Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Samples that generate absorbance values A₂ – A₁ of 0.3 should be diluted in distilled water prior to the Sample Preparation (section A, page 7) and the second incubation of step 2 increased (glucose oxidase / catalase) to 30 min. 

Some critical steps of the assay are as follows:
A₂ should be read after approximately 10 min and you should ensure that the reaction has finished, i.e. measure the absorbance until it stops increasing.  (Slight increases in absorbance of 0.001/min or less are acceptable).
The supernatants from both steps (1 and 2) of A. Sample Preparation should be clear. 

The K-LACTUL kit will measure lactulose in most samples however it is the sample preparation prior to the Enzymatic Determination Reaction that is important. Megazyme has only tested milk-based samples, however most samples that do not contain high protein levels may work using the same standard procedure as described in the K-LACTUL data booklet. Samples containing very high levels of free fructose may not work.

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme ([email protected]). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
3. State the kit lot number being used (this is found on the outside of the kit box).
4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
6. State the sample type and describe the sample preparation steps if applicable.

If it is suspected that the measurements of K-LACTUL are not correct and there is doubt regarding the performance of the kit then the following steps should be checked.
1. Check that the cuvettes are 1.5 mL microcuvettes and that the volume of the liquid in the cuvettes is high enough for the spectrophotometer.
2. Check the temperature of the reactions is correct.
Using the standard lactulose/fructose solution (bottle 8) that is supplied with the kit will help determine where issues are occurring with the measurement of lactulose samples.  The obvious steps where issues may occur are: A. Sample Preparation (page 7 K-LACTUL booklet) and B. Enzymatic Determination Reaction (page 8 K-LACTUL booklet).
3. The performance of K-LACTUL can be tested as follows:
(A. Sample Preparation (page 7 K-LACTUL booklet)
Use 0.5 mL of the standard lactulose /fructose solution (Bottle 8) which contains 0.1 mg/mL lactulose and 0.05 mg/mL fructose.  The typical individual absorbance values are: A1 = 0.2, A2 = 0.2, A3 = 1.0.  This should generate a final absorbance difference of (A3-A2) of approximately 0.8 (Note: this measurement includes the lactulose and fructose measurement and is not just lactulose content only).
Note: If the correct values are obtained for the performance of K-LACTUL then there is no need to check the performance of the Enzymatic Determination Reaction step separately.
4. The performance of the Enzymatic Determination Reaction step can be tested separately as follows:
B. Enzymatic Determination Reaction (page 8 K-LACTUL booklet)
This test uses 0.1 mL of the standard lactulose /fructose solution (Bottle 8) which contains 0.05 mg/mL fructose. This is equivalent to 5 μg of fructose added to the cuvette and should generate an absorbance difference (A3-A2) of approximately 0.3. If this absorbance difference is obtained then it can be concluded that the step is performing correctly.
B. ENZYMATIC DETERMINATION REACTION:
Wavelength: 340 nm
Cuvette: 1 cm light path (glass or plastic; 1.5 mL semi-micro)
Final volume: 1.16 mL
Sample solution: 0.65-65 μg of lactulose per cuvette (in 0.1-1.0 mL sample volume)
Read against air (without cuvette in the light path) or against water Pipette

* for example with a plastic spatula or by gentle inversion after sealing the cuvette with a cuvette cap or Parafilm®. 

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.