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Measurement and Stability of Aspartame in a Fruit Spread
Can. Ins. Food Sci. Techno/. J. Vol. 19, No.2, pp. 86-88. 1986
RESEARCH NOTE
Measurement and Stability of Aspartame in a Fruit Spread M.e. Dever, H.J.T. Beveridge, D.B. Cumming and D.R. MacGregor Agriculture Canada Research Station Summerland, B.C. YOH IZO
.and a cyclization reaction forming diketopiperazine (Dwivedi, 1978). Influences of temperature and pH on the stability of aspartame in model food systems have been determined. Mazur (1976) found that stability was primarily a function of pH and predicted a halflife of 6-10 mo for aspartame in an aqueous solution over a pH range of 3.0 to 5.0, at 25°C. Stamp and Labuza (1983) found the Maillard reaction involving aspartame to be temperature sensitive, resulting in a significant loss of aspartame at higher process temperatures (80-100° C). A literature search revealed little information on the retention of aspartame in a food medium such as a low calorie fruit spread. In this study, an HPLC method for determining aspartame levels in such a medium was developed and used to examine the retention of aspartame in an apricot spread stored at 25°e.
Abstract A procedure was developed for extraction and quantitation of aspartame from a fruit spread. Quantitation was by HPLC using a reverse phase column with ion pairing and detection at 260 nm. Coefficients of variation for paired extractions at different aspartame levels ranged from 2.7 to 7.1. The standard curve prepared in fruit media had R2 values of 0.98 or 0.99 (p ~ .01). The method was applied in a 6-month storage trial of an aspartame sweetened apricot spread (pH 3.2). Aspartame storage losses were modeled as a first order reaction with an average half life of 168 days.
Resume Une procedure fut developpee pour I'extraction et la determination de I'aspartame dans une tartinade aux fruits. La determination fut faite par HPLC munie d'une colonne a phase inversee avec accouplage ionique et detection a 260 nm. Les coefficients de variation des extractions couplees a differents niveaux d'aspartame varierent de 2.7 a 7.1. La courbe de reference etablie avec un milieu aux fruits eut des valeurs R 2 de 0.98 ou 0.99 (p ~ 0.01). La methode fut appliquee a une tartinade d'abricot sucree a I'aspartame et entreposee jusqu'a 6 mois. Les pertes de I'aspartame a l'entreposage furent representatives d 'une reaction de premier ordre avec une demi-vie moyenne de 168 jours.
Materials and Methods
Fruit Spread Preparation The solubility of aspartame in water-based products at 25°C and isoelectric point pH 5.2 is 1%, rising to a maximum of 8070 at pH 2.2 (Dwivedi, 1978). This limited solubility, combined with a tendency to clump on addition to aqueous media required that aspartame be dissolved in water before incorporation into the spread. To minimize the effects of heat, the aspartame solution was added to a fruit spread concentrate (Table 1) after the cooking process. The aspartame (Equa 200) was obtained from Searle Biochemics (Arlington Heights, Illinois). A fill ratio of 20 parts of 1% aspartame solution to 80 parts concentrate (by weight) gave a final concentration of 0.2% aspartame of 2 mg/g of product. The I % solution was dispensed by a calibrated automatic pipette into 150 mL white enamel lined cans and the concentrate was hot filled to a previously determined level. Cans were inverted for 30 sec to sterilize the headspace and lids, cooled 5 min in a
Introduction Aspartame is the name given to the chemical compound I-methyl N-L aspartyl-L-phenylalanine. It has a clean taste with a sweetening potency 150-200 times that of sucrose, depending on the food system sweetened (Mazur, 1976). Consequently, there exist many potential applications in the formulation of calorie reduced foods. At moisture levels above 8070, aspartame is unstable producing breakdown products which are not sweet so that during storage, foods formulated with aspartame demonstrate a decrease in sweetness. Decomposition reactions include hydrolysis of the dipeptide ester to methanol and aspartylphenylalanine
Contribution No. 630
Copyright
<0
1986 Canadian Institute of Food Science and Technology
86
Chromatography
Table I. Composition of an apricot concentrate Component Apricots (diced and mashed) Water Citric Acid Ca. Lactate Sorbate L.M. Pectin ILevels set before cooking the mixture.
070 1
64.0 33.9 0.6 0.25 .40
1.25
water bath (45°C) and then shaken to thoroughly mix the contents.
Extraction of Aspartame For each analysis, 5 cans of aspartame sweetened spread were blended in a Waring blender for 1 min and extractions were made from the blended sample. For each extraction, 15 g of sample was combined with 15 g of methanol and mixed on an orbital shaker at 250 RPM for 15 min. The samples were centrifuged in a Sorval RC-5 (Dupont Instruments; Newtown, Conn.) at 12,000 RPM (17300 g) and the supernatant decanted into a vacuum flask. A second extraction of the same sample, using 10 g of methanol, was made in· an identical manner. The combined methanol extracts were evaporated to dryness under vacuum (68 cm Hg) at 30°C using a Brinkman Rotavapor-E 1 [Brinkman Instruments (Canada) Ltd.]. Water (15 mL) was added and the mixture swirled until the residue was dissolved. The resulting solution was filtered through a 0.2 j.tm filter into 1 mL sample vials.
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Analysis of aspartame content in the extracts was done on a Hewlett Packard (HP) 1084B liquid chromatograph with an HP 798508 recording integrator.The column was a HP reverse phase C-8 (10 j.tm). The mobile phase was 70010 buffer and 30% (v/v) methanol (HPLC grade). The buffer (90% double distilled H 20, 10% acetic acid vlv, pH 2.3) contained 1 giL ammomium chloride as an ion pairing agent. This solvent was filtered through a 0 .2 j.t filter and degassed before it was used. Solvent and oven temperatures were 40°C and flow rate was 1.5 mL/min. The variable wavelength V.V. detector was set at 260 nm. A slope sensitivity of .1 and tangent skim integration produced the most consistent results.
Standard determination A standard curve was determined at each sampling date from extracts of fruit spreads with aspartame levels ranging from 0 to 2 mglg. These fruit spreads were prepared by adjusting the percentage of aspartame in a water solution and then combining with a canned apricot concentrate in a 1 to 4 ratio by weight. No heat was applied and extractions were done immediately.
Storage Trial An aspartame sweetened spread was prepared as previously described and stored at 25°C. The retention of aspartame was determined throughout the 6-mo storage period. Five canned samples were taken at 2 w intervals for the first 2 mo and then at monthly intervals. The samples were blended and 5 extractions were made from the blended sample with a separate aspartame analysis for each extraction.
Results and Discussion An unindentified substance was found to elute at the same time as aspartame (Figure 1). The resulting peak had a low area count but was sufficient to pre~
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LOG AR: 0.240 - 1.79 X 10-
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Fig. I. A) A typical chromatogram of an extract of fruit spread sweetened with 2 mg/g aspartame. Retention time (min) for aspartame shown at peak apex is 4. J 8. B) Chromatogram of a fruit spread which does not contain aspartame showing a small peak eluting at the same time (4.17) as aspartame. Can. {nSf. Food Sci. Techno!. J. Vol. 19. No.2. 1986
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120
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TIME (DAYS)
Fig. 2. Plot of the log of the average (n = 5) aspartame content for apricot spread extracts against days of storage at 25°C. Dever et al. / 87
vent using aspartame/water solutions for the standard curve. Attempts to remove this peak by sample extraction with chloroform, butanol or hexane were unsatisfactory since varying amounts of aspartame were also removed. At each sampling date a standard curve with R2 values of 0.98 or 0.99 was determined from 2-4 extractions of apricot spreads having aspartame concentrations of .75 mg/g, 1.0 mg/g, 1.25 mg/g, 1.5 mg/g and 2.0 mg/g. The coefficients of variation for paired extractions at each aspartame level were 7.11, 5.56, 4.48, 4.69 and 2.76. The relationship described in Figure 2 (log of the average aspartame content for 5 extractions plotted against time) demonstrates a rate constant of 4.12 x 10- 3 ± 5.2 x 10- 4 day-I (± value is a 95070 confidence interval) and indicates an average half-life of 168 d for aspartame in a fruit spread stored at 25°C. The decomposition of aspartame in acidic aqueous solution is thought to be predominantly a hydrolysis reaction (Crosby, 1976) following 1st order reaction kinetics (Searle, 1982). Mazur (1976) found stability at 25°C to be pH dependent with other food components having little effect. His pH stability profile, which plotted half-life of aspartame against pH values
88 / Dever et al.
ranging from I to 8, indicates a 260 d maximum halflife at pH 4.3. At pH 3.2 (the pH of our apricot spread measured before dilution) the· predicted half-life was approximately 170 d. The 168 d average half-life of aspartame in an apricot spread as determined by this storage trial is in excellent agreement with those results. This would indicate that the Maillard reaction found by Stamp and Labuza (1983) to contribute to aspartame loss at higher storage temperatures is not a factor at 25°C.
References Crosby, G.A. 1976. New Sweeteners. Critical Reviews in Food Science and Nutrition. 8:297. Dwivedi, B.K. 1978. Low Calorie and Special Dietary Foods, CRC Press Inc. West Palm Beach, Florida. Mazur, R.H. 1976. Aspartame a Sweet Surprise. J. Toxicol. Environ. Health. 2:243. Searle. 1982. The Nutra Sweet T.M. Breakthrough. Technical Bulletin. G.D. Searle & Co. Stamp, J.A. and Labuza, T.P. 1983. Kinetics of the Maillard Reaction between Aspartame and Glucose in Solution at High Temperatures. J. Food Sci. 48:543. Submitted July 31, 1985 Revised September 28, 1985 Accepted October 15, 1985
J. Ins!. Can. Sci. Technal. Aliment. Vol. 19. No.2. 1986
RESEARCH NOTE
Measurement and Stability of Aspartame in a Fruit Spread M.e. Dever, H.J.T. Beveridge, D.B. Cumming and D.R. MacGregor Agriculture Canada Research Station Summerland, B.C. YOH IZO
.and a cyclization reaction forming diketopiperazine (Dwivedi, 1978). Influences of temperature and pH on the stability of aspartame in model food systems have been determined. Mazur (1976) found that stability was primarily a function of pH and predicted a halflife of 6-10 mo for aspartame in an aqueous solution over a pH range of 3.0 to 5.0, at 25°C. Stamp and Labuza (1983) found the Maillard reaction involving aspartame to be temperature sensitive, resulting in a significant loss of aspartame at higher process temperatures (80-100° C). A literature search revealed little information on the retention of aspartame in a food medium such as a low calorie fruit spread. In this study, an HPLC method for determining aspartame levels in such a medium was developed and used to examine the retention of aspartame in an apricot spread stored at 25°e.
Abstract A procedure was developed for extraction and quantitation of aspartame from a fruit spread. Quantitation was by HPLC using a reverse phase column with ion pairing and detection at 260 nm. Coefficients of variation for paired extractions at different aspartame levels ranged from 2.7 to 7.1. The standard curve prepared in fruit media had R2 values of 0.98 or 0.99 (p ~ .01). The method was applied in a 6-month storage trial of an aspartame sweetened apricot spread (pH 3.2). Aspartame storage losses were modeled as a first order reaction with an average half life of 168 days.
Resume Une procedure fut developpee pour I'extraction et la determination de I'aspartame dans une tartinade aux fruits. La determination fut faite par HPLC munie d'une colonne a phase inversee avec accouplage ionique et detection a 260 nm. Les coefficients de variation des extractions couplees a differents niveaux d'aspartame varierent de 2.7 a 7.1. La courbe de reference etablie avec un milieu aux fruits eut des valeurs R 2 de 0.98 ou 0.99 (p ~ 0.01). La methode fut appliquee a une tartinade d'abricot sucree a I'aspartame et entreposee jusqu'a 6 mois. Les pertes de I'aspartame a l'entreposage furent representatives d 'une reaction de premier ordre avec une demi-vie moyenne de 168 jours.
