An important facet of a forensic investigation can be the analytical examination of fluids such as blood and urine for the presence of compounds that are not normally present. These can include excessive levels of prescription drugs, illicit drugs , and toxic compounds. The latter can be naturally occurring inorganic or microbial toxins or can be synthetic in origin.
Toxicological analysis is concerned with over 150 different compounds, depending on the circumstances of the crime or accident, and the observations of the physical appearance of the victim or suspect. As just a few examples, these include stimulants (amphetamine, caffeine, cocaine), alkaloids and amines (dextromethorphan, ephedrine, quinine), narcotics and analgesics (codeine, morphine), hallucinogens (LSD, PCP), antidepressants, sedatives (barbiturates ), tranquilizers, marijuana, and amyl nitrate.
Toxicological analysis is done in several different ways, depending on the target compound. Typically, the fluids that are of forensic interest are blood and urine. At a crime or accident scene, or even later, collection of the fluid is all that is necessary. The actual analysis is done in a dedicated laboratory using sophisticated equipment and trained personnel.
Collection for analysis needs to be done under controlled conditions using collection reservoirs especially designed for the purpose. For example, collection of a urine sample in a non-sterile container without a lid could result in contamination of the urine and would be grounds for subsequent legal inadmissibility of the results. Fortunately, protocols for sample collection and transport are relatively easy to observe.
Toxicological analysis is geared towards the detection of the presence of a compound (qualitative analysis) rather than determining the amount that is present (quantitative analysis). Other than alcohol, determining the actual amount of a compound is of little value. For many illicit drugs, there are really no beneficial levels. Thus, for example, merely demonstrating the presence of marijuana or cocaine is sufficient.
Often, a toxic or illicit compound is present in blood or urine along with other substances. To identify the target compound, it must be physically separated from the other compounds.
One tried and true method of physical separation is chromatography . In the various forms of chromatography, compounds are separated from one another based on their tendency to prefer either a solid material that is packed in the volume of the chromatography column (the stationary phase) or the fluid or gas that percolates through or over the stationary phase (the mobile phase).
There may be several compounds in a sample that show a preference for the stationary or mobile phases. By tailoring the chemistry of the stationary phase (commonly by judicious selection of the stationary phase material and the composition of the chemical side groups that protrude from the material or the thin coating of fluid that chemically clings to the solid) and the composition of the mobile fluid, different compounds will move through the column at different speeds.
Sample molecules can move through the matrix passively, under the force of gravity or via capillary action as liquid is drawn upwards into chromatographic paper, or can involve the use of a pump to drive the sample through the matrix at high pressure.
As the fluid emerges from the chromatographic column, it is collected in defined amounts. Thus, the separated compounds in the mobile phase will be collected in different reservoirs for their subsequent analysis.
The compounds that have been more tenaciously retained in the solid material in the column can then be chemically driven off of the material by the addition of fluid that differs in chemistry from that present initially. This step is known as elution. Elution can be tailored so that the compounds are released at different times.
In gas chromatography, the mobile phase is an inert gas such as helium, while in liquid chromatography this phase consists of a liquid. The latter form of chromatography ranges in sophistication from the dipping of one end of a strip chromatographic paper in liquid, with the separation of compounds occurring as the liquid moves upward through the paper via capillary action, to high-performance liquid chromatography (HPLC), in which compounds are powered through the matrix at high pressure.
Ion exchange chromatography is also useful in forensic toxicology . This relies on the net charge (the balance of positive charges and negative charges) of the target molecule. If a compound has an overall net negative charge, it will be retained more so by a positively charged material than by a negatively charged matrix. The opposite is true for a compound that has a positive net charge.
Chromatography can be combined with mass spectrometry to reveal very detailed information about the separated compounds. For example, the use of mass spectrometry can reveal the molecular weight of each separated compound. When two mass spectrometers are connected in series (tandem mass spectrometry), the arrangement of amino acid build blocks of the separated proteins can be determined, as can the types of fatty acids that comprise a lipid sample.
Other detection methods include the absorption of ultraviolet radiation by the sample molecules (evident by a change in absorbance on a plotted graph), the different refraction of light by different molecules (which can be quantified as a refractive index), and the reaction of certain sample chemical groups with light that results in the emission of light of a different wavelength (fluorescence ).
A different, and very efficient and economical, way to separate various proteins in blood and urine is electrophoresis . The technique is based on the migration of charged molecules in a solution in response to an electric field. The differing rates at which proteins migrate depends on the strength of the electric field, the protein's net charge, the size and shape of the protein molecules, and on the properties of the support matrix through which the molecules move.
The support matrix is typically paper, cellulose acetate, starch gel, agarose (which is purified from various species of seaweed), or polyacrylamide gel. The latter two are used most commonly.
Agarose and polyacrylamide are prepared as molten suspensions, which are poured into a mold. As the suspension cools, a gel forms. Depending on the concentration of the agarose or polyacrylamide, the gel will contain spaces that vary in size. Thus, agarose and polyacrylamide gel electrophoresis provide ways of separating different protein species and even nucleic acid fragments based on their different sizes.
By the inclusion of the appropriate controls, nucleic acid electrophoresis can even reveal the sequence of nucleotide building blocks that make up deoxyribonucleic acid (DNA ). Indeed, prior to the advent of computer technology and gene sequencers, the determination of DNA sequences was routinely done this way.
In electrophoresis, the separated compounds will typically form "bands" in the gel, which can be detected using special stains. As well, since under some electrophoretic conditions regions on the separated proteins can retain their ability to react with antibodies, the latter can bind to the protein. Then, tagging the bound antibody with a fluorescent probe allows a specific protein to be detected fluorescently.
More recently, the technique of capillary electrophoresis has proved useful in toxicologal analysis. Instead of a gel, compounds move through a tube of extremely small diameter (the capillary). The charge on the capillary wall retards the motion of the compounds to varying degrees.
Capillary electrophoresis is quite efficient; compounds that are very similar in character can be separated this way. As well, very small sample volumes are used and the separation is completed very quickly.
When the target compound is a protein, the molecule can be distinguished from other proteins by antibodies that have been formed in response. Generically, this approach is known as antibody capture. The binding of an antibody to its corresponding antigen can cause formation of a complex that becomes so large that it precipitates out of solution or suspension. Even more sophisticated applications of antibodies are available. For example, antibodies can be bound to magnetic particles. Once binding of the antibody-magnetic particle complex to a protein has occurred, the protein can be magnetically separated from other proteins in the mixture.
No matter what the nature of the toxicological analysis procedure, all are conducted using standard protocols and with the inclusion of the appropriate controls to ensure that the equipment is operating properly, that what is supposed to be detected is indeed being detected, and that extraneous or interfering compounds are excluded from detection.
These rigorous quality control procedures helps strengthen the validity and legal admissibility of the results of the toxicological analysis.
see also Analytical instrumentation; Antibody; Biosensor technologies; Chromatography; Fourier transform infrared spectrophotometer (FTIR); Gas chromatograph-mass spectrometer; Pathogens; Spores; Thin layer chromatography.