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backs. Liquid-liquid extraction methods have the ability to extract several compounds at once, but they can be time consuming and usually require greater quantities of solvents compared to other methods. This increases the overall cost per sample for laboratories. Solid phase extraction methods tend to be quicker and more cost effective, however, if samples are not homogenised properly, column clogging and inconsistent flow rates can lead to inconsistent results for analysts.
Experimental Sample Preparation
Blank bovine liver samples were homogenised using a Robot-Coupe Blixer ® at a 1:4 ratio by weight with deionised water. Blank liver homogenate (2 mL) was fortified with appropriate amounts of working standards prior to being added to 15 mL centrifuge tubes containing QuEChERS salts (800 mg magnesium sulphate (MgSO4
) and
200 mg sodium chloride (NaCl)) and 2 mL of acetonitrile containing 5% ammonium hydroxide. Samples were briefly vortexed to break up any salt agglomerates prior to shaking for 5 minutes at a rate of 1000 strokes/minute using a Spex Geno/ Grinder®. After shaking, samples were placed into a centrifuge and spun for 10 minutes at a speed of 3000 rcf. Further sample cleanup was performed by adding 1 mL of the centrifuged supernatant to 2 mL micro-centrifuges tubes containing 150 mg of MgSO4
and 50 mg, silica based C18
endcapped sorbent (UCT LLC, Bristol PA, USA. Part Number CUMC182CT). Samples were vortexed at a rate of 100 strokes/ minute for 1 minute, then placed into a centrifuge and spun for 5 minutes at a rate of 3000 rcf. A 500 µL aliquot of the final extract was then dried to completion for concentration purposes and reconstituted in 100 µl of the appropriate mobile phase for instrumental analysis; however, a simple dilution of the final extract with water may be performed dependant on the sensitivity of the analytical instrument being used.
Chromatographic Analysis
Analysis was performed using an Agilent 1200 HPLC system combined with an ABSciex 4000 Q Trap. UCT’s Selectra® DA 50 mm x 2.1 mm, 3 µm column was used for separation. The column temperature was kept at 40° C and the injection volume was 10 µL. The mobile phases consisted of solvent A: water containing 0.1% formic acid and solvent B: methanol containing 0.1% formic acid with a flow rate of 0.4 mL/ min. Analytes were chromatographically separated using the following gradient: A 0.5 minute initial hold at 15% solvent B
followed by a linear ramp to 95% solvent B in 12 min. The gradient was held at 95% B for 1 minute after which it was lowered to 15% B in 0.1 min and held at this level for 4 minutes. The entire cycle time was 17 minutes (Figure 1).
The MS was operated in positive ESI mode, analyte MRM transitions can be found in Table 1. Data was acquired and analysed using Analyst Software (version 1.5.2).
Results and Discussion
Excellent recoveries were achieved for the range of analytes included in this study. Recoveries were evaluated by fortifying samples at two varying concentrations. On average, the recovery for samples spiked at 75 ng/g was 81% and for samples spiked
extraction. It is important to account for any residual matrix components which may lead to analyte enhancement or suppression upon analysis.
As laboratories all over the world explore the uses of QuEChERS, several modifications from the traditional food safety approach will need to be made to improve analyte recovery when being applied to a forensic toxicological setting. One of the obvious changes that needs to be accounted for is the adjustment of salt and solvent amounts due to smaller sample sizes. Food testing laboratories are accustomed to working with large volumes (the original method started with 10 g of starting material), while in the forensic toxicology realm, sample amount is often limited and once it is consumed, there is rarely, if ever a chance to obtain more.
Figure 1
at 300 ng/g it was 83%. Recoveries were calculated by dividing the chromatographic peak area of samples spiked prior to extraction by the peak area produced by samples that were spiked into a pre- extracted blank matrix. A full list of recoveries can be found in Table 2. The responses for the representative compounds were linear with R2 values ranging from 0.93 to 0.99 over a concentration range of 0-500 ng/g, with the lowest spiked calibrator being 25 ng/g for all analytes. Internal standards were not used in this study; however, implementing isotopically labelled internal standards always aid in compensating for any analyte loss and remaining matrix components that were unable to be removed via the
Table 1 The 4:1 MgSO4 : NaCl salt blend ratio was
maintained, however since the amount of starting sample was reduced five-fold, the salt ratio was reduced from 4 g MgSO4
: 1 g
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