The pig blood sample (25ml) was mixed with dichloromethane (25ml) in the ratio of 1:1 to form an emulsion. This emulsion was subjected to a solvent assisted flavor evaporation using a   SAFE apparatus (Bahr, Manching , Germany ) (Figure 1, Engel et al. 1999) and a vacuum pump ( Pfeiffer , Germany ) for careful and efficient isolation of volatile constituents.

Food substances usually contain many compounds, of which only a small number contribute to the characteristic odor or flavor of that particular substance. In order to distinguish potent odor active compounds from less odor active compounds an aroma extraction dilution analysis (AEDA) is performed.

AEDA is a procedure for determining flavor dilution (FD) factors of odor active compounds in food extracts (Grosch 2001). The flavor dilution factor of a substance can be defined as the highest dilution at which a substance can still be smelled using HRGC-O (Grosch 2001). Any extract may contain many compounds, the compound with the highest FD factor has the highest impact on the final odor impression of that particular extract, where as the compound with lowest FD factor has the lowest impact and does not contribute much to the final odor impression. The obtained extract of the blood and the blank sample were diluted in series with dichloromethane (1:1) starting with a flavor dilution (FD) factor of 1 and continued until a FD factor of 512. The AEDA was carried out on two different capillary columns, a non-polar DB 5 column and a polar DB-FFAP column. Four panellists evaluated the different odorants in each extract of the blood and the blank sample. In total eight extracts (blood and blank) were investigated. Initially, FD 1 was determined by HRGC-O and then the analysis was continued in a series of increasing FD factors until no odor was further detected in the diluted extracts via HRGC-O. The linear retention index (RI) of each odorant was calculated as described in Dool and Kratz (1963) and Kovats (1958). The confirmation of the detected odorants was performed by comparing the RI and the odor of all detected compounds with that of reference substances

The mass spectrometry analysis of the blood extracts were performed by a 2D-HRGC-MS consisting of two high resolution gas chromatographs (GC) 3800 (Varian, Darmstadt , Germany ) in combination with a Saturn 2200 mass spectrometer (Fig. 3) (Varian). Two types of capillary columns were used of which the first oven was equipped with a DB-FFAP column (15 m x 0.25 mm, film thickness 0.25 µm; Factor four, Varian, Germany) and the second oven with a DB-5 column (30 m x 0.25 mm, film thickness 0.25 µm; Restek, Bad Homburg, Germany).  The mass spectra of the odorants in the blood extract were compared with the mass spectra of reference substances measured at similar conditions. The main advantage of the 2D-HRGC-MS is the higher degree of the chromatographic resolution and the greatly enhanced separation power because the analyte of interest along with possible impurities are transferred to the second oven. Impurities having the same retention on the first column may have a different retention on the second column and will not co-elute with the analyte of interest, hence impurities are cleaned out to give mass spectra of specific compounds .