6. AOACSPIFANMethods-2018Awards

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2 H ostetler : J ournal of AOAC I nternational V ol . 100, N o . 3, 2017 liquids, 25 g sample plus 200 g water for powder samples, or diluted 1:1 by weight for liquid concentrates] in accordance with Standard Method Performance Requirement (SMPR ® ) 2014.014 (21). The test sample concentrations calculated in H(f, g) are expressed per 100 g on an as is basis for all samples, allowing each laboratory to apply appropriate reconstitution factors.

(d)  n-Hexane .—HPLC grade. (e)  Potassium hydroxide (KOH) .—Pellets, ACS grade. (f)  Reagent alcohol (ROH) .—Denatured ethanol, HPLC grade. (g)  α-Tocopherol (vitamin E) .—95%. (h)  Pyrogallic acid (pyrogallol) .—ACS grade. (i)  IPA .—HPLC grade. (j)  THF .—99.9%, stabilized with BHT. (k)  Ammonium acetate .—HPLC grade, 98%. (l)  Potassium hydroxide solution, 50% (w/w).— Add 50 mL water to a 250 mL beaker. Weigh 50 g KOH and slowly transfer to the beaker under constant stirring. When dissolved and cooled, transfer to a media bottle and store at room temperature for up to 6 months. (m)  MTBE/vitamin E.— Dissolve 2.2 g α-tocopherol in 500 mL MTBE. Store in the refrigerator for up to 1 month. (n)  Pyrogallol solution (0.2 M pyrogallic acid in ethanol).— Dissolve 6.3 g pyrogallic acid in 250 mL ROH. Store in the refrigerator for up to 1 month. Solution should be clear at room temperature; discard if colored. (o)  Extraction solution [10 mM vitamin E in MTBE–THF (1 + 1)].— Dissolve 2.2 g α-tocopherol in 250 mL MTBE and 250 mL THF. Store in the refrigerator for up to 1 month. (p)  Sample solvent (10 mM vitamin E in IPA).— Dissolve 4.4 g α-tocopherol in 1000 mL IPA. Store in the refrigerator for up to 1 month. (q)  Mobile phase for LC system .—( 1 ) Phase A .—20 mM ammonium acetate in methanol–water (98 + 2). Combine 980 mLMeOH, 20 mLwater, and 1.54 g ammonium acetate and mix to dissolve. Store at room temperature for up to 1 month. (2) Phase B .—MTBE. (a)  Lutein .—ChromaDex (Part No. ASB-00012453; Irvine, CA), or equivalent. (b)  β-Carotene .—USP (Part No. 1065480; Rockville, MD), or equivalent. (c)  Apocarotenal (β-apo-8′-carotenal) .—USP Part No. 1040854, or equivalent. (d)  Lutein containing approximately 10% zeaxanthin .—USP Part No. 1370804. (e)  β-Carotene system suitability reference standard .—USP Part No. 1065491. D. Standards Standard solution preparation is summarized in Table 2016.13A and detailed below. (a)  Carotenoid stock solutions (20000 μg/100 mL).— Weigh (to 0.01 mg) approximately 10 mg each of the lutein, D(a) ; β-carotene, D(b) ; and apocarotenal, D(c) reference standards into separate 50 mL volumetric flasks. Add approximately 40 mL MTBE/vitamin E, C(m) , to each, sonicate for 2 to 3 min, and dilute to volume with MTBE/vitamin E. Store stock solutions at −20°C for up to 6 months and check their purity each time new standard solutions are made from them. (b)  Standard measuring solutions for UV-Vis spectroscopy potency check (200 μg/100 mL).— Transfer 1.0 mL aliquots of lutein and β-carotene standard stock solutions, E(a) , to E. Standards Preparation

A. Principle

Test samples (reconstituted powders, liquid RTFs, and liquid concentrates) are 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 2-propanol (isopropyl alcohol; IPA). Separation is performed by reversed-phase chromatography on a C30 column. All- trans -lutein and β-carotene are separated from their major cis isomers, as well as from zeaxanthin, α-carotene, and lycopene. Note : Carotenoids are light sensitive, and all work should be done under protection from UV light. (a)  Ultra-HPLC (UHPLC) system .—Consisting of binary 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 × 250 mm (Part No. CT99S03-2502WT; YMC, Kyoto, Japan). (c)  Guard column .—C30 guard column, 3 μm, 2.1 × 10 mm (Part No. CT99S03-01Q1GC; YMC). (d)  Guard cartridge holder .—Part No. XPGCH-Q1 (YMC). (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 0.01 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. B. Apparatus

(q)  HPLC vials .—Amber, with 300 μL inserts. (r)  Class A volumetric pipets .—Various sizes.

C. Reagents

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

(a)  Laboratory water .—>18 megohm-cm. (b)  Methanol (MeOH) .—HPLC grade. (c)  MTBE .—HPLC grade.