Materials and Methods
Fruit Spread Preparation The solubility of aspartame in water-based products at 25°C and isoelectric point pH 5.2 is 1%, rising to a maximum of 8070 at pH 2.2 (Dwivedi, 1978). This limited solubility, combined with a tendency to clump on addition to aqueous media required that aspartame be dissolved in water before incorporation into the spread. To minimize the effects of heat, the aspartame solution was added to a fruit spread concentrate (Table 1) after the cooking process. The aspartame (Equa 200) was obtained from Searle Biochemics (Arlington Heights, Illinois). A fill ratio of 20 parts of 1% aspartame solution to 80 parts concentrate (by weight) gave a final concentration of 0.2% aspartame of 2 mg/g of product. The I % solution was dispensed by a calibrated automatic pipette into 150 mL white enamel lined cans and the concentrate was hot filled to a previously determined level. Cans were inverted for 30 sec to sterilize the headspace and lids, cooled 5 min in a
Introduction Aspartame is the name given to the chemical compound I-methyl N-L aspartyl-L-phenylalanine. It has a clean taste with a sweetening potency 150-200 times that of sucrose, depending on the food system sweetened (Mazur, 1976). Consequently, there exist many potential applications in the formulation of calorie reduced foods. At moisture levels above 8070, aspartame is unstable producing breakdown products which are not sweet so that during storage, foods formulated with aspartame demonstrate a decrease in sweetness. Decomposition reactions include hydrolysis of the dipeptide ester to methanol and aspartylphenylalanine
Contribution No. 630
Copyright
<0
1986 Canadian Institute of Food Science and Technology
86
Chromatography
Table I. Composition of an apricot concentrate Component Apricots (diced and mashed) Water Citric Acid Ca. Lactate Sorbate L.M. Pectin ILevels set before cooking the mixture.
070 1
64.0 33.9 0.6 0.25 .40
1.25
water bath (45°C) and then shaken to thoroughly mix the contents.
Extraction of Aspartame For each analysis, 5 cans of aspartame sweetened spread were blended in a Waring blender for 1 min and extractions were made from the blended sample. For each extraction, 15 g of sample was combined with 15 g of methanol and mixed on an orbital shaker at 250 RPM for 15 min. The samples were centrifuged in a Sorval RC-5 (Dupont Instruments; Newtown, Conn.) at 12,000 RPM (17300 g) and the supernatant decanted into a vacuum flask. A second extraction of the same sample, using 10 g of methanol, was made in· an identical manner. The combined methanol extracts were evaporated to dryness under vacuum (68 cm Hg) at 30°C using a Brinkman Rotavapor-E 1 [Brinkman Instruments (Canada) Ltd.]. Water (15 mL) was added and the mixture swirled until the residue was dissolved. The resulting solution was filtered through a 0.2 j.tm filter into 1 mL sample vials.
<-
.;,'"
:!:
",.
OJ
'"
~ ~ <-
OJ
.;,
Analysis of aspartame content in the extracts was done on a Hewlett Packard (HP) 1084B liquid chromatograph with an HP 798508 recording integrator.The column was a HP reverse phase C-8 (10 j.tm). The mobile phase was 70010 buffer and 30% (v/v) methanol (HPLC grade). The buffer (90% double distilled H 20, 10% acetic acid vlv, pH 2.3) contained 1 giL ammomium chloride as an ion pairing agent. This solvent was filtered through a 0 .2 j.t filter and degassed before it was used. Solvent and oven temperatures were 40°C and flow rate was 1.5 mL/min. The variable wavelength V.V. detector was set at 260 nm. A slope sensitivity of .1 and tangent skim integration produced the most consistent results.
Standard determination A standard curve was determined at each sampling date from extracts of fruit spreads with aspartame levels ranging from 0 to 2 mglg. These fruit spreads were prepared by adjusting the percentage of aspartame in a water solution and then combining with a canned apricot concentrate in a 1 to 4 ratio by weight. No heat was applied and extractions were done immediately.
