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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.
Ultrasonic bath.
Ultrasonic bath able to sonicate (glass) tubes.
Suction device.
Vacuum pump with flask to remove solvent.
Water bath.
Shaking water bath (85⁰C).
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.
Rac-5,7-dimethyltocol (DMT). Matreya
art.nr. 1074 or equivalent.
Ethanol (EtOH). Absolute, Nedalco 100% technical grade 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.
Sodium ascorbate (NaAsc). Merck
art.nr.50076 or equivalent.
Sodium chloride (NaCl). P.a., Merck
art.nr.106404 or equivalent.
Sodium sulfide (Na
2
S). Analar Normapur, 58-64 %, VWR
art.nr.83756.290 or equivalent.
Tetrahydrofuran (THF). HPLC grade, Biosolve
art.nr. 20220601 or equivalent.
Zeaxanthine. All-trans, Fluka
art.nr.14681 or equivalent.
CAROT-01
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE