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Maltose/Sucrose/
D-Glucose
UV-method
for the determination of maltose, sucrose and D-glucose in
foodstuffs and other materials
BOEHRINGER MANNHEIM / R-BIOPHARM
Enzymatic BioAnalysis / Food Analysis
For use in in vitro only Store at 2-8°C
For recommendations for methods and standardized procedures see
references (2)
Cat. No. 1 113 950
Test-Combination for approx. 12 determinations each
Principle (Ref. 1)
Maltose and sucrose are hydrolyzed in the presence of the enzyme -
glucosidase (maltase) at pH 6.6 to two molecules D-glucose or to D-glucose
and D-fructose, respectively (1,2).
Moreover, sucrose is also hydrolyzed by the enzyme -fructosidase
(invertase) at pH 4.6 to D-glucose and D-fructose (3).
At pH 7.6 the enzyme hexokinase (HK) catalyzes the phosphorylation of
D-glucose by adenosine-5’-triphosphate (ATP) under simultaneous formation
of adenosine-5’-diphosphate (ADP) (4).
The formed D-glucose-6-phosphate (G-6-P) is oxidized by nicotinamideadenine
dinucleotide phosphate (NADP) in the presence of glucose-6-
phosphate dehydrogenase (G6P-DH) to D-gluconate-6-phosphate with the
formation of reduced nicotinamide-adenine dinucleotide phosphate
(NADPH) (5).
The amount of NADPH formed in this reaction is stoichiometric to the
amount of sucrose, D-glucose and half the amount of maltose. The increase
in NADPH is measured by means of its light absorbance at 334, 340 or 365 nm.
The Test-Combination contains
1. Bottle 1 with approx. 0.2 g lyophilizate, consisting of:
citrate buffer, pH approx. 6.6; -glucosidase, approx. 210 U
2. Bottle 2 with approx. 0.5 g lyophilizate, consisting of:
citrate buffer, pH approx. 4.6; -fructosidase, approx. 720 U
3. Bottle 3 with approx. 7.2 g powder mixture, consisting of:
triethanolamine buffer, pH approx. 7.6; NADP, approx. 110 mg; ATP,
approx. 260 mg; magnesium sulfate
4. Bottle 4 with approx. 1.1 ml suspension, consisting of:
hexokinase, approx. 320 U; glucose-6-phosphate dehydrogenase, approx.
160 U
Preparation of solutions
1. Dissolve contents of bottle 1 with 6 ml redist. water.
2. Dissolve contents of bottle 2 with 10 ml redist. water.
3. Dissolve contents of bottle 3 with 45 ml redist. water.
4. Use contents of bottle 4 undiluted.
Stability of reagents
The contents of bottles 1, 2 and 3 are stable for at 2-8°C (see pack label).
Solution 1 is stable for 4 weeks at 2-8°C, for 2 months at 15 to 25°C.
Bring solution 1 to 20-25°C before use.
Solution 2 is stable for 4 weeks at 2-8°C, for 2 months at 15 to 25°C.
Bring solution 2 to 20-25°C before use.
Solution 3 is stable for 4 weeks at 2-8°C, for 2 months at 15 to 25°C.
Bring solution 3 to 20-25°C before use.
The contents of bottle 4 are stable at 2-8°C (see pack label).
1 The absorption maximum of NADPH is at 340 nm. On spectrophotometers, measurements
are taken at the absorption maximum; if spectralline photometers equipped with a mercury
vapor lamp are used, measurements are taken at a wavelength of 365 nm or 334 nm.
2 If desired, disposable cuvettes may be used instead of glass cuvettes.
3 See instructions for performance of assay
4 Available from Roche Molecular Biochemicals
(1) Maltose + H2O
-glucosidase
2 D-glucose
(2) Sucrose + H2O
-glucosidase
D-glucose + D-fructose
(3) Sucrose + H2O
-fructosidase
D-glucose + D-fructose
(4) D-Glucose + ATP
HK
G-6-P + ADP
(5) G-6-P + NADP+ G6P-DH
D-gluconate-6-phophate + NADPH + H+
Procedure
Wavelength1: 340 nm, Hg 365 nm or Hg 334 nm
Glass cuvette2: 1.00 cm light path
Temperature: 20-25°C
Final volume: 3.020 ml
Read against air (without a cuvette in the light path) or against water
Sample solution: 4-100 μg maltose + sucrose + D-glucose/assay3
(in 0.100-0.700 ml sample volume)
* Pipette solution 1, solution 2 and sample solution, each, onto the bottom of the cuvette and
mix by gentle swirling. When using a plastic spatula, remove it from the cuvette only directly
before measuring absorbance A1.
** Rinse the enzyme pipette or the pipette tip of the piston pipette with sample solution before
dispensing the sample solution.
*** For example, with a plastic spatula or by gentle swirling after closing the cuvette with
Parafilm (trademark of the American Can Company, Greenwich, Ct., USA)
If the absorbances increase constantly, extrapolate the absorbances A2 to
the time of addition of suspension 4 (HK/G6P-DH).
Determine absorbance differences (A2-A1) for blanks and samples. Subtract
the absorbance differences of the blanks from the absorbance differences of
the corresponding samples.
A = (A2-A1)sample - (A2-A1)blank
It follows:
The measured absorbance differences should, as a rule, be at least 0.100
absorbance units to achieve sufficiently precise results (see "Instructions for
performance of assay" and “Sensitivity and detection limit”, pt.4).
Pipette into
cuvettes
Blank
maltose
sample
Maltose
sample
Blank
sucrose
sample
Sucrose
sample
Blank
D-glucose
sample
D-Glucose
sample
solution 1*
solution 2*
sample
solution**
0.200 ml
-
-
0.200 ml
-
0.100 ml
-
0.200 ml
-
-
0.200 ml
0.100 ml
-- -
--
0.100 ml
Mix*, and incubate for 20 min at 20-25°C. Add:
solution 3
redist. water
1.000 ml
1.800 ml
1.000 ml
1.700 ml
1.000 ml
1.800 ml
1.000 ml
1.700 ml
1.000 ml
2.000 ml
1.000 ml
1.900 ml
Mix***, read absorbances of the solutions after approx. 3 min (A1). Start
reaction by addition of:
suspension 4 0.020 ml 0.020 ml 0.020 ml 0.020 ml 0.020 ml 0.020 ml
Mix***, wait for the completion of the reaction (10-15 min) and read
absorbances of the solutions (A2).
If the reaction has not stopped after 15 min, continue to read the absorbances
at 2 min intervals until the absorbance increases constantly.
Amaltose sample, Asucrose sample and AD-glucose sample.
The difference of Amaltose sample and Asucrose sample gives Amaltose.
The difference of Asucrose sample and AD-glucose sample gives Asucrose.
0101.1. 1748 327 rb bz
2
Calculation
According to the general equation for calculating the concentration:
V = final volume [ml]
v = sample volume [ml]
MW = molecular weight of the substance to be assayed [g/mol]
d = light path [cm]
ε = extinction coefficient of NADPH at:
340 nm = 6.3 [l × mmol-1 × cm-1]
Hg 365 nm = 3.5 [l × mmol-1 × cm-1]
Hg 334 nm = 6.18 [l × mmol-1 × cm-1]
It follows for maltose:
for sucrose:
for D-glucose:
If the sample has been diluted on preparation, the result must be multiplied
by the dilution factor F.
When analyzing solid and semi-solid samples which are weighed out for
sample preparation, the result is to be calculated from the amount weighed:
1. Instructions for performance of assay
The amount of maltose + sucrose + D-glucose present in the assay has to
be between 8 g and 100 g (measurement at 365 nm) or 4 g and 50 g
(measurement at 340, 334 nm), respectively. In order to get a sufficient
absorbance difference, the concentration of maltose (inclusive sucrose and
D-glucose) in the sample solution used for the assay should range between
0.10-1.0 g/l (measurement at 365 nm) or 0.05-0.5 g/l (measurement at 340,
334 nm). Dilute, if necessary.
Dilution table
If the measured absorbance difference (A) is too low (e.g. < 0.100), the
sample solution should be prepared again (weigh out more sample or dilute
less strongly) or the sample volume to be pipetted into the cuvette can be
increased up to 0.700 ml (maltose assay), 1.800 ml (sucrose assay), 2.000 ml
(D-glucose assay), resp.. The volume of water added must then be reduced
so as to obtain the same final volume in the assays for sample and blank.
