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Enzymatic Method for Determining D-Sorbitol (D-Sorbitol Assay)

This assay protocol is suitable for the colorimetric detection of D-sorbitol in samples such as foods, fruits, fruit juices, cosmetics, pharmaceuticals, and paper using the D-Sorbitol Assay Kit (MAK010). D-Sorbitol is oxidized to fructose with the proportional development of intense color with an absorbance maximum at 560 nm. The linear range of detection for this assay is between 0.2–1.0 nmole.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents

Sorbitol Assay Buffer                  25 mL
   Catalog Number MAK010A

Sorbitol Probe                           0.2 mL
   Catalog Number MAK010B

Sorbitol Enzyme Mix                      1 vl
   Catalog Number MAK010C

Sorbitol Developer                          1 vl
   Catalog Number MAK010D

Sorbitol Standard, 100 mM         0.1 mL
   Catalog Number MAK010E

Reagents and Equipment Required but Not Provided:

96 well flat-bottom plate – It is recommended to use clear plates (Catalog Number M4436 or equivalent) for colorimetric assays.

Spectrophotometric multiwell plate reader

Preparation Instructions

Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

Sorbitol Assay Buffer – Allow buffer to come to room temperature before using

Sorbitol Enzyme Mix – Reconstitute in 220 µL of water. Mix well by pipetting, then aliquot and store, protected from light and moisture, at –20 °C.

Sorbitol Developer – Reconstitute in 1.0 mL of water. Mix well by pipetting. Keep on ice while in use. Aliquot and store protected from light and moisture at –20 °C.

Storage/Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

Procedure

All samples and standards should be run in duplicate.

D-Sorbitol Standards for Colorimetric Detection
Dilute 10 µL of the 100 mM Sorbitol Standard Solution with 990 µL of water to prepare a 1 mM standard solution. Mix well. Add 0, 2, 4, 8, 10 µL of the 1 mM Sorbitol standard solution into a 96 well plate, generating 0 (assay blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Sorbitol Assay Buffer to each well to bring the volume to 50 µL.

Sample Preparation
Food product samples should be dissolved in water and centrifuged to remove insoluble material. Liquids such as juice should be diluted 10-fold (1:9) with water and centrifuged. Add 1–50 µL of sample to wells. Bring samples to a final volume of 50 µL with Sorbitol Assay Buffer.

Notes:
For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

Interfering substances present in the sample can generate background. To control for background, include a blank sample for each sample by omitting the Enzyme Mix in the Reaction Mix.

Assay Reaction

1. Set up the Reaction Mixes according to the scheme in Table 1. 50 µL of the appropriate Reaction Mix is required for each reaction (well).

Table 1.

Reagent Sample Blank Samples and Standards
Sorbitol Assay Buffer 38 µL 36 µL
Sorbitol Enzyme Mix 2 µL
Sorbitol Developer 10 µL 10 µL
Sorbitol Probe 2 µL 2 µL


2. Add 50 µL of the appropriate Reaction Mix to each of the wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 30 minutes at 37 °C. Protect the plate from light during the incubation.

3. Measure the absorbance at 560 nm (A560).

Results

Calculations
The background for the assay is the value obtained for the 0 (assay blank) Sorbitol standard. Correct for the background by subtracting the 0 (assay blank) value from all readings. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate Sorbitol standards to plot a standard curve.
Note: A new standard curve must be set up each time the assay is run.

Subtract the blank sample value from the sample reading to obtain the corrected measurement. Using the corrected measurement, the amount of Sorbitol present in the sample may be determined from the standard curve.

Concentration of Sorbitol

              Sa/Sv x D = C (nmole/µL or mM)

Sa = Amount of Sorbitol in unknown sample (nmole) from standard curve
Sv = Sample volume (µL) added into the wells
D = Dilution factor, if necessary
C = Concentration of Sorbitol in sample

Sorbitol molecular weight: 182.17 g/mole

Sample Calculation
Amount of Sorbitol (Sa) = 2.4 nmole
         (from standard curve)
Sample volume (Sv) = 50 µL

Concentration of Sorbitol in sample

2.4 nmole/50 µL = 0.048 nmole/µL

0.048 nmole/µL x 182.17 ng/nmole = 8.74 ng/µL

Materials

     
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