AOAC 2017 First Action Methods

Table 2017.04B. Gradient time Time, min

Table 2017.04C. Peak resolution requirements Peak

A, %

B, %

Minimum resolution

0.0

100 100

0 0

13- cis -Lutein and 13′- cis -lutein 13′- cis -Lutein and trans -lutein

1.3 1.3 7.0 1.3

10.0 10.1 18.0 18.1 27.0 27.1 30.0

65 65 25 25

35 35 75 75

13- cis -β-Carotene and trans -β-carotene

trans -Lycopene and 5- cis -lycopene

where C t -Lut = trans -lutein concentration in µg/100 g; C DS = lutein concentration extrapolated from the calibration curve in µg/L; D 1 = first dilution volume in milliliters (10 mL extraction solution); D 2 = second dilution volume in milliliters (0.25 mL dilution solution); R TW = total reconstitution weight in grams, if applicable; A 1 = aliquot of the first dilution pipetted, in milliliters; SW = sample weight in grams; R PW = weight of powder reconstituted in grams, if

100 100

0 0

( 3 ) Flow rate. —1 mL/min. (4) Injection volume. —50 µL. (5) Column temperature. —25°C. ( 6 ) Run time. —30 min. ( 7 ) Detection wavelength. —445 ± 4 nm. ( 8 ) Gradient.—See Table 2017.04B .

applicable; and 10 = conversion to 100 g. ( 2 ) cis-Lutein sample concentration. —

(b) Peak resolution. —Inject a sample containing all carotenoids of interest and confirm acceptable resolution between isomers using the guidelines in Table 2017.04C and the following equation: R t t W W = − + 2 2 1 2 1 ( ) ( ) where R = resolution; t 1 = retention time of peak 1 in minutes; t 2  = retention time of peak 2 in minutes; W 1 = peak width of peak 1 in minutes; and W 2 = peak width of peak 2 in minutes ( see Table  2017.04C ). (c) Standard and sample analysis. —Once the system has equilibrated, inject each working standard, inject samples, and inject another set of working standards. (d) System shutdown. —After all samples and standards have been analyzed, store the column in mobile phase A. G. Calculations (a) Standard curve preparation. —( 1 ) Visually inspect each standard and sample chromatogram and verify peak resolution from other peaks and correct identification of peaks. ( 2 ) Peak areas are measured with a data system. Before calculating the concentration of all - trans -β-carotene, cis isomers of β-carotene, all - trans -lutein, cis isomers of lutein, and all - trans - lycopene and lycopene isomers, verify that sample peak responses are within the range of standard peak responses. ( 3 ) Prepare standard curves by averaging the peak areas of standards injected at the beginning of a set of samples with the peak areas of standards of the same concentration injected at the end of the set of samples and performing linear least-squares (regression) on concentration versus averaged peak areas. Astandard curve must have a correlation of at least 0.999 to be considered acceptable for sample calculations. At each working standard concentration, the peak areas of standards injected at the beginning and end of a set of samples should not increase or decrease by more than 10%. (b) Sample concentration. — Note : All cis -lutein isomer concentrations are calculated from the trans -lutein curve. All cis- β-carotene isomer concentrations are calculated from the trans- β-carotene curve, and all cis- lycopene isomer concentrations are

C C cis − Lut (

D D R A SW R × ) ( ×

= × × × × . 1 43

×

10

)

DS

1

2

TW

1

PW

where C cis -Lut = cis -lutein concentration in µg/100g; C DS = lutein concentration extrapolated from the calibration curve in µg/L; 1.43 = correction factor when calculating cis -lutein from the trans -lutein curve; D 1 = first dilution volume in milliliters (10 mL extraction solution); D 2 = second dilution volume in milliliters (0.25 mL dilution solution); R TW = total reconstitution weight in grams, if applicable; A 1 = aliquot of the first dilution pipetted; SW = sample weight in grams; R PW = weight of powder reconstituted in grams, if applicable; and 10 = conversion to 100 g. ( 3 )  cis- and trans-β-carotene and lycopene sample concentration. — where C cis,trans -BC,Lyc = cis- or trans -β-carotene or lycopene concentration in µg/100 g; C DS = cis- or trans -β-carotene or lycopene concentration extrapolated from the calibration curve in µg/L; D 1 = first dilution volume in milliliters (10 mL extraction solution); D 2 = second dilution volume in milliliters (0.25 mL dilution solution); R TW = total reconstitution weight in grams, if applicable; A 1 = aliquot of the first dilution pipetted; SW = sample weight in grams; R PW = weight of powder reconstituted in grams, if applicable; and 10 = conversion to 100 g. ( 4 ) Total lutein, β-carotene, or lycopene concentration.— Total lutein, β-carotene, or lycopene concentration (µg/100 g) = concn of each cis isomer (µg/100 g) + concn of trans isomer (µg/100 g) C C D D R A SW R ) ( = × × × cis trans , ( ) − × × × BC,Lyc DS TW PW 1 2 1 10

References: J. AOAC Int . 100 , 264(2018) DOI: 10.5740/jaoacint.17-0287 AOAC SMPR 2014.014 J. AOAC Int. 98 , 1077–1078(2015) DOI: 10.5740/jaoac.int.SMPR2014.014

calculated from the trans- lycopene curve. ( 1 ) trans-Lutein sample concentration. —

C C D D R A SW R t − = × × × × × × Lut DS TW PW ( ) ( ) 1 2 1 10

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