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Measure each standard measuring solution from E.(b) against an MTBE blank at its absorbance
maximum (444 nm for lutein and 450 nm for β-carotene). Calculate the spectrophotometric
purity of each reference standard as the observed absorbance over the expected absorbance:
SP = (Abs
MS
x 100 x 1000)/(E
1%,1cm
x W)
Where Abs
MS
= absorbance of the standard measuring solution
100 = dilution factor for stock solution to standard measuring solution
1000 = factor for g to mg
E
1%,1cm
= extinction coefficient (from Craft and Soares, 1992)
Lutein in MTBE: 2589 at 444 nm
β-Carotene in MTBE: 2588 at 450 nm
W = weight of reference standard, in mg
(2)
Chromatographic Purity (CP)
Inject standard working solutions E.(c) at least three times. The CP is calculated as:
CP = (area of the all-trans-carotenoid peak)/(sum of areas of all relevant peaks)
Relevant peaks include all peaks in the HPLC chromatogram with the exception of solvent peaks.
(3)
Reference Standard Purity (P)
Calculate the purity of each reference standard:
P(%) = SP x CP x 100
Where 100 is the conversion of decimal to percent
(b)
Calculate the concentration of each carotenoid analyte (e.g. lutein, C
L
) in the all-
trans
form, in
μg/100 mL, in the working solution:
C
L
= W
Lut
x P
Lut
x (1000/10,000)
Where W
Lut
= the weight of lutein used to make the stock solution, in mg
P
Lut
= the reference standard purity of all-
trans
-lutein calculated in section H.(a) above
1000 = the conversion of mg to μg
10,000 = the factor for the dilution of 1 mL stock in 100 mL working solution and for the
conversion of percent to decimal
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE