High Fructose Corn Syrup
DEFINITION
High Fructose Corn Syrup is a sweet, nutritive saccharide mixture prepared as a clear, aqueous solution from high-dextrose-equivalent corn starch hydrolysate by the partial enzymatic conversion of dextrose to fructose, using an insoluble glucose isomerase enzyme preparation that complies with 21 CFR 184.1372. It is available in two types, 42% and 55%, based on fructose content. High Fructose Corn Syrup 42% contains NLT 97.0% of total saccharides, expressed as a percentage of total solids, of which NLT 92.0% consists of monosaccharides (fructose and dextrose), including NLT 41.5% and NMT 44.8% of fructose, and NMT 8.0% consists of other saccharides. High Fructose Corn Syrup 55% contains NLT 95.0% of total saccharides, expressed as a percentage of total solids, of which NLT 95.0% consists of monosaccharides (fructose and dextrose), including NLT 54.5% and NMT 56.5% of fructose, and NMT 5.0% consists of other saccharides.
IDENTIFICATION
•  A.
Analysis:  Add a few drops of a solution (1 in 10) of Syrup to 5 mL of hot, alkaline cupric tartrate TS.
Acceptance criteria:  A copious, red precipitate of cuprous oxide is formed (distinction from sucrose).
ASSAY
•  Procedure
Mobile phase:  Water
Standard solution:  Prepare a solution in water containing a total of 10% saccharide solids of USP Dextrose RS, USP Fructose RS, and USP Maltose Monohydrate RS, in which the USP Dextrose RS and USP Fructose RS percentage concentrations are in the same ratio as those in the Sample solution, based on the labeled nominal fructose percentage for the Syrup under test.
Calculate the percentage of USP Maltose Monohydrate RS:
Result = 100 (F + D)
F== labeled nominal fructose percentage for the Syrup under test
D== difference between the specified minimum percentage concentration of total monosaccharides for the Syrup and F
Sample solution:  Dilute a known weight of Syrup, determined from the results of the test for Total Solids and on the nominal total saccharides content, with water to a total saccharides concentration of 10% (w/v), and mix.
Chromatographic system 
Mode:  LC
Detector:  Refractive index
Column:  7.8-mm × 30-cm; packing L19
Temperature 
Detector:  45
Column:  85
Flow rate:  0.6 mL/min
Injection size:  10 µL
System suitability 
Sample:  Standard solution
[Note—The relative retention times for maltose, dextrose, and fructose are about 0.83, 1.0, and 1.32, respectively. ]
Suitability requirements 
Resolution:  NLT 1.2 between maltose and dextrose
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of fructose and of dextrose in the portion of Syrup taken:
Result = (rU/rS) × CS × {VA/[WS × (0.01 × S1) × (0.01 × S2)]} × 100
rU== peak area of fructose or dextrose from the Sample solution
rS== peak area of fructose or dextrose from the Standard solution
CS== concentration of USP Fructose RS or USP Dextrose RS in the Standard solution (mg/mL)
VA== volume of the Sample solution (mL)
WS== weight of Syrup taken to prepare the Sample solution (mg)
S1== percentage of total saccharides in the Syrup as specified on the label
S2== percentage of total solids in the Syrup as determined in the test for Total Solids
Calculate the percentage of other saccharides, expressed in terms of maltose, in the portion of Syrup taken:
Result = (rU/rS) × CS × {VA/[WS × (0.01 × S1) × (0.01 × S2)]} × 100
rU== sum of all peak areas from the Sample solution, except those of fructose and dextrose
rS== peak area of maltose from the Standard solution
CS== concentration of USP Maltose Monohydrate RS in the Standard solution (mg/mL)
VA== volume of the Sample solution (mL)
WS== weight of Syrup taken to prepare the Sample solution (mg)
S1== percentage of total saccharides in the Syrup as specified on the label
S2== percentage of total solids in the Syrup as determined in the test for Total Solids
Acceptance criteria 
For High Fructose Corn Syrup 42% 
Total saccharides:  NLT 97.0%, expressed as a percentage of total solids. Total saccharides contain monosaccharides and other saccharides as follows.
Monosaccharides:  NLT 92.0% (fructose and dextrose)
Fructose:  41.5%–44.8%
Other saccharides:  NMT 8.0%
For High Fructose Corn Syrup 55% 
Total saccharides:  NLT 95.0%, expressed as a percentage of total solids. Total saccharides contain monosaccharides and other saccharides as follows.
Monosaccharides:  NLT 95.0% (fructose and dextrose)
Fructose:  54.5%–56.5%
Other saccharides:  NMT 5.0%
IMPURITIES
•  Residue on Ignition 281: NMT 0.05%
•  Heavy Metals, Method II 231: NMT 5 ppm, using an ignition temperature of 500
•  Limit of Lead
[Note—For the preparation of all aqueous solutions and for the rinsing of glassware before use, use water that has been passed through a strong-acid, strong-base, mixed-bed ion-exchange resin. For digestion, use acid-cleaned, high-density polyethylene, polypropylene, polytef, or quartz tubes. Select all reagents to have as low a content of lead as practicable, and store all reagent solutions in borosilicate glass containers. Cleanse glassware before use by soaking in warm 8 N nitric acid for 30 min and rinsing with deionized water. Store final diluted solutions in acid-cleaned plastic or polytef tubes or bottles. ]
Modifier solution:  200 mg/mL of magnesium nitrate. Just before use, transfer 1.0 mL of this solution to a 10-mL volumetric flask, and dilute with 5% nitric acid to volume.
Standard stock solution:  Transfer 10.0 mL of Lead Nitrate Stock Solution, prepared as directed in Heavy Metals 231, to a 100-mL volumetric flask, add 40 mL of water and 5 mL of nitric acid, and dilute with water to volume. Transfer 1.0 mL of this solution to a second 100-mL volumetric flask, dilute with 5% nitric acid to volume, and mix. This solution contains 0.1 µg/mL of lead.
Standard solutions:  Transfer portions of Standard stock solution to four suitable containers, and dilute with 5% nitric acid to obtain Standard solutions having lead concentrations of 100, 50, 25, and 10 ng/mL, respectively.
Sample solution:  [Note—Perform this procedure in a fume hood. ] Transfer 1.5 g of Syrup to two digestion tubes, labeled “Sample solution” and “Temperature monitor solution”, and add 0.75 mL of nitric acid to each tube. Warm both solutions slowly to 90–95 to avoid spattering. Heat until all brown vapors have dissipated and any rust-colored tint is gone from the tube labeled “Sample solution” (20–30 min). Cool, add 0.5 mL of 50% hydrogen peroxide dropwise to both solutions, heat to 90–95 for 5 min, and cool. Add a second 0.5-mL portion of 50% hydrogen peroxide dropwise to each solution, and heat to 90–100 for 5–10 min until the tube labeled “Sample solution” is clear. Cool, and transfer the Sample solution to a 10-mL volumetric flask. Rinse the tube labeled “Sample solution” with 5% nitric acid, add the rinsing to the volumetric flask, dilute with 5% nitric acid to volume, and mix.
Standard blank:  5% nitric acid
Sample blank:  Transfer 1.5 g of water to a digestion tube, and proceed as directed for the Sample solution, beginning with “add 0.75 mL of nitric acid”.
Instrumental conditions 
Mode:  Graphite furnace atomic absorption with pyrolytically coated graphite tubes and adequate means of background correction
Lamp:  A lead hollow-cathode
Analytical wavelength:  Lead emission line of 283.3 nm
Furnace program:  See Table 1. [Note—The temperature program may be modified to obtain optimum furnace temperatures. ]
Table 1
Step Dry Ash Purge Atomize
Temperature () 200 750 Cool down, and purge the air from the furnace
20
1800
Ramp time (s) 20 40 0
Hold time (s) 30 40 60 10
Argon flow rate
(mL/min)
300 300 300 Argon gas flow stopped
Injection volume:  20 µL
Analysis 
[Note—Use peak area measurements for all quantitations. ]
Samples:  Add 5 µL of the Modifier solution to 20 µL each of the Standard solutions, the Sample solution, the Standard blank, and the Sample blank, and mix.
