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From: Food Quality & Safety magazine, August/September 2013

Sample Preparation Selection

by Sueki Leung and Allen Misa

In The Lab

With the increasing awareness of food safety in both developed and developing countries, analysis of a variety of imported and exported commodities is a priority concern for the national competent authorities. Sample preparation of foods such as vegetables, fruits, dairy, and meats followed by downstream high-pressure liquid chromatography (HPLC), liquid chromatography with tandem mass spectrometry (LC/MS/MS), or gas chromatography with mass spectrometry (GC/MS) analysis is a practice that helps ensure the safety of consumers. Sample preparation is a huge challenge in this analytical process for two primary reasons. First, typical food testing assays can include many analytes with widely varying chemical properties, and second, sample matrices are complex and often contain compounds that can interfere with analysis. For instance, the avocado matrix is rich in lipids that cause ion suppression in mass spectrometry (MS) analysis, leading to inaccurate results when tested for a particular set of analytes.

All analysis begins with sample preparation, whether it's a simple dilution or filtration or uses more targeted techniques such as LLE (liquid-liquid extraction), QuEChERS (quick easy, cheap, effective, rugged, and safe), or SPE (solid phase extraction). Because accurate analysis is required in food safety testing, sample preparation plays an integral role and directly affects downstream analytical results.

Sample preparation helps ensure that accurate and reproducible results are produced across a wide variety of food sample matrices. As regulations change and become more strict, analysts are challenged to develop robust analytical methods that reach even lower detection and quantitation levels. More selective sample preparation methods are employed in some cases; while in other situations, a less specific technique focusing on simple matrix removal may be more effective.

Matrix interferences such as lipids, proteins, and carbohydrates have become a limiting factor because they can cause ion suppression or enhancement, making it difficult to accurately identify and quantify the target analytes. Further, without adequate cleanup, these troublesome components can damage or shorten the lifetime of laboratory instrumentation. Thus, sample preparation for matrix interference removal in food samples is extremely important in order to achieve proper performance requirements and to improve the “shelf life” of instrument systems.

Selecting the most effective sample preparation technique to achieve your overall analysis goals saves time that would otherwise be spent on trying different techniques based on trial and error.

Sample preparation can also represent a major bottleneck in the analytical laboratory. It’s estimated that the sample preparation step can make up 60 to 70 percent of the total time required for analysis. Estimates also show that 30 percent of analytical errors originate from the sample preparation step. Most food testing labs follow approved official/standard methods that often specify a validated sample preparation procedure. In some cases, when special needs must be met, analysts are granted flexibility to deviate from the official method and select a more appropriate sample preparation technique.

Straightforward, non-specific sample preparation techniques such as weighing, dilution, or filtration typically work well and can adequately achieve the goals of sample preparation for simple sample matrices.

For more complex and dirty sample matrices, more intricate and selective extraction/cleanup methods such as LLE, QuEChERS, and SPE are required to transform samples into compatible formats for GC/MS and LC/MS/MS analyses. (See Figure 1.)

Each approach has its own benefits, drawbacks, and specific uses and no one approach is superior to the other. However, choosing the correct approach can significantly help in analysis goals.

Figure 1
Figure 1

LLE

Probably the simplest and most typical sample preparation approach for a variety of sample matrices is LLE. In LLE, homogenized sample is added to a biphasic system containing an aqueous phase and an organic phase. The target analytes will partition into either layer, which can then be isolated for further analysis.

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.

The flexibility and strength of SPE comes from the users’ ability to choose the sorbent that selectively interacts with the analyte(s) of interest or with the matrix interferences that could affect the recovery. Thus, SPE cartridges can also be used in a nonretentive approach as a “chemical filter” that removes interference from the sample while target analytes pass through the sorbent and are collected for further analysis. Unwanted matrix interferences will remain in the SPE cartridge while the analytes of interest are collected.

SPE is performed by using a tube filled/packed with a chemically derivitized sorbent. By varying the chemical nature of the sorbent and the buffer conditions used during the loading, washing, and elution stages, a method can be developed that can be very selective to clean and isolate the target analytes from complex sample matrices.

The steps for SPE include:

  • Pretreat sample (via LLE, homogenization, buffering, etc.);
  • Choose appropriate SPE sorbent and protocol;
  • Condition sorbent to prepare for interaction with sample;
  • Load pretreated sample onto SPE sorbent (target analyte will be retained on sorbent);
  • Wash sorbent to remove unwanted interferences that are not retained on the sorbent;
  • Elute target analytes (using a combination of organic strengths and buffers); and
  • Analyze clean eluent by GC or HPLC.

Although SPE is highly selective, produces high recoveries, and provides repeatable results, there are a few drawbacks that prevent labs from implementing the technique. SPE requires method development, special equipment, and is much more expensive than its alternatives due to sorbent packing and media costs. When the analysis of complex sample is required, SPE would be the ideal choice of sample preparation technique because it reliably and repeatedly provides high recovery.

Real-World Instances

The following are some real-world examples on how each approach was chosen as the most effective technique.

Example 1: Multiresidue Pesticide Analysis in Spinach using QuEChERS AOAC Kits. With the strong presence of pesticides in the food cycle, the purpose of this analysis is to detect concentrations of pesticides below the maximum residue limits because global legislations are quickly becoming more concerned. It is imperative that sensitive and efficient analytical techniques are used to detect low levels of the variety of pesticides.

The primary challenge in this analysis is to eliminate the naturally occurring pigments, fatty acids, nutrients, and fats that are present in spinach samples in order to achieve lower limits of detections (LOD) of pesticides. Although a traditional technique such as LLE can be used, it employs the use of hazardous solvents and cannot remove all matrix interferences that can prevent reaching the desired LOD.

Sample preparation helps ensure that accurate and reproducible results are produced across a wide variety of food sample matrices.

roQ QuEChERS Kits (Phenomenex) were employed. The combination of buffering salts, magnesium sulfate, and organic solvent induced delivered clean separation and extracted all pesticides, while PSA and GCB dSPE sorbents were used to remove the remaining matrix interferences

(Figure 2 and Figure 3). Because the AOAC 2007.01 method had already been validated and established, this procedure was the best alternative to achieve a combination of low limits of detection, easy and quick processing, and analysis of a wide range of pesticides. SPE would also be an acceptable cleanup option for this work, however, because pesticides are of varying polarities, SPE method development would have been quite intensive and may not have produced the high recoveries of all of the varying pesticides that can be achieved with QuEChERS, which provides a wide analyte screening.

Spinach extracts after liquid partitioning
Figure 2. Spinach extracts after liquid partitioning step with 1 percent acetic acid in ­acetonitrile and magnesium sulfate. The organic phase was heavily pigmented in dark green.
Spinach extracts after dSPE cleanup
Figure 3. Spinach extracts after dSPE cleanup. GCB removed a majority of the pigment from the sample matrix and the extracts were clear with a light green tint.

Example 2: Sulfonamides Extraction from Honey Using Strata-X-C Polymeric SPE Sorbent. Two bacterial species, Paenibacillus larvae and Melissococcus pluton are known to cause American and European Foulbrood from honey bees. Honey is a widely used sweetener and is highly produced and tested. Although antibacterial agents such as sulfa drugs (sulfonamides) are effective in controlling their growth, high residues of these antibacterial agents found in consumers’ honey have become a huge concern. A reproducible and highly selective method is required for their analysis.

A strong cation-exchange polymeric SPE sorbent, Strata-X-C (Phenomenex) was chosen to perform sample cleanup due to its specificity to extract sulfonamides from honey. Honey is saturated with a variety of classes of compounds including carbohydrates, aliphatic acids, amino acids, proteins, and minerals. Simpler extraction methods such as LLE or QuEChERS are not specific enough for the extraction and concentration of sulfonamides without co-extracting matrix interferences.

Using SPE allowed target of sulfonamides based on their basic properties, which formed interactions with the strong cation-exchange sorbent. A strong organic wash could then be used to rinse off all interferences from the cartridge prior to elution, producing the cleanest and most concentrated extract of the target drug residues.

Sample preparation is an integral step in any analysis. The lack of proper sample preparation in food testing can lead to instrumentation and analytical challenges. It also adds substantial effort and time to the complete analysis. Knowing which sample preparation technique to use for the goals at hand is quite beneficial in achieving the desired outcome. In foods, LLE, QuEChERS, and SPE are the three most commonly used approaches to sample preparation. No one approach is better than another; rather each approach has its own strengths in achieving the desired outcome of the analysis.


Leung holds a Masters of Science degree in Organic Chemistry from the University of California and is a sample preparation technical specialist at Phenomenex. Reach her at suekil@phenomenex.com. Misa holds a Masters of Science degree in Health Care Administration from California State University along with a Microbiology degree from California State Polytechnic University. He is a sample preparation and food industry brand specialist for Phenomenex and can be reached at allenm@phenomenex.com.

 

References Furnished Upon Request

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