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Eurofins Forensics Belgium

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Samenvatting bachelorproef 2014-2015: Determination of ethanol in biological samples using static headspace and gas chromatography with flame ionization detector

The existing method for quantitative determination of ethanol in biological samples using static headspace and gas chromatography with flame ionization detection (HS-GC-FID) with capillary column has been optimized. The goal of this optimization is to obtain higher ethanol signals without observing an increased signal of other by-products such as acetaldehyde. The different parameters of the headspace: equilibrating temperature, incubation time, sample volume and the nature of the added inorganic salt, have been examined. Different temperatures, times, volumes and salts have been tested. The headspace settings that provided the highest ethanol signals and did not increase the signal received from by-products, are the settings that will be used for the determination and quantification. The method for quantification of ethanol in biological samples was validated. The system suitability was evaluated as well. The injection precision, the evaluation of relative retention times of standards, the autosampler stability and column oven temperature have to meet certain criteria.
The headspace parameters which resulted in the highest ethanol signals were: headspace equilibration temperature of 50°C, 15 minutes incubation time, sample volume of 0,250 ml and 1 ml of internal standard (0,005% t-butanol) with the addition of an inorganic salt, 1M ammonium sulfate, for the salting out process.
The quantification of ethanol is performed using linear regression analysis. Therefore, a calibration model must be determined.  The method is linear (r² > 0,9995) within the concentration range of 0 – 3 g/L and sensitive (cut-off value 0,05 g/L), precise (bias < 10%, coefficient of variation within-run precision and between-run precision is less than 5%). There is no carry-over (observed signal in blank sample after a high concentrated sample (6,0 g/L) is less than 10% of the lowest calibrator (0,5 g/L)).
The system is suitable for ethanol determination and quantification. The relative standard deviation measured on six injections is less than 15% (2,8%). The relative standard deviation of the relative retention time of standards is less than 0,5% (0,08%). The autosampler is stable, the maximum difference measured on two identical samples is 8%. The difference between the set column oven temperature and the measured temperature is less than +/- 5%. The method and system have been validated and are ready for use.


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