JCAM No.

114-R1

Japan Customs Analysis Methods

No. 114

Quantitative Analysis of Reducing Sugars in Sugar Preparations

consisting of Sugar and Dextrin
(Issued in June 1999)
(Updated in May 2001)

1. Scope water.

This analysis method is applied to sugar 3.2. Preparation of standard dextrose solution
preparations which consist of sugar and dextrin and for standard-addition
which require the determination of their “reducing Accurately weigh about 4 g of dextrose, transfer to a
sugar contents, expressed as dextrose on dry 100 mL volumetric flask, and dilute to volume with
substances,” as referred to in Note 2 to Chapter 35 in water.
Customs Tariff Law (Appendix Table–Customs Tariff
3.3. Preparation of standard invert sugar
Schedule).
solution and other reagents
2. Outline of Test Method (1) Standard invert sugar solution

This analytical method is applied for products Accurately weigh 4.75 g of sucrose, transfer

containing sucrose and dextrin (starch degradation with 90 mL of water to a 500 mL volumetric flask,

products) to determine reducing sugars, e.g. dextrose and add 5 mL of hydrochloric acid (specific gravity,

and maltose, contained in the dextrin. The procedure is 1.18). After leaving to stand at 20–30°C for three

summarized below. days, dilute the solution to volume with water and
store in a cool dark place.
(1) Determination of moisture content
Transfer a 50 mL portion of the solution above
(2) Determination of direct reducing sugars by the
to a 200 mL volumetric flask, neutralize with 1
Lane-Eynon method
mol/L sodium hydroxide aqueous solution using
(3) Determination of sucrose by the Lane-Eynon
phenolphthalein as an indicator, and dilute to
method
volume with water.
(4) Determination of the content of dextrin
Use the solution as standard invert sugar
(5) Calculation of DE value solution for the standardization of Fehling’s
Solution.
3. Reagents
(2) 1% Methylene Blue solution
All chemicals must be JIS special reagent grade or
Dissolve 1 g of methylene blue in water to
equivalent, unless otherwise specified.
make 100 mL.
3.1. Preparation of standard dextrose solution (3) Fehling’s Solution
for making a calibration curve
Solution A: Dissolve 34.639 g of copper sulfate
Accurately weigh about 1 g of dextrose, transfer to a (CuSO4•5H2O) in water to make
1,000 mL volumetric flask, and dilute to volume with

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 JCAM No.114-R1
exactly 500 mL, leave it for two days, 3.5.3. Invertase solution
and then filter. Dissolve invertase in the 0.1M acetic acid buffer
Solution B: Dissolve 173 g of potassium sodium (pH 4.6) so that the concentration of invertase becomes
tartrate (KNaC4H4O6•4H2O) and 50g 400 units/mL.
of sodium hydroxide in water to make
3.5.4. Enzyme solution for determination of
exactly 500 mL, leave it for two days,
dextrin
and then filter.
Dissolve glucoamylase (1,4-α-D-Glucan
3.4. Standardization of Fehling’s Solution glucohydrolase EC 3.2.1.3) and α-amylase (α-1,4-Glucan
Put 5.0 mL of Fehring’s Solution A and 5 mL of 4-glucanohydrolase EC 3.2.1.1) in the 0.2M acetic acid
Fehring’s Solution B into a 200 mL Erlenmeyer flask buffer (pH 4.8) so that their concentrations become 20
containing a few glass beads and add from a 50 mL units/mL(3) and 80 units/mL,(4) respectively. (5)
burette 19.5 mL of the standard invert sugar solution.
Note 3) One unit represents the amount of enzymes
After boiling it on an electric stove (heater) for two
able to produce 10 mg of glucose from soluble
minutes, add four drops of the methylene blue solution.
starch (as substrate) every 30 minutes at 40°C
Complete titration within a total boiling time of three
and pH 4.5.
minutes by dropwise addition of the standard invert
Note 4) One unit represents the amount of enzymes
sugar solution—without preventing boiling—until the
able to produce 0.18 mg of reducing sugar,
blue color disappears. Repeat titration twice and
expressed as glucose, from soluble starch (as
calculate the mean of three parallel titrations. (1)
substrate) per minute at 40°C and pH 6.0.
Obtain the factor of the Fehring’s Solution from the
Note 5) When using enzymes whose potencies or units
following formula:
have been determined based on different
Factor (2) = 20.36 / A definitions, confirm in advance that their
Where– A: Volume (mL) of the standard invert recovery rates in digesting 50 mg of corn starch
sugar solution required are 100%.

