Blood, sweat and tears - GC/MS in forensic toxicology

When it comes to forensic toxicology, it’s all about separating and identifying poisons from a wide range of biological samples – and it is a laboratory favourite that has proved ideal for the task

FORENSIC toxicology is the branch of forensic science that seeks to identify and quantify the presence of toxins (poisons) primarily in the human body. Samples examined for toxicological purposes can include blood, urine, various other biological fluids, hair, nails and other tissues. Toxicological investigations are typically carried out in forensic laboratories under the jurisdiction of government agencies, or in private laboratories certified for such practices.

Agents screened in a toxicological assay can include carbon monoxide, cyanide, heavy metals, ethanol (grain alcohol), methanol (wood alcohol), huffing agents (aerosol propellants), drugs of abuse and other controlled substances such as prescription drugs, as well as a host of poisonous industrial and household chemicals. A negative result eliminates the need for further testing while a positive result must be confirmed by a more rigorous analysis based on a chemical principle different from the initial screen. The confirmation requirement helps eliminate false positives resulting from defects in test materials or the possibility that a non-target substance has produced a positive response.

For post-screening confirmation, gas chromatography/mass spectrometry (GC/MS) is generally recognised as the gold standard. Unlike individual screens that detect the presence or absence of a target set of substances, GC/MS can separate out, identify and measure virtually any component in a sample; the primary constraint being that the sample must first be vaporised. The separating action takes place in the GC column, a fused silica tube 25-60 meters (83-200 feet) in length with an inner diameter measuring only tenths of a millimeter (1 mm = 0.0054 in). The inner wall of the column is coated with a material (the stationary phase) that interacts with the sample components as they travel through the tube propelled by a flow of carrier gas. The entire column, in a coiled form, is placed inside the GC oven, and heated to progressively higher temperatures. A sample is injected into a chamber at the head of the column. Alternative sample introduction methods include solid phase microextraction (SPME) - evaporation from a special sampling rod, or headspace sampling - drawing the vapors above a liquid sample directly into the column.

Figure 1. Illustration of gas chromatographic separation. A) The carrier gas moves a mixed sample into the column. B) As the sample moves through the column, its various components (represented as the coloured spheres) begin to separate based on their interaction with the coating of the column (stationary phase). C) Like components exit the column in groups.

Helium is the most frequently used carrier gas. It has high separation efficiency and is inert, making it safe to work with and suitable for a wide range of detectors. As the oven temperature increases, different sample components vaporise, enter the gas stream and travel through the column. Interaction with the stationary phase coating differentially retards the forward progress of the sample components, causing them to become separated (Figure 1). The separated components exit the far end of the column and enter a detector that registers their presence. This response is recorded as a rising and falling trace or peak. The entire train of recorded peaks is termed the sample chromatogram and the exit or retention time for each peak is characteristic for that component. Modern GCs have the capability to reproduce retention times at accuracies down to hundredths of a second.

Under tightly controlled GC conditions, the retention time may be sufficiently specific to identify a substance; however, this is not considered adequate to positively confirm a toxicological screen. That objective is met by coupling the GC to a mass spectrometer or mass selective detector (MSD) (Figure 2). Unlike other detectors that merely register a GC peak, the MSD bombards each component entering from the GC column with high energy electrons. This creates a set of ions (charged molecules) of characteristic mass, which are measured and recorded. The unique combination of GC retention time and mass peaks generated by a substance constitutes a spectral fingerprint or signature of identity.
 
Over the years, GC/MS has become an indispensable tool in forensic toxicology because it can identify a very large number of substances using the same equipment and a similar analytical approach. Increasing reliance on GC/MS by the forensics community has caused an explosion in the number of toxicological tests being ordered and created a serious bottleneck in GC/MS sample processing and data management. Instrument manufacturers have responded to this need for greater productivity and simpler operation with new technology and automation.

The recent innovation of capillary flow technology provides greater control of column flow, saving time and improving performance. For example, preparing a column for reuse following an analysis typically requires a time-consuming, high temperature 300 0C (572 0F) “bake out” and subsequent cool down before the next sample can be run. Capillary flow technology makes this step unnecessary. Following the analysis, the carrier gas flow is reversed and any residual material is backflushed out of the column in as little as two minutes. Time saved in this way has enabled labs to process almost twice as many samples in a work cycle.

Capillary flow technology also saves time when columns are changed between analyses. Normally, a column swap requires venting of the MSD vacuum followed by a lengthy “pump down.” Capillary flow technology delivers pure “makeup” gas to the MSD inlet during column exchange, preventing contamination from the outside atmosphere, and  eliminating the need to vent and restore the MSD vacuum.

Capillary flow technology also allows the GC flow to be split among a number of  detectors, multiplying the amount of information that can be obtained in a single run without any sacrifice in throughput. This capability makes it possible to select detectors with sensitivities targeted to specific trace-level substances that might otherwise go undetected.

'Increasing reliance on GC/MS by the forensics community has caused an explosion in the number of toxicological tests being ordered and created a serious bottleneck in GC/MS sample processing and data management'

In addition to accelerating sample processing, laboratory managers must keep their instruments operating at peak performance. To help meet this requirement, instruments are equipped with automation that simplifies and streamlines operations and data handling. For example, setting up an automated GC/MS for a particular analysis now typically consists of selecting from a menu of methods. Instrument settings are archived along with the analytical method; so that recalling the method automatically sets up the instrument. It is the ability to electronically capture and restore instrument settings as well as recent advances in flow control that have made it possible to precisely reproduce retention times. What’s more, this information can be transmitted electronically and downloaded to comparably configured instruments. This means that the capability to perform identical analyses can be transferred between instruments irrespective of location. For the first time, perhaps, everyone can now truly be on the same analytical page.

A GC/MS analysis can generate a bewildering amount of information. The data from a single run can take an experienced analyst as much as an hour to review, interpret and confirm. As throughput increases, the volume of information generated is making timely interpretation of results more difficult. Now there is light at the end of this tunnel. Recently introduced deconvolution reporting software makes it possible to automatically extract and employ pertinent information from complex sample mass spectra to search and match against an electronic library of GC/MS data containing almost 150,000 unique entries. Using this tool, unambiguous identification of a sample can be accomplished in as little as two minutes with the results printed out in a simple one-page report. To deal with the increasing flow of information, enterprise content management (ECM) systems are being used to simplify data management in regulated environments such as forensic laboratories. This advanced form of electronic record keeping can organise and store original data generated by various instruments as well as other records, in an electronic format, at a single secure location. Access is granted to authorised users linked to the site via a secure network but the original data cannot be altered.

As workload and throughput increase, tolerance for instrument downtime diminishes. To ensure that instruments are up and running as often as possible, self-monitoring automation is being employed to register and report when consumables are low or when specific components are nearing the end of their service life and require replacement. Additional monitoring ensures that instrument performance remains within certain specifications. If these specifications are exceeded, the system issues an alert along with prescriptive advice for restoring performance. This proactive approach minimises system failures and the extensive downtime often needed for repairs. Other malfunctions can often be handled with interactive diagnostics that walk a user through a troubleshooting Q&A that produces suggestions for a quick fix. For more complex problems, some vendors provide expert-staffed call centers. In some cases, technical support staff have the ability to remotely interrogate instruments linked to the vendor servicing network in order to extract the detailed information needed to resolve a problem. This multitiered approach to support and service ensures that problems great and small are dealt with in the most efficient and cost-effective manner.
 
Advances in GC/MS technology have led to dramatic improvements in sample throughput and analytical performance. Now the forensic application of an alternate technology, liquid chromatography/mass spectrometry (LC/MS), is also attracting attention. The most significant limitation of GC/MS applications is the need to convert all compounds to a vaporisable form. LC/MS has no such requirement since samples are run in a liquid medium. On the other hand, GC/MS produces a very consistent ionization that makes possible very large electronic libraries of verified standards for sample matching and confirmation of identity.  Accordingly, one can see that these techniques are likely to be deployed in tandem, so as to extend the range of analytical applications in forensic toxicology.
               

By Tom Gluodenis. Tom is Forensics Marketing Manager, Agilent Technologies.