The gas chromatography/electron impact mass spectrometry (GC/EI-MS) is carried out using an Agilent 5973 Network mass selective detector connected to an Agilent 6890 gas chromatograph equipped with a capillary DB-23 column (30 m x 0.25 mm; 0.25 µm coating thickness; J&W Scientific, Agilent). Helium is used as a carrier gas at a flow rate of 1 ml/min. The temperature gradient is as follows: 150 °C for 1 min, 150 – 200 °C at 4 K/min, 200 – 250 °C at 5 K/min and 250 °C for 10 min. Electron energy of 70 eV, an ion source temperature of 230 °C, and a temperature of 260 °C for the transfer line are used.
Hydroxy fatty acids are measured as trimethylsilyl ether/methyl ester derivatives. For derivatization, 1 µl of BSTFA solution is added to the sample. For quantification, the ions m/z 324 (D 3 -2HOT), 337 (2-HHT), 367 (2-HOD), 321 (2-HOT), 273 (D 6 -9,10,11-TriHOD), 271 (9,10,11-TriHOE), 271 (9,10,11-TriHOD), 173 (9,12,13-TriHOE), 171 (9,12,13-TriHOD), 259 (9,12,13-TriHOE) and 259 (9,12,13-TriHOD) are used, respectively. As internal standards 500 ng of deuterium-labeled compounds (D 3 -2HOT and D 6 -9,10,11-TriHOD) are added to the sample at the beginning of the extraction. Before analysis, the column is characterized by generating standard curves plotting the intensity (m/z) ratios of [non-labeled standard]/[deuterium-labeled standard] versus the molar concentrations of the respective non-labeled standard. The amounts of endogenous compounds present in samples are then calculated according to the standard curves:
standards are available from Sigma (Germany) Larodan (Sweden), Cayman Chemical (USA), kindly provided by Mats Hamberg (Stockholm, Sweden) and Otto Miersch (Halle/Salle, Germany) or enzymatically synthesized, respectively
BSTFA solution :
N ,O-bis(trimethylsilyl)trifluoroacetamide [ >99 % (GC), SIGMA]
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