Introduction
BAC analysis, one of the most common tests performed in forensic testing laboratories, is used by law enforcement officials to determine intoxication levels in individuals. While on-site, officers test sobriety using a breathalyzer, along with an assortment of additional tests. However, if the offender refuses to take the breathalyzer test or other issues emerge with on-site testing, officials bring in the individual for a BAC. Given the severe repercussions of a DUI charge, such as suspension of a driver’s license, paying large fines, insurance surcharges, sobriety program requirements, disqualification from certain jobs, lifelong history of the offense, and maybe even jail time, forensic laboratories need to ensure accurate and reliable results.
Moreover, as the number of potential DUI cases in a region can sharply increase due to weekend activities, seasonal events, and changing demographics, local forensic laboratories need to offer higher-throughput capabilities and deliver results in a timely and robust manner to allow appropriate and prompt legal action. Therefore, the adopted methodology must not only be fast but also precise, accurate, and reliable to produce defensible results.
Current technologies used for BAC analyses may suffer from a range of limitations that can compromise the results, and subsequently, have serious effects on an individual’s social and economic standing if wrongfully convicted of a DUI. Issues such as carryovers from previous injections, poor instrument performance or inadequate optimization can all result in false positives. The lack of automation capabilities in legacy instruments and the long turnaround times due to sample backlog, especially in a high-throughput laboratory, can delay case proceedings. Additionally, the need to repeat BAC assays due to their inconsistencies can increase operating costs.
On the other hand, these well-known limitations in analytical methods can also be unfairly exploited by defense attorneys to purge DUI charges against true offenders. Sample carryovers, wrongful labeling, improper sample handling, and data inaccuracies are all reasons that lawyers can currently cite to reverse DUI convictions.
Forensic laboratories, therefore, have the responsibility to re-evaluate and upgrade the technologies used for BAC analysis to develop a robust, error-free, and reliable method that eliminates false positives and provides a trustworthy report in every analysis. In this article, we discuss the recent advances in headspace sampling technology that have been designed to meet the pressing needs of forensic laboratories for reliable and high-throughput BAC analysis.
BAC Analysis: Under the Influence of Carryovers and False Positives
To measure BAC, most forensic laboratories use headspace gas chromatography (GC) coupled with a flame ionization detector. The headspace injection method, a solvent-less extraction technique, is most suitable to measure volatile analytes, such as ethanol, in complex and less volatile matrices, like blood. Individual blood samples, loaded into sealed vials, are heated at a constant temperature to allow the volatile compounds to rise into the gaseous phase above the sample, referred to as the headspace, and allowed to equilibrate. Based on the partition coefficient, the concentration of the volatiles in the headspace is proportional to the concentration of the volatiles in the sample. An aliquot of the headspace is then injected for GC analysis.
Using the headspace sampling technology eliminates complicated sample preparation steps, making workflows faster and simpler, and prevents the build-up of matrix-related contaminants on the column, thereby, extending column lifetime. However, despite its widespread use, limitations with BAC workflows and methods are frequently recounted to dispute DUIs. Analyte carryover from previous tests, poor reproducibility, low precision in pressure and temperature control, software defaults, and even uncalibrated instruments are among the reasons lawyers have used to disprove BAC results.
The injection methodology used in headspace analysis can inherently result in carryovers or poor repeatability of the response. For instance, injection port contamination, dead volumes or a long sample path increasing the probability of analyte adsorption, are all factors that can directly skew BAC results. These, in turn, can increase the risk of false positive/negative results in back-to-back measurements.
Moreover, as the cases pile up in busy forensic laboratories, the ability to carry out unattended operations and automate sample tracking, coupled with the use of compliant software that upholds data integrity and offers traceability will ensure that the efficiency of the laboratory is maintained without influencing data quality.
It is clear that, while the fundamental aspects of headspace sampling coupled with GC are beneficial for BAC measurement, forensic scientists need robust and reliable technology that better support the headspace analysis, and address the pressing needs of accuracy, repeatability, and timely response.