Separately inject equal volumes (about 20 µL) of the Samples into the instrument for analysis.
Using the Standard blank to set the instrument to zero, determine the integrated absorbances of the Standard solutions. Plot the integrated absorbances of the Standard solutions versus their contents of lead, in ng/mL, and draw the line best fitting the four points to determine the calibration curve. Similarly determine the integrated absorbances of the Sample solution and the Sample blank. Correct the absorbance value of the Sample solution by subtracting from it the absorbance value obtained from the Sample blank.
Calculate the concentration of lead, in ppm (µg/g), in the portion of Syrup taken:
Result = (V × CL/W) × F
V== volume of the Sample solution, 10 mL
CL== concentration of lead in the Sample solution, as determined from the calibration curve (ng/mL)
W== weight of Syrup taken to prepare the Sample solution (g)
F== conversion factor, 10-3 µg/ng
Acceptance criteria:  NMT 0.1 ppm (µg/g)
•  Limit of Sulfur Dioxide
Starch indicator solution:  Mix 10 g of soluble starch with 50 mL of cold water. Transfer to 1000 mL of boiling water, stir until completely dissolved, cool, and add 1 g of salicylic acid preservative. [Note—Discard the solution after 1 month. ]
Sample:  100 g
Titrimetric system 
Mode:  Direct titration
Titrant:  0.005 N iodine VS
Blank:  200 mL of water
Endpoint detection:  Visual
Analysis:  Transfer the Sample to a 250-mL conical flask, add 100 mL of water, and mix. Cool to 5–10. While stirring with a magnetic stirrer, add 10 mL of cold (5–10) 1.5 N sodium hydroxide. Stir for an additional 20 s, and add 10 mL of Starch indicator solution. Add 10 mL of cold (5–10) 2.0 N sulfuric acid, and titrate immediately with Titrant until a light blue color persists for 1 min. Perform a blank determination, and make any necessary correction.
Calculate the concentration, in ppm (µg/g), of sulfur dioxide (SO2) in the Sample taken:
Result = {[(VS VB) × N × F1]/W} × F2
VS== Titrant volume consumed by the Sample (mL)
VB== Titrant volume consumed by the Blank (mL)
N== actual normality of the Titrant (mEq/mL)
F1== equivalency factor, 32.0 mg/mEq
W== Sample weight (g)
F2== conversion factor, 103 µg/mg
Acceptance criteria:  NMT 30 ppm
SPECIFIC TESTS
•  Microbial Enumeration Tests 61 and Tests for Specified Microorganisms 62: The total aerobic microbial count does not exceed 103 cfu/g, and the total combined molds and yeast count does not exceed 102 cfu/g.
•  Total Solids: Determine the refractive index of the Syrup at 20 or 45 (see Refractive Index 831). Use Table 2 for calculating the percentage of dry substance (percentage of total solids on a weight/weight basis).
Table 2
Fructose Content
(%)
Dry Substance (DS)
(%)
Refractive Index at 20 Refractive Index at 45
42 70.5 1.4632 1.4577
71.0 1.4643 1.4589
72.0 1.4667 1.4612
73.0 1.4691 1.4635
55 76.5 1.4774 1.4716
77.0 1.4786 1.4728
78.0 1.4811 1.4752
79.0 1.4835 1.4776
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight containers. No storage requirement specified.
•  Labeling: Label it to state, as part of the official title, the nominal percentage of fructose, based on the specified minimum percentage concentration of total saccharides. Label it to indicate the presence of sulfur dioxide if the residual concentration is greater than 10 ppm (µg/g).
•  USP Reference Standards 11
USP Dextrose RS Click to View Structure
USP Fructose RS Click to View Structure
USP Maltose Monohydrate RS Click to View Structure
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USP35–NF30 Page 1768
Pharmacopeial Forum: Volume No. 34(2) Page 329