It’s official: Sample and standards preparation are significant bottlenecks in high-throughput laboratories, particularly those partly or fully automated. Improved instrument sensitivity—and the need for speed and accuracy—have made sample prep a top priority.
Yet discussing sample prep as a monolithic operation is impossible because prep steps, analyses, and matrices are highly variable among industries. Measuring protein levels in energy drinks is not the same as for granola; food safety analyzes for pesticide residues and microbial contamination; still other tests quantify trace nutrients. Matrix removal is a great idea, but even the “matrix” differs for all the tests mentioned.
Sensitivity allows analysts to probe for low-abundance or dilute compounds. This is usually a good thing, but sensitivity also uncovers more contaminants and interferences. As with analytical instruments, users are looking for simpler, faster, solvent-sparing prep methods. “New sorbent chemistries are allowing analysis of a wider range of analytes more rapidly than ever,” says Sueki Leung, technical specialist for sample preparation at Phenomenex (Torrance, CA). Selecting the right prep method can save thousands of dollars per week in solvent and solvent disposal costs and can be “greener” than traditional methods.
The major challenge, according to Leung, is striking a balance between sample cleanup (removing the matrix and contaminants) and target analyte recovery.
Where traditional methods relied on extraction, today’s methods aim more at matrix removal that spares target compounds. Rapid cleanup methods may not produce samples that are quite as clean as those produced by more time-consuming protocols. “Laboratories need to look hard at their overall goal,” Leung adds. “Can you compromise cleanliness for the speed of newer techniques, or will that approach generate samples that do not meet your objectives? Labs may need to rethink their sample preparation methods and adopt ones that take more time.”
Diverse samples, diverse methods
Pesticide testing in foods has been simplified by the QuEChERS (pronounced “ketchers,” a portmanteau for “quick, easy, cheap, effective, rugged, and safe) method. QuEChERS is a dispersive solid-phase extraction method that isolates basic, acidic, and neutral pesticides. Developed at the USDA in 2003 and adopted by AOAC in 2007, QuEChERS greatly improves pesticide analysis by removing interfering species. “Traditionally, pesticides were analyzed individually by concentrating analytes. QuEChERS removes species that interfere with chromatography or suppress ionization in mass spectrometry,” Leung explains.
Several companies sell proprietary QuEChERS extraction kits, many of which contain a capital “Q” in their trademark name. Phenomenex’s roQ QuEChERS kit debuted in October 2012.
Sample prep for biological samples is a topic in and of itself. Think about the diversity of biological analytes: organisms, organs, tissues, molecule types, etc. Proteins, for example, exist over a concentration dynamic range of approximately 1014. For analysis of very low-concentration proteins, removing the matrix must be done with extreme care so as not to deplete the analyte as well.
Assuming that efficient cleanup is possible, lowabundance proteins face other issues between the source and the HPLC system. Proteins are known to stick to a variety of surfaces, including most glass and plastics. Protein adhered to surfaces is forever removed from the usable sample.
“The inherent danger of standard polypropylene tubes is that proteins and nucleic acids stick to surfaces through hydrophobic interactions,” comments Matt Lieber, product manager for centrifugation and sample prep at Eppendorf North America (Hauppauge, NY). Conventionally, analysts swamped dilute protein solutions with bovine serum albumin (BSA) to stabilize the molecule of interest. Another approach is to apply a thin coating of silicone oil during the manufacture of sample tubes.
The first approach is impractical because BSA tends to swamp out signals from the analyte, particularly if it resembles the stabilizing protein. Moreover, silicone is known to separate from surfaces and contaminate samples. Constructed from a proprietary polypropylene, Eppendorf ’s LoBind tubes practically eliminate protein binding to tube surfaces. The company claims maximum protein recovery of 97 percent, which is comparable to the performance of siliconized tubes, without the possibility of silicone oil leaching into the sample.
According to Tony Mamone, senior market manager at Tecan Group (Männedorf, Switzerland), throughput needs are the most common, obvious triggers for automating sample preparation. Then an entirely new world opens up. Customers may not realize it initially, but arguably the greatest benefits to automating are consistency; error reduction; more efficient utilization of solvents and reagents; an audit-worthy paper trail; and the practical elimination of tedious, repetitive work. “Automation greatly reduces the potential for someone who is tired mixing up samples at the end of [a] shift.”
For example, most assays require the use of standards and generation of standard curves. When the end point is a mass spectrum, the standards need to be “right and tight,” Mamone explains. “Questionable results can be questioned in court. If the curves aren’t straight you may have to explain it.” To avoid those uncomfortable situations, technicians will discard standards and start fresh, wasting both time and reagents. “The goal is making processes more reproducible and secure, even if you don’t have a million samples.”
Traceability becomes a huge factor when a lab manager must face a disappointed pharmaceutical customer or an inquisitive attorney. At the basic level, all data generated through sample prep robotics enters a log file. “What you do with that data is up to you,” Mamone tells Lab Manager. “You can ignore it or use built-in reporting capability to generate a report. Or you can pull the data into a LIMS, compile it, and generate a report consisting only of the information you care about. It’s even possible to use tracking functions as a timereporting tool for human resources.”
For additional resources on sample preparation for chromatography, including useful articles and a list of manufacturers, visit www.labmanager.com/sample-prep