Measurement of squalene in olive oil by fractional crystallization or headspace solid phase microextraction coupled with gas chromatography

Squalene is the most abundant component in the unsaponifiable fraction of olive oil with strong antioxidant properties. Its concentration in olive oils varies between 0.2 and 16.2 g/kg depending on the cultivar(s) used. The propose of this work was to determine the effectiveness of two different extraction methods for squalene determination by gas chromatography (GC) coupled to a flame ionization detector (FID) or to mass spectrometry (MS). In a first approach, oil samples were dissolved in methanol/acetone mixture 7:3 (v/v) and triglycerides separated by fractional crystallization at −20°C. The organic layer was removed, reduced to dryness and the residue reconstituted in n-heptane (containing squalane as external standard) and analyzed by GC-FID. A headspace (HS) solid phase microextraction (SPME) GC-MS method has been also developed in order to have an environmentally friendly (i.e. solventless) extraction procedure. The linear range investigated with both methods was 1.0-10 g/kg. Within-day and between days precision values, expressed as RSD%, were 4 and 7% (GC-FID), and 3 and 6% (GC-MS), respectively. The limit of detection (LOD) at a signal-to-noise (S/N) ratio of 3 were 0.019 (GC-FID) and 0.003 (GC-MS) g/kg; the limit of quantification (LOQ) calculated at S/N = 10 were 0.063 (GC-FID) and 0.008 (GC-MS) g/kg, well below the typical squalene concentration levels found in olive oils. The obtained percentage recoveries were 70 ± 2 (GC-FID) and 98 ± 3 (GC-MS), and were not concentration dependent. The potential of the method has been demonstrated by the analysis of several different olive oil samples produced from different cultivars and different locations.