High-Throughput Blood Alcohol Analysis Determination Using Headspace Gas Chromatography

10 February 2012 Accuracy and precision are critical parameters in blood alcohol concentration (BAC) measurements, providing forensic toxicologists with optimal confidence in analytical results and allowing them to be fully prepared to withstand tough cross examination by defense attorneys. There…

10 February 2012

Accuracy and precision are critical parameters in blood alcohol concentration (BAC) measurements, providing forensic toxicologists with optimal confidence in analytical results and allowing them to be fully prepared to withstand tough cross examination by defense attorneys. There are five different bodily samples that can be screened to determine a person’s BAC: namely urine, saliva, hair follicles, blood, and breath. Currently, breath analysis and blood screening are the most commonly used BAC measurement methods implemented by law enforcement agencies to gather evidence.

Blood alcohol testing is one of the most accurate methods for measuring a person’s BAC, enabling forensic toxicologists to determine the amount of alcohol that is in the blood at the time a blood sample is taken. However, blood alcohol testing is one of the most intrusive methods and is mainly used when a suspect has refused a breath test or following a serious accident.

Modern forensic toxicology laboratories need productivity and reliability to enable continuous 24/7 operations and allow the dependable quantitative determination of alcohol content in blood. In addition, this type of testing is strictly regulated, posing a further challenge to forensic toxicology laboratories that require advanced technology in order to comply with industry regulations.

Regulatory Outlook
In the U.S. all 50 states and the District of Columbia impose regulations, according to which it is a crime to drive with a BAC at or above a specified level which is currently 0.08 g alcohol per 100 ml blood. Conviction for alcohol impaired driving is normally followed by license suspension, while licenses can also be taken before conviction when a driver fails or refuses to take a chemical test. This is called administrative license suspension and is enforced in 41 states and the District of Columbia.

Additionally, more than half of the states require driving under the influence (DUI) and driving when intoxicated (DWI) offenders to install ignition interlocks on their vehicles into which they must exhale before being allowed to drive during a license suspension. In 15 states and four California counties, such a restriction is applied to all offenders, while another 16 states apply the restriction to offenders with a high BAC (typically 0.15% or higher) and to repeat offenders. Finally, six states apply the restriction only to repeat offenders.1

Each of the countries in the European Union has regulations in place to restrict the amount of alcohol allowed in the blood when driving. Driving under the influence of alcohol is a crime and can be punished by a fine and driving ban for a specified amount of time. The average BAC permitted across the majority of European countries, including Spain, Italy, and Ireland, is 0.05%, with some countries such as the United Kingdom allowing 0.08%.

With regard to blood alcohol testing, different regulations are enforced in each one of the U.S. states. In California, for example, the California Code of Regulations for Forensic Alcohol Analysis and Breath Alcohol Analysis2 mandates that every laboratory performing forensic alcohol analysis should have a valid license issued in accordance with certain provisions and all analyses must be performed only by persons who meet specific qualifications. Blood samples must be collected by venipuncture from living individuals as soon as feasible after an alleged offense and only by authorized persons. Sufficient blood should be collected to permit duplicate determinations. The regulation also specifies certain standards of performance for the methods used for forensic alcohol analysis. According to these standards, the method must be capable of analyzing a reference sample of known alcohol concentration within accuracy and precision limits of plus or minus 5% of the value. These limits should be applied to alcohol concentrations which are 0.10 grams per 100 milliliters or higher.

Analytical Approach 
The analysis of blood and other body fluids for alcohol is most commonly performed using headspace gas chromatography due to its simplicity and the number of samples that normally run daily. The quality of GC results depends on many factors, including the stability of the gas chromatograph, the ruggedness of the injection system, and the sensitivity of the detector. Within this process, sample preparation and introduction provide the foundation for repeatability and reliability that are essential for the generation of quality data. Robustness and easy maintenance are also paramount to ensure continuous operation.

An experiment was performed to demonstrate the superior analytical capabilities of modern GC instruments used in conjunction with advanced autosamplers. Leading autosampler technology, such as that used in this study, is strongly recommended when dealing with dirty matrices because it does not use any sample loops, which may require cleaning, or transfer lines that may create active sites for these polar compounds. In this application, the sample was injected with an automated headspace autosampler using a heated gas-tight syringe. To eliminate carry-over, the heated syringe is also cleaned automatically between injections with a nitrogen purge.

To achieve the maximum throughput, and for confirmatory purposes, two narrow bore columns are used for the gas chromatographic isothermal analysis of the headspace sample. In fact, every sample is injected into the GC’s split/splitless injector, which is connected to a pre-column, and the effluent is then split into two capillary columns with different stationary phases. The effluent of each column is connected to a flame ionization detector (FID) for quantification purposes.

The correspondence of analytes and internal standard retention times on the two columns and the analytes and internal standard peak area ratios are used for the qualitative and quantitative determination of blood alcohol content.

The autosampler technology used in this study allows the continuous and automatic running of blood alcohol analysis due to the system’s high number of sample positions. Up to 300 samples can be loaded and run in a sequence, allowing around 20 hours of continuous operation in this specific application.

A gas chromatograph (TRACE GC 1310, Thermo Fisher Scientific) equipped with digital electronic flow control and dual FIDs with fast response. The gas chromatograph, although of small bench size, features a GC oven able to accommodate multiple capillary columns for confirmation analysis and to provide rapid analysis of alcohol in blood samples by headspace injection. This was combined with an autosampler (TriPlus RSH Autosampler, Thermo Fisher Scientific), using a high temperature resistant gas-tight syringe injection technique for direct headspace injection.

Instrument Setup Parameters
The GC instrument was configured with a pre column (0.32 mm lD, 30 cm) and two columns (TraceGOLD TG-ALC1 26074- 3390 30m x 0.32mm x 1.80μm and TraceGOLD TG-ALC2 26073-2260 30m x 0.32mm x 1.20μm) for confirmatory analysis into dual FIDs (Figure 1).

The sample was placed into a 20 mL vial for analysis by headspace together with an n-propyl alcohol solution (used as internal standard). Then, 1.0 mL of headspace was removed by the autosampler from the vial with a gas-tight syringe and injected into the GC split/splitless injector with a 1.2 mm ID liner. Data collection was accomplished using a Chromatography Data System, (Chromeleon, Thermo Fisher Scientific). The instrument setup parameters are listed below.

GC conditions 
Oven temp: 40°C isothermal (3.5min)
SSL injector: 220°C, split, split flow 80 ml/min
Carrier: constant pressure, 80 kPa
FID: 230°C, H2 flow = 35 ml/min,
air = 350 ml/min, make-up = 30 ml/min

Agitator temperature: 80°C
Syringe temperature: 110°C
Incubation time: 10 min
Injection speed: 60 ml/min

The two capillary columns demonstrated excellent resolution for all analytes with no distortion of peak shape (Figure 2). The FIDs showed more than adequate linearity and sensitivity; figures 3 and 4 show the calibration results over a concentration range from 0.01 to 0.10 g/dL for ALC1 and ALC2 respectively. Detection limits of <0.005 g/dL were easily achievable and blanks ran after a 0.3 g/dL standard showed a non detectable carry-over.

Reproducibility tests were run on a sample containing ethanol at a concentration of 0.05 g/dL blood. Results are shown below:

Combining a GC instrument with a headspace autosampler has been proven to offer a reliable tool for the analysis of alcohol in blood. Confirmatory analysis is accomplished in the concentration range usually investigated with excellent linearity and reproducibility. The high number of samples that can be simultaneously loaded on the sample tray (up to 300, for 20 hours of unattended operation) and the gas chromatograph robustness and ease of use significantly extended the overall laboratory throughput when running this application.


  1. Insurance Institute for Highway Safety, DUI/DWI Laws, November 2011, http://www.iihs.org/laws/dui.aspx
  2. California Code of Regulations Title 17. Public Health Division 1. State Department of Health Services Chapter 2. Laboratories Subchapter 1. Service Laboratories Group 8. Forensic Alcohol Analysis and Breath Alcohol Analysis, http://www.california-duilawyers.org/showthearticle.asp?idArticle=26
  3. SOFT/AAFS Forensic Toxicology Laboratory Guidelines 2002,
    http://www.1800dialdui.com/CM/DUIDWIPublications/SOFT%20 Drug%20Lab%20Guidelines%202002%20final.pdf

Silvia Gemme is presently Gas Chromatography Product Specialist at Thermo Fisher Scientific. She joined Thermo Fisher Scientific in 2008, initially as product support of GC/MS with a focus on MS products while now she’s working mainly with GC and sample preparation techniques, developing new applications. Silvia has a master degree in Chemistry from the Università degli studi di Pavia, Italy.

Massimo Santoro is currently Gas Chromatography Product Manager at Thermo Fisher Scientific. He received his Master Degree in Chemistry at the Universita’ degli Studi Di Pavia, Italy. He has since continued his work in chromatography and mass spectrometry in a wide breadth of countries and industries including forensic and environmental, while he spent most of his career in the instrumentation research, design, and manufacturing business where his job has kept him at the cutting edge of chromatography and mass spectrometry.