30 February / March 2017
A Modified QuEChERS Approach for the Extraction of Common Prescription and Illicit Drugs from Liver Prior to LC-MS/MS Analysis
by Tina Fanning and Jody Searfoss
United Chemical Technologies, 2731 Bartram Rd., Bristol, PA 19044. USA
tfanning@unitedchem.com
Traditional sample extraction methods tend to focus on a small number of compounds or a single drug class to achieve optimal results. Unfortunately, with enhanced specificity comes an increase in overall sample preparation time and cost. The high demands placed on toxicology laboratories to produce accurate results quickly has fuelled a transition towards universal sample preparation techniques. These broad approaches are fast and efficient as they are able to encompass more analytes and drug classes within a given panel; however, since many of these methods are not as selective, analysts may be forced to compromise on analyte recovery and sample cleanliness.
Preliminary studies have shown that the QuEChERS technique should be considered as a reliable alternative when extracting multiple drug classes from challenging biological matrices. The project described here, explores a modified universal QuEChERS approach for sample clean-up in post-mortem liver, coupled with LC- MS detection, for the analysis of several common prescription and illicit drugs.
Introduction
The sensitivity and resolving power of analytical instrumentation being used in the forensic toxicology community has vastly evolved in the past decade. Once samples are received, analysts are tasked with identifying and quantitating a wide variety of drugs and toxicants from an assortment of matrix types. Even with improvements to instrumentation, this is no easy feat. The sample preparation technique on these given matrices largely dictates the quality and validity of the final results. Many new instruments feature enhanced sensitivity and regulatory reporting limits as a result are being lowered. The number of compounds needing to be identified and quantitated is also steadily on the rise. Unfortunately, if samples are not handled appropriately, it can curtail the ability for analysts to achieve reliable results, often times leading to a compromise on the sample preparation approach in order to save time and money.
One possible solution toxicology laboratories are beginning to explore that provides an optimum balance between producing reliable results and saving time
and cost on analysis is the QuEChERS approach. Crossing over from the food safety industry, QuEChERS (pronounced ‘catchers’) is an acronym for Quick, Easy, Cheap, Effective, Rugged and Safe. This technique was originally developed for multi-residue pesticide analysis in fruits and vegetables in 2003 by Anastassiadies et al [1,2]. When QuEChERS were first developed the typical procedure for sample clean-up in the food safety industry was time consuming and required large amounts of solvents.
With the QuEChERS approach, these arduous methods are reduced to three simple steps:
1. A liquid micro-extraction 2. Solid phase clean-up 3. GC-MS or LC-MS analysis.
Initially, QuEChERS salts (magnesium sulphate (MgSO4
) and sodium chloride
(NaCl)) are added to an aqueous based matrix that has been mixed with acetonitrile. The use of acetonitrile makes final extracts amenable to both GC-MS and LC-MS analysis. The magnesium sulphate serves to bind large amounts of water while the sodium chloride increases the ionic strength of the aqueous phase in addition to inducing phase separation. Following this step, dispersive SPE (dSPE) can then be executed to provide additional matrix removal if this is required. This secondary clean-up also serves to eliminate any residual water that remains from step one and also allows for extraction salts to diffuse homogenously throughout the entire sample. The end result is a more thorough, condensed overall extraction when compared to traditional SPE protocols [3].
Since its conception, this technique has been utilised for sample clean up on a wide variety of matrices to include animal-based products (meat, fish, kidney, chicken, milk, honey), cereals and grains, and other food produce market sectors (wines, juices, fruit and vegetables) [2,3]. The QuEChERS method has not only proven to be simple to perform, but also is rugged enough to withstand any necessary modifications that make it amenable to complex matrices such as those commonly encountered in a forensic toxicological setting.
Various types of specimens are acquired during an autopsy for toxicological analysis. While blood and urine are by far the more desirable biological fluids of choice for analysts to work with, other matrices are often tested to either substantiate the concentrations found in blood and urine or in instances of limited sample [4]. Liver is the primary alternative tissue used for toxicological analysis based on the biological role it plays in the metabolism of drugs and toxicants in the body. Drugs become concentrated in this vital organ and can be found even when there are no detectable quantities present in the blood. This additional information becomes very critical when trying to determine the cause of death.
While the benefits for analysing liver are clear, the one major drawback is the amount of sample preparation needed in order to get specimens ready for analysis. After liver samples are homogenised, they must undergo further extraction methods such as solid phase or liquid-liquid extractions. While neither of these techniques are particularly difficult, they do have their draw
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