Sample Preparation Selection

LLE is cheap, relatively quick to perform, and fairly simple. It also provides short method development times and is easily transferable to other labs because it is a simple approach that requires no special equipment or consumable products. When developing a LLE extraction, a few simple considerations are: In what solvents will the targets most likely solubilize (i.e. what is the log P of my target analytes?); and are the extraction solvents compatible with the analytical approach?

Although LLE is known to be simple, quick, and cheap, the major tradeoff is that LLE is not analyte specific. Co-extraction of interferences with the target compounds is a very common problem, leading to inaccurate results caused by ion suppression/enhancement. Another common problem is that LLE often requires large volumes of hazardous solvents such as petroleum ether, dichloromethane, and other organic solvents, which raise undesired environmental and health concerns. Because two immiscible solvents must be used to create a biphasic system, the choice of solvent is often limited. Additionally, the formation of emulsion from the biphasic system can produce inconsistent data.

Thus an example where LLE would be a preferred option is when a wide range of organic-soluble compounds must be analyzed from a small volume of aqueous-based sample.

QuEChERS

A widely used method, QuEChERS was first introduced in 2002 at the European Pesticide Residues Workshop in Rome. It was developed by Lehotay, et al. to extract and analyze multi-residue pesticides from food samples and was published in the Journal of AOAC in 2003. In 2005, the USDA reported a validation study for 229 analytes of varying polarities. In 2007, QuEChERS was designated as an official AOAC Method 2007.01 for pesticide residues. The main advantage of QuEChERS is its ability to remove a large quantity of unwanted interferences from a large variety of food matrices in a quick, easy, cheap, effective, rugged, and safe process. Since its creation, QuEChERS has been used with a variety of food matrices and is slowly being evaluated for other uses.

The QuEChERS method is broken down into two main steps.

Step 1: Extraction. The purpose of the extraction step is to extract analytes from any given sample matrix by using a combination of solvents, magnesium sulfate (to induce phase separation and LLE partitioning), and buffering salts (to stabilize base sensitive analytes). Analytes of interest will partition into the organic solvent, and physical matrix interferences are eliminated during this extraction step. Sample matrices can be solids, semisolids, small volumes of liquid, or viscous liquids. To summarize, the following events take place during the extraction step:

• Sample is homogenized;
• Sample is transferred to an extraction tube and organic solvent and salts are added, the sample is then shook by hand;
• Extraction tube is centrifuged to pellet homogenate; and
• Top layer of solvent is extracted and is further cleaned up during Step 2.

Step 2: Dispersive Solid Phase Extraction (dSPE). The main purpose of the dSPE step is to remove from the sample undesired chemical matrix interferences such as lipids, organic acids, sugars, and pigments. These chemical matrix interferences are damaging to instrumentation and can lead to inaccurate results. Typically, end-capped C18 (C18E), primary secondary amine (PSA), and graphitized carbon black (GCB) SPE sorbents are used to remove these interferences. To summarize, the following events take place during the dSPE step:

• Solvent extracted from Step 1 is added to a dSPE tube that contains a combination of dSPE sorbents and salts;
• Tube is shaken by hand and centrifuged; and
• Supernatant is ready for analysis by GC and LC/MS/MS.

Although QuEChERS is a quick multi-matrix solution, it still has its drawbacks in covering specific single-class analytes that are difficult to extract or clean up from persistent interferences. In addition, because QuEChERS is mainly a manual process, automation of the procedure is not very effective. Because QuEChERS does make use of SPE sorbents, some method development to determine the best sorbent combinations is required, which can take additional time.

SPE

One of the most selective sample preparation techniques employed in food safety testing, SPE is a technique in which intermolecular interactions between a solid stationary phase and the target analyte results in the removal of contaminant and the concentration of the analyte. SPE addresses the three primary goals of sample preparation including analyte extraction, concentration, and solvent switching. It is used in a wide variety of industries and can be utilized to clean up a multitude of sample matrices and target analytes.