Responding to advances in complementary technologies like GC-MS and LC-MS, researchers from Boston University School of Medicine's Biomedical Forensic Sciences program have authored a study review of current sample preparation techniques and considerations for different sample types that are typically encountered in forensic toxicology cases.
"Researchers have a number of biological samples to choose from when trying to identify what substances may be in the human body, however, it is important that they choose the right one to help answer their research question. As equally important is the choice of how to prepare the sample for analysis,” said corresponding author Sabra Botch-Jones, assistant professor of anatomy and neurobiology at Boston University School of Medicine.
According to the study, published in WIRES Forensic Science, liquid-liquid extractions and solid phase extractions are still the most popular sample preparation techniques. However, other techniques, such as supported liquid extraction, Phospholipid depletion and dilution are also employed on a regular basis in a typical crime laboratory.
Liquid-liquid extractions (LLE) is the standard technique for the analysis of acidic, neutral and alkaline drugs from biological fluids. The method is based on an analyte's chemical properties. For example, a common LLE extraction is protein precipitation, which uses inorganic acids or solvents (i.e., acetonitrile, chloroform, or methanol) to physically remove proteins from the matrix.
Solid phase extraction (SPE) is also a choice method for drug analysis, but is better suited for targeted analysis in unknown screenings. According to the study authors, when analytes of interest have similar polarities or acid/base properties, SPE, rather than LLE, is the preferred method. While SPE is precise and accurate, it is labor- and time-intensive, and can be costly columns, vacuums, manifolds, etc.
As with GC-MS and LC-MS, sample prep has advanced over time. Supported liquid extraction (SLE), which removes potential interferences and isolates a wide range of compounds, is an up-and-coming technique. The method allows unwanted materials in a biological sample to be retained on a solid surface comprising natural materials, such as silica. Because of this, SLE is ideal for the recovery of a large variety of drugs, which can help improve efficiency for labs working with poly-drug cases. This can be especially important for lab located in areas where the opioid crisis continues to increase.
Additionally, SLE has demonstrated success in the extraction of THC and metabolites from both whole blood and oral fluid. Cannabis is legalized for recreational use in 18 states and for medical purposes in 36 states. Trends in the last few years have shown increasing support for legal cannabis use at the recreational level—meaning SLE’s success with cannabis metabolites may be another reason its popularity is steadily increasing.
Meanwhile, according to the authors, phospholipid depletion is being used successfully with hydrophilic interaction liquid chromatography (HILIC). Biological fluids contain phospholipids, which can interfere with some chromatographic methods as “phospholipids can be retained on hydrophobic columns, suppress ionization in electrospray ionization sources and have an adverse effect on reproducibility.” However, in an unaffiliated paper published this year, scientists successfully employed HybridSPE for sample preparation on plasma specimens to reduce the interference of phospholipidmatrix in HILIC LC-MS analysis.
While “dilute and shoot” carries challenges and potential risks such as matrix components or interfering substances entering the analytical system, it is a very popular method for doping control. Coupled with LC-MS, dilution can quickly and accurately detect and quantify stimulants and narcotics in human urine. It can do the same with horse urine—and blood—where it has been used for the detection of polar drugs. The dilute and shoot method has also been used to extract a commonly prescribed antihypertensive medicine from human urine.
As with everything else—inside the lab and out—the authors conclude by acknowledging the valued role automation plays in sample handling and preparation. In sample prep, liquid handling units are by far the most popular complement, but tracking systems and consumable supplies are also high on the list for researchers.
Regardless of the type of automation, ultimately the technology helps to “increase the number of samples prepared for analysis within a given period of time, decrease human error, closely control resources, sample track with the use of barcoding…[and] minimize specimen usage by reducing the volume of sample needed for downstream analysis,” the authors conclude.