EnzytecTM Liquid Maltose/Sucrose/D-Glucose Art. No.

E8170
Version 1 / 2023-02-27 Page 1 / 2

UV assay for the determination of maltose, sucrose and D-glucose in foodstuffs and other sample materials For in vitro use only
Test combination for 50 determinations Store between 2 - 8 °C

1. Test principle 4. Assays performance

The enzyme α-glucosidase (maltase) cleaves maltose and sucrose Wavelength: 340 nm
into two molecules D-glucose and D-glucose and D-fructose, Temperature: 20 - 37 °C (during the measurement)
respectively. Furthermore, sucrose is also hydrolyzed to D-glucose Measurement: against air (without cuvette) or water
and D-fructose by the enzyme β-fructosidase (invertase): Sample: 10 - 1100 mg/L
Maltose + H2O  α-glucosidase  2 D-glucose
Reagent blank Sample / control
Sucrose + H2O  α-glucosidase  D-glucose + D-fructose
Sucrose + H2O  β-fructosidase  D-glucose + D-fructose Reagent 1 2000 µL 2000 µL
Sample / control - 100 µL
D-glucose is phosphorylated with ATP in the presence of the enzyme
Dist. water 100 µL -
hexokinase (HK) to D-glucose-6-phosphate (G-6-P), simultaneously
producing ADP: Mix, incubate approx. 30 min at 20 °C, 20 min at 25 °C or 15 min at
37 °C. Read absorbance A1, then add:
D-glucose + ATP  HK  D-glucose-6-phosphate + ADP
Reagent 2 500 µL 500 µL
In the presence of a glucose-6-phosphate dehydrogenase (G6P-DH),
D-glucose-6-phosphate is oxidized to D-gluconate-6-phosphate: Mix, incubate approx. 5 min at 20 - 37 °C and read absorbance A2.
G-6-P + NAD+  G6P-DH  D-gluconate-6-phosphate + NADH + H+
The reagent blank value must be determined once for each run and
In this process, nicotinamide adenine dinucleotide (NAD) is reduced subtracted from each sample result.
to NADH. The amount of NADH formed is proportional to the amount
of D-glucose formed and is measured at 340 nm. The result is
To achieve sufficiently precise results, the absorbance differences
expressed as total maltose [g/L].
measured should usually be at least 0.100 absorbance units

2. Reagents 5. Calculation of results

2.1. Content & composition 5.1. Calculation of sample solutions

The test is suitable for manual and automated processing. With
manual processing, the reagents are sufficient for 50 determinations.
The number of determinations for automated processing is increased 5.1.1. Total concentration maltose/sucrose/D-glucose
by a multiple; however it depends on the device. ΔA = (A2 - df x A1)sample - (A2 - df x A1)RB
 Reagent 1: 2 x 50 mL with buffer, maltase, invertase, NAD, ATP df: dilution factor
 Reagent 2: 2 x 12.5 mL with buffer, HK, G6P-DH RB: Reagent blank

sample volume + R1
2.2. Reagent preparation dfbasic application = = 0.808
test volume
The reagents are ready-to-use and be allowed to reach room
Increasing the sample volume (up to max. 1000 μL) with unchanged reagent volumes
temperature (20 - 25 °C) before use. Do not interchange components requires conversion of the reagent dilution factor (df).
between kits of different batches.
(V × MW × ΔA)
Ctotal maltose [g/L] = = 0.7063 × ΔA
2.3. Storage & stability (Ɛ × d × v × 1000 × 2)
The reagents are stable until the end of the month of the indicated The factor 2 results from the two glucose molecules formed during the hydrolysis of maltose.
shelf life (see label) even after opening at 2 - 8 °C if handled properly. Take the sample dilution factor into account in the calculation.
Do not freeze reagents.
V: Test volume basic application [mL] = 2.600
MW: Molecular weight [g/mol] = 342.3
d: Optical path [cm] = 1.00
2.4. Safety & disposal v: Sample volume [mL] = 0.100
ε: Extinction coefficient NADH [L/mmol x cm] = 6.3 (at 340 nm)
The general safety rules for working in chemical laboratories should
be applied. Do not swallow! Avoid contact with skin and mucous
membranes. 5.1.2. Calculation of the real maltose concentration after
subtraction of sucrose/D-glucose
This kit may contain hazardous substances. For hazard notes on the
contained substances, please refer to the appropriate safety data The result of the E8170 test additionally includes the amounts of free
sheets (SDS) for this product, available online at sucrose and free D-glucose that might be present in the sample.
www.r-biopharm.com. After use, the reagents can be disposed of with It is calculated using the molecular weight of maltose (342.3 g/mol)
the laboratory waste. Packaging materials may be recycled. and is referred to as total maltose.
To determine the real maltose concentration, the sum of sucrose
including free D-glucose must be determined using the Enzytec™
3. Sample preparation Liquid Sucrose/D-Glucose assay (E8180).
 Use liquid, clear and almost neutral sample solutions directly or The result is expressed as total sucrose (342.3 g/mol) and subtracted
after dilution with dist. water. Water to a concentration within the from total maltose for differentiation:
measuring range (see performance data).
 Filter or centrifuge turbid solutions. Cmaltose [g/L] = Ctotal maltose E8170 − 0.5 × Ctotal sucrose E8180
 Degas samples containing carbonic acid.
Example: Enzytec™ Liquid Multi-sugar standard low E8440
 Grind and homogenize solid or semi-solid samples, weigh in
suitable sample quantity and extract with water. If necessary,
Total maltose (E8170) = 1.225 g/L
centrifuge, filter or perform Carrèz clarification.
Total sucrose (E8180) = 1.450 g/L
 Weigh samples with a high fat content into a volumetric flask and
extract with hot water; allow sample solution to cool down for fat Maltose = 1.225 g/L - 0.5 × 1.450 g/L = 0.500 g/L
separation (e.g. 15 min in an ice bath); fill volumetric flask up to
the mark with water, filter aqueous solution before testing.

R-Biopharm AG, An der neuen Bergstraße 17, 64297 Darmstadt, Germany, www.r-biopharm.com
EnzytecTM Liquid Maltose/Sucrose/D-Glucose Art. No. E8170
Version 1 / 2023-02-27 Page 2 / 2

5.1.3. Calculation of the real maltose concentration after 6.2. Interferences

differentiation of sucrose and D-glucose Citric acid and ascorbic acid showed no interference at or below
50 g/L. In the case of SO2, at a concentration of approximately 2 g/L,
a slightly increased recovery can be expected.
5.1.3.1. Differentiation of sucrose und D-glucose
In the case of D-mannose, strong interference was observed at levels
If differentiation of all three types of sugar is preferred, the free above 7.5 g/L.
D-glucose must also be determined separately using the Enzytec™
Liquid D-Glucose test (E8140) and subtracted from the result of the
Enzytec™ Liquid Sucrose/D-Glucose test (E8180). The ratio between 6.3. Linearity, measuring range & sensitivity
the molecular weights of the two sugars must be taken into account Linearity is given up to 1300 mg/L maltose, whereas the
(MW sucrose 342.3 g/mol, MWglucose 180.16 g/mol). recommended measuring range is between 10 and 1100 mg/L.
For further information, please refer to the corresponding package The lower limit of detection (LoD) and limit of quantification (LoQ) were
inserts for D-Glucose (E8140) and Sucrose/D-Glucose (E8180). determined according to method DIN 32645:2008-11 in buffered
aqueous solution for a sample volume of v = 100 μL. This results in
Csucrose [g/L] = Ctotal sucrose E8180 - 1.9 × Cglucose E8140 an LoD of 5 mg/L and an LoQ of 10 mg/L maltose.
The lowest absorbance difference that the method can distinguish is
Example: Enzytec™ Liquid Multi-sugar standard low E8440 ΔA = 0.005, resulting in an LoD of 0.5 mg/L for a sample volume of
v = 1000 μL. Based on ΔA = 0.010, an LoQ of 1.0 mg/L was
Total sucrose (E8180) = 1.450 g/L calculated.
D-Glucose (E8140) = 0.500 g/L
Sucrose = 1.450 g/L - 1.9 × 0.500 g/L = 0.500 g/L
7. Supporting documents
On request, we offer the following documents:
5.1.3.2. Calculation of the maltose concentration
 Enzytec™ Liquid Validation reports
For this calculation, the glucose amount from the calculated real  Enzytec™ Liquid Sample preparation guide
sucrose value (D-glucose + D-fructose) and the measured glucose  Enzytec™ Liquid Excel templates for results calculation
value adjusted for water content (0.95) is subtracted from the total  Enzytec™ Liquid Troubleshooting guide
maltose value.
Safety data sheets (SDS) und certificates of analysis (CoA) are
Cmaltose [g/L] = Ctotal maltose E8170 - (Csucrose E8180 ÷ 2) - (Cgluc. E8140 × 0.95) available in digital form on the website https://eifu.r-biopharm.com/:

Example: Enzytec™ Liquid Multi-sugar standard low E8440

Total maltose (E8170) = 1.225 g/L

Sucrose (E8180) = 0.500 g/L ÷ 2 (1x D-glucose) = 0.250 g/L
D-Glucose (E8140) = 0.500 g/L (D-glucose) × 0.95 (H2O) = 0.475 g/L
Maltose = 1.225 g/L - (0.500 g/L ÷ 2) - (0.500 g/L × 0.95) = 0.500 g/L

5.2. Calculation of solid samples 8. Services & technical support

On request, we offer the following services:
Cmaltose [g/L sample solution]
Contentmaltose [g/100 g] = × 100  Customized troubleshooting
weightsample in g/L sample solution  Data & results analysis
 Customer workshops & webinars
5.3. Controls & acceptance criteria  Automation: application support and technical service
Controls or reference samples should be carried along for quality
control during each run. For this purpose, we recommend the use of
the Enzytec™ Liquid Multi-sugar standard low (Art. No. E8440) with 9. Haftungsausschluss
0.5 g/L each of maltose, sucrose and D-glucose. This information corresponds to our present state of technology and
The recovery of maltose control solutions should be 100 ± 5 % and provides information on our products and their uses. R-Biopharm
for extracted samples within 100 ± 10 %. makes no warranty of any kind, either expressed or implied, except
that the materials from which its products are made are of standard
quality. Defective products will be replaced. There is no warranty of
6. Performance data merchantability of this product, or of the fitness of the product for any
purpose. R-Biopharm shall not be liable for any damages, including
6.1. Specificity & side activities special or consequential damage, or expense arising directly or
indirectly from the use of this product.
The α-glucosidase specifically hydrolyzes α-1,4-glycosidic bonds in
maltose, sucrose, maltotriose, maltotetraose and in maltodextrins as
well as in other oligo-glucosides such as turanose or maltitol. The
β-fructosidase specifically hydrolyzes the β-fructosidic bond of
sucrose.
In case of maltotriose, the assay shows a high side activity of 92 %.
Maltotetraose reacted at 24 % and maltodextrin at 40 %.
Consequently, it is not possible to distinguish whether maltose,
sucrose, D-glucose or other oligosaccharides with α-1,4-glycosidic or
β-fructosidic bond are contained in a sample. For differentiation, a
parallel test for sucrose and, if necessary, glucose must always be
performed (see calculation of results). Starch and disaccharides with
β-glycosidic bonds such as lactose, lactulose, cellobiose and raffinose
as well as disaccharides with α,α-glycosidic bonds such as trehalose
and with α-1,6-bonds such as isomaltose and isomaltulose do not
react. In the presence of sucrose and D-fructose, a creep reaction
may occur if the second incubation time of 5 minutes is exceeded.

R-Biopharm AG, An der neuen Bergstraße 17, 64297 Darmstadt, Germany, www.r-biopharm.com