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272 S CHIMPF ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 1, 2018

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. ( ii ) b -Carotene (approximately 20 mg/L). — Accurately weigh approximately 1 mg b -carotene standard, transfer to a 50 mL volumetric flask, and dilute to volume with hexane. Sonicate to dissolve b -carotene. ( iii ) 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) ]. — ( i ) Lutein (approximately 1.2 mg/L). — Dilute 6 mL stock standard to 100 mL with methanol. ( ii ) b -Carotene (approximately 1.2 mg/L). — Dilute 6 mL stock standard to 100 mL with hexane. ( iii ) Lycopene (approximately 0.4 mg/L). — Dilute 10 mL stock standard to 100 mL with hexane. ( 3 ) Working standard preparation. — ( i ) High, mixed (approximately 300 µg/L lutein, 500 µg/L b -carotene, and 100 µg/L lycopene). — Pipet 2.0 mL lycopene intermediate standard and 3.0 mL b -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 of 10% BHT in methanol into the container. Cap and mix well. Store at less than – 50°C. Expiration 2 weeks. ( ii ) Middle, mixed (approximately 150 µg/L lutein, 250 µg/ L b -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. ( iii ) Low, mixed (approximately 30 µg/L lutein, 50 µg/L b -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. ( iv ) Very low, mixed (approximately 15 µg/L lutein, 25 µg/L b -carotene, and 5 µg/L lycopene). — Pipet 0.5 mL middle, mixed standard into a 5 mL volumetric flask and dilute to

Table 2017.04A. Absorbance settings for standard concentration determinations

Absorbance wavelength, nm

Absorption coefficient, dL/g cm

Blank solvent

Standard

Lutein

445 452 472

2550 2592 3451

Methanol Hexane Hexane

b -Carotene

Lycopene

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 ð C Þ , µg = L=Measured absorbance = absorption coefficient × 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: Purity ð P Þ = trans peak area = ð sum of areas of all peaks except solvent front Þ Average the results from triplicate injections. ( 3 ) Intermediate standard concentrations. — Multiply the concentration, C , calculated in step D(f) ( 1 ) by the average standard purity, P , calculated in step D(f) ( 2 ). ( 4 ) Working standard concentrations. — ( i ) High. — Concentration (µg/L) = Intermediate standard concentration [step D(f) ( 3 ) above] × milliliters of standard pipetted in step D(e) (3)(i) ÷ 8. ( ii ) Middle. — Concentration (µg/L) = High standard concentration [step D(f) (4)(i) ] ÷ 2. ( iii ) Low. — Concentration (µg/L) = High standard concentration [step D(f) (4)(i) ] ÷ 10. ( iv ) Very low. — Concentration (µg/L) = Middle standard concentration [step D(f) (4)(ii) ] ÷ 10.

Table 8. trans - b -Carotene calibration curve data

Relative error, % a

R

Curve

Date

Slope

Intercept

L 1

L 2

L 3

L 4

Avg.

4.32 × 10 0 8.66 × 10 4 4.81 × 10 0 4.26 × 10 0 4.46 × 10 0 4.24 × 10 0 4.44 × 10 0 4.18 × 10 0 3.94 × 10 0

4.23 × 10 0 – 9.51 × 10 2 – 3.33 × 10 0 2.56 × 10 0 – 2.94 × 10 0 9.87 × 10 0 7.47 × 10 0 8.31 × 10 0 1.06 × 10 0

1 2 3 4 5 6 7 8 9

Feb 7, 2017 Feb 6, 2017 Feb 1, 2017 Jan 31, 2017 Jan 30, 2017 Jan 27, 2017 Jan 26, 2017 Jan 26, 2017 Jan 25, 2017

0.999983 0.999975 0.999938 0.999943 0.999964 0.999912 0.999877 0.999926 0.999934 0.99988

– 1.562 – 0.066

1.613 1.426

0.064

– 0.115

0.000 0.000 0.000 0.315 0.000 0.000 0.000 0.000 0.000

– 2.442

1.082 0.017 0.630 0.052

2.823 1.888 2.761

– 3.155 – 2.911 – 3.042

0.315 1.653 0.229 0.707 0.878

– 1.843 – 3.202

1.552 2.892 4.453 2.833 0.629

– 0.417 – 0.568 – 0.046 – 0.477

– 4.01

– 0.397

– 3.048 – 0.695

0.691 0.189

Avg.

0.018

a L 1 – 4 corresponds to b -carotene concentrations of ~25, 50, 250, and 500 µg/L.