Pigment Reduction in Corn Gluten Meal and Its Effects on Muscle ...

kept for 30 m<strong>in</strong> at room temperature, <strong>and</strong> homogenized aga<strong>in</strong> for 30 s. The mixture wascentrifuged at 10,000 x g for 5 m<strong>in</strong>, <strong>and</strong> an aliquot of the supernatant (0.5 mL) was filteredthrough a 0.45 µm Nylon Acrodisc syr<strong>in</strong>ge filter (Cole-Parmer Canada Inc., Montreal, QC). Thefirst two drops of the filtrate were discarded, <strong>and</strong> the rem<strong>in</strong>der was collected for HPLC analyses.Carotenoids from corn gluten meal samples were separated <strong>and</strong> quantified accord<strong>in</strong>g toAbdel-Aal et al. (2007) us<strong>in</strong>g a 1100 series liquid chromatographer (Aligent, Mississauga, ON).<strong>Pigment</strong>s separation was performed on a short (10 cm x 4.6 mm, pack<strong>in</strong>g 3 µm) C30 column,YMC Carotenoid (Waters, Mississauga, ON, Canada), operated at 35°C. The mobile systemgradient used for elution was conducted us<strong>in</strong>g solutions of (A) methanol/methyl tert-butilether/nanopure water (81:15:4, v/v/v) <strong>and</strong> (B) methyl tert-butyl ether/methanol (90:10, v/v), <strong>and</strong>programed as follows: 0-9 m<strong>in</strong>, 100 - 75% A; 9 - 14 m<strong>in</strong>, 75 - 20% A; 14 - 15 m<strong>in</strong>, 25 - 0% A;17 - 18 m<strong>in</strong>, hold at 0% A; 17 - 18 m<strong>in</strong> 0 - 100% A; 18 -20 m<strong>in</strong> hold at 100% A. Afterseparation, detection <strong>and</strong> measurement of carotenoids was conducted at 450 nm (all-trans-lute<strong>in</strong>,all-trans zeaxanth<strong>in</strong>, all-trans-β-cryptoxanth<strong>in</strong> <strong>and</strong> all-trans-β-carotene,) <strong>and</strong> 478 nm (all-transastaxanth<strong>in</strong>),respectively. The correspondence of the retention times <strong>and</strong> UV/vis spectra ofanalysed samples <strong>and</strong> those showed by pure authentic st<strong>and</strong>ards was utilized for identification ofcarotenoids.For identification <strong>and</strong> quantification purposes five pure authentic st<strong>and</strong>ards carotenoids(all-trans-astaxanth<strong>in</strong>, all-trans-lute<strong>in</strong>, all-trans-β-carotene, all-trans zeaxanth<strong>in</strong>, all-trans-βcryptoxanth<strong>in</strong>)were utilized. Five concentrations for each carotenoid were prepared <strong>in</strong> butanol <strong>in</strong>order to assess l<strong>in</strong>earity of the response. The regression analysis (response area v/s <strong>in</strong>jectedamount) showed a l<strong>in</strong>ear relationship for all-trans-lute<strong>in</strong>, all-trans zeaxanth<strong>in</strong>, all-trans-βcryptoxanth<strong>in</strong>,<strong>and</strong> all-trans-β-carotene with the coefficient of determ<strong>in</strong>ation (R 2 ) 0.9996, 0.9996,111