_1.jpg?ext=.jpg)
QuEChERS: Beyond Convention - Part 2
In this second of a four-part series, Smithers Study Director, Sidney Bluemink, discusses the method and application of QuEChERS.
QuEChERS: Beyond Convention - Part 1
.jpg?ext=.jpg)
In this first article of a four-part series, Smithers Study Director Sidney Bluemink discusses the wide-ranging application of QuEChERS and its variants.
The term QuEChERS was first coined in 2002, and subsequently published in 2003, as an acronym of Quick, Easy, Cheap, Effective, Rugged, and Safe and describes a method of extracting multiple analytes from biological samples (1). While the scientific concepts that underpin the technique were not particularly new, the success of QuEChERS came from combining these into one relatively simple sample analysis approach coupled with improvements in instrumentation.
QuEChERS has been embraced by the residue monitoring industry, since fast and reliable high-throughput analysis is of paramount importance based on the need to monitor the levels of, or contamination by, pesticides in food commodities. QuEChERS methods (there are multiple adaptations) can accommodate multiple compounds in one analysis making them a suitable extraction technique for multiresidue methods (MRMs). Before the advent of QuEChERS, MRMs were labor intensive, complex, time-consuming, and used a significant quantity of reagents, and as such, were expensive, error prone, and environmentally problematic.
Despite its benefits, QuEChERS is not always seen as a go-to methodology outside of residue analysis. The throughput benefits of QuEChERS are not always required, notably for those studies conducted in a research setting. This series will explore the application of QuEChERS beyond residue analysis and offer discussion on the pros and cons in each case, and review some of the available variants and their applications.
Extraction and analytical principles
The history and detailed theory of QuEChERS is described in detail elsewhere (2). The process of extraction is to separate analyte from the matrix followed by clean-up of potentially interfering matrix components to allow accurate and precise quantification. In simplest terms the goal is to enhance the signal (analyte) to noise (matrix) ratio (S/N) by removing enough of the matrix. Sufficient matrix removal is of key importance since it is not always necessary in terms of time and resources to over optimize a method when it is not needed.
A sufficiently precise and accurate method (extraction + instrument) where there is a high degree of confidence that the correct analyte(s) are being quantitated and that matrix components are not introducing bias can be said to be suitably selective. The selectivity of a method refers to the extent to which it can determine analyte(s) in a complex mixture without interference from other components in the mixture (3).
QuEChERS at Smithers
Smithers labs perform a broad array of methods that require sample extraction and analysis. The number of samples to analyze in each study can vary greatly from a handful to thousands. QuEChERS methods prove to be especially valuable tools when there are many samples to analyse. Crop residue packages routinely consist of several hundreds of samples, with field dissipation and livestock feeding studies having a significant but lower sample load. Other tests where QuEChERS has been employed at Smithers include ecotoxicology studies such as fish early lifecycle, fish bioconcentration, and field pollinator studies.Metabolism and Triggered MOR Studies
Due to the low sample number, and limited requirement for extensive sample clean-up, QuEChERS is not usually considered for metabolism studies since there are limited economic or analytical benefits. Due to the requirement for total radioactive recovery (TTR), tissue samples are extensively extracted with various solvents and techniques so that every significant radioactive component (parent + metabolites) is identified. The extraction must consider the efficiency of the solvent and the energy that can be delivered to the sample without destroying it. Vigorous shaking or grinding with (e.g. a Geno/Grinder), Ultra-Turrax® blending, microwave-assisted extraction (MAE) and accelerated solvent extraction (ASE) are additional tools employed to enhance the energy delivered and therefore the extraction efficiency in metabolism studies.
Where QuEChERS is beneficial is when metabolism study results trigger further magnitude of residue (MOR) non-radiolabeled studies. Such studies can generate high numbers of samples and involve independent laboratory validations to facilitate use as a post-approval monitoring method. Care must be taken to ensure that analyte levels are not underestimated through incomplete extraction, and typically, regulators require that the MOR samples undergo the same extensive extraction regime as the metabolism samples. Such extensive extractions are impractical at the expanded scale of MOR studies. Many of the metabolites identified in the metabolism studies will not form part of the residue definition for the MOR studies due to their levels being below the required thresholds. Due to these potentially reduced identification requirements, the MOR studies might not require such extensive extraction.
