AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

Carot-02 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Determination of Lutein and β-Carotene in Infant Formula and Adult Nutritionals

Reversed-Phase High Performance Liquid Chromatography

Greg Hostetler, Perrigo Nutritionals, Georgia, VT USA

(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 and α-carotene. B. Apparatus (a) HPLC System. – Consisting of pump, autosampler, thermostatted column compartment, UV-Vis detector, and data acquisition software (b) Analytical column. – YMC C30 Carotenoid column, 3 μm, 2.0 x 250 mm (c) Guard column. – YMC C30 Guard column, 3 μm, 2.0 x 10 mm (d) Spectrophotometer. – Wavelength range of 200-700 nm, with 1 cm quartz cells

(e) Top loading balance. – Capable of weighing to 0.1 g (f) Analytical balance. – Capable of weighing to 0.1 mg (g) Ultrasonic water bath. – 40 kHz

(h) Reciprocating shaker. – Capable of 200 rpm (i) Evaporator. – With pure nitrogen supply (j) Laboratory centrifuge. – With adapters for 50 mL centrifuge tubes (k) Centrifuge tubes. – 50 mL, polypropylene (l) Syringes. – 1 mL, disposable (m) Syringe filters. – 0.2 μm, PTFE (n) Class A volumetric flasks. – Various sizes, clear and amber (o) Scintillation vials. – 12 mL, amber

(p) HPLC vials – amber, with 300 μL inserts (q) Class A volumetric pipettes. – Various sizes C. Reagents (a) Deionized water. – >18 MΩ·cm (b) Methyl tert butyl ether (MTBE). – HPLC grade (c) n-Hexane. – HPLC grade (d) Potassium hydroxide (KOH). – pellets, ACS grade