AOAC 2017 First Action Methods

(b) BHT. —American Chemical Society (ACS). (c) Alcohol, dehydrated undenatured 200 proof or reagent alcohol. —HPLC grade. (d) Hexane. —HPLC grade. (e) Lutein. —Sigma standard 07168 or equivalent. (f) Lycopene. —Sigma standard 75051 or equivalent. (g) MtBE. —HPLC grade. (h) Methanol. —HPLC grade. (i) Potassium hydroxide, 45% (w/w). —ACS. (j) Sodium ascorbate. —ACS. (k) THF with BHT preservative. —HPLC grade. (l) Water. —Resistivity of ≥15 mega-ohm/cm. D. Standard and Solution Preparation All solutions can be scaled up or down for convenience, provided good laboratory practices are observed. All solutions can be stored at 2–30°C and are good for 6 months unless otherwise noted; however, discontinue use of any chemical or solution regardless of expiration dates whenever indications of contamination, chemical degradation, or changes in concentration are evident. (a) Mobile phase. —Methanol–MtBE (85 + 15); mix 850 mL methanol and 150 mL MtBE. (b) Extraction solution. —Hexane–MtBE (75 + 25); mix 375 mL hexane and 125 mL MtBE. (c) 10% (w/v) BHT in methanol. —Weigh 5.0 g BHT and transfer to a 50 mL volumetric flask. Dissolve in and dilute to volume with methanol. (d) Dilution solution. —10% BHT in methanol–MtBE (75 + 25); add 12 mL of 10% BHT in methanol and 4 mL MtBE to a small glass container and tightly cap. Mix well. (e) Standard preparations. —Prepare all standards under UV- shielded fluorescent lights and store in tightly stoppered volumetric flasks or glass containers at less than –50°C. ( 1 ) Stock standard preparation. —( a ) Lutein (approximately 20 mg/L). —Accurately transfer 1 mg Sigma standard to a 50 mL volumetric flask and dilute to volume with alcohol. Sonicate to dissolve lutein. ( b ) β-Carotene (approximately 20 mg/L). —Accurately weigh approximately 1 mg β-carotene standard, transfer to a 50 mL volumetric flask, and dilute to volume with hexane. Sonicate to dissolve β-carotene. ( c ) Lycopene (approximately 4 mg/L). —Accurately weigh approximately 1 mg lycopene standard, transfer to a 250 mL volumetric flask with 1 mL THF and hexane, and dilute to volume with hexane. Sonicate to dissolve lycopene. ( 2 )  Intermediate standard preparation. —Determine concentration each time new working standards are made, D(f) . ( a ) Lutein (approximately 1.2 mg/L). —Dilute 6 mL stock standard to 100 mL with methanol. ( b ) β-Carotene (approximately 1.2 mg/L). —Dilute 6 mL stock standard to 100 mL with hexane. ( c ) Lycopene (approximately 0.4 mg/L). —Dilute 10 mL stock standard to 100 mL with hexane. ( 3 )  Working standard preparation. —( a ) High, mixed (approximately 300 µg/L lutein, 500 µg/L β-carotene, and 100 µg/L lycopene). —Pipet 2.0 mL lycopene intermediate standard and 3.0 mL β-carotene intermediate standard into a small glass container and evaporate the solvent with nitrogen in a 37°C water bath. After all of the solvent has evaporated, pipet 2.0 mL MtBE into the container. Cap and mix well with a vortex mixer. When all residue has dissolved, pipet 2.0 mL lutein intermediate standard and 4.0 mL

AOAC Official Method 2017.04 cis- and trans- Lutein, cis- and trans -β-Carotene, and cis- and trans -Lycopene in Infant, Pediatric, and Adult Nutritionals Reversed-Phase HPLC with UV-Visible Detection First Action 2017 Caution: Refer to Material Safety Data Sheets (MSDS) of chemicals prior to use and follow the suggestions for personal protective equipment. A. Principle This reversed-phase HPLC method uses C30 chromatography and UV-Vis spectroscopy to determine cis and trans isomers of lutein, β-carotene, and lycopene in infant, pediatric, and adult nutritionals. Carotenoids are extracted from samples with hexane– methyl tertiary butyl ether (MtBE) (75 + 25) after saponification with a mixture of potassium hydroxide, tetrahydrofuran (THF), and methanol. After extraction, a portion of the organic layer is evaporated to dryness, and the residue is dissolved in methanol with 10% butylated hydroxytoluene (BHT)–MtBE (75 + 25). Prepared samples are injected into a C30 HPLC column where cis and trans isomers of lutein, β-carotene, and lycopene are separated with a methanol–MtBE gradient and detected with UV-Vis spectroscopy at 445 nm. B. Apparatus and Materials (a) Balance, analytical. —Minimum weighing capacity of 0.00001 g. (b) Balance, top-loading. —Minimum weighing capacity of 0.01 g. (c) Beakers. —Assorted sizes. (d) Bottle-top dispenser. —Capable of dispensing 10 mL. (e ) Centrifuge . (f) Centrifuge tubes. —50 mL glass with Teflon-coated caps. (g) Cylinders. —Graduated glass, assorted sizes. (h) Column. —C30; 3 μm, 4.6 × 250 mm (YMC Carotenoid Part No. CT99S032546WT or equivalent). (i) Guard column. —C30; 3 µm, 4.6 × 10 mm [YMC Part No. CT99S03-0104GC or equivalent (optional)]. (j) Guard column holder. —YMC Part No. XPGCH-Q1. (k) HPLC system. —Binary or quaternary gradient pump with degasser, autosampler capable of injecting 50 µL, thermostatted column compartment, and UV-Vis detector (Agilent 1290 Infinity II with 60 mm flow cell or equivalent). (l) HPLC vials. —Amber with 300 µL inserts. No split septa. (m) Light shields. —Yellow with cutoff of 440 nm. (n) N-evap. —With water bath or equivalent nitrogen evaporator. (o) Ice.— For cooling . (p) Pipettor. —Variable volume, 250 μL. (q) Pasteur pipets. (r) Spectrophotometer. (s) Stir plate with stir bars or mechanical shaker. (t) Ultrasonic bath. (u) Volumetric flasks. —Assorted sizes. (v) Volumetric pipets. —Class A, assorted sizes. (w) Vortex mixer. (x) Water bath. C. Chemicals and Solvents (a) β-Carotene. —U.S. Pharmacopeia standard 1065480 (Sigma PHR123, or equivalent).

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