HEMP-SEED AND OLIVE OILS 233 For non-rancid virgin oils, D..K is below 0.010. Pure oil analysis. To simulate the degradation of oil over time, an accelerated stability test was performed: spectrophotometric analysis was carried out on oil samples before and after heating to 40°C for 24 hours. Oil-in-emulsion analysis. To evaluate the antioxidant effect of excipients, namely Sepigel 305® and Carbopol 1382®, on the oil dispersed in an 0/W emulsion, simple formula­ tions containing these two products were prepared, (Table I). Prior to spectrophotometric examination the oil had to be extracted from the emulsion. It was thus necessary to break the system by adding NaCl crystals under stirring. The dispersion was then subjected to centrifugation at 13000 rpm for 10 minutes to separate the oil from the water phase. The fat supernatant phase was collected and analyzed spectrophotometricall y. The procedure employed to prepare the oil samples was as follows: A 0.1 % oil-in- 1-butanol solution was prepared, placing 27 µl of oil (oil density being 0.92 g/ml) in a 25-ml volumetric flask and diluting it to volume with 1-butanol. The solution was stirred on vortex and subjected to spectrophotometric analysis. To calculate D..K the absorbance values (E,.) detected by the spectrophotometer were converted into specific extinctions (E 1 % lcm or K) from the following equation: KA= EA.IC (7) where C is the solution concentration expressed in g/100 ml. IRRADIATION TEST Spectrophotometric determination of conjugated dienes and trienes is also useful to evaluate the photostability of an oil. As a consequence of photodegradation, the number of conjugated systems considerably increases. In order to evaluate photostability, oil samples were irradiated under UVB lamps. Conjugated dienes and trienes were determined after two hours' irradiation, as described above. Three grams of oil were placed in Pyrex containers with caps and irradiated for two hours under magnetic stirring, at 10 cm from the lamp. The mean surface area exposed to radiation was 10.17 cm2. The radiation intensity of the lamp was measured with a CO.FO.ME.GRA measurer with a probe sensitive to radiation in the range of 290-400 nm. The radiation intensity recorded for the UVB lamp was 2.6 x 10-4 W cm- 2 this is double the intensity ofUVB solar radiation (1.3 x 10-4 W cm- 2 ) (12). Two variables were taken into account: the cap on the Pyrex containers might act as a Ingredients Vegetable (hemp-seed, olive) oil Sepigel 305 ® Carbopol 1382® NaOH 10% in water Water Table I Composition of Formulations Emulsion 1 (w/w %) 5 94 Emulsion 2 (w/w %) 5 0.25 0.27 94.98

234 JOURNAL OF COSMETIC SCIENCE radiation screen, and during emission the lamp might dissipate thermal energy, thereby heating the samples. Both phenomena can influence the formation of conjugated dienes and trienes. Thus, to evaluate the method qualitatively, the photostability study was done in three different ways: 1. The capped Pyrex containers were placed in a closed solar box. 2. The open Pyrex containers were placed in a closed solar box. 3. The capped Pyrex containers were placed in an open solar box, to dissipate thermal energy from the lamp. MALONDIALDEHYDE AND MONOALDEHYDE DETERMINATION (11) Malondialdehyde (MDA) and monoaldehyde (MONO) are formed from oxidative reac­ tions of unsaturated fatty acids followed by the decomposition of tryglicerides they produce an unpleasant smell. The presence of MDA and MONO, an indication of rancidity, could be revealed through colorimetry with thiobarbituric acid (TBA). TBA reacts with the two aldehydes to give two colors that can easily be determined spectrophotometrically. To detect the presence of MDA and MONO in the oils, samples were prepared as follows: an aqueous solution ( 100 ml) was prepared containing 15 % w /w of trichloroacetic acid (TCA), 0. 3 7 5 % w /w of thiobarbituric acid (TBA) in hydrochloric acid (0.25 mol/1), and 18.8 µl of oil. The solution was placed in a thermostatic water bath (at 100°C), incubated for 15 minutes, and then transferred to an ice bath. Three milliliters of 1-butanol were added under stirring the solution was then transferred to a separating funnel to extract any pink or yellow coloration that had formed from the reaction of TBA with the aldehydes. The reactions are: OHC-CHrCHO + TBA -----t TBA-MDA-TBA (pink) R-CH 2 -CHO + TBA -----t TBA-MONO (yellow) The supernatant organic phase was extracted and analyzed spectrophotometrically at 5 3 5 nm and 450 nm to detect, respectively, the pink and the yellow coloration. DETERMINATION OF CHLOROPHYLL Chlorophyll is a lipophilic yellowish-green pigment present in the non-saponifiable matter of an oil it influences taste and smell and determines color. It is a relatively unstable molecule: it easily degrades under the influence of light and heat. To quantify the chlorophyll content in hemp-seed oil, a calibration curve was made against a reference pigment (a standard solution of 4.0% chlorophyll in maize germ oil). Non-pigmented maize germ oil was used both as a reference for spectrophotometric analysis and as a solvent for dilutions. Three dilutions of the standard pigmented solution were prepared, containing, respectively, 0.0016%, 0.008%, and 0.004% chlo­ rophyll. The three standard solutions were analyzed spectrophotometrically in the 300- 600 nm range. The same procedure was applied to the 1999 hemp-seed oil and extra­ virgin olive oil samples to determine their percentage chlorophyll content.