Can Mass Spec Really Do That?

Unexpected applications of a technique best known as a proteomics powerhouse.

Mass spectrometry (MS) is not just about proteomics, though its star in that field certainly shines bright. As anyone in the drug discovery, food science, petroleum, or chemistry industries can tell you, mass spec is considerably broader than that.

“Traditionally it was a technique for looking at relatively small compounds across environmental issues, industrial applications, contaminants, and so on,” says John Callahan, Chief of the Spectroscopy and Mass Spectrometry Branch of the Division of Analytical Chemistry, Office of Regulatory Science, at the US Food and Drug Administration.

Partly that’s because existing ionization techniques were too harsh for biomolecular work; only the relatively recent discovery of techniques such as MALDI and ESI, which can ionize proteins without shattering them in the process, has enabled the shift towards life science applications that we see today....

Yet despite this shifting focus, MS continues to play key roles in everything from nanotech process development to environmental science. The Scientist spoke with four researchers using MS to answer questions biologists may not have remembered the technology could address. Here’s what they said.

Sizing Nanoparticles

RESEARCHER: Anderson Marsh, Assistant Professor of Chemistry, Lebanon Valley College, Annville, Pa.

PROJECT: Synthesizing colloidal platinum nanoparticles, which are used as catalysts

PROBLEM: In the world of nanoparticles, size is key. Particles typically are sized via x-ray diffraction or transmission electron microscopy (TEM), but at his small liberal arts college, Marsh lacked the former, and the latter was both low resolution and inconvenient (requiring the development of film negatives). The question was, how could he measure the size of his synthesized particles?

SOLUTION: Lebanon Valley College had neither an x-ray source nor TEM, but it did have a linear MALDI-TOF mass spectrometer. Marsh says nanoparticles had previously been subjected to MALDI-TOF MS, but only occasionally, and generally as a matrix material, not as the analyte. Marsh wanted to know whether the instrument could actually resolve platinum nanoparticles by their diameter, and if it could quantify the capping agent coupled to the particles during synthesis to prevent aggregation.

The answer to both questions turned out to be yes (Anal Chem, 81:6295–99, 2009). “MALDI-TOF seems just as good as electron microscopy or x-ray diffraction,” Marsh says—in fact, in some ways it is even better, because data analysis is simpler.

And the technique also provided a little extra. If you assume nanoparticles are spheres, and you know their mass, then calculating their density is trivial, Marsh says; it’s just that no one had ever thought to use a mass spec in this way. “That’s one thing that has come out of this research: we can maybe get the density of metal nanoparticles,” he says.

Surprisingly, when he ran that calculation, Marsh found that nanoparticulate platinum is about 20% denser than its bulk counterpart. “I don’t know if that’s significant,” Marsh says, “but it could help explain some properties of metal nanoparticles, such as melting behavior and thermal expansion.”

IMPLICATIONS: As Marsh’s experience shows, you don’t necessarily need top-of-the-line, next-generation equipment to try something new. “Push your instruments to the limit,” he advises—you never know what they are capable of. In this case, some nanoparticles had apparent masses of 500,000 mass units or more, which for some MALDIs is really pushing the instrument’s resolving power, he says.

Illuminating Manuscripts

RESEARCHERS: Marc Walton and Catherine Schmidt, Assistant Scientists; and Karen Trentelman, Senior Scientist, Getty Conservation Institute, Los Angeles, Calif.

PROJECT: Analysis of lapis lazuli—a blue pigment made from stone mined mostly in Afghanistan—in illuminated manuscripts

PROBLEM: Spectral analysis of the 14th-century Laudario of Sant’Agnese and the 15th-century Hours of Louis XII revealed features not normally associated with lapis. GCI researchers wanted to track down the source of those features, and use them to provenance and fingerprint the pigments.

SOLUTION: For conservation scientists, lapis illuminates not only manuscripts but the technology, trade, and economy of medieval Europe, Walton says. He, Schmidt, and Trentelman put nine reference lapis samples through their analytical paces in a battery of analytical techniques. The goal: to characterize the samples’ mineral content and spectral characteristics, and to determine whether those features could help identify the origin of the pigments.

One technique the team used was Raman microspectroscopy, a nondestructive method that reveals a material’s molecular structure. Another was more aggressive: laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS), a “brute-force method” that reveals a material’s elemental “fingerprint” by cooking femtogram samples in plasma prior to mass analysis, says Walton.

In this case, the team introduced nine reference samples into the plasma by zapping them with a laser—essentially the same principle that drives MALDI mass spectrometers—and identified them by their flight time to the detector. The analysis, Walton says, demonstrated subtle differences in the chemical makeup of different lapis samples including several candidate trace elements (including chromium, vanadium, and titanium) that, in conjunction with a common lapis accessory mineral called diopside, could potentially explain the unexpected spectral characteristics the team observed (Anal Chem, 81:8513-8, 2009) and may eventually help scientists pinpoint the geologic source for lapis specimens in medieval artwork. “Now, other people can look at their illuminated manuscripts and see if they produce a similar Raman pattern, and can say they are probably associated with the deposits we identified,” Walton says.

IMPLICATIONS: ICPMS is too harsh for most biological samples, not to mention precious objets d’art. Manuscripts at the Getty and elsewhere will thus continue to be analyzed by nondestructive Raman spectroscopy. But ICPMS, says Walton, can provide the foundational knowledge to interpret Raman spectra. “It gives another tool to conservation scientists to identify where these materials are coming from,” he says. It also, he adds, can reveal the inorganic content of biological samples—a fact Walton is exploiting to study the sulfur forms and content in rubber associated with contemporary art and design collections. “ICPMS is the perfect technique for that,” he says.

Pesticide Profiling

RESEARCHER: Douglas G. Hayward, Research Chemist, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Office of Regulatory Science, College Park, MD

PROJECT: Detecting pesticides in dietary supplements

PROBLEM: Literally hundreds of pesticides can make their way into food and supplements. To make safety testing efficient, researchers require a universal method to extract and identify all pesticides simultaneously.

SOLUTION: Hayward is a methods development guy. Focusing on ginseng root, a popular nutritional supplement, he and FDA colleague Jon Wong developed an ethyl acetate–based procedure to extract pesticides from dried powders, which they then cleaned up on columns prior to gas chromatographic separation. They analyzed the extracts by two different mass spec techniques, both to assess the efficacy of the extraction procedure and to compare the relative strength of the two MS methods (Anal Chem, 81:5716–23, 2009).

According to Hayward, each MS approach produce very different data. The first, called selected ion monitoring via single quadrupole MS (qMS-SIM), is a low-resolution technique that looks specifically for preselected ions and ignores the rest; as a result, it provides excellent sensitivity for compounds it’s programmed to find, but cannot detect anything unexpected (such as new pesticides). The second—high-resolution time-of-flight (HR-TOF) MS—is a higher-end, high-mass accuracy (and also more expensive) approach that collects full mass spectra, meaning it provides higher confidence data that may be subsequently reanalyzed as new materials are discovered.

"Even if you had an immunoassay, could you look at 170 or 500 or 600 compounds? I don't think so."

Nevertheless, the two methods—tasked with finding some 170 pesticides in both spiked reference and commercial ginseng samples—were more or less equally effective, Hayward says. “What we have is a clean-up method that cleans up the ginseng very well, and we got accurate results down to low levels,” he says. “Using this procedure gives a clean enough extract to use in either SIM on a single quad, which everyone has, or on the high-resolution TOF.”

IMPLICATIONS: According to Hayward, there really is no other way besides MS to detect multiple pesticides—or, for that matter, any small organic molecules—in the low part-per-billion range. ELISA might work, he says, “but even if you had an immunoassay, could you look at 170 or 500 or 600 compounds? I don’t think so.” Actually, the task was even harder. Hayward looked for 170 compounds based on three or four fragment ions apiece; that means the instruments had to be looking for between 500 and 700 ions per GC run. Now he’s upping the ante to look for 600 pesticides. For that, he says, “We need other MS techniques,” not to mention better analytical software and reference libraries. “We are going at unknowns,” he says. “We are approaching a sample and we are not sure what might show up, so we are going to try to make the thing tell us everything, and then we can mine it.”

Testing Toothpaste

RESEARCHER: Huanwen Chen, Professor of Analytical Chemistry, East China Institute of Technology, Fuzhou, Jiangxi Province, China

PROJECT: Detecting diethylene glycol (DEG), a toxic compound sometimes used as a sweetener in Chinese toothpastes

PROBLEM: China has prohibited use of DEG-containing toothpastes since July 2007. But how to test for it? Toothpaste is a highly complex, viscous, and sticky mixture, making sensitive high-throughput analysis difficult. Traditional approaches to DEG analysis use chromatography and chemical derivitization prior to mass spec, but that adds time. Chen wanted to develop an approach that was fast, efficient, sensitive, and required no preparation.

SOLUTION: The ideal test method would allow simply squirting a dab of each batch of toothpaste and analyzing the samples directly. To make that possible, Chen and his colleagues modified a technique called neutral desorption-extractive electrospray ionization (ND-EESI) mass spectrometry.

In Chen’s method, a concentrated jet of nitrogen gas is directed at a sample, liberating a small amount of the toothpaste in the form of an aerosol, which is directed into the EESI source to produce analyte ions for subsequent mass spec analysis. Typically, material sampled by the gas jet is ionized by the EESI source directly, but that approach doesn’t work for DEG, as it cannot be efficiently protonated. So, Chen tweaked the system to inject ammonium acetate, which ionizes the DEG instead. The resulting technique, called reactive ND-EESI-MS, is both efficient (with recovery rates exceeding 90%) and sensitive (as low as 0.00002% DEG by weight; Anal Chem, 81:8632–38, 2009).

“ND-EESI-MS provided analytical performances, in terms of sensitivity, throughput, and specificity, similar or even better than existing methods or national standard methods,” Chen wrote in an email.

And, he adds, it should prove to be broadly applicable to the biotech, pharma, chemical, and food industries. “Trace impurities such as HMF [hydroxymethylfurfural] in milk and honey, [and] hormones in cosmetics are being detected by ND-EESI-MS, and showed promising results,” he says.

IMPLICATIONS: In the world of MS, on-site testing always beats taking samples back to the lab—it’s faster and easier that way. Chen’s technique isn’t yet field-ready—its use of a neutral gas spray is particularly problematic, he says—but he is working to optimize it. “[It] will be suitable for in situ analysis eventually,” he says. In the meantime, Chen says he’s seen some interest in his technique from government and testing bodies in China, “but not so many at this moment” since official reports, based on limited testing, say DEG is no longer present in commercial toothpastes.

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