VITAMIN A FORTIFIed PEANUT BUTTER - The Official Website of ...

mixture to 250 mL and mixed thoroughly. Twenty mL of sample solution was transferred to a 250 mL
separatory funnel containing 8 mL and 20 mL hexane (HPLC grade, Merck Darmstadt, Germany), and
partitioned by vigorously shaking the solution for 1 min. Sufficient time was allowed for the layer to
separate, and the hexane layer (yellow) was extracted and washed with distilled water. Vitamin A retinol
was eluted with 15% acetone (analytical grade) in a chromatographic tube (C18, 200 mm x 12 mm ID)
packed with adsorbent (alumina, activity grade 1, No. A-950, Fisher Scientific).
Beta-carotene from chocolate-peanut spread samples fortified with 10% beta-carotene was
extracted using the same procedure for vitamin A extraction with modifications. From the extracted
hexane layer, beta-carotene was eluted in the chromatographic tube using 4% acetone in hexane solution
(15%) as eluant.
Preparation of Standards
The retinol and beta-carotene standards were obtained from Sigma (St. Louis, Mo., U.S.A.) and
E. Merck (Darmstadt, Germany, respectively. To prepare the retinol stock solution, one capsule of USP
vitamin A (retinol) reference standard (approximately 0.2-0.3 g) was added with 20 mL 70% KOH and 80
mL 95% ethanol and saponified for 30 min. The cooled standard solution was placed in an amber 250
mL solution flask. Ethanol and water (3+1) was added up to the mark. From the stock solution, an
intermediate solution of 10 μg/mL was prepared. From the intermediate solution three to four series of
standard with different concentrations depending on the concentration of retinol in the sample, were
prepared. For the chocolate-peanut spread samples, the retinol standards used ranged from 0.01 to 0.5
µg/mL.
For beta-carotene stock solution, 0.0026 g of the standard was weighed in a 25 mL volumetric
flask, dissolved and diluted to volume with hexane. An intermediate solution was prepared by diluting 5
mL of the stock solution with hexane up to the mark of a 50 mL volumetric flask. Depending on the
approximate concentration of the beta-carotene in the sample, 3 to 4 series of standard solutions were
prepared from the intermediate solution. For the determination of beta-carotene in stabilized chocolatepeanut
spread, the standards used had concentrations of 0.125, 0.25 and 0.5 µg/mL.
HPLC quantification of vitamin A
Twenty µL of eluate was injected to HPLC (Shimadzu 10AVP, Shimadzu Corp., Columbia, Md.,
U.S.A.). Separation was achieved through a reverse phase column (Purospher RP 18e, 5 µm, 15-4,
Merck, Darmstadt, Germany) equipped with column oven at 28 C and UV detector set to 326 nm. The
analysis was carried out with 92% methanol (HPLC grade, Merck, Darmstadt, Germany) as the mobile
phase at a flow rate of 1 mL/min. The concentration of retinol was calculated using the average peak
areas compared between standards and samples. Results were reported as µg RE.
UV spectrophotometric quantification of beta-carotene
The resulting eluate from peanut butter samples fortified with beta-carotene was monitored to UV
Spectrophotometer (Model Lambda 20, Perkin Elmer, Norwalk, Connecticut, U.S.A.) at 436 nm.
Absorbance was compared with the standard carotene curve read at the same wavelength and
concentration of beta-carotene was calculated. Results were reported as µg ß-carotene/g. All betacarotene
analyses were run in duplicate. ß-carotene was converted to vitamin A (retinol) using the
equation:
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