371 / 696
3/6
(6) Mobile phases and gradient. A. 7.5 g/L ammonium acetate in MeOH. B. ACN. C. IPA. D. ACN/water (1/1,
V/V).
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, zeaxanthin = 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%).
Table 3 summarizes the results for carotenoids obtained for the samples from the (second) SLV Kit. Please note
that none of the samples contain Lycopene and only one of the samples contains alpha-carotene.
Figure 1 shows an example chromatogram of lutein and beta-carotene in an Infant Formula sample. Figure 2
shows an example chromatogram of carotenoids in a vegetable mixture which is used since none of the samples
in the SLV Kit contain all carotenoids and standards are both expensive and instable. Zeaxanthin co-elutes with
cis-isomers from lutein. Cis-isomers from alpha-carotene co-elute with cis-isomers from beta-carotene. This is the
best chromatographic method available at this moment. Zeaxanthin will not be present in IF or AN and if so, it can
be discriminated from lutein using the spectrum and elution time. With optimal chromatographic separation and
careful identification of peaks, cis-isomers from alpha-carotene can be discriminated from cis-isomers from beta-
carotene.
Carot-01 (February 2016)
FOR ERP USE ONLY
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