U. pinnatifida seaweeds were harvested on a monthly basis from June 2011 to November 2011. The seaweeds were obtained from two farms located in Port Underwood; farm PE327 (41° 20′ 53.05″ and 174° 07′ 20.96″) and farm 106 (41° 19′ 37.74″ and 174° 08′ 57.54″), and two farms from the Pelorus Sound; farm 122 (41° 06′ 30.89″ and 173° 54′ 58.05″) and farm 353 (41° 01′ 56.95″ and 173° 56′ 12.55″) located in the Marlborough Sounds, New Zealand ( Fig. 2). Samples collected from each farm were washed several times with seawater on the boat to remove foreign matters and sand, and were then dried with paper towels. Blade and sporophyll parts were separated and kept in individual bags. These samples were then frozen overnight and air-freighted to Vitaco Limited, Avondale, Auckland, to be lyophilised in bulk within 48 h of frozen storage. All freeze-dried U. pinnatifida samples were pulverized (Breville CG2B Coffee ‘n’ Spice Grinder) and sieved (600 μm sieve) to obtain a fine powder. Each sample was stored in individual 200 ml PET bottles and kept in the dark at room temperature until use.
All fucoxanthin extraction was carried out away from direct sunlight to reduce the possibility of oxidation by sunlight. The freeze dried sample (100 mg) was mixed with methanol (15 ml) and stirred using a magnetic bar for an hour at room temperature. Solids were removed using a Whatman No.1 filter paper (Thermofisher, New Zealand). Hexane (15 ml) and water (15 ml) were added to the methanol extract and vortexed for 1 min and left to partition into two distinct layers using a separation funnel. The upper phase was discarded and the lower phase was vortexed for 1 min with 10 ml of chloroform to extract fucoxanthin. The mixture was centrifuged (Sorvall RC5C instruments, Fiberlite F21–8x50y rotor, Thermofisher USA) at 17,000×g for 15 min at 4 °C. The aqueous phase was removed, leaving the organic layer undisturbed.
The organic phase was then dried at 30 °C using a rotary evaporator. Methanol (5 ml) was added to the dried extract. The resulting solution was transferred into a glass vial, flushed with argon gas and stored at −80 °C until further use. Seven U. pinnatifida samples (comprising both blade and sporophyll) from each farm were selected for further analysis.