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.

The 55.6-fold dilution factor is the overall dilution of the sample (after extraction) throughout the Standard Assay Procedure of K-PHYT (Part A step 2, and Part B).

Yes. Liquid samples can be tested for phytic acid using K-PHYT.

At Step 1 of the procedure (Sample Extraction), dilute a given volume of the liquid sample with an appropriate volume of hydrochloric acid (0.66 M), e.g. 1 mL liquid sample plus 19 mL hydrochloric acid (0.66 M) and proceed with the extraction incubation as for solid samples. The level of sample dilution can be adjust accordingly depending on the phytic acid content of the sample. This will need to be determined experimentally.

In the calculation for liquid samples, the sample weight (g) is replaced with sample volume (mL), e.g. if 1 mL of sample was added to 19 mL of hydrochloric acid (0.66 M) in Step 1 of the procedure (Sample Extraction) then in the calculation the sample volume is 1 and extraction volume is 20. The results of phosphorus and phytic acid content for liquid samples are given as g/100 mL instead of g/100 g.

The K-PHYT MegaCalc spreadsheet contains a separate worksheet for the calculation of results using liquid samples.

Unprocessed samples originating from grains usually contain ~ 97% of the total phosphorus content as phytic acid and therefore can be accurately measured using K-PHYT.
When analysing more pure forms of  phytic acid (inositol hexakisphosphate) (e.g. Sigma product P5681) the Phytic Acid Kit (K-PHYT) is very accurate and is able to generate results within ~ 2% of the value stated for the product after taking into account the purity and moisture content of the dry sample.

Firstly the definition of measurement should be clearly defined prior to the analyses, e.g. are the analyses for total phosphorus (this includes enzymatically released phosphorus from sources other than phytic acid), or phytic acid?
The K-PHYT Kit measures the total phosphorus released by phytase and alkaline phosphatase.  The measurement is given as grams of phosphorus / 100 g original material.
When measuring for phytic acid the assumption is that all of the phosphorus that was measured is released from phytic acid (IP6) and therefore the amount of phosphorus released is then converted to an amount of phytic acid where phosphorus comprises 28.2% of phytic acid.

When processed samples, which may contain various monophosphates not associated with phytic acid, are analysed the phytic acid content may be overestimated since the alkaline phosphatase can release phosphate from various monophosphates in addition to inositol-monophosphate and the calculation of phytic acid assumes that all of the phosphorus is released from phytic acid.  For such samples this may be overcome by performing the “Free Phosphorus” sample test exactly as described in the K-PHYT data booklet except that the alkaline phosphatase is included in the second part of the dephosphorylation reaction step (in place of the water addition).  This sample is not treated with phytase but is treated with alkaline phosphatase and will give the amount of enzymatically released phosphorous which is not associated with phytic acid.  Subtracting this value of alkaline phosphatase released phosphorus from the Total Phosphorus value for the same sample and then calculating the phytic acid would be more accurate for phytic acid determination.

The certificate that Megazyme currently supplies for K-PHYT is the certificate of analysis for the kit (available where the product is located on the Megazyme website).
Measurements using K-PHYT rely on accurate determination of phosphorus.  The performance of this kit requires that a phosphorus standard curve is performed for each batch of tests and the phosphorus standard that is supplied with the kit is standardised against a certified phosphorus standard obtained from SIGMA (cat no. 79409).  If required this certified standard may be used in place of the phosphorus standard that is supplied with K-PHYT.
The oat sample supplied with the kit is to be used as a control and should generate phosphorus / phytic acid values within 10% of the values supplied on the sample label.

Yes.  Solid samples such as grain can be tested for phytic acid using K-PHYT.  For solid samples, perform step 1 of the Sample Extraction using the following modification:
1. Accurately weigh approximately 1 g of sample material into a 75 mL glass beaker. Add 20 mL of hydrochloric acid (0.66 M) and homogenise the sample using a standard laboratory homogeniser or mortar and pestle, cover the beaker with foil and stir vigorously for a minimum of 3 hours at room temperature (preferably overnight for convenience).  Follow the full Standard Assay Procedure as described in the K-PHYT data booklet from step 2 of the Sample Extraction procedure.
Alternatively mill cereal, plant or food product to pass a 0.5 mm screen, then follow the full Standard Assay Procedure as described in the K-PHYT data booklet from step 1 of the Sample Extraction procedure.

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

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).

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

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.

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results.