The new sample volume v must be taken into account in the calculation
* Only when analyzing maltose
c =
V × MW
× A [g/l]
ε × d × v × 1000 (× 2)*
c =
3.020 × 342.3
× Amaltose =
5.169
× Amaltose
[g maltose/ε × 1.00 × 0.100 × 1000 × 2 ε l sample solution]
c =
3.020 × 342.3
× Asucrose =
10.34
× Asucrose
ε × 1.00 × 0.100 × 1000 ε [g sucrose/l sample solution]
c =
3.020 × 180.16
× AD-glucose =
5.441
× AD-glucose
ε × 1.00 × 0.100 × 1000 ε [g D-glucose/l sample solution]
Contentmaltose =
cmaltose [g/l sample solution]
× 100 [g/100 g]
weightsample in g/l sample solution
Contentsucrose =
csucrose [g/l sample solution]
× 100 [g/100 g]
weightsample in g/l sample solution
ContentD-glucose =
cD-glucose [g/l sample solution]
× 100 [g/100 g]
weightsample in g/l sample solution
Estimated amount of
maltose + sucrose + D-glucose
per liter
measurement at
Dilution
with water
Dilution
factor F
340 or 334 nm 365 nm
0.5 g
0.5-5.0 g
5.0-50 g
> 50 g
1.0 g
1.0-10 g
10-100 g
> 100 g
-
1 + 9
1 + 99
1 + 999
1
10
100
1000
2. Technical information
If the ratio maltose + sucrose to D-glucose in the sample is higher than e.g.
10:1, the precision of the determination of maltose and sucrose is impaired.
In this case, as much as possible of the D-glucose should be removed by
means of glucose oxidase in the presence of oxygen from the air. (For details
see pt. 10)
3. Specificity (Ref. 1)
-Glucosidase hydrolyzes -glucosidic bonds in maltose, maltotriose,
sucrose, turanose, 2-O--D-glucosido-D-erythrose and maltitol (4-O--Dglucopyranosyl-
D-sorbitol). Other -glucosides such as ,-trehalose,
maltopentaose and higher oligo-glucosides, dextrins and starch do not
react. Under the given reaction conditions, maltotetraose reacts to approx.
5%, isomaltose to 15% and palatinose (O--D-glucopyranosyl(1→6)-Dfructofuranose)
to 40%. Carbohydrates with -glucosidic bonds (lactose,
lactulose, cellobiose), as well as raffinose are not hydrolized.
-Fructosidase hydrolyzes the -fructosidic bond in sucrose and other
oligoglucosides. If the sample contains only sucrose, it is measured
specifically via D-glucose. Even in the presence of fructosanes, sucrose can
be measured specifically if after enzymatic hydrolysis with -fructosidase Dglucose
and D-fructose are determined and the ratio of these monosaccharides
is 1:1. If the D-fructose rate dominates 2 -oligofructosanes are
contained in the sample . "Polyfructose" (e. g. inulin) is not split.
The determination of D-glucose is specific.
In the analysis of commercial sucrose, results of have to be expected.
In the analysis of water-free D-glucose (molecular weight 180.16) resp.
D-glucose monohydrate (molecular weight 198.17) and of maltose, results of
< have to be expected because the materials absorb moisture.
(Commercial maltose may also contain D-glucose.)
4. Sensitivity and detection limit (Ref. 1.2)
The smallest differentiating absorbance for the procedure in the determination
of D-glucose is 0.005 absorbance units. This corresponds to a maximum
sample volume v = 2.000 ml and measurement at 340 nm of a D-glucose
concentration of 0.2 mg/l sample solution (if v = 0.100 ml, this corresponds
to 4 mg/l sample solution).
The detection limit of 0.4 mg D-glucose/l is derived from the absorbance
difference of 0.010 (as measured at 340 nm) and a maximum sample volume
v = 2.000 ml.
The smallest differentiating absorbance for the procedure in the determination
of maltose is 0.010 absorbance units (in the presence of D-glucose). This
corresponds in the determination of maltose to a maximum sample volume v =
0.700 ml and measurement at 340 nm of a maltose concentration of 1 mg/l
sample solution (if v = 0.100 ml, this corresponds to 8 mg/l sample solution).
