1 - ALKANES - ALKENES
Among several reported techniques of squalene analysis by HPLC, we have selected an efficient method valuable as well for oil mixtures than for cell extracts (Lu HT et al., Chromatographia 2004, 59, 367).
Materials and reagents
HPLC system, C18 column(150x3.9 mm Symmetry from Watres), UV or diode array detector, acetone, acetonitrile, ethanol, chloroform, methanol, squalene standard.
A squalene stock solution is prepared in dissolving an appropriate amount in ethanol and stored at -20°C. Working solutions are prepared by dilution with acetonitrile (from 0.1 to 40 mg/l).
Vegetal lipids are extracted or dissolved in methanol/acetone (7/3, v/v) or chloroform/methanol (1/2, v/v), after filtration and evaporation, the residue is dissolved in mobile phase.
A fractional crystallization may be used to avoid the interference of high amounts of cholesterol or phytosterols. Lipid solutions are kept at -20°C for 30 h and interferences are eliminated by filtration.
The column is maintained at 30°C and acetonitrile is pumped at a flow rate of 1.5 ml/min. Squalene is detected at 195 nm and is eluted at around 15 min. Even for complex lipid samples, there is no interference of other sompounds at the elution time of squalene. Linearity is excellent from 0.1 to 40 mg/l and the detection limit is about 0.04 mg/l. The recovery (from 90 to 95%) may be determined by addition of a known amount of standard in the sample.