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Gradient:
time (min)
% A
% B
% C
% D
0
51.1
36.2
1.9
10.8
10
51.1
36.2
1.9
10.8
20
54
44
2.0
0
36
54
44
2.0
0
36.1
51.1
36.2
1.9
10.8
41
51.1
36.2
1.9
10.8
(b) System suitability test.
Equilibrate the chromatographic system for ≥0.5 h. Inject a working standard solution at
least three times and check peak retention times and responses (peak area). Inject working standard solutions on
a regular basis within a series of analyses.
(c) Calibration.
Make single injections of HPLC standard solution at least at the beginning and the end of each
analytical series. Establish the calibration curve (one point through zero) by plotting peak response (area) ratios of
beta-carotene/DMT versus beta-carotene concentration. Perform linear regression. Calculate the slope (S) of the
calibration curve. Other carotenoids are determined using relative response factors.
(d) Analysis.
Make single injections of sample solutions.
(e) Identification.
Identify the carotenoid peaks in the chromatograms of the sample solutions by comparison with
the retention times and UV spectra of the corresponding peaks in the standard solution.
H. Calculations.
Summarize peak areas of trans and cis isomers and calculate the content of carotenoids in µg/ 100 g product “as
is”, as follows:
C = ((Ap / AISp) / (As / AISs)) x (Cs / CIS) x (aIS / w) x RRF x 1000
Where Ap = peak area of the carotenoid in the sample, AISp = peak area of DMT in the sample, As = peak area of
the carotenoid in the standard , AISs = peak area of DMT in the standard, Cs = concentration of the carotenoid in
the HPLC standard in mg/L, CIS = concentration of DMT in the HPLC standard in mg/L, aIS = amount of DMT added
to the sample in µg, w = weight of the sample in g, RRF = relative response factor (see below), and 100 is a
conversion factor.
Relative response factors of carotenoids relative to beta-carotene are: Lutein = 1.06, zeaxanthine = 0.98, lycopene
= 1.21 and alpha-carotene = 0.90.
Results
A summary of (preliminary) validation results is shown in tables 1 and 2. Calibration was performed with DMT at
50 mg/L and beta-carotene at 0.01 – 10 mg/L. The area-ratio is linear through the intercept with R
2
= 0.9993.
Validation results for lutein show that the area-ratio is linear through the intercept from 0.02 – 3.3 mg/L with R
2
=
0.9999. Accuracy of beta-carotene and lutein (total of trans and cis isomers) is between 97 and 109%.
Repeatability ranged from 0.9 to 4.5% (RSD
r
), and intermediate reproducibility results will be available shortly
(RSD
R
). Since SRM 1849A does not contain beta-carotene and lutein, the Global control sample from Wyeth was
analyzed. Results are: total beta-carotene = 1230 mcg/kg (DV = 3.3%) and total lutein = 1180 mcg/kg (DV = 5.3%).
Figure 1 shows an example chromatogram.
CAROT-01
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE