields about a 10-fold improvement in signal to noise over the 5500, resulting in a three to five fold boost in limit of quantification (LOQ). (The 6500 and 5500 also sport an upfront ion-mobility system called Selexion, which adds an additional degree of separation prior to mass spectrometry for greater selectivity, Aiello says.)
Most AB SCIEX mass specs are available in triple-quad and hybrid quadrupole-linear ion trap (QTRAP) configurations, Aiello notes (all but the 5000). Technically, a QTRAP is just a modified triple quad, and so it also supports MRM, he adds. But they can do a bit more: Using a workflow called MRM3, researchers can refragment interesting fragment ions—that is, perform MS/MS/MS experiments—and increase their confidence in molecular identification.
“Adding another level of fragmentation dramatically improves your selectivity,” Aiello says.
Agilent Technologies' product line includes four options: the 6420, 6430, 6460 and 6490, says Waddell. These also differ mostly in sensitivity, he says, with the 6460 offering a five-fold boost in sensitivity over the 6420 and 6430 thanks to its use of the Agilent JetStream ionization source, and the 6490 offering another 10-fold increase on top of that via its IonTunnel focusing technology.
Thermo Fisher Scientific’s triple-quad line includes the TSQ Quantum Access MAX, the TSQ Quantum Ultra and the TSQ Vantage, with the latter offering the best sensitivity of the group. According to Tichy, the Access MAX, popular in the food and environmental safety industries, “is the best price-performance triple quad we offer, [providing] the best value for the buck.”
Waters’ Xevo line of triple quads includes the Xevo TQD, Xevo TQMS and Xevo TQS. According to Worrall, the TQS, featuring an orthogonal ion guide to reduce noise from neutral and solvent molecules, is the most sensitive of the three, and “can get you down to zeptomole [sub-femtogram] levels of detection in the best cases.”
Worrall says Waters’ Xevo triple quads are used in applications from food safety and environmental testing to biopharmaceuticals testing—drug bioavailability, metabolism, pharmacokinetics and so on. And in that latter area, he says, triple quads increasingly are being called upon to handle peptides. “We are seeing some shift from small-molecule to large-molecule work as the [types of] drugs that are being developed are changing,” he says, noting that pharmaceutical companies increasingly are moving away from small-molecule drugs to protein therapeutics.
All these triple-quad systems use liquid chromatography-coupled electrospray ionization or related sources. But Bruker Daltonics’ Scion TQ™ is a gas chromatography-based system instead. Recipient of the 2012 Pittcon Editors Silver Award, the Scion TQ features a lens-free design and curved ion path, which serve to simplify maintenance and increase sensitivity, respectively, says Sandy Yates, Western Region Application Group leader at Bruker Daltonics.
According to Yates, gas chromatography (GC) traditionally has been coupled to single-quadrupole instruments. But with the Scion TQ (and other GC-coupled triple quads), he says, “MRMs will be the future of GC.”
(Waters’ Xevo triple quads can be fitted with an optional atmospheric-pressure GC interface as well, says Worrall.)
Although it is possible to use a triple quad to search for a single molecule, users often configure their SRM experiments to scan for whole panels. Some, for instance, must scan foodstuffs for hundreds or even thousands of banned or regulated pesticides. In the laboratory of John Yates (no relation to Sandy), a mass spectrometry expert at the Scripps Research Institute in La Jolla, Calif., researchers sometimes interrogate samples for 50 peptides at a time. “It gets very complicated,” he says.
To solve this problem, researchers use something of an analytical heuristic. This trick has several names—AB SCIEX calls its approach Scheduled MRM™; Agilent’s is called Dynamic MRM; Thermo’s is dubbed “timed-SRM”—but they all work essentially the same.
Researchers know that a given analyte will elute off the liquid chromatography (LC) column at a specific point, say five minutes into the chromatographic run. Another analyte might elute at 10 minutes, and a third at three minutes. The instrument doesn’t need to be looking for all three analytes all the time, but only around the time those peaks will elute into the electrospray ionization source. In other words, these algorithms factor peak retention time into their scheduling, thereby maximizing the time needed to look for more pressing ions.
In practice, triple quads can easily handle dozens or even hundreds of MRM transitions at a time, rapidly switching from one to the next like tuning a radio. The more times it makes that switch, however, the less time the instrument can devote to any given MRM (this is called the “dwell time”) and the more sensitivity suffers. With Scheduled MRM, says Aiello, “you can maximize the duty cycle of your instrument around your particular analyte.”
Mass spec vendors often work to simplify this problem with databases of canned methods—essentially lists of MRM transitions, retention times and so on, for an array of interesting compounds. Bruker Daltonics, for instance, has “hundreds” of so-called “compound-based scanning” methods, enabling its users to concentrate on the compounds they wish to identify rather than the minutiae of retention times, collision energies and MRM transitions. “This is a compound-centric way of constructing an MRM,” says Sandy Yates. “The user thinks about the compound of interest. The instrument and software take care of the rest.”
But for John Yates and others, even this approach may not suffice. “MRM is very difficult to set up,” he says. “You basically have to do all this leg work to establish the [transitions and retention times] for each and every peptide, and that winds up being a boatload of work.”
His lab is now considering an alternative approach called targeted MS/MS, using a Thermo Fisher Scientific Orbitrap-based Q Exactive. With this approach, rather than looking for particular ions and their diagnostic transitions, researchers can collect full MS/MS spectra and interrogate them after the fact for analytes of interest.
Sandy Yates notes that some researchers are complementing or even replacing triple-quad MRMs with time-of-flight (TOF) or quadrupole-time-of-flight hybrids for another reason, too: as a way to quantify not only what they anticipate finding but the unexpected molecules, too.
One application, he says, is in animal racing, where regulators are constantly battling with those who hope to game the system. New performance-enhancing compounds might slip through a targeted MRM analysis, he notes. But full-scan TOF data retains those data and can be analyzed post hoc to determine if a suspect animal may have been doped.
“MRM does a great job finding things you already know, whereas the full-scan data allows you to look for things you hadn’t thought of before,” says Sandy Yates.
According to Aiello, the first variable to consider when purchasing a triple quad is, obviously, its ability to quantify your analyte of interest. “Reproducibility and accuracy are the ultimate tests of whether you can quantify an analyte.”
Consider also speed (and dwell time), linear dynamic range (the bigger the better) and robustness, ease of use and reproducibility with your complex samples. “You don’t want an instrument that will run a couple times and then require cleaning,” Aiello says.
But don’t forget about the up-front separation, says Tichy. “In order for SRM to work well, you need to have good LC separation in front of a good working mass spectrometer.” After all, timed-SRM depends on reproducible retention times. “It’s an LC-MS, and they both rely on each other.”
The image at the top of this page is Agilent Technologies' 6400 Series Triple Quadrupole LC/MS.