Traceability refers to connection to the analysis of certified reference materials, and the uncertainty referred to is 'measurement uncertainty'. Measurement uncertainty is defined under ISO/IEC 17025 and methods are described there for its estimation. Essentially it's an estimate (cv), of what used to have several different names.

Mr. Wu,

In addition to the comments above you should carry out independent measurements using other method, for example MS.

As Paul mentioned about the construed meaning of the terms traceability and uncertainty measurement. Take some representative samples of yours and quantify them using the standard methods laid down by the apex bodies in reference to above terms.

First of all, thank you for all of your kindly comments.

@Paul Milham

I try to figure out your comment on traceability: National standard method proposed immunoaffinity column was used in my manuscript to demonstrate the superiority of lab-made SPE column.

CV, also known as RSD was described in a table of spiked samples, so as to as one of aspects to validate the method .

So, as you can see, I feel confused about the reviewer's comment.

@Bojidarka B. Ivanova

Yes, UPLC-MS/MS was applied to demonstrate the selectivity of the lab-made column.

@Shrikaant Kulkarni

In this manuscript, the novelty was the synthesis of a new adsorbent. Real samples were processed by lab-made column and IAC (The national standard proposed column) based on the national standard, and then was analyzed by HPLC whose method was also based on the national standard.

It seems all you mentioned points were involved in my manuscript if I, as a non-native speaker do not misunderstand all of your comments. This is why I do not know how to react to this reviewer's comments.

Again, thank you for your help!

If all of our points are there in your manuscript in your knowledge then the other connotation of the reviewer's comment is to compare the given method with others using other adsorbents and to check the variation in the recovery in terms of concentration and uncertainty level.

I have previously found that leaking is occurring in SPE (solid phase extraction) using commercial cartridge columns such as Sep-Pak Cartridge.

I have usually made hand-made HPLC columns, and have found that the HPLC columns should not have chink or crack in order to load the sample molecules completely (please see file; Netherlands biotin).

Therefore, I have always used the sample pretreatment for HPLC such as filtration by Ekicrodisc 13 (pore diameter 0.2 μm; Nihon Pall Ltd., Tokyo, Japan) (please see file; Wide Range of Biotin). Centrifugation at 13,000 x g for 20 min is similarly OK (please see file; JCB Fucoidan Transport).

By the way, HPLC-photometric method is quantitative, but HPLC-MS method is not quantitative. MS is depended on gasification of each molecules, and detects extremely small area.

Good question and useful answers.