Improvements in BAC Analysis: Fast, Sensitive and Reliable
When approaching the headspace sampling technique, there are several ways to transfer the headspace sample from the vial to the gas chromatograph. The valve-and-loop approach is typically preferred as it offers a reliable and repeatable way for injecting samples. However, other contributing factors such as the pressure and temperature control during the sampling process and the inertness of the sample path are all critical to ensure repeatability and preserve sample integrity during transfer. Recent advances in headspace technology have aimed to directly focus on these critical issues facing forensic analysts. Below, we highlight some of the latest innovations that have boosted confidence in BAC analysis results:
Precise pressure and temperature control: Electronic pressure and temperature control during the headspace sampling process are critical to ensure that repeatable sample amounts are transferred into the GC column. The latest headspace sampling technology offers highly precise and sustained pressure control in the vial and during the loop filling stage. This helps achieve higher injection repeatability and increased reliability with minimal standard deviations, speeding up internal validation procedures. The advanced pneumatic controls also allow effective purging of the sample path, in addition to the effective heating controls that minimize carryover effects.
Direct GC column interface: Instead of the long, external transfer lines connected to the GC injector as seen in conventional systems, modern headspace GC systems bear a short and integrated GC interface where the sample path from the vial to the column is much shorter. The short sample path, made possible with a direct column connection design, reduces dead volumes along the flow passage, thereby, minimizing the risk of analyte carryover. Sample degradation, a possible issue with using longer paths, is eliminated as samples maintain their integrity during transfer. A single, compact thermal control also ensures uniform heating of the valve manifold up to the GC column, addressing any concerns over possible cold spots affecting sample recovery.
An application test performed on the Thermo Scientific TriPlus 500 HS autosampler connected to the Thermo Scientific Trace 1310 GC showed area counts of relative standard deviation (RSD) variation below 1% over a long sequence of 120 injections of a 50 ppm solution of ethanol in water. To clearly define the level of intoxication, and accurately determine compliance against legal levels, accurate quantitative data and recovery values are critical in BAC analysis. In this test, the excellent recovery of ethanol from whole blood certified control samples was achieved, with recovery rates between 93% and 107%. Moreover, there were no detectable carryovers in the blank samples analyzed after repeated injections of the whole blood control samples, even at the highest concentration level of 0.3 g/dL of ethanol.
Continuous, unattended operation: A surge in sample demand should, in no way, compromise the capability of a forensic laboratory. The latest headspace systems offer options for scalability with 120-vial and 240-vial capacities to extend the sample sequence capability without requiring additional bench space. The vial trays are added on top of the existing instrument and a robotic arm loads the vials in a programmed sequence, enabling unattended workflows. Moreover, vial trays can be swapped out with new samples without interrupting the current sequence, allowing continuous operation and higher throughputs. To measure the overall stability of the system over time, the long-term stability ‘K’ factor was tested on the TriPlus system over one month of continuous operation. The resulting ‘K’ factor was <1% of the mean value, well within the quality control criteria specified by the State of California. This suggests that even with continuous operation over an extended period of time, the instrument does not deviate from its specifications, maintaining data reliability in high-throughput environments.
Sample traceability: Modern headspace autosamplers are designed for full sample traceability with barcode readers that eliminate laborious manual sample tracking. Before the analysis, the vials are automatically scanned, the barcodes are read, and all the relevant information is transferred into a data system. This can significantly save time in busy laboratories and improve efficiencies when tracking samples or recording information for legal purposes.
Figure 1: Vial Tray Loader
Modernizing BAC Methods for Defendable Results
As one of the most regularly performed forensic tests, laboratories need to re-evaluate or upgrade traditional BAC analysis workflows to eliminate longstanding issues of assay carryover and instrument inconsistencies. By modernizing GC sampling systems, forensic testing facilities can deliver accurate, reproducible BAC results, keep up with the increasing demands for higher sample throughput without compromising data quality, and, ultimately, defend their own results with assured confidence for legal and medical cases.