Chromatography Forum

Hi Guys,

I have to quantitate free amino acid in my sample using LC-MS/MS. My sample mainly is an assay for checking the enzyme activity of phenylalanine hydroxylase in converting phenylalanine to tyrosine. Mostly researchers are using ez-faast kit but I want to know about AccQ tag from waters or any other alternative.
Can you please help me find a kit that suits my assay? Iam completely new to this field..

Thank you in advance

Waters AccQ-Tag:
https://www.waters.com/webassets/cms/li ... 930494.pdf
https://www.waters.com/webassets/cms/su ... 052881.pdf
Waters AccQ-Tag Ultra:
https://www.waters.com/webassets/cms/su ... 001331.pdf

AccQ-Tag is an HPLC-UV or an HPLC-FLD variant. AccQ-Tag Ultra is a UPLC-UV variant (smaller particle size, higher pressure, higher speed of analysis). Both variants use essentially the same derivatization procedures and reagents. The derivatization is manual, but fairly simple. Use of internal standard is desirable to obtain accurate results. Derivatization kit for the HPLC variant is labeled as AccQ-Fluor because the reagents are tested for fluorescent impurities by the manufacturer. Eluent concentrates and columns are marketed by Waters. UPLC variant uses non-linear gradient in order to separate Lys and Tyr. Hence, Waters Acquity UPLC system is desirable. Agilent Infinity 1290 with ISET function in the software (emulation of other LC instruments) can be used instead. HPLC variant is relatively sensitive to the design of the eluent mixing unit of the instrument, which influences the stability of retention times of the early eluting amino acids.

Derivatizaton with OPA. HPLC-UV or HPLC-FLD. Solutions from Agilent. Variants with Hypersil AA-ODS column and with Zorbax Eclipse-AAA column. Derivatization is carried out in the autosampler of the HPLC instrument. FMOC reagent is added only when proline is needed.
https://www.agilent.com/cs/library/slid ... 0Steed.pdf

With Hypersil AA-ODS, it is sometimes difficult to obtain acceptable separation of Ala/Arg and His/Gly and to obtain acceptable peak shapes for Asp and Glu (addition of THF is the cure), but this is not a problem for your task. Phe and Ile are eluted close to each other, but the separation is usually acceptable.

If necessary, centrifugal filters can be used to remove the enzyme from your samples before the derivatization.
https://www.merckmillipore.com/RU/ru/li ... plvyxn,nav

Thank you so much for your reply.

I am going to use HPLC so Waters AccQ taq is gonna be useful for me.
But is this compatible with cell culture medium. My samples also contain protein from mammalian cell which are lysed with detergent. Will any of the component hinder with the kit solution?

And can pls tell me what is the difference between 930494 and 0052881.
Does it depend on column dimensions. I am new to this field so kindly help me understand this.

And can pls tell me what is the difference between 930494 and 0052881.

These are just two links describing one thing (AccQ-Tag).

I am going to use HPLC so Waters AccQ taq is gonna be useful for me. But is this compatible with cell culture medium. My samples also contain protein from mammalian cell which are lysed with detergent. Will any of the component hinder with the kit solution?

Look here:
https://www.waters.com/webassets/cms/li ... 6130en.pdf
In general, the AccQ-Tag procedure is compatible with cell culture medium with only minor sample pretreatment (dilution). Anyway, you will have to do some method development and validation work to be sure that you get accurate results with your specific samples. Additional sample pretreatment (e.g. protein removal) will extend the lifetime of the column.

Thank you so much for your help.

If you are doing LC/MS analysis, then you don't need to do any derivatization and can anaalyze amino acids directly using either RP cation-exchange mixed-mode or HILIC/cation-exchange mixed mode.
Here are few applications:
https://helixchrom.com/applications/hpl ... hd-column/
https://helixchrom.com/applications/hpl ... de-column/

contact us if you have questions