40 February / March 2016


Navigating the Vast Array of Sample Preparation Techniques for Biological Samples – Whole Blood


by Matt Brusius, Phenomenex, Torrance, CA USA


Work place drug and pain management testing has historically been performed in urine samples. However, urine is typically more indicative of what was previously introduced into the body as opposed to the more complete and real time indicator that whole blood testing offers which explains its popularity in the world of Forensic Toxicology. Unfortunately, whole blood is significantly more complex and requires thorough sample preparation even prior to ultra-selective cleanup techniques like solid phase extraction (SPE), let alone before analysis by GC-MS/MS or LC-MS/MS.


The appropriate pretreatment of whole blood prior to SPE is vital for accurate quantification. Two challenges that must be overcome are ensuring complete disruption of analyte protein interactions (not normally found in urine) and release into the liquid portion of the blood. This pretreatment usually involves a haemolysis step to release the drugs that may have been taken up by the erythrocytes followed by a subsequent protein precipitation, or other form of sample preparation, which must ensure that analytes do not co-precipitate out of solution. Implementing such a pretreatment for a wide range of chemically diverse analytes can prove challenging as their intrinsic physiochemical properties have a significant impact on what solvents they can be extracted into.


Determining the Most Effective Pretreatment Step


To determine the most effective approach to whole blood testing, an experiment was designed that evaluated different pretreatment options to prepare whole blood for an SPE method with the goal of determining which option(s) resulted in the highest recovery for each analyte class. Since the effectiveness of the sample preparation is determined in part by the chemical properties of the analytes, testing a wide range of forensically relevant compounds (Table 1) is necessary, and there may not be one pretreatment that is best across all classes of compounds. The scope of this study includes mostly basic compounds with the exception of some neutrals like carisoprodol and some benzodiazepines. These compounds range from moderately


hydrophobic (methadone, Log P = 5.01) [4] to relatively polar (benzoylecognine, Log P = -0.59) [4]. However, overall Log D will be more effective at predicting solubility and consequently the recovery for a particular precipitating solvent.


In this method, whole blood pretreatments are broken down into two important steps: haemolysis and protein precipitation (followed by centrifugation).


Haemolysis – osmotic pressure and metal induced protein denaturation


Osmotic breakdown:


In order to reproducibly quantify the total drug and metabolites present it in a blood sample, it is necessary to lyse the red blood cells to account for any drug taken up by the erythrocytes in addition to the surrounding plasma.


Figure 1: Lysed Blood (left) vs Unprepped blood (right) [1].


One of the most popular lysing approaches is done with water, often times referred to as osmotic breakdown. The method is simple and requires a 1:1 dilution of the whole blood with water followed by simple


shaking (vortex or sonication


is preferred). Figure 1 shows a side-by- side comparison of an unprepared blood sample and one that has been lysed via osmotic breakdown. On the left, the whole blood sample that has been lysed is easily


distinguished because the lysed blood cells do not stick to the side of the culture tube.


Inorganic denaturing:


The second pretreatment evaluated was the use of zinc sulphate (ZnSO4


denaturant where 100 µL of 5% ZnSO4 added to 500 µL of whole blood. Zn2+


) as a protein was


binds


to proteins in the blood, forming insoluble metal-protein salt complexes which cause the membrane proteins of the erythrocytes to precipitate, lysing the cell. In addition, as Zn2+


binds to the coordinated amino acids,


protons are displaced, thus decreasing the pH of the sample [5], which can decrease Log D values and improve solubility in acetonitrile. As the red blood cells lyse, the haemoglobin enters the liquid part of the blood causing the bright colour change providing visual confirmation.


Comparing the two methods:


Figure 2 shows the difference between the ZnSO4


haemolysis


and the osmotic breakdown


Figure 2: ZnSO4


Precipitation vs Osmotic Breakdown [1].


product. ZnSO4 seems to be more effective at lysing the red blood cells and thus produces a much brighter red solution, indicating a more effective lysing step [1].


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