Note 1) Use the mean value of the three parallel 3.5.5. Glucose determination kit
titrations as “A”; duplicate titrations must Use a commercially available enzyme-based assay
agree to within 0.1 mL in the volume of the kit for the determination of glucose (dextrose).
sugar solution required
3.6. Deproteinizing agent
Note 2) Calculate factor by rounding off fractions to the
Solution A: Dissolve 2 g of zinc sulfate
third decimal place; the factor must be within a
(ZnSO4•7H2O) in 100 mL of water.
range of 1±0.02.
Solution B: Dissolve 1.8 g of barium hydroxide
3.5. Preparation of buffers and enzyme solutions
[Ba(OH)2•8H2O] in 100 mL of water.
3.5.1. 0.1M acetic acid buffer (pH 4.6)
4. Preparation of samples
Weigh 6 g of glacial acetic acid in a beaker, dissolve
in 100 mL of water and adjust the pH to 4.6 using a 5% Prepare and collect analysis samples in appropriate

sodium hydroxide aqueous solution. Transfer the manners, e.g. sample reduction methods, etc.,

solution to a 1,000 mL volumetric flask and dilute to depending on their conditions presented. For powder or

volume with water. crystal mixtures, grind them with a grinder or a mixer.
For pasty or wet materials, homogenize them by mixing
3.5.2. 0.2M acetic acid buffer (pH 4.8)
in mortars. In any case, collect relatively large amounts
By adding 120 mL of a 0.2M sodium acetate to 80 of samples randomly, and grind or mix them to
mL of 0.2M acetic acid, adjust the pH of the mixed uniformity.
solution to 4.8.

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 JCAM No.114-R1
5. Procedure compositions, change the amount of sucrose
added so that the sucrose concentrations in the
5.1. Determination of moisture content
test solution and the blank solution become
Accurately weigh about 2 g of the sample almost the same.
homogenized in 4. in a weighing bottle which has been
5.3.3. Titration
previously dried to a constant weight. Dry it in a
vacuum oven at a temperature of 70–75°C for four hours, Put 5.0 mL of Fehring’s Solution A and 5 mL of

cool to room temperature in a desiccator and weigh. Fehring’s Solution B into a 200 mL Erlenmeyer flask.

Repeat vacuum drying until the loss in weight does not Add from a 50 mL burette 15 mL of the test solution

exceed 2 mg per hour in the drying period. prepared in 5.3.1., and titrate as described in 3.4
(preliminary titration).
Calculate the moisture content in the sample from
Then, put 5.0 mL of Fehring’s Solution A and 5 mL
the following formula. Round off fractions to the first
of Fehring’s Solution B into another 200 mL Erlenmeyer
decimal place.
flask. Add from a 50 mL burette the test solution within
1 mL of the anticipated end point from the result of the
%, moisture content = {(W0 − W1 ) / W0 }× 100
preliminary titration above, and titrate in the same
manner as in the preliminary titration.
Where–
Multiply by the factor of the Fehling’s Solution the
W0: Amount (g) of sample collected
volume (mL) of the test solution required in order to
W1: Weight (g) of sample after drying
obtain the corrected titre, X (mL). Obtain the
5.2. Preparation of sample solution concentration of the direct reducing sugars, Ds
Accurately weigh 15 g of the sample homogenized (mg/100mL) from the titre, X (mL), by reference to the
in 4., and dissolve in water. Transfer the solution to a appended Lane-Eynon’s Table (dextrose). Similarly,
500 mL volumetric flask and dilute to volume with perform titration with the blank solution and obtain the
water. concentration of the direct reducing sugars, Ds’
(mg/100mL), from the corrected titre, X’ (mL), by
5.3. Determination of Direct Reducing Sugar in
making reference to the appended Lane-Eynon Table
Sample
(dextrose).
5.3.1. Preparation of test solution Using Ds and Ds’, calculate the content (%) of direct
Put a 100 mL portion of the sample solution reducing sugar from the following formula:
prepared in 5.2. in a 200 mL volumetric flask, add 10
mL of the standard dextrose solution for %, direct reducing sugar =
(Ds − Ds' ) × 500 × 200 × 100
S 100 100
standard-addition prepared in 3.2., and dilute to volume
with water.
Where–
5.3.2. Preparation of blank solution Ds: Concentration (mg/100mL) of direct reducing
Put a 10 mL portion of the standard dextrose sugar in test solution, obtained by reference
solution for the standard-addition prepared in 3.2 in a to the appended Lane-Eynon Table
200 mL volumetric flask, add about 2.5 g(6) of sucrose (dextrose).
and dissolve by adding a small amount of water. Dilute Ds’: Concentration (mg/100mL) of direct reducing
the solution to volume with water. sugar in blank solution, obtained by
Note 6) This is a reference on the amount of sucrose reference to the appended Lane-Eynon’s
added specifically for samples consisting of Table (dextrose).
83 % of sucrose and 17 % of dextrin. Thus, S: Weight (mg) of sample collected in 5.2.
when analyzing samples with different

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 JCAM No.114-R1
500 200 DRs: Concentration (mg/100mL) of direct
(Ref.) Dilution rate = ×
100 100 reducing sugar in test solution, obtained by
reference to the appended Lane-Eynon
5.4. Determination of sucrose in sample Table [Invert sugar (without sucrose)].

5.4.1. Inversion reaction DRs’: Concentration (mg/100mL) of direct

reducing sugar in blank solution, obtained
Put a 20 mL portion of the sample solution
by reference to the appended Lane-Eynon’s
prepared in 5.2. into a 200 mL volumetric flask and add
Table [Invert sugar (without sucrose)].
1 mL of the invertase solution. Hydrolyze the sample
solution by placing the flask in a water bath at a S: Weight (mg) of sample collected in 5.2.
constant temperature of 37°C for 30 minutes, add 5 mL
each of the deproteinizing solutions A and B, and mix %, Sucrose content = {invert sugar content (%) –
thoroughly. Dilute the deproteinized solution to volume A (%)} × 0.95
with water and filter. Use the filtrate as test solution for
the determination of sucrose. Note 7) The reason why two types of direct reducing
5.4.2. Titration and calculation of sucrose sugar contents in dextrin are calculated is that
content the content of direct reducing sugar, expressed
as invert sugar, is needed for the determination
Titrate with the test solution prepared in 5.4.1. in
of sucrose content, whereas that expressed as
accordance with the procedure in 5.3.3. Obtain the
dextrose is needed for the determination of DE
concentration of the invert sugar, T (mg/100mL), by
value.
reference to the appended Lane-Eynon’s Table [invert
sugar (without sucrose)] and calculate the invert sugar 5.5. Determination of dextrin content
content (%) from the following formula. 5.5.1. Enzymatic digestion
Put a 2 mL portion of the sample solution prepared
T 500 200
%, invert sugar = × × × 100 in 5.2. in a 100 mL volumetric flask and add 5 mL of the
S 20 100
enzyme solution for determination of dextrin. Place the
Where– flask in a water bath at a constant temperature of 37°C
T: Concentration (mg/100mL) of invert sugar in for two hours for enzymatic digestion, add 5 mL each of
test solution, obtained by reference to the the deproteinizing solutions A and B, and mix
appended Lane-Eynon’s Table [invert sugar thoroughly. Dilute the solution to volume with water
(without sucrose)]. and filter. Use the filtrate as test solution for the
determination of dextrin.
S: Weight (mg) of sample collected in 5.2.
5.5.2. Preparation of calibration curve for
Next, from the titres (X and X’) obtained in 5.3.3,
dextrose
obtain the contents of the direct reducing sugar in the
Put 5, 10, 15, 20 and 25 mL of the standard
sample and blank solutions, DRs (mg/100mL) and DRs’
dextrose solutions prepared in 3.1. in 100 mL
(mg/100mL), by reference to the appended Lane-Eynon
volumetric flasks, respectively, and dilute the solutions
Table [Invert sugar (without sucrose)]. Calculate from the
to volume with water. Use them as standard solutions
following formula the content of the direct reducing sugar,
for constructing a calibration curve.
expressed as invert sugar, in dextrin contained in the
sample, A (%).(7) Using a commercially available dextrose
determination kit, e.g. glucoxidase-peroxidase-based
A (%) =
(DRs − DRs') × 500 × 200 × 100 assay kits, described in 3.5.5., prepare a calibration
S 100 100
curve by plotting absorbance against dextrose
concentration (mg/ml) for each of the standard solutions
Where–

