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Lactose/D-Galactose BOEHRINGER MANNHEIM / R-BIOPHARM Lactose/D-Galactose Enzymatic BioAnalysis / Food Analysis UV-method for the determination of lactose and D-galactose in food- stuffs and other materials For use in in vitro only Store at 2-8° C Cat. No. 10 176 303 035 For recommendations for methods and standardized procedures see Test-Combination for 32 determinations references (A 2, B 2) Principle (Ref. A1) Lactose is hydrolyzed to D-glucose and D-galactose at pH 6.6 in the Pipette into Blank Lactose Blank4 D-Galactose presence of the enzyme ␤-galactosidase and water (1). cuvettes lactose sam- sample D-galactose sample4 ␤-galactosidase ple sample (1) Lactose + H O D-glucose + D-galactose 2 solution 1* 0.200 ml 0.200 ml 0.200 ml 0.200 ml D-Galactose is oxidized at pH 8.6 by nicotinamide-adenine dinucleotide ␤ suspension 2 0.050 ml 0.050 ml - - (NAD) to D-galactonic acid in the presence of the enzyme -galactose sample solution** - 0.100 ml - 0.100 ml dehydrogenase (Gal-DH) (2). Mix* and incubate for 20 min at 20-25°C. Add: + Gal-DH + (2) D-galactose + NAD D-galactonic acid + NADH + H solution 3 1.000 ml 1.000 ml 1.000 ml 1.000 ml The amount of NADH formed in reaction (2) is stoichiometric to the amount redist. water 2.000 ml 1.900 ml 2.050 ml 1.950 ml of lactose, and D-galactose, resp. The increase in NADH is measured by Mix***, after approx. 2 min read absorbances of the solutions (A ). Start reac- means of its light absorbance at 334, 340 or 365 nm. 1 tion by addition of: The Test-Combination contains suspension 4 0.050 ml 0.050 ml 0.050 ml 0.050 ml 1. Bottle 1 with approx. 600 mg lyophilizate, consisting of: citrate buffer, pH approx. 6.6; NAD, approx. 35 mg; magnesium sulfate Mix***, wait until reaction has stopped (approx. 15 min with 50 ␮g lactose/ 2. Bottle 2 with approx. 1.7 ml ␤-galactosidase suspension, approx. 100 U assay and approx. 30 min with 200 ␮g lactose/assay) and read absorbances 3. Bottle 3 with approx. 34 ml solution, consisting of: of the solutions (A2). potassium diphosphate buffer, pH approx. 8.6 If the reaction has not stopped after 15 min, continue to read the absorb- 4. Bottle 4 with approx. 1.7 ml galactose dehydrogenase suspension, ances at 5 min intervals until the absorbance increases constantly over approx. 40 U 5min. 5. Bottle 5 with lactose assay control solution for assay control purposes (measurement of the assay control solution is not necessary for calculat- * Pipette solution 1, suspension 2 and sample solution, each onto the bottom of the cuvette, ing the results.) Use the assay control solution undiluted. (Expiry date: mix by swirling. When using a plastic spatula, remove it from the cuvette only directly before measuring absorbance A1. see pack label) ** Rinse the enzyme pipette or the pipette tip of the piston pipette with sample solution before Preparation of solutions dispensing the sample solution. *** For example, with a plastic spatula or by gentle swirling after closing the cuvette with 1. Dissolve contents of bottle 1 with 7.0 ml redist. water. Parafilm (trademark of the American Can Company, Greenwich, Ct., USA) 2. Use suspension of bottle 2 undiluted. 3. Use solution of bottle 3 undiluted. If the absorbance A2 increases constantly, extrapolate the absorbance to the 4. Use suspension of bottle 4 undiluted. time of the addition of suspension 4 (ß-galactose dehydrogenase). Stability of reagents Determine the absorbance differences (A2-A1) for both, blanks and samples. The contents of bottle 1 are stable at 2-8°C (see pack label). Subtract the absorbance difference of the blank from the absorbance differ- Solution 1 is stable for 3 months at 2-8°C. ence of the corresponding sample. Bring solution 1 to 20-25°C before use. ⌬ A = (A2-A1)sample - (A2-A1)blank The contents of bottles 2, 3 and 4 are stable at 2-8°C (see pack label). ⌬ Bring solution 3 to 20-25°C before use. It follows AD-galactose (from “D-galactose sample”) and ⌬ Procedure Alactose + D-galactose (from “lactose sample”) ⌬ Wavelength1: 340 nm, Hg 365 nm or Hg 334 nm The difference of these values stands for Alactose. Glass cuvette2: 1.00 cm light path The measured absorbance differences should, as a rule, be at least 0.100 Temperature: 20-25°C absorbance units to achieve sufficiently precise results (see “Instructions for Final volume: 3.300 ml performance of assay“ and “Sensitivity and detection limit”, pt. 4). Read against air (without a cuvette in the light path) or against water Calculation Sample solution: 4-200 µg lactose and D-galactose/assay3 in 0.100-0.500 ml sample volume According to the general equation for calculating the concentrations: V × MW c = × ⌬A [g/l] ε × d × v × 1000 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 NADH at: 340 nm = 6.3 [l × mmol-1 × cm-1] Hg 365 nm = 3.4 [l × mmol-1 × cm-1] Hg 334 nm = 6.18 [l × mmol-1 × cm-1] It follows for lactose (calculated as anhydrous lactose): 1 The absorption maximum of NADH is at 340 nm. On spectrophotometers, measurements 3.300 × 342.3 11.30 c = × ⌬A = × ⌬A [g lactose/l are taken at the absorption maximum; if spectralline photometers equipped with a mercury ε lactose ε lactose vapor lamp are used, measurements are taken at a wavelength of 365 nm or 334 nm. × 1.00 × 0.100 × 1000 sample solution] 2 If desired, disposable cuvettes may be used instead of glass cuvettes. 3 See instructions for performance of assay 4 The performance of blank D-galactose sample and D-galactose sample can be omitted if the sample does not contain free D-galactose. 0703.10249106y for lactose (calculated as lactose monohydrate): resp. 1 mg D-galactose/l sample solution (if v = 0.100 ml, this corresponds 3.300 × 360.32 11.89 to 10 mg lactose/l, resp. 5 mg D-galactose/l sample solution). c = × ⌬A = × ⌬A [g lactose × ε × 1.00 × 0.100 × 1000 lactose ε lactose The detection limit of 7 mg lactose/l, resp. 4 mg D-galactose/l is derived H2O/l sample from the absorbance difference of 0.020 (as measured at 340 nm) and a solution] maximum sample volume v = 0.500 ml. for D-galactose: 3.300 × 180.16 5.945 5. Linearity c = × ⌬A = × ⌬A [g D- ␮ ε × 1.00 × 0.100 × 1000 galactose ε D-galactose Linearity of the determination exists from approx. 4 g lactose + D-galactose/ galactose/l sample assay (7 mg lactose + D-galactose/l sample solution; sample volume v = solution] 0.500 ml) to 200 ␮g lactose + D-galactose/assay (2 g lactose + D-galac- If the sample has been diluted during preparation, the result must be tose/l sample solution; sample volume v = 0.100ml). multiplied by the dilution factor F. 6. Precision When analyzing solid and semi-solid samples which are weighed out for sample preparation, the result is to be calculated from the amount weighed: In a double determination of D-galactose using one sample solution, a difference of 0.005 to 0.010 absorbance units may occur. With a sample clactose [g/l sample solution] volume of v = 0.100 ml and measurement at 340 nm, this corresponds to a Contentlactose = weight in g/l sample solution × 100 [g/100 g] D-galactose concentration of approx. 5-10 mg/l. (If the sample is diluted sample during sample preparation, the result has to be multiplied by the dilution cD-galactose [g/l sample solution] factor F. If the sample is weighed in for sample preparation, e.g. using 1 g ContentD-galactose = × 100 [g/100 g] sample/ 100 ml = 10 g/l, a difference of 0.05-0.1 g/100 g can be expected.) weightsample in g/l sample solution In a double determination of lactose using one sample solution, a difference of 0.010 to 0.015 absorbance units may occur in the presence of D-galac- 1. Instructions for performance of assay tose in the sample. With a sample volume of v = 0.100 ml and measurement The amount of lactose and D-galactose present in the assay has to be at 340 nm, this corresponds to a lactose concentration of approx. 15-25 mg/ between 10 ␮g and 200 ␮g (measurement at 365 nm) or 4 ␮g and 100 ␮g l. (If the sample is diluted during sample preparation, the result has to be (measurement at 340, 334 nm), respectively. In order to get a sufficient multiplied by the dilution factor F. If the sample is weighed in for sample absorbance difference, the sample solution is diluted to yield a lactose and preparation, e.g. using 1 g sample/100 ml = 10 g/l, a difference of 0.15- D-galactose concentration between 0.3 and 2 g/l or 0.2 and 1 g/l, respectively. 0.25 g/100 g can be expected.) Dilution table The following data for the determination of lactose and D-galactose have been published in the literature: Estimated amount of Dilution Dilution Lactose in chocolate: lactose and D-galactose per liter with water factor F x = 6.01 g/100 g s = 0.04 g/100 gCV = 0.7 % (Ref. A 1.1) 25 ␮g D-galactose/assay CV = 0.95 % measurement at 85 ␮g lactose/assay CV = 1.13 % 340 or 334 nm 365 nm 50 ␮g D-galactose/assay CV = 0.64 % ␮ 1.0 g 2.0 g - 1 170 g lactose/assay CV = 0.78 % (Ref.
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