Research on small molecules relies on methods to analyze the components. In many cases, scientists use liquid chromatography (LC) and mass spectrometry (MS) in this work. With LC/MS, getting the best results depends on the most effective form of sample preparation. Although small-molecule research often takes place in the pharmaceutical industry, other areas of science also rely on similar kinds of experiments. In all applications, getting the best results from MS depends on starting with the best prepared sample.

A team of scientists from the University of Toledo College of Medicine and Life Sciences, used LC/MS to study 3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (6-MOMIPP), which disrupts microtubules.1 The researchers tested 6-MOMIPP on cultured U251 glioblastoma cells using LC/MS assays. As the team noted: “Pharmacokinetic studies in mice show that concentrations of 6-MOMIPP in the brain mirror those in the plasma, indicating that 6-MOMIPP readily penetrates the blood-brain barrier.” Overall, they concluded that 6-MOMIPP “warrants further preclinical evaluation as a potential small-molecule therapeutic that may have advantages in treating primary and metastatic brain tumors.”

MS can also be used to study other aspects of cancer. A team of scientists from the South-Central University for Nationalities (Wuhan, China) used LC/MS-MS when studying focal adhesion kinase (FAK), which is a non-receptor tyrosine kinase that plays a role in cell migration, spreading, and proliferatiion in early stages of some cancers.2 “Small-molecule inhibitors of FAK are effective to inhibit its activation in the process of tumor formation,” the scientists wrote. Working with three FAK inhibitors applied to rat-liver microsomes, the scientists used LC/MS-MS to characterize metabolites of the inhibitors. The researchers noted: “The structures and fragmentation patterns of these metabolites were elucidated, and phase I metabolic pathways for FAK inhibitors were proposed.”

At Janssen Research and Development, scientists applied MS to untargeted metabolomics in hopes of “reducing the significant attrition rate for discovering and developing drugs in the pharmaceutical industry.”3 As these scientists pointed out, untargeted metabolomics could be used “for the advancement of drug candidates in drug discovery/development including potentially identifying and validating novel therapeutic targets, creating alternative screening paradigms, facilitating the selection of specific and translational metabolite biomarkers, identifying metabolite signatures for the drug efficacy mechanism of action, and understanding potential drug-induced toxicity.”

Still, MS studies of small molecules extend beyond the pharmaceutical industry. When asked about the main application of studying small molecules with LC/MS, Manuel Bauer, senior market and product manager at Tecan, says that it “has a plethora of applications—from pesticide and contaminants testing in food to quantification of clinically relevant parameters, such as vitamins, therapeutic drugs, antibiotics, steroids and drugs of abuse to metabolite and fatty acid analysis, which is currently mainly performed in the research environments.”

To explore this wide range of samples with MS, scientists work with material from various sources. “The complex sample matrices are challenging,” says Kim Haynes, principal product marketing manager at Waters. So, scientists must prepare the samples in the best way.

Proper preparation

The key to accurate analysis in many methods depends on isolating the key components as much as possible. “The main challenge is the specific and reproducible clean-up of the analyte from the matrix it’s in,” Bauer explains. “This is a combination of the extraction method and the chemistry used.”

Plus, that combination must be used across a broad collection of samples and matrices. “Depending on the sample you’re working with, you could be dealing with a wide variety of complexity,” Haynes notes. Sample matrices range from plasma and whole blood to soil, tissues, and water. “In each matrix, there are components that compete for signal in MS analysis,” she says. “By removing the competing matrix components, you get better sensitivity, because that increases the signal of the analytes of interest.” Getting rid of any extra matrix material also leads to longer LC column life and keeps the MS up and running more.

Depending on the application, the constraints on methods vary. “Especially in clinical environments, the sample traceability is a key requirement,” Bauer says. “A main challenge in the manual preparation of such samples is that this is very repetitive and dull work, which is prone to inadvertent mistakes.”

So, Tecan developed its RESOLVEX A200. It’s key features, according to Bauer, include unattended positive pressure solid phase extraction (SPE), greater ease of use, and enhanced performance. “Eight-channel dispensing delivers a threefold increase in speed compared to single-channel dispensing, and the intuitive touchscreen control enables straightforward integration into laboratory protocols for immediate productivity,” he says.

The Oasis Sample Extraction SPE products from Waters also simplify sample preparation for MS. “These products come with simple three-step methods that are designed to remove extra sample matrix and concentrate the analyte of interest,” Haynes explains. The automation of this process also improves the outcome. “You save time and money, plus you get more robust analysis,” she says.

That three-step method makes it easy to begin preparing a sample for MS. “It’s a great starting place,” Haynes says. “A little optimization might be required for some samples.”

The key for almost any small-molecule sample is building a workflow that recovers as much of the key analytes as possible, and analyzes them with high sensitivity and repeatability. “That requires a good sample–clean-up protocol,” Haynes says. “Without repeatability, a result could change even with the same sample.”

No matter why a lab needs to analyze small molecules with MS, some requirements stay the same. One of them is getting the cleanest sample possible before analysis, and that applies to any level of throughput. “Sample cleanliness is key when working with a lot of samples or just a few,” Haynes says. Consequently, labs that run MS on small molecules use some form of sample clean-up ahead of the analysis. The analysis that works the best depends on the sample and the application or required results. As MS gets even more sensitive, sample-preparation steps need to provide even more pure samples.

Hero image: Tecan’s RESOLVEX A200 performs solid-phase extraction with eight-channel dispensing, which speeds up sample clean-up. Image courtesy of Tecan.

References

1. Du, S; Sarver; JG; Trabbic, CJ; et al.6-MOMIPP, a novel brain-penetrant anti-mitotic indolyl-chalcone, inhibits glioblastoma growth and viability. Cancer Chemother. Pharmacol. 2019. 83(2):237–254. [PMID: 30426158]

2. Chi, Q; Wang, L; Xie, D; et al. Characterization of in vitro metabolism of focal adhesion kinase inhibitors by LC/MS/MS. J. Pharm. Biomed. Anal. 2019. 168:163–173. [PMID: 30807921]

3. Caldwell, GW; Leo, GC. Can untargeted metabolomics be utilized in drug discovery/development? Curr. Top. Med. Chem. 2017. 17(24):2716–2739. [PMID: 28685690]