If your HPLC procedure is just optimized, you need to perform the following experiments:

- linearity

- LOD and LOQ

- precision and trueness

- recovery

- stability (short and long-term)

For each point you need to perform 6 independent experiments.

For linearity are necessary at least 5 non zero points at different concentration levels.

Additionally for precision and trueness you need to analyse 6 times 3 QC levels (low, medium, and high and the concentrations need to be different respect the concentration levels used in linearity) obtained by spiking (with known amount) blank matrix.

All these procedures are deeply discussed at the following links:

http://www.ich.org/products/guidelines/quality/quality-single/article/validation-of-analytical-procedures-text-and-methodology.html

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002662.pdf

If your HPLC procedure is just optimized, you need to perform the following experiments:

- linearity

- LOD and LOQ

- precision and trueness

- recovery

- stability (short and long-term)

For each point you need to perform 6 independent experiments.

For linearity are necessary at least 5 non zero points at different concentration levels.

Additionally for precision and trueness you need to analyse 6 times 3 QC levels (low, medium, and high and the concentrations need to be different respect the concentration levels used in linearity) obtained by spiking (with known amount) blank matrix.

All these procedures are deeply discussed at the following links:

http://www.ich.org/products/guidelines/quality/quality-single/article/validation-of-analytical-procedures-text-and-methodology.html

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002662.pdf

If you are using the AOAC HPLC procedure you may only have to verify that 'it is appropriate in your hands for your sample'. Thus, the previous suggestion.

You can follow ICH guidelines for method validation.

ICUMSA (International Commission for the Unified Methods of Sugar Analysis) may also have a method for honey.

Dear Marcello, Bruce and Atul

Thank you for your time.

I have a query, How can i prepare samples to calculate Accuracy /%Recovery of the method?

Directly spike lowest and highest calibration level in water

or Spike lowest and highest calibration level in honey?

Thanks & Regards

Dear Bruce,

Thanks for your reply but i didn't find any method for honey analysis in ICUMSA

Regards

Dear Ankita Sood

I would like to expand Marcello Locatelli's idea

If standard methods have not been established, the guideline mentioned below may helpful for method validation.

1 Selection of suitable column and solvent system

· C18 column has shown good separation for this type of compounds

· The mobile phase, water-methanol with a buffer (you can use gradient/Isocratic) – gradient elution is good for better separation

2 Select suitable wavelength for quantification

First you have to run stander solutions (HMF standard are available at Sigma-Aldrich) to find out suitable wavelength for quantification (285 nm is commonly used).

It is better to use UV/Visible spectrometer or DAD detector to find out λ max for quantification

3 Linear range

Inject standards solutions (from signal to nose ration is 3:1) to higher levels to get the linear range for the calibration curve. (In accordance the guideline attached)

4 Meted of Quantification

It is beret to use External calibration procedure (using peak area or high)

5 Determination of Analytical Limits

you have to determine the Limit of detection (LOD) and The Limit of quantification (LOQ) or Minimum Detection Limit (MDL) – you can calculate those valves using the method attached.

6 Recovery study

You have to conduct recovery study (in accordance the guideline attached). You can start from estimated LOQ.

7 Confirmation test

You can use spectrum of DAD of your sample at the retention time (only for positive samples) to get some confirmation evidences. If it is possible to use LC/MS, It is much better.

thank you

Piyal

Pls find the attachment

Piyal

Dear A. G. Piyal Aravinna ,

Thank you for your reply.

The method which i am using is already an established method.

Regards

Dear A. G. Piyal Aravinna ,

Thanks for sharing the link.

Regards

I would like to read the method that you are going to apply.

thank you

piyal

"HARMONISED METHODS OF THE INTERNATIONAL HONEY COMMISSION"

"Determination of hydroxymethylfurfural by HPLC"

Regards

Dear Ankita,

I would recommend checking the following document:

DIN 10751-3:2018-09 Analysis of honey - Determination of content of hydroxymethylfurfural - Part 3: High performance liquid chromatographic method

(https://www.beuth.de/de/norm/din-10751-3/290897116)

It looks like the final draft of an official DIN method to replace older standards.

As usual such methods include the validation data for the method chosen, and this might serve as a starting point for your laboratory validation. The benefit of having such a standard is that once the method is accordingly established in your lab, you would only need to run the lab validation, rather going through the hassle of a basic and fundamental evaluation of the entire method.

Hope this helps.

Best regards,

Detlef.

Dear Detlef,

Thank you for your reply. I would like to have a look into the document specified by you.

If possible, kindly share the document as i don't have access to it.

Regards

Dear Ankita,

many thanks for your message.

I am afraid to say, but I cannot share this document, as copyrights heavily protect it. For this reason, I provided you with the link to the publisher site.

Regards

Detlef.

Dear Detlef,

I can totally understand.

Anyways thank you so much.

I really appreciate your quick response and valuable guidance.

Regards

I have found a gradient elution method (please see file; HMF determine).

Gradient elution by using methanol/sulphuric acid seems to be difficult. Therefore, we have developed an Acidic-phosphate buffer (0.1 M, pH 2.0)/2-propanol/ethylene-glycol system (please see file; Lysozyme by RP-HPLC). In this HMF case (non protein molecule), column temperature should be elevated to 40 - 45 C in order to reduce the column inlet-pressure. In protein case, we used column temperature at 15 C in order to get high-recovery of proteins (non-denatuted form) from the Reversed-Phase (RP) column.

Dear Kou Hayakawa,

Thank you for your reply but i cannot change my SOP as i am working on isocratic elution i.e. (10% methanol throughout the run)

Regards