To overcome this apparent contradiction (same method of extraction used as the metabolism study, while being not necessarily required or scalable), radio- or cross-validations using a QuEChERS technique can be performed. QuEChERS extraction followed by quantitation is compared to a selection of incurred tissue samples where the concentrations of the significant components are known. If the results are comparable the QuEChERS technique is appropriate to analyze the incurred samples from the MOR study.
If the initial cross-validation is unsuccessful using a QuEChERS technique, the approach can be modified, while retaining the elements of time and cost effectiveness. For example, if it is determined that sample extraction needs to be more extensive than the standard QuEChERS, higher energy extraction can be carried out before the salting out step. An aliquot of the extract would be manipulated to have the right composition of organic solvent and water so that partitioning would occur after the salt mixture is added for salting out. At that point a normal QuEChERS workflow would continue. If planned and implemented correctly, this considerably reduces the effort and cost expended on these types of studies.
Residue Chemistry Validation Studies
With reference to development of the correct extraction conditions as referenced above, Smithers scientists have employed QuEChERS approaches to validate pesticide methods in a variety of matrices such as animal tissues/blood, soils, and plant commodities. While these are usually smaller-scale studies across multiple matrices, the use of QuEChERS does provide a standardized approach to analysis across a variety of studies, and therefore benefits in terms of laboratory standardization for method development.
QuEChERS in Terrestrial and Aquatic Ecotoxicology Studies
.jpg)
Ecotoxicology sample analysis presents probably some of the most varied challenges in terms of the combination of analyte and matrix. Diverse analytes can be pesticides, biocides, industrial chemicals, human/veterinary pharmaceuticals, and Unknown or Variable composition, Complex reaction products or Biological materials (UVCBs). Matrices include various water-based media, sediment, soils, numerous tissues, honey, royal jelly, and plant commodities. Many of the studies are of small scale in terms of sample analysis requirements, so QuEChERS may not immediately present an economic or scientific benefit to a single study, however, a uniform approach employed across the various analytes and matrices can still bring long-term value based on a standard workflow for method development and application.There are some ecotoxicology studies with a significant sample analysis requirement: higher-tier, long-term fish and amphibian tests, pollinator field and semi-field studies and non-radiolabeled fish bioconcentration studies.
Even using QuEChERS protocols for water analysis is practical in some cases. Water is commonly analyzed by liquid/liquid partitioning (LLP), solid phase extraction, direct injection and dilution before injection (dilute and shoot). In cases where liquid/liquid partitioning is required a QuEChERS adaptation can be made where the water to be analyzed is mixed with the QuEChERS salt mixture and acetonitrile in the right proportions for the water and acetonitrile to separate. This is actually a scaled down version of a common LLP technique called “salting out” (a hydrophobic analyte’s partition into the acetonitrile phase by eliminating the water) that has been used for many decades before QuEChERS was first described. If the matrix load in the water sample is particularly high, a further dSPE step could clean up the sample before analysis.
Another example of adapting QuEChERS methodologies at Smithers has been in the regular validation and analysis of bee diet samples during terrestrial ecotoxicology studies. The adapted workflow consists of shaking royal jelly (or sucrose solution) with a mix of water and acetonitrile. A QuEChERS salt mixture is then added, and an aliquot of the upper acetonitrile phase is subjected to dSPE before LC-MS/MS analysis.
In part 2 of the series, Sidney will discuss the core QuEChERS approach and the benefits and drawbacks of this powerful methodology.
.jpg)
Author:Sidney Bluemink
Study Director
Harrogate, UK
References:
1. M. Anastassiades, S.J. Lehotay, D. Štajnbaher, and F.J. Schenck, J. AOAC Int. 86, 412–431 (2003)
2. QuEChERS: Home (www.quechers.eu)
3. The IUPAC Compendium of Chemical Terminology (https://goldbook.iupac.org)
.jpg?ext=.jpg)
.jpg?ext=.jpg)
-(644-×-350-px).jpg?ext=.jpg)
.jpg?ext=.jpg)