6. AOACSPIFANMethods-2018Awards

279

( d ) Very low. —

Table 2017.04A. Absorbance settings for standard concentration determinations

Concentration, µg/L = middle standard concentration [ D(f) (4)(b) ] ÷ 10

Absorbance wavelength, nm

Absorption coefficient, dL/g cm

Standard

Blank solvent

Lutein

445 452 472

2550 2592 3451

Methanol Hexane Hexane

E. Sample Preparation (a) Saponification. —( 1 ) Liquid samples. —Thoroughly mix or stir products prior to sampling. Homogenize if necessary. Accurately weigh 1.0 g ± 10% into a 50 mL centrifuge tube and record the weight to the nearest 0.0001 g. Proceed with step E(a) (3) . ( 2 ) Powder samples. —If powder sample homogeneity is unknown, assume it is nonhomogeneous and proceed as for dry- blended/nonhomogeneous powder. ( a ) Dry-blended/nonhomogeneous powder. —Accurately weigh approximately 25 g powder and add approximately 200 g water. Mix until homogeneous suspension is obtained. Ahomogenizer can be used when necessary. Accurately weigh 1 g ± 10% homogeneous suspension into a 50 mL centrifuge tube and record the weight to the nearest 0.0001 g. Proceed with step E(a) (3) . ( b ) Wet blended powder. —Accurately weigh approximately 0.2 g powder into a 50 mL centrifuge tube and record the weight to the nearest 0.0001 g. Proceed with step E(a) (3) . ( 3 ) Add 3 mL laboratory water to liquid samples and 4 mL laboratory water to powder samples. Swirl to mix or suspend powder. ( 4 ) Add approximately 0.5 g or 1/8 tsp sodium ascorbate and swirl each tube gently to mix. ( 5 ) Add 10 mL methanol to each tube. ( 6 ) Add 1.0 mL 45% potassium hydroxide to each tube and swirl to mix. ( 7 ) Add 1.0 mL THF to each tube. ( 8 ) Cap tubes and mix for 30 s. If any of the tubes leak, reweigh the sample. ( 9 ) Heat tubes in a 60 ± 2°C water bath for 15 ± 2 min shaking tubes for ~10 s every 5 min. ( 10 ) After 15 min, immediately place tubes in an ice bath to cool. (b) Extraction and concentration. —( 1 ) Add 10.0 mL extraction solution (hexane–MtBE; 75 + 25) to each cooled tube and immediately cap each tube after adding the extraction solution to prevent evaporation. ( 2 ) Stir, shake, or mix on a vortex mixer each tube for 5 min (if stirring, add stir bar to tube before adding the extraction solution). ( 3 ) After 5 min, add 15 mL water and stir, shake, or mix on a vortex mixer each tube for an additional 5 min. ( 4 ) Centrifuge until a complete separation of the aqueous/ methanol and organic layers is achieved. ( 5 ) Quantitatively pipet 4.0 mL organic layer into an appropriate container and evaporate the organic solvent with a gentle stream of nitrogen in a 37 ± 2°C water bath. After evaporating the solvent, there should be a thin slightly cloudy layer of residue on the bottom of the container and no droplets of oil. If there are any oil droplets in the bottom or on the sides of the tube, the preparation should be repeated. ( 6 ) Remove one container at a time from the water bath and quantitatively add 0.25 mL dilution solution (10% BHT in methanol–MtBE; 75 + 25), cover the container, mix on the vortex mixer for 30 s, and immediately transfer contents of container to an autosampler vial fitted with a 300 μL insert and cap vial. F. HPLC Analysis (a) Instrument operating conditions. —( 1 ) Mobile phase A. — Methanol–MtBE (85 + 15). (2) Mobile phase B .—MtBE.

β-Carotene Lycopene

of 10% BHT in methanol into the container. Cap and mix well. Store at less than –50°C. Expiration 2 weeks. ( b ) Middle, mixed (approximately 150 µg/L lutein, 250 µg/L β-carotene, and 50 µg/L lycopene). —Pipet 2.0 mL high, mixed standard and 2.0 mL dilution solution into a small glass container. Cap and mix well. Store at less than –50°C. Expiration 2 weeks. ( c ) Low, mixed (approximately 30 µg/L lutein, 50 µg/L β-carotene, and 10 µg/L lycopene). —Pipet 0.5 mL high, mixed standard into a 5 mL volumetric flask and dilute to volume with dilution solution. Mix well. Transfer to a small glass container. Cap. Store at less than –50°C. Expiration 2 weeks. ( d ) Very low, mixed (approximately 15 µg/L lutein, 25 µg/L β-carotene, and 5 µg/L lycopene). —Pipet 0.5 mL middle, mixed standard into a 5 mL volumetric flask and dilute to volume with dilution solution. Mix well. Transfer to a small glass container. Cap. Store at less than –50°C. Expiration 2 weeks. (f) Determination of standard concentrations. —( 1 ) Measure the absorbance of each intermediate standard at the appropriate wavelength ( see Table 2017.04A ) after zeroing the spectrometer with the appropriate solvent and calculate the concentration using the information in Table 2017.04A and the following equation: Concn µ Measured absorbance absorption coefficient ( ), C g L = × 10 000 000 ( 2 ) Determine standard purity, P , by injecting each intermediate standard in triplicate and dividing the trans peak area by the total area of all peaks in the chromatogram except the solvent front using the following equation: Average the results from triplicate injections. ( 3 ) Intermediate standard concentrations. —Multiply the concentration, C , calculated in D(f) ( 1 ), by the average standard purity, P , calculated in D(f) ( 2 ). ( 4 ) Working standard concentrations. —( a )  High. — Concentration, µg/L = intermediate standard concentration [ D(f) ( 3 )] × mL standard pipetted in D(e) (3)(a) ÷ 8 Purity peak area sum of areas of all peaks except solven ( ) ( P trans = t front)

( b ) Middle. —

Concentration, µg/L = high standard concentration [ D(f) (4)(a) ] ÷ 2

( c ) Low. —

Concentration, µg/L = high standard concentration [ D(f) (4)(a) ] ÷ 10

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