AOAC SPIFAN Review Team (August 2016)

Page

Determination of Lutein and β-Carotene in Infant Formula and

Adult Nutritionals

Reversed-Phase Ultra-High Performance Liquid Chromatography

(Applicable to the determination of all-

trans

-lutein,

cis

isomers of lutein, all-

trans

-β-carotene, and

cis

isomers of β-carotene in infant formula and adult nutritionals.)

A. Principle

Powder samples are reconstituted in water, and liquid sample is first spiked with an internal standard

and saponified with potassium hydroxide. Samples are then extracted with MTBE and THF, followed by

hexane. The supernatants from the liquid-liquid extraction are dried under nitrogen and reconstituted in

IPA. Separation is done by reversed-phase chromatography on a C30 column. All-

trans

lutein and β-

carotene are separated from their major

cis

isomers, as well as zeaxanthin, α-carotene, and lycopene.

B. Apparatus

(a) UHPLC System. – Consisting of pump, autosampler, thermostatted column compartment, UV-Vis

detector with 60 mm flow cell, and data acquisition software

(b) Analytical column. – C30 Carotenoid column, 3 μm, 2.0 x 250 mm (YMC p/n CT99S03-2502WT)

(d) Guard cartridge holder. – YMC p/n XPGCH-Q1

(e) Spectrophotometer. – Wavelength range of 200-700 nm, with 1 cm quartz cells

(f) Top loading balance. – Capable of weighing to 0.1 g

(g) Analytical balance. – Capable of weighing to 0.1 mg

(h) Ultrasonic water bath. – 40 kHz

(i) Reciprocating shaker. – Capable of 200 rpm

(j) Evaporator. – With pure nitrogen supply

(k) Laboratory centrifuge. – With adapters for 50 mL centrifuge tubes

(l) Centrifuge tubes. – 50 mL, polypropylene

(m) Syringes. – 1 mL, disposable

(n) Syringe filters. – 0.2 μm, PTFE

(o) Class A volumetric flasks. – Various sizes, clear and amber

(p) Scintillation vials. – 12 mL, amber

(q) HPLC vials – amber, with 300 μL inserts

(r) Class A volumetric pipettes. – Various sizes

C. Reagents

Note: Reagent volumes may be scaled up or down provided good laboratory practices are followed.

(a) Deionized water. – >18 MΩ·cm

(b) Methanol (MeOH). – HPLC grade

(d) n-Hexane. – HPLC grade

METHOD

FOR ERP USE ONLY

DO NOT DISTRIBUTE