Materials and methods
The following materials were purchased: peroxide test strips, 0–100 ppm (Merckoquant, EM Science, Gibbstown, NJ); ascorbic
acid (AA), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), citric acid (CA), methionine (METH), sodium ascorbate
(SA), sucrose (SU), and tocopherol (vitamin E [VE]), 2-propanol, high performance liquid chromatography grade and 3% w/w hydrogen
peroxide (Spectrum Chemicals, Gardena, CA); and ELPG (Riba-Fairfield, Decatur, IL).
ELPG stability study
. Partially oxidized ELPG (30 ppm peroxide) was dispensed (15 mL) into 20 glass scintillation vials (VWR International, West
Chester, PA). The following antioxidants were selected from the GRAS list: AA, BHA, BHT, CA, METH, SA, SU, and VE. Each antioxidant
(3% w/v) was weighed individually into a separate scintillation vial. This concentration was above the saturation solubility
for AA, CA, METH, SA, and SU (i.e., residual undissolved powder was observed in each of these containers at 25 °C). Additionally,
two control groups were prepared: a control group with an ambient-air head (C1) and one control group containing a nitrogen
head (N2). A replicate study of the remaining 10 scintillation vials was also prepared for storage at 40 °C. During the six-month
stability test, each vial was opened daily for the first seven days, and peroxide levels were determined using peroxide test
strips. N2 vials were sealed with a septum, and nitrogen was flushed through to obtain a fresh nitrogen head daily. After
seven days, the samples were checked at one, two, four, and six months to determine peroxide levels. Additionally, samples
were observed and photographed during the same sampling periods, and a scoring system of 1–5 was applied for yellow discoloration
(i.e., 5 being the most discolored and 1 being a colorless solution). Ethyl pyruvate, the degradation product from the oxidation
process, is known to have a yellow color.
Solubility of AA in ELPG
. An ultraviolet–visible (UV–vis) spectrophotometric determination of AA concentration in ELPG was used in a method developed
from Memon et al. (12). A solution was prepared by dissolving 1,10-phenanthroline monohydrate (1.97 g) into 1.0 N HCl (10
mL). Iron (III) ammonium sulfate (1.67 g) was added to the solution before dilution with deionized water to make a 1-L stock
solution. This iron (III)-tris-1,10-phenanthroline complex (I3PC) has no absorption peak in the visible spectrum (at 510 nm)
until it is reduced to the iron (II)-tris-1,10-phenanthroline complex (I2PC).
To determine the maximum wavelength of absorption, I2PC was prepared by adding a 5-mg/mL ascorbic-acid solution (5 mL) to
a 5% v/v solution of the I3PC stock solution (5 mL) prepared as previously described. A scan of the resultant solution revealed
a maximum absorption wavelength (λ max) of 510 nm. Absorption of this solution was checked at 0.5, 1, 5, 10, 15, 20, 30, and
60 min to determine the stability of the I2PC formed in solution.
Aliquots of 5% v/v I3PC stock solution (3.5 mL) were added to separate aliquots of AA standard solutions (6.5 mL) prepared
at 1, 2, 3, 4, 5, 7, and 10 mg/L. Each of the seven samples was allowed to react for precisely 10 min, prior to analysis.
A calibration curve was constructed at 510 nm. AA (1.5 g) was weighed into 10 separate brown-glass scintillation vials (because
AA is photosensitive), and ELPG (5 mL) was added. The vials were sealed and shaken at 25 °C (five vials) and at 37 °C (five
vials) for 48 h. An aliquot (1 mL) of each vial was dispensed into 1.5-mL centrifuge tubes, and the ELPG was evaporated to
dryness in a vacuum desiccator for 24 h. Each dried sample was reconstituted into 1 L of deionized water, and the same procedure
for determination of the calibration curve as previously described was used to ascertain each unknown concentration.
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