AOAC Official Method 980.

13
Fructose, Glucose, Lactose,
Maltose, and Sucrose in Milk Chocolate
Liquid Chromatographic Method
Final Action
(Caution: See Appendix B, safety notes on acetonitrile.)
A. Apparatus
(a) Liquid chromatograph.—With Waters Associates, Inc.
M6000A pump, R401 refractive index detector, or equivalent, and
10 mV recorder.
(b) Column packing.—Waters Associates, Inc. m-Bondapak carbohydrate
column, 300 ´ 4 (id) mm.
Column must meet following criteria:
Capacity factor for fructose = K¢ = (tR - t0)/t0 ³ 5
where tR = retention time for fructose = time from injection to
maximum peak height of fructose; t0 = retention time for solvent =
time from injection to maximum peak height of first baseline distortion
or solvent peak.
Resolution factor (distance between 2 band centers divided by
average band width) =
Rs = (t2 - t1)/0.5(tw1 + tw2)
where t2 and t1 = times from injection to maximum peak heights of
second peak (glucose) and first peak (fructose), respectively; and tw1
and tw2 = baseline widths (in time units) of first and second peaks,
respectively. For fructose: glucose ratios of 2.0–0.5, Rs ³ 1.0; for
ratios ³2, Rs ³ 1.25.
Replace column when either or both criteria are not met.
(c) Injection valve.—Waters Associates, Inc. 7120 LC injector
with 50 mL loop, or equivalent.
(d) Ancillary equipment.—Bransonic 12 ultrasonic bath (Branson
Ultrasonics Corp., Eagle Rd, Danbury CT 06810-1961, No.
B1210 MT), or equivalent, to degas solvents; Corning PC 353
stirrer (replaced by PC 510); and filtration apparatus for solvent
purification.
B. Reagents
(a) Sugar standard solution.—10 mg/mL. Dry individual sugar
standards (fructose, glucose, sucrose, lactose, and maltose; available
from Sigma Chemical Co.) 12 h at 60° under vacuum. Dissolve in
H2O and serially dilute to concentration of 10 mg/mL. Prepare daily.
(b) Mobile phase.—CH3CN (No. 2442, Mal linckrodt
Nanograde, or equivalent) + H2O (charcoal filtered) (80 + 20). Filter
through Whatman GF/F 0.7 mm glass fiber filter, and degas in
ultrasonic bath before use.
C. Preparation of Sample
Weigh 10.0 g finely divided milk chocolate into ³100 mL centrifuge
bottle and add 50 mL petroleum ether. Centrifuge ca 15 min at
ca 1800 rpm. Decant and discard supernate. Repeat extraction.
Pulverize residue with glass rod, add 100 g H2O, and weigh.
Place in 85–90° H2O bath 25 min. Cool to room temperature and
add H2O to original weight. Centrifuge 10 min at 2000 rpm,
withdraw portion of clear supernate, and filter through 0.45 mm
Swinney syringe filter.
D. Determination
Fill 50 mL injection loop with sample solution and inject into column
with mobile solvent flowing at 1.5–2.0 mL/min. Calculate concentrations
of each sugar by comparing peak heights or areas of each sugar
peak from sample with corresponding height or area of standard. Use
same method of measurement (area or height) throughout.
Reference: JAOAC 63, 595(1980).
CAS-57-48-7 (fructose)
CAS-50-99-7 (glucose)
CAS-63-42-3 (lactose)
CAS-69-79-4 (maltose)
CAS-57-50-1 (sucrose)