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 JCAM No.114-R1
prepared above. Construct the calibration curve 6. References
simultaneously during the procedure in 5.5.3.
(1) 中村道徳, 貝沼圭二 「澱粉･関連糖質実験法」学術出
5.5.3. Determination of dextrose and 版センター (1986)
calculation of dextrin content (2) 三国二郎 監修 「澱粉化学ハンドブック」 朝倉書店
Utilizing the same assay kit described in 3.5.5., (1977)
quantify dextrose in the test solution prepared in 5.5.1.
and determine the concentration (mg/ml) of dextrose in
the test solution using the calibration curve constructed
in 5.5.2. Calculate the dextrin content (%) in the sample
from the following formula.(8, 9)

Dextrose (%) × 0.9 × Dilution rate

%, dextrin = × 100
S

Where–

S: Amount (mg) of sample collected in 5.2.

Note 8) In the calculation, the dextrin content is

deemed to be equal to “dextrose (%) × 0.9.”

Note 9) Dextrin content may be calculated as a

balance. In that case, calculate the dextrin
content from the following formula:

Dextrin content (%) = 100 – {sucrose content

(%) +moisture content (%)}

However, when the calculated value looks

doubtful due to the differences from those in
the attachments and composition table
provided, determine the exact value with
assay.

5.6. Calculation of DE
Calculate the content of reducing sugars, expressed
as dextrose on the dry substance, in dextrin contained
in the sample by applying the following formula with
the values obtained in 5.3.3. and 5.5.3. Round off
fractions to the first decimal place.

Direct reducing sugar content (%) in dextrin

DE = × 100
Dextrin content (%) in sample

Where–

DE: Content (%) of reducing sugar, expressed as

dextrose on the dry substance, in the
dextrin contained in the test sample.

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 JCAM No.114-R1
Appendix

Lane-Eynon Table (Invert sugar and Dextrose)

Saccharides
mL sugar solution Invert sugar Dextrose
required (without sucrose) (anhydrous)
mg/100ml mg/100ml
15 336 327
16 316 307
17 298 289
18 282 274
19 267 260
20 254.5 247.4
21 242.9 235.8
22 231.8 225.5
23 222.2 216.1
24 213.3 207.4
25 204.8 199.3
26 197.4 191.8
27 190.4 184.9
28 183.7 178.5
29 177.6 172.5
30 171.7 167
31 166.3 161.8
32 161.2 156.9
33 156.6 152.4
34 152.2 148
35 147.9 143.9
36 143.9 140
37 140.2 136.4
38 136.6 132.9
39 133.3 129.6
40 130.1 126.5
41 127.1 123.6
42 124.2 120.8
43 121.4 118.1
44 118.7 115.5
45 116.1 113
46 113.7 110.6
47 111.4 108.4
48 109.2 106.2
49 107.1 104.1
50 105.1 102.2

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