|
|
Profile 
JacquelineJoey 18 Ngee Ann Poly Disclaimer In this disclaimer, I wanted to welcome you in my blog and thanks for passing by or visiting by any chance in here. This blog is purely created for our INAC project. Hoep you have a great time here! :D Tagboard Archives  Recent posts:Credits Layout: Nicole and Dirah. Resources: Soonei and Sugarpink.  |
Results & Discussions
The chromatograms are recorded at λ = 295 nm which shows the
elution profile of δ, γ, and α-tocopherol in spiked and unspiked human milk are shown in Fig. 1.
The native amount of δ-tocopherol in human milk is negligible in
comparison with the amount of δ-tocopherol added as the internal standard.
The major forms of
vitamin E found in milk were α and γ-tocopherol and tocopherol acetate was present in minor amounts in
some milk samples.
Fig 2A. shows the recovery of the internal standard
(δ-tocopherol spike) from human milk using the two methods of extraction.
Key: Shaded
circles: Method I,
white circles: Method II.
A. Recovery of δ-tocopherol spike from pooled milk sample using two
methods of extraction. Regression analysis (Method I, y(x) = 17311x + 255, r2 =
0.999; Method II, y(x) = 10593x, r2 = 0.998) was performed by
plotting the integrated peak areas for δ-tocopherol against the concentration
of the spike.
In Fig. 2, the ranges of the amount of δ-tocopherol spike used in both methods are
reasonably comparable. In Method I, 0.7–28 μg/mL of δ-tocopherol was detected while in Method II,
1.49–37.3 μg/mL of δ-tocopherol was detected.
Recovery of the δ-tocopherol spike from pooled milk sample was within the physiological range of concentrations for both extraction methods. The correlation coefficients (r) obtained for assay of δ-tocopherol standard in milk samples by use of both methods were indicative of very good linearity of recovery of δ-tocopherol standard. This shows that there is a directly proportional relation between the δ-tocopherol spike and δ-tocopherol peak area. The ratio of the slope coefficients of the regression lines for Methods 1 and 2 was 1.6342, which shows that the they are in good agreement with the value of the ratio for average δ-tocopherol recovery in Methods 1 and 2 (100 and 60%, respectively;). The signal to noise ratio (S/N) for more than 3 the overall limit of detection of Method I (including saponification, extraction, and HPLC analysis) was 0.65 μg/mL δ-tocopherol in milk. (The higher the signal to noise ratio, the more accurate the results will be as there are less interferences from other compounds.) The percentages for recovery of the internal standard for the two methods of extraction are shown in Fig. 2A. When extraction was performed directly (without saponification), the recovery was approximately 60% and varies from range 19%–82% depending on the milk sample. On the other hand, the recovery after saponification was nearly 100% within a more reproducible range. There are two possible reasons for this improvement for the method with saponification. First, after saponification the milk matrix does not affect the recovery. Second, the conditions of liquid–liquid extraction with hexane from saponified milk are better optimised than in direct extraction. Reduced recovery of the internal standard leads to less accurate measurement. ----------------------------------------------------------------------------------------------------------------
Fig. 3 (below) shows the correlation of
levels of α
and γ-tocopherol in
milk samples measured after direct extraction and extraction after
saponification.
Fig. 3.
Correlation of the levels of
native α and γ-tocopherol in human milk samples, measured after use of the two
methods of extraction.
A. Regression analysis (y(x) = 0.063
+ 0.952x; r2
= 0.868; P < 0.001) was performed by plotting integrated peak
areas for native α-tocopherol in twenty milk samples, measured after extraction
using Method I, against integrated peak areas for native α-tocopherol in the
same samples measured after extraction using Method II.
B. Regression analysis for γ-tocopherol (y(x) = 0.01 + 1.07x; r2 = 0.851, P < 0.01) was performed in the same way as for α-tocopherol. Average and standard deviation of three measurements for the same milk specimen with the two methods are shown.
There was a significant relationship between the two methods of
extraction for both α-tocopherol and γ-tocopherol. Levels of the major form of vitamin E, α-tocopherol, in
milk samples were variable within the range 2.8–15 μg mL−1 (Method I: 7.2 ± 4.2 μg mL−1 , Method II: 7.0 ± 4.1 μg mL−1 ).
For determination of native concentration of α-tocopherol in human milk by Methods I and II, δ-tocopherol was used as the internal standard. Although we were able to demonstrate a significant correlation between results obtained with the two methods, the method including saponification gave higher and more consistent recoveries for the internal spike and, therefore, more reliable data. The proposed saponification step was a simple addition to the method, because it was performed in the same tube as the extraction, and did not involve use of an inert gas. |
