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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

#

, Edith de Haan

*

, Arjan de Vries

*

, and Fariha Bouraada

*

.

#

Ausnutria Hyproca Analytics, Lelystad, The Netherlands, to whom correspondence should be addressed.

*

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

(a)

Balances.

With readability of 0.1 mg and 0.01 g.

(b)

Centrifuge.

Laboratory centrifuge able to centrifuge 50 mL screw cap (Greiner) tubes.

(c)

Evaporation device (nitrogen).

Zymark TurboVap LV or equivalent.

(d)

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.

(e)

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.

(f)

Shaker.

Laboratory shaker able to shake 250 mL bottles.

(g)

Spectrofotometer.

Spectrofotometer able to measure from 445 – 472 nm using a 1 cm quartz cuvette.

(h)

Ultrasonic bath.

Ultrasonic bath able to sonicate (glass) tubes.

(i)

Suction device.

Vacuum pump with flask to remove solvent.

(j)

Water bath.

Shaking water bath (85⁰C).

C. Reagents and standards

(a) Acetonitrile (ACN). HPLC grade, Biosolve 01200701 or equivalent.

(b) Ammonium acetate. Merck,

art.nr.

1116 or equivalent

(c) Butylated Hydroxy Toluene (BHT). Sigma

art.nr.

B-1378 or equivalent.

(d) Carborundum (SiC). Silicon carbide, nr 12, circa 1.44 mm, Boom

art.nr.

76054102 or equivalent.

(e) beta-Carotene. All-trans, Fluka art.nr.22040 or equivalent.

(f) Dichloro methane (DCM). AR, Biosolve

art.nr.

13790501 or equivalent.

(g) Diisopropyl ether (DIPE). Reinst, stabilized with BHT, Merck

art.nr.

800866 or equivalent.

(h) Rac-5,7-dimethyltocol (DMT). Matreya

art.nr

. 1074 or equivalent.

(i) Ethanol (EtOH). Absolute, Nedalco 100% technical grade or equivalent.

(j) Glycerol. Circa 86.5 %, Boom

art.nr.

50771 or equivalent.

(k) n-Hexane. AR, Biosolve

art.nr

. 08290501 or equivalent.

(l) Isopropyl alcohol (IPA). Baker

art.nr.

8068 or equivalent.

(m) Lutein. All-trans, Sigma

art.nr.

X-6250 or equivalent.

(n) Lycopene. All-trans, Sigma

art.nr

. L-9879 or equivalent.

(o) Methanol (MeOH). Absolute LC-MS grade, Biosolve

art.nr

. 13687801 or equivalent.

(p) Potassium hydroxide (KOH). P.a., Merck

art.nr.

5033 or equivalent.

(q) Purified water. Milli-Q or equivalent.

(r) Sodium ascorbate (NaAsc). Merck

art.nr.

50076 or equivalent.

(s) Sodium chloride (NaCl). P.a., Merck

art.nr.

106404 or equivalent.

(t) Sodium sulfide (Na

2

S). Analar Normapur, 58-64 %, VWR

art.nr.

83756.290 or equivalent.

(u) Tetrahydrofuran (THF). HPLC grade, Biosolve

art.nr

. 20220601 or equivalent.

(v) Zeaxanthin. All-trans, Fluka

art.nr.

14681 or equivalent.

Carot-01 (February 2016)

FOR ERP USE ONLY

DO NOT DISTRIBUTE