SPIFAN Nutrients ERP Book_9-29-15

Carot-01 FOR ERP USE ONLY DO NOT DISTRIBUTE

Determination of Carotenoids in Infant Formula and Adult/Pediatric Nutritional Formula using High-Performance Liquid Chromatography with Photo Diode Array Detection.

Martijn Vermeulen, Arjan de Vries, and Fariha Bouraada. TNO Triskelion, Department of Analytical Chemistry, Utrechtseweg 48, Zeist, The Netherlands.

A. Principle Carotenoid esters are saponified using potassium hydroxide in ethanol/water in the presence of sodium ascorbate, sodium sulfide and glycerol to prevent oxidation. Dimethyltocol (DMT) is added as an internal standard and carotenoids are extracted using diisopropyl ether. The extract is subsequently washed and concentrated. Carotenoids are determined by HPLC-UV at 450 nm and DMT is measured at 292 nm. Separation is performed using 2 suplex pkb-100 columns of 25 cm and a gradient of methanol (with ammonium acetate), acetonitrile, isopropyl alcohol and water. B. Apparatus Balances. With readability of 0.1 mg and 0.01 g. Centrifuge. Laboratory centrifuge able to centrifuge 50 mL screw cap (Greiner) tubes. Evaporation device (nitrogen). Zymark TurboVap LV or equivalent. HPLC system. Waters Alliance 2695 HPLC system equipped with a Waters 996 PDA detector (292 nm and 450 nm) and a column oven (20⁰C) or equivalent. LC columns. Supelcosil Suplex pkb-100, pre-column: 4.0x20 mm, 5 µm and two analytical columns in series: 4.6x250 mm, 5 µm or equivalent. Shaker. Laboratory shaker able to shake 250 mL bottles. Spectrofotometer. Spectrofotometer able to measure from 445 – 472 nm using a 1 cm quartz cuvette. C. Reagents and standards Acetonitrile (ACN). HPLC grade, Biosolve 01200701 or equivalent. Ammonium acetate. Merck, art.nr. 1116 or equivalent Butylated Hydroxy Toluene (BHT). Sigma art.nr. B-1378 or equivalent. Carborundum (SiC). Silicon carbide, nr 12, circa 1.44 mm, Boom art.nr. 76054102 or equivalent. beta-Carotene. All-trans, Fluka art.nr.22040 or equivalent. Dichloro methane (DCM). AR, Biosolve art.nr. 13790501 or equivalent. Diisopropyl ether (DIPE). Reinst, stabilized with BHT, Merck art.nr. 800866 or equivalent. Glycerol. Circa 86.5 %, Boom art.nr. 50771 or equivalent. n-Hexane. AR, Biosolve art.nr. 08290501 or equivalent. Isopropyl alcohol (IPA). Baker art.nr. 8068 or equivalent. Lutein. All-trans, Sigma art.nr. X-6250 or equivalent. Lycopene. All-trans, Sigma art.nr. L-9879 or equivalent. Methanol (MeOH). Absolute LC-MS grade, Biosolve art.nr. 13687801 or equivalent. Potassium hydroxide (KOH). P.a., Merck art.nr. 5033 or equivalent. Purified water. Milli-Q or equivalent. Rac-5,7-dimethyltocol (DMT). Matreya art.nr. 1074 or equivalent. Ethanol (EtOH). Absolute, Nedalco 100% technical grade or equivalent. Ultrasonic bath. Ultrasonic bath able to sonicate (glass) tubes. Suction device. Vacuum pump with flask to remove solvent. Water bath. Shaking water bath (85⁰C).

Sodium ascorbate (NaAsc). Merck art.nr. 50076 or equivalent. Sodium chloride (NaCl). P.a., Merck art.nr. 106404 or equivalent. Sodium sulfide (Na 2 Tetrahydrofuran (THF). HPLC grade, Biosolve art.nr. 20220601 or equivalent. Zeaxanthine. All-trans, Fluka art.nr. 14681 or equivalent.

S). Analar Normapur, 58-64 %, VWR art.nr. 83756.290 or equivalent.