Storage Trial An aspartame sweetened spread was prepared as previously described and stored at 25°C. The retention of aspartame was determined throughout the 6-mo storage period. Five canned samples were taken at 2 w intervals for the first 2 mo and then at monthly intervals. The samples were blended and 5 extractions were made from the blended sample with a separate aspartame analysis for each extraction.
Results and Discussion An unindentified substance was found to elute at the same time as aspartame (Figure 1). The resulting peak had a low area count but was sufficient to pre~
-c 0
w
z C
~
.;
I-
w
0.3
LOG AR: 0.240 - 1.79 X 10-
R2
:
3
DAYS
0.986
0.2
ll:
A ~
'" 0:
:o---! .,:;::
"'..;
'"
W
..
'."
B ~
.,'" 0:
~
.,z
:I
U
",M
0-: N
f,. _
•
... ". "
Fig. I. A) A typical chromatogram of an extract of fruit spread sweetened with 2 mg/g aspartame. Retention time (min) for aspartame shown at peak apex is 4. J 8. B) Chromatogram of a fruit spread which does not contain aspartame showing a small peak eluting at the same time (4.17) as aspartame. Can. {nSf. Food Sci. Techno!. J. Vol. 19. No.2. 1986
C Ill: C
lL III
c
0.1
•
•
•
0.0
IL
0 <:l 0
-0.1
~
40
120
200
TIME (DAYS)
Fig. 2. Plot of the log of the average (n = 5) aspartame content for apricot spread extracts against days of storage at 25°C. Dever et al. / 87
vent using aspartame/water solutions for the standard curve. Attempts to remove this peak by sample extraction with chloroform, butanol or hexane were unsatisfactory since varying amounts of aspartame were also removed. At each sampling date a standard curve with R2 values of 0.98 or 0.99 was determined from 2-4 extractions of apricot spreads having aspartame concentrations of .75 mg/g, 1.0 mg/g, 1.25 mg/g, 1.5 mg/g and 2.0 mg/g. The coefficients of variation for paired extractions at each aspartame level were 7.11, 5.56, 4.48, 4.69 and 2.76. The relationship described in Figure 2 (log of the average aspartame content for 5 extractions plotted against time) demonstrates a rate constant of 4.12 x 10- 3 ± 5.2 x 10- 4 day-I (± value is a 95070 confidence interval) and indicates an average half-life of 168 d for aspartame in a fruit spread stored at 25°C. The decomposition of aspartame in acidic aqueous solution is thought to be predominantly a hydrolysis reaction (Crosby, 1976) following 1st order reaction kinetics (Searle, 1982). Mazur (1976) found stability at 25°C to be pH dependent with other food components having little effect. His pH stability profile, which plotted half-life of aspartame against pH values
88 / Dever et al.
ranging from I to 8, indicates a 260 d maximum halflife at pH 4.3. At pH 3.2 (the pH of our apricot spread measured before dilution) the· predicted half-life was approximately 170 d. The 168 d average half-life of aspartame in an apricot spread as determined by this storage trial is in excellent agreement with those results. This would indicate that the Maillard reaction found by Stamp and Labuza (1983) to contribute to aspartame loss at higher storage temperatures is not a factor at 25°C.
References Crosby, G.A. 1976. New Sweeteners. Critical Reviews in Food Science and Nutrition. 8:297. Dwivedi, B.K. 1978. Low Calorie and Special Dietary Foods, CRC Press Inc. West Palm Beach, Florida. Mazur, R.H. 1976. Aspartame a Sweet Surprise. J. Toxicol. Environ. Health. 2:243. Searle. 1982. The Nutra Sweet T.M. Breakthrough. Technical Bulletin. G.D. Searle & Co. Stamp, J.A. and Labuza, T.P. 1983. Kinetics of the Maillard Reaction between Aspartame and Glucose in Solution at High Temperatures. J. Food Sci. 48:543. Submitted July 31, 1985 Revised September 28, 1985 Accepted October 15, 1985
J. Ins!. Can. Sci. Technal. Aliment. Vol. 19. No.2. 1986