The detection limit of 2 mg maltose/l is derived from the absorbance
difference of 0.020 (as measured at 340 nm) and a maximum sample volume
v = 0.700 ml.
The smallest differentiating absorbance for the procedure in the determination
of sucrose is 0.010 absorbance units (in the presence of D-glucose). This
corresponds to a maximum sample volume v = 1.800 ml and measurement
at 340 nm of a sucrose concentration of 1 mg/l sample solution (if v = 0.100 ml,
this corresponds to 15 mg/l sample solution).
The detection limit of 2 mg sucrose/l is derived from the absorbance
difference of 0.020 (as measured at 340 nm) and a maximum sample volume
v = 1.800 ml.
5. Linearity
Linearity of the determination exists from 4 g maltose + sucrose + D-glucose/
assay (2 mg maltose + sucrose + D-glucose/l sample solution; sample volume
v = 1.800 ml) to 100 g maltose + sucrose + D-glucose/assay (1 g maltose +
sucrose + D-glucose/l sample solution; sample volume v = 0.100 ml).
6. Precision
In a double determination of D-glucose using one sample solution, a
difference of 0.005 to 0.010 absorbance units may occur. With a sample
volume of v = 0.100 ml and measurement at 340 nm, this corresponds to a
D-glucose concentration of approx. 4-8 mg/l. (If the sample is diluted during
sample preparation, the result has to be multiplied by the dilution factor F. If
the sample is weighed in for sample preparation, e.g. using 1 g sample/
100 ml = 10 g/l, a difference of 0.04-0.08 g/100 g can be expected.)
3
In a double determination of maltose, resp. sucrose using one sample
solution, a difference of 0.010 to 0.015 absorbance units may occur in the
presence of D-glucose in the sample. With a sample volume of v = 0.100 ml
and measurement at 340 nm, this corresponds to approx. 8-12 mg maltose/l
or 15-25 mg/sucrose/l. (If the sample is diluted during sample preparation,
the result has to be multiplied by the dilution factor F. If the sample is
weighed in for sample preparation, e.g. using 1 g sample/100 ml = 10 g/l, a
difference of 0.08- 0.12 g maltose/100 g or 0.15-0.25 g sucrose/100 g can be
expected.)
The following data for the determination of maltose have been published in
the literature:
CV = 1.7-2.1 % maltose solutions (Ref. 1.2)
Rusks for children:
x = 2.9 g/100 g r = 0.260 g/100 g s(r) = ± 0.092 g/100 g
R = 0.461 g/100 g s(R) = ± 0.163 g/100 g (Ref. 2.3)
7. Recognizing interference during the assay procedure
7.1 If the conversion of D-glucose has been completed according to the
time given under ”Procedure”, it can be concluded in general that no
interference has occurred.
7.2 On completion of the reaction, the determination can be restarted by
adding D-glucose (qualitative or quantitative): if the absorbance is
altered subsequent to the addition of the standard material, this is also
an indication that no interference has occurred.
The reaction cannot be restarted with maltose and sucrose as, subsequent
to altering the reaction conditions from pH 6.6 to pH 7.6, resp. from pH 4.6
to 7.6 (“change of the buffer”), maltose and sucrose are no longer
cleaved.
7.3 Operator error or interference of the determination through the
presence of substances contained in the sample can be recognized by
carrying out a double determination using two different sample
volumes (e.g. 0.100 ml and 0.200 ml): the measured differences in
absorbance should be proportional to the sample volumes used.
When analyzing solid samples, it is recommended that different quantities
(e.g. 1 g and 2 g) be weighed into 100 ml volumetric flasks. The
absorbance differences measured and the weights of sample used
should be proportional for identical sample volumes.
The use of “single” and “double” sample volumes in double determinations
is the simplest method of carrying out a control assay in the determination
of maltose and sucrose.
7.4 Possible interference caused by substances contained in the sample
can be recognized by using an internal standard as a control: in
addition to the sample, blank and standard determinations, a further
determination should be carried out with sample and assay control
solution in the same assay. The recovery can then be calculated from
the absorbance differences measured.
7.5 Possible losses during the determination can be recognized by carrying
out recovery tests: the sample should be prepared and analyzed with
and without added standard material. The additive should be recovered
quantitatively within the error range of the method.
