Introduction

Quantitative determination of blood alcohol (BAC) is one of the most common analyses performed in the forensic toxicology laboratory. GC with FIR (flame ionization detection) is the preferred technique.

Blood samples must be drawn by medical personnel and transported to the lab, where they are stored in a refrigerator. The blood tubes used for collection generally contain sodium fluoride, an anti-glycolytic, which inhibits enzyme reactions with glucose (recall that fermentation of glucose can produce alcohol!). Sample preparation is minimal and will vary by laboratory. Nearly all labs, however, will require use of an internal standard (discussed further below).

Quantitative analysis by GC is typically done using an autosampler. Even with this, the very small sample injection volumes and potential small changes in instrumental conditions, such as gas flow, might introduce other variations. One method commonly used to compensate for these difficulties is the use of internal standards.

The internal standard method involves spiking an exactly known quantity of a substance into every sample and standard. The area of the internal standard and the area of the analyte are determined, and then a ratio of these two is calculated by dividing the area of the analyte peak by the area of the internal standard peak. The result is called a peak area ratio (PAR). The idea is that even though the peak areas for a given sample may vary from one test to the next due to injection differences or instrumental variations, the ratio of the two peaks will be constant, since the variations will affect both substances equally. Then, when preparing the calibration curve, the PAR is plotted on the y-axis rather than the simple peak area.

A good internal standard has the following characteristics:

ü Yields a peak that is well resolved from other peaks

ü Has a retention time close to the analyte’s retention time

ü Normally, some structural similarity between the IS and analyte is desirable

ü Is a compound not readily available to the public and this not typically ingested

o This can be confirmed by using two Internal Standards (e.g., N-Propanol and Isobutanol)

§ The area ratio between the two internal standards should be a constant

There are two ways of introducing an internal standard into the analysis. One way is to dissolve the internal standard into the solvent used to dilute both samples and standards. A second way is to add an accurate and precise volume of concentrated internal standard solution to the samples and standards. The Internal Standard solution may contain salt (i.e., sodium chloride) to enhance the headspace analyses.  Addition of salt reduces the solubility of alcohol in an aqueous solution.

For blood alcohol quantitative determinations, n-propanol is a commonly used internal standard. In the data set below, you are provided with peak areas for both ethanol and propanol. When you make the calibration curve, plot the PAR vs the concentration.

Quality control samples (QC) are a critical component of a forensic BAC. QC samples typically include:

Negative control (contains no ethanol)

Positive control (contains a known amount of ethanol)

Quality control requirements typically include:

Agreement between the calculated value found for a control and its true, assigned value

Agreement between the calculated ethanol concentrations in duplicate samples

Agreement between the retention times of calibrator and sample ethanol peaks and internal standard peaks

For this “dry lab” you will be given a set of data from a BAC run. You will need to determine the PAR, plot a calibration curve, determine the concentrations of the unknowns, and calculate the QC results to be sure they are within the pre-established limits.

Data

In a real world forensic BAC run, you would have two separate columns running these samples simultaneously. You would also determine acetone, isopropanol, and methanol. For this dry lab, however, we will only do calculations for ethanol on one column.

 

Concentration (g   ethanol / 100 mL)

Peak area for ethanol

Retention time ethanol, min

Peak area for n-propanol (IS spiked at 0.05g/dL)

Retention time n-propanol min

 

Blank

0

1259

1.141

25649

1.991