Technology Forum: Sample Prep

This month, Chromatography Online's Technology Forum looks at the topic of Sample Prep and the trends and issues surrounding it. Joining us for this discussion is Michael Freeney of Agilent, Tom Hall of Horizon, Bill Hudson of Varian, Kelly Johnson of Zirchrom, and Joan Stevens of Gilson.

Sample preparation is a major challenge in the development and application of an analytical method. It is the most time-consuming and error prone step. LCGC columnist Ron Majors has stated, "Many of the currently used sample preparation techniques are much slower and have created a bottleneck which slows down overall productivity. Unfortunately, automation of some of these sample preparation techniques has not kept up with the analysis, nor is even integrated."

Some exciting trends in sample preparation include high-throughput techniques, automation, solid phase microextraction, molecular imprinted polymers, high abundance protein depletion sample preparation columns, selective phases for environmental contaminants, chiral separations, dispersive SPE techniques, positive pressure SPE, increasingly smaller sample sizes requirements, and environmentally friendly techniques.Which three trends, technologies, or techniques are the most important? Why are these three developments the most beneficial to researchers?

Freeney: Solid phase extraction, in all of its forms (including high abundance protein depletion, dispersive SPE, positive pressure SPE, and SPME), is the only sample prep technology that has both a great deal of future potential for automation as well as wide spread current acceptance. New chemical phases will continue to be developed for specialized analyses (such as protein depletion phases) and automation will continue to advance so that method development and routine use becomes faster and less chemist intensive.

A trend toward discrete automation at the point of analysis is occurring. Systems such as the CTC/PAL auto-sampler allow for simple automation at the point of analysis. This trend is important because it signals the beginning of a new way to look at sample preparation automation. Instead of trying to automate an entire sample preparation process, only the last common steps of analysis are automated. This greatly simplifies the automation process while decreasing errors associated with simple repetitive tasks (such as diluting to volume and internal standard addition). A greater degree of automation will be available to the researcher without the investment in time and effort in a classical (robotic) automated system. The benefit to the chemist is more automation at a lower initial investment (in both time and capital equipment).

A third trend is for less sample preparation and more technology on the analysis. The increasing use of MS/MS for detection is only increasing the drive toward less sample preparation and placing increasing pressure on injection systems. Less sample preparation without regard to the increased amount of non-volatiles introduced to the system very quickly leads to disaster in the form of increased system maintenance. Various vendors offer systems that include "back flush" and large volume injection that allow for lower detection limits along with the promise of less instrument maintenance. These instrumental advances may not remove the need for sample preparation, but they can decrease the amount of sample preparation needed drastically.

Hall: Sample preparation techniques have increasingly become a critical issue for many laboratories today. For example, contract labs have large numbers of samples that require analysis everyday for the lab to remain profitable, so the shortening of the time required for the overall analysis is key. Other labs may have issues with spikes in sample throughput, such as a potential Homeland Security threat, so it is imperative that they are able to rely on effective and efficient sample prep techniques, to ensure reliable and consistent identification and quantification of analytes of interest in the samples.

In an attempt to solve these, and other similar problems, many labs increased their budget and added more chromatographic instruments and technicians - but discovered that sample throughput did not increase, nor did the work get done quickly enough. Adding more technicians also lead to several other potential issues, such as inconsistent results from technician to technician that are technique sensitive. Lab managers realized that what they needed was samples prepared at a much faster rate, with consistent results; to keep up with the latest advances in automated chromatographic instrumentation.

Enter - automated sample preparation - a robust, new trend that has starting replacing the older, manual, and potentially error prone techniques. Environmental labs and others are moving to automated SPE, extract drying and concentration to obtain the highest throughput, and most consistent results, at the lowest cost. These systems are also considered "green solutions" as they use and emit less solvent, thus helping to protect our environment.

Once methods are developed and saved, they can be rerun by any technician in the laboratory, with minimal knowledge of the instrumentation, reducing the need for training and operator variances in technique.

There are now automated SPE systems with web-based control software for methods development and graphical viewing of the status of the sample extraction which allows researchers to develop, validate and implement methods throughout their organization faster.

Hudson: When developing an analytical method, analysts want fast, trouble free, generic techniques. The three most important trends are improvements in high throughput automation, method robustness and the removal of matrix interferences. Automated analysis decreases sample preparation labor, allowing the researcher to generate more information for less money. Method robustness equates to simple and generic method development and is critical to streamlining the analytical setup. The analyst must be able to generate methods for new compounds quickly, in order to maximize laboratory efficiency. The removal of matrix interferences prevents instrument downtime and improves data accuracy, reproducibility and sensitivity.

High throughput automation fulfills the desire for faster sample processing with minimal operator interaction. 96 well plates have been very successful but still require intermittent user intervention. New OMIX SPE pipette tips for robots have been introduced to eliminate this user intervention. The tips are high flow so no sample clogging occurs and they eliminate the need for a vacuum manifold. In an automated setup, the user can walk away from the SPE process; there is no need to check well flows and manually turn the vacuum on or off.

Method robustness is decreased by flow inconsistencies and sample blockages which lead to irreproducible results and loss of sample. A robust method needs to include a generic sorbent with high flow capabilities. The new generation of Plexa SPE products have been developed with a much narrower particle size distribution to eliminate sample clogging while offering the versatility of a polymeric sorbent. More consistent particles result in higher, more reproducible flow, reducing downtime and user intervention. Removal of matrix components that cause ion suppression is also critical to generating quality data. Protein elimination is one aspect of matrix removal, but phospholipid removal has also received a lot of attention lately. The Plexa products have been designed with a specially engineered pore structure and surface chemistry to more effectively remove these interferences. These cleaner extracts improve accuracy and sensitivity by minimizing baseline noise, matrix variability, and ion suppression. These advances help the analyst overcome sample prep bottlenecks, increase laboratory throughput and maintain high quality data.

Johnson: Solid-phase micro-extraction (SPME) has great potential as a general technique because it is very compatible with today's autosampler (needle and vial) technology and is well suited to trace analysis. Highly selective solid-phase enrichment products based on molecular imprinted polymers (MIPS) to extract analyte types of universal interest from interferences should become more important. Improved solid-phase products to remove abundant interfering proteins from biological samples, without removing analytes, should also become generally useful. While short columns or tubes filled with particles have been the workhorse tools, dispersive SPE approaches may prove to be advantageous in that they can be integrated more easily into current GC and HPLC autosampler designs.

Stevens:I think it is difficult to choose three of the above mentioned techniques over the others because each has its own place in sample preparation considering the wide variety of analyte-matrix combinations that analysts encounter. However Molecular Imprinted Polymers (MIPs), Immobilized Liquid Extraction (ILE) - and Dispersive SPE (QuEChERS) are unique and selective sample prep techniques. As stated by R.E. Majors, LCGC Magazine, 1991, 1997 and 2002, "the major source of chromatographic error is sample processing, greater than 30%, with 61% of the analysis process time being spent on sample prep." With this in mind, it is imperative that trends, technologies and techniques must focus on minimizing the error and sample prep time while still maintaining consistent and extremely reproducible results.

Molecular Imprinted Polymers (MIPs) are a polymer-based sorbent that have a predetermined selectivity for a particular analyte or group of analytes. The highly cross-linked polymer is prepared in the presence of a template molecule (analyte of interest or analog of analyte of interest). The functional monomers interact with the template non-covalently prior to or during polymerization. After polymerization using a cross linker, the template is removed through exhaustive wash steps. This process leaves specific cavities or imprints in the polymers that are chemically and sterically complementary to the template, producing selective target recognition essentially mimicking an antibody or receptor. The benefit of MIPs is that it is a highly selective sample prep technique, which results in lower LLOQ (Lower Limit of Quantification) from very difficult matrices and reduced ion-suppression in MS-MS analysis. Since the methodology is robust and rapid there's a substantial savings in time, money and frustration.

Preparation of samples (plasma, serum, urine, semi-volatile in water), prior to GC-MS/ LC-MS analysis has traditionally been accomplished by two techniques: liquid-liquid extraction (LLE) and solid phase extraction (SPE). Each technique involves laborious procedures and LLE includes steps that are difficult to automate as well as both are plagued by complications and therefore poorly suited for high throughput sample prep.

Immobilized liquid extraction- (ILE) separations are fundamentally very similar to traditional liquid-liquid extractions. However, ILE extractions utilize a thin layer of polymer rather than an organic solvent (as in LLE), to extract the compound from the sample. When an aqueous sample is directly exposed to the immobilized liquid polymer, compounds partition between the sample and polymer based on their affinity for each, at a predictable rate. The partitioning process is accelerated by agitating the ILE well plate with a vortexer, sonicator or other appropriate method. Proper agitation disrupts a boundary layer which forms at the sample-polymer interface, a consequence which may alternatively be accomplished by repeatedly dispensing and aspirating a sample in to and out of each well. Analytes which partition into the polymer are "back-extracted" or desorbed into a small amount of LC-GC solvent for analysis.

ILE well plates are immune to clogging, formation of emulsions and require neither vacuum/pressure. Derivatizing reagents has no adverse effect on the polymer; therefore back-extraction and derivatization may be performed simultaneously. Little to no human attention or interaction is required to use ILE well plates, especially on a well-to-well basis; hence ILE well plates are a very practical and efficient method for high throughput bioanalytical applications.

Dispersive SPE or QuEChERS (quick, easy, cheap, effective, rugged and safe) method is growing in popularity around the world as a method of choice for food testing. QuEChERS ("catchers") method is based on work done and published by the USDA Eastern Regional Research Center, Anastassiades, et. al. Researchers were looking for a simple, effective and inexpensive way to extract and clean pesticide residues from the numerous varied sample matrices with which they routinely worked. The Modified Luke Extraction Method was the standard, which is highly effective and rugged, although labor and glassware intensive and lead to high cost per sample. SPE has also been effective, but the complexity of the matrices required various cartridges and packing media in order to remove the various classes of interferences and leads to increased cost and complexity to the sample prep process. This new method must be able to remove sugars, lipids, organic acids, sterols, proteins, pigments and excess water, any of which could be present, while still being easy to use and inexpensive.