8. Reagent hazard
The reagents used in the determination of maltose, sucrose and D-glucose
are not hazardous materials in the sense of the Hazardous Substances
Regulations, the Chemicals Law or EC Regulation 67/548/EEC and
subsequent alteration, supplementation and adaptation guidelines.
However, the general safety measures that apply to all chemical substances
should be adhered to.
After use, the reagents can be disposed of with laboratory waste, but local
regulations must always be observed. Packaging material can be disposed
of in waste destined for recycling.
9. General information on sample preparation
In carrying out the assay:
Use clear, colorless and practically neutral liquid samples directly, or
after dilution according to the dilution table, and of a volume up to 2.000 ml
(D-glucose), resp. up to 0.700 ml (maltose), resp. up to 1.800 ml (sucrose);
Filter turbid solutions;
Degas samples containing carbon dioxide (e.g. by filtration);
Adjust acid samples to pH 8 by adding sodium or potassium hydroxide
solution (determination of D-glucose);
Adjust acid and weakly colored samples to approx. pH 8 by adding
sodium or potassium hydroxide solution and incubate for approx. 15 min
(determination of D-glucose);
Measure “colored” samples (if necessary adjusted to approx. pH 8)
against a sample blank (= buffer or redist. water + sample), adjust the photometer
to 0.000 with the blank in the beam (determination of D-glucose);
Treat “strongly colored” samples that are used undiluted or with a higher
sample volume with polyvinylpolypyrrolidone (PVPP) or with polyamide, e.g.
1 g/100 ml;
Crush or homogenize solid or semi-solid samples, extract with water or
dissolve in water and filter if necessary; resp. remove turbidities or dyestuffs
by Carrez clarification;
Deproteinize samples containing protein with Carrez reagents;
Extract samples containing fat with hot water (extraction temperature
should be above the melting point of the fat involved). Cool to allow the fat
to separate, make up to the mark, place the volumetric flask in an ice bath
for 15 min and filter; alternatively clarify with Carrez-solutions after the
extraction with hot water.
Carrez clarification:
Pipette the liquid sample into a 100 ml volumetric flask which contains
approx. 60 ml redist. water, or weigh sufficient quantity of the sample into a
100 ml volumetric flask and add approx. 60 ml redist. water. Subsequently,
carefully add 5 ml Carrez-I-solution (potassium hexacyanoferrate(II) (ferrocyanide),
85 mM = 3.60 g K4[Fe(CN)6] × 3 H2O/100 ml) and 5 ml Carrez-IIsolution
(zinc sulfate, 250 mM = 7.20 g ZnSO4 × 7 H2O/100 ml). Adjust to pH
7.5-8.5 with sodium hydroxide (0.1 M; e.g. 10 ml). Mix after each addition. Fill
the volumetric flask to the mark, mix and filter.
Samples containing protein should not be deproteinized with
perchloric acid or with trichloroacetic acid in the presence of sucrose
and maltose as these disaccharides are fully or partially hydrolized
with the release of D-glucose. The Carrez clarification is recommended
for normal use.
10. Special preparation of sample for the determination of maltose
and sucrose in the presence of a large excess of D-glucose
The precision of the maltose and sucrose determination is impaired if the
ratio of D-glucose to maltose and sucrose is higher than e.g. 10:1. In this
case, as much as possible of the D-glucose should be removed. In the
presence of glucose oxidase (GOD) and oxygen from the air, D-glucose is
oxidized to D-gluconate:
Hydrogen peroxide is destroyed by catalase:
Reagents
Glucose oxidase (GOD), from Aspergillus niger, 200 U/mg (25°C; D-glucose
as substrate); amylase and β-fructosidase < 0.01% each, e.g. Cat. No.
2208 1134
Catalase, Cat. No. 106 8104
Triethanolamine hydrochloride,
MgSO4 × 7 H2O
NaOH, 4 M
Preparation of solutions for 10 determinations
Enzyme solution:
Dissolve 5 mg (approx. 1000 U) GOD in 0.750 ml redist. water, add 0.250 ml
catalase, and mix.
Buffer solution:
Dissolve 5.6 g triethanolamine hydrochloride and 0.1 g MgSO4 × 7 H2O in 80 ml
redist. water, adjust to pH 7.6 with NaOH (4 M), and fill up to 100 ml with
redist. water.
Stability of solutions
The enzyme solution must be prepared freshly daily.
The buffer solution is stable for 4 weeks when stored at 2-8°C.
Performance of D-glucose oxidation
D-Glucose + H2O + O2
GOD
D-gluconate + H2O2
2 H2O2
catalase
2 H2O + O2
Pipette into 10 ml volumetric flask
buffer solution
sample solution (up to approx. 0.5% D-glucose)
enzyme solution
2.000 ml
5.000 ml
0.100 ml
Pass a current of air (O2) through the mixture for 1 h; during the oxidation
process check the pH with indicator paper and, if necessary, neutralize
the formed acid with NaOH.
To inactivate the enzymes GOD and catalase, keep the volumetric flask in a
boiling water-bath for 15 min, allow to cool, and dilute to the mark with
water. Mix and filter, if necessary. Use the clear solution for the determination
of maltose and sucrose. Determine the residual D-glucose in a parallel assay
and subtract as usual.
11. Further applications
The method may also be used in research when analyzing biological
samples. For sample treatment see Ref. 1.2.
For further information see instructions for
Test-Combination D-Glucose Cat. No. 0 716 251
Test-Combination D-Glucose/D-Fructose Cat. No. 0 139 106
Test-Combination Sucrose/D-Glucose Cat. No. 0 139 041
Test-Combination
Sucrose/D-Glucose/D-Fructose Cat. No. 0 716 260
Test-Combination D-Sorbitol/Xylitol Cat. No. 0 670 057
Test-Combination Starch Cat. No. 0 207 748
References
1.1 Gutmann, I. (1974) in Methoden der enzymatischen Analyse (Bergmeyer, H. U., Hrsg.)
3. Aufl., Bd. 2, S. 1230-1233, Verlag Chemie, Weinheim, and (1974) in Methods of
Enzymatic Analysis (Bergmeyer, H. U., ed.) 2nd ed., vol. 3, pp. 1185-1188, Verlag
Chemie, Weinheim/Academic Press, Inc., New York and London
1.2 Beutler, H.-O. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.) 3rd ed.,
vol. VI, pp. 119-126, Verlag Chemie, Weinheim, Deerfield Beach, Florida, Basel
2.1 Gombocz, E., Hellwig, E., Vojir, F. & Petuely, F. (1981) Deutsche Lebensmittel-Rundschau
77, 10-11 (Maltose), 11 (Saccharose) und 3 (D-Glucose)
2.2 Brautechnische Analysenmethoden, Band III, S. 589-592 (1982), Methodensammlung
der Mitteleuropäischen Brautechnischen Analysenkommission (MEBAK), herausgegeben
von F. Drawert im Selbstverlag der MEBAK, Freising
2.3 Amtliche Sammlung von Untersuchungsverfahren nach § 35 LMBG; Untersuchung von
Lebensmitteln: Bestimmung von Maltose in Kinder-Zwieback und Zwiebackmehl,
48.02.07-2 (Mai 1985)
3.1 Drawert, F. & Hagen, W. (1970) Enzymatische Analysenmethoden zur Bestimmung von
Würze- und Bierinhaltsstoffen: II. Bestimmung von Maltose neben Saccharose, Glucose
und Fructose in Würze und Bier; Verhalten der Zucker während der Gärung und
Lagerung, Brauwissenschaft 23, 95-101
3.2 Postel, W., Drawert, F. & Hagen, W. (1977) Enzymatische Differenzierung der Zucker in
Malz-, Nähr- und Süßbieren: I. Enzymatische Analyse von Glucose, Fructose,
Saccharose und Maltose, Deutsche Lebensmittel-Rundschau 67, 107-110 und 195-202
(II. Zusammensetzung und Beurteilung)
For further references, see Test-Combination Sucrose/D-Glucose
(Cat. No. 0 139 041).
R-BIOPHARM GmbH
Dolivostraße 10
64293 Darmstadt/Germany
Telefon + 49 61 51 / 81 02-0
Fax + 49 61 51 / 81 02-20
麦芽糖/蔗糖/D-葡萄糖检测试剂盒