In recent years, mass spectrometry (MS) instrumentation has advanced tremendously, placing considerable demands on analytical laboratories and generating a need for high-throughput sample preparation processes. However, sample preparation methodologies have progressed much more slowly, and have largely remained manual activities, creating a major bottleneck. Time-consuming manual sample processing has not only resulted in a lower throughput for analytical laboratories—accompanied by concerns about data quality—but also restricted the uptake of MS innovation in the life science industry in general. End-to-end process automation for even the most challenging sample preparation protocols is the need of the hour, relieving scientists from the tedium of manual processing and accelerating the further adoption of MS technology in the life science industry.
Why Use Automation?
Until comparatively recently, MS was very much a research tool rather than a mainstream technology. Today, MS is widely used across many industry segments, including clinical and toxicology, pharma, food and environmental, and academia. Manual sample preparation has emerged as a bottleneck for each of these segments despite the differing applications of interest, and laboratories are increasingly turning to automated MS protocols to improve throughput and turnaround times, as well as to reduce the number of false-positive and -negative results. Automated sample preparation provides much needed high throughput while still meeting laboratories’ stringent quality demands.
MS is broadly used for two types of analysis: proteomics and the currently more dominant small molecules market. A variety of different techniques—solid phase and liquid-liquid extraction, enzymatic hydrolysis, protein precipitation, "dilute and shoot" and protein purification—are available for MS sample preparation, and are selected according to the application, sample matrix, and the analyte under investigation. A typical drugs of abuse screen may use a combination of these techniques, requiring several pipetting and sample transfer steps, a process which is both tedious and time consuming when performed manually, and which can easily occupy an entire day. MS analysis and data interpretation often cannot take place until the second day, prolonging sample turnaround times. Manual sample preparation also has the potential for processing errors to be introduced which, without stringent process security, are very difficult to trace. Automated liquid handling holds the key to efficient high-throughput mass spectrometry sample preparation. It offers walkaway operation, releasing skilled analysts to focus on more demanding tasks such as data interpretation, while maximizing sample throughput and improving turnaround times. And, with sample tracking and barcode reading capability, full traceability is assured.
Small Molecule Sample Preparation
Automation of small molecule sample preparation processes on a liquid handling workstation offers many advantages, allowing a diverse range of analytes and laboratory protocols to be accommodated, increasing versatility and analytical flexibility. Typically, throughput is enhanced considerably, with one liquid handling system able to prepare sufficient samples for up to 10 mass spectrometry systems on a daily basis. Additionally, the adoption of built-in security features, such as sample tracking, satisfies even the most stringent quality requirements.
Laboratories working in the small molecule field generally analyze large numbers of blood, urine, plasma, and serum samples, with the choice of sample preparation method dependent on the matrix, analyte of interest, and the sensitivity required. For example, solid phase extraction (SPE) is a popular means of purifying and concentrating samples prior to liquid chromatography-mass spectrometry (LC-MS) analysis. However, when performed manually, it is cumbersome, time consuming, and prone to errors, resulting in reduced productivity. Compared to manual SPE, automation offers significant benefits, enabling parallel extractions to be performed in 96-well plates, increasing speed of preparation and sample throughput for maximal productivity.
Liquid-liquid extraction (LLE) is another popular purification technique which, although effective, can show variability in results according to the operator when performed manually. It also exposes users to large volumes of organic solvents. Automated LLE provides fast, robust analyte purification and quantification, increasing operator safety by reducing exposure to organic solvents.
For the analysis of blood, plasma, and serum, a protein precipitation step is generally also necessary to remove the proteins that would otherwise prevent MS detection of the analyte, while some conjugated compounds may exhibit poor ionization efficiencies and require enzymatic hydrolysis prior to analysis; when performed manually, both these techniques involve a great deal of laborious pipetting. Similarly, the straightforward "dilute and shoot" technique—sample dilution and addition of an internal standard—requires numerous serial dilutions, increasing the likelihood of introducing manual errors and inconsistencies. These simple pipetting tasks can be carried out reliably and consistently, irrespective of the operator, using automated liquid handling.
Other crucial considerations are the flexibility and versatility of the workstation; laboratories must plan for the future, anticipating the changing demands of the marketplace. Today’s liquid handling workstations can be designed to perform a single extraction technique in high throughput, or a combination of techniques. This enables laboratories to future-proof their investment by specifying not only the sample preparation method currently in use, but also those likely to be required as new applications are introduced.
Biomarker research and the pharmaceutical industry’s growing interest in biologics have made protein analysis routine in many laboratories, and the bioprocessing market in particular is growing steadily in response to the increasing number of protein-based medicines.
Time to market is crucial, and many companies developing and manufacturing therapeutic agents have discovered the advantages of automation for the miniaturization and parallelization of high-throughput bioprocesses, overcoming the restrictions of limited availability of sample material. Automation of protein digestion maximizes peptide recovery and assay throughput, and a broad range of protein purification systems can also be automated—including chromatography in columns, tips, and microplates—allowing batch processing of samples. With end-to-end process automation, the most cumbersome digestion and/or purification tasks are seamlessly performed, ensuring the correct results are achieved first time, every time. Throughput is improved and analysts are freed to focus on downstream LC-MS analysis, helping to ensure cost-effective development and reduce the time-to-market for new medications.
Sample traceability is vital in any laboratory, but maintaining the chain of custody is very hard to do manually, even with staff dedicated to the task. Without full sample traceability, troubleshooting becomes extremely difficult; a vial may have been placed in the wrong position, or a test may need repeating, and tracing the source of the problem becomes almost impossible. This has huge time and cost implications. Automation allows laboratories to incorporate sample tracking features such as barcode readers and LIMS traceability, simplifying the troubleshooting process and helping to reduce analysis times and costs.
Automation—The Way of the Future
For laboratories performing high-throughput analysis by MS, the advantages of automated sample preparation are enormous. Analytical laboratories typically require maximum throughput without compromising on excellent data quality, and must ensure that the number of false-positive and -negative results and repeat analyses are minimal. Exploiting the power of automation enables laboratories to benefit from full sample traceability, increased throughput, improved turnaround times, and enhanced reproducibility, as well as the virtual elimination of manual errors, resulting in fewer repeat analyses. With end-to-end process automation, scientists are freed from tedious manual sample preparation, leaving them to focus on the crucial task of data interpretation.
Rohit Shroff and Isabel Patocchi-Tenzer are applications specialists at Tecan Schweiz.
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