Scientists in many fields—basic research, pharma, forensics, and more—must isolate small molecules from biological samples to analyze them. Collecting the right small molecules for analysis can be done in many ways, including solid-phase extraction (SPE). The analysis is often done using liquid chromatography-mass spectrometry (LC-MS). With the right expertise and tools, which often include automation, mostly any sample can be handled efficiently.
The key applications of small-molecule extraction for LC-MS detection include “all fields where you need to concentrate an analyte or remove background to increase the signal-to-noise ratio and reduce matrix effects for easier detection,” Manuel Bauer, market manager at Tecan, explains. Examples include testing for drugs of abuse, determining steroid levels, or measuring levels of immunosuppressants in a transplant patient.
For accurate and repeatable results, these measurements depend on the right sample preparation and automation, which adds consistency and can be more affordable over time.
A collection of challenges
The variety of possible small molecules adds considerable complexity to these studies. “The biggest challenge is that not one solvent can extract all metabolites present in every biological sample,” says Ute Roessner, professor of plant biochemistry at the University of Melbourne in Australia. “Therefore, a number of different extraction protocols need to be deployed to capture as many metabolites as possible for subsequent analysis.”
According to Mark Boggeri, senior field application specialist at Tecan, customers often need a streamlined and automated sample-preparation workflow. He adds that you need to “minimize the need for high-level training.”
Achieving that depends on several factors. In addition to good advice, Jack Andrews, senior field application specialist at Tecan, says that a good extraction requires quality instrumentation. “It’s a marriage of consumables that are automation-friendly and automation equipment,” he says.
Then, a second marriage is required—preparation to analysis. “LC-MS/MS today is one of the most used analytical approaches to measure small molecules, such as metabolites, due to its superior sensitivity and ability to measure very many different chemistries,” Roessner explains. “The versatility of LC-MS/MS is therefore of great benefit for metabolite analysis since metabolites are chemically very diverse representing compounds with different chemical and physical properties, such as size, polarity, stability, etcetera.”
Selecting the extraction
Before jumping into options, the first question, according to Bauer, is pretty simple: Is an extraction needed? “With some drugs, only a dilute-and-shoot is necessary,” he says.
So, some samples can just be diluted and then injected in an MS platform. Although this method works in some cases, dilution does not concentrate a target analyte enough. Plus, dilution doesn’t remove any of the matrix, which can reduce the sensitivity and accuracy of later analysis and puts higher strain on the analytical equipment.
In other cases, a liquid-liquid extraction can be used. “If you have a working liquid-liquid extraction” Bauer says, “it’s cheaper than solid-phase extraction.” Nonetheless, SPE allows easier automation, because it can be used with a 96-well format and requires no centrifugation. “There’s also a wide variety of sorbents, for all kinds of different analytes,” Bauer notes.
So, how does a scientist pick the right method of extraction? “It’s chemistry,” Andrews explains, “simple chemistry.” He adds that “a good synthetic chemist can look at the molecules and know exactly how to extract them.”
In some cases, scientists will consider more than one extraction option. “Usually, they consult with peer-reviewed articles, run tests with their samples, and then make an empirical determination,” Boggeri says. “From that, they just see which extraction works the best.”
Real-world work
In Roessner’s lab, scientists use LC-MS/MS to analyze metabolites from many kinds of biological samples. “One of the examples would be that we are aiming to determine the metabolic basis of communication between beneficial microbes and crop roots under a range of environmental stresses,” she explains. “Plant roots partner with a number of microbes beneficial for nutrient and water update, however at this stage we do not know how plant roots attract these beneficial microbes while repelling pathogenic microbes.”
It’s possible that roots release compounds that attract the desirable microbes. To find out, Roessner and her colleagues “inoculate plant roots with microbes known to be beneficial for plant growth and determine the metabolic signatures of interaction,” she says. “In addition, we then treat those plants with different abiotic stresses—such as drought, salinity, or temperature stress—and then determine if the metabolic signatures are altered thus leading to a breakdown in communication with beneficial microbes.”
Beyond such exciting work in basic research, some clinical applications continually evolve. One example is tracking abused pain medications. “It’s a changing target, depending on what’s prescribed or what’s available at the street level,” says Boggeri. “We get calls often to add new compounds to existing screening panels, and that takes a lot of sample prep and LC-MS expertise.”
Beyond testing for new compounds in an existing area, other scientists or businesses want to put platforms to work in new ways. Although the details of such requests often remain confidential, Andrews says that one lab wanted to diversify its capabilities by adding a panel of small peptides to its testing options. “We sent scientists there and developed a brand new extraction column,” he says. That diversified the lab’s abilities and gave them a business edge. “They are the only ones able to use this matrix and provide this turnaround for this panel of compounds,” Andrews adds, “because it’s automated with Tecan liquid handling and Tecan SPE extraction materials.”
The small-molecule challenge at hand—from basic research through medical applications and beyond—determines the best method of sample preparation to select ahead of LC-MS detection. In some situations, manual dilute-and-shoot can solve a problem. More likely, some form of extraction will be needed, and finding the best method can take experimentation, which can be streamlined with advice from an expert chemist. In pharmaceutical labs and clinical labs, high throughput can be crucial, which increases that value of automating small-molecule extraction. In all cases, the key comes down to optimizing the extraction that sets up the best analysis, and the ultimate results depend on that.