Poor AAA Reproducibilty

<p><span>I am dealing with amino acid analysis onto my UPLC. I introduced Waters' application with AccqTag Derivatisation Kit, column and mobile phases. However, I have poor reproducibility and accuracy during method validation. I am using PLNO, injection reproducibility is ok, so I was thinking that sample prep is a critical step. I use calibrated automatic micropippetor during derivatisation. There is no pattern that can lead me to the conslusion: AA peaks are not <u>all </u>smaller or greater than in previous probe, actually some peaks are greater, some smaller. Maybe it is derivatisation process. Please, if someone have an idea- help me!</span></p>


  • ...For additional info, I tried to introduce L-norvaline as internal standard, but results were not better

  • lizh

    Bear with us as we will need to get an AAA specialist in touch with you. Do the starndards work or are there problems with all the analayses?


  • Thnx Liz,

    I am having the same situation with standards too.



  • Dear Perakojot,

    I assume you are running the Masstrak AAA system. What is the area precision over 10 injections of the Std and 10 extractions of the Std? How did you measure injection reproducibility? Which amino acids have the poorest reproducibility?


    LC Section

  • I am using UPLC with PDA detector. Area precision of standards is below 5%. Area precision of 5 preparation (extractions) of standards is poor 10-20%. Injection reproducibility is measured via peak areas of consecutive injections of the same standard solution. Tyrosine has the poorest RSD due to its low conc ina my samples (its area is about 400), but other AA are having much greater concs and responses.
  • lizh


    Apologies for taking so long, but I do have the best advice listed here from our experts. Please work through this list and please let us know, as follows:

    - Can you confirm whether you are running the method on MassTrak or UPLC AAA Solution? The question is necessary because the detector being used is a PDA, this is not a supported configuration for the MassTrak system. Are you working with an officially installed UPLC AAA Solution system? Can you confirm the configuration, what is the column management used, have you added or changed any tubing on the system? This is important as the dispersion and dwell volume of the system is critical to ensure performance with this demanding application.

    - Please can you send a chromatogram of that demonstrates the issue. Has the system performed this analysis successfully in the past? Knowing how the standard chromatogram looks would be very useful in troubleshooting.

    - How much sample is being derivatized and injected on the column? This will also help in assessing whether the user is operating well within the dynamic range of the method.

    - Are you following the method as given in the total system solution? We are asking this because of your statement about using PLNO. No settings in the method should need to be adjusted when working in the total system solution, so PLNO should be a given.

    - Are you performing the 10 minute heating step for the derivatization? We are concerned here, as the accurate and complete quantitation of Tyrosine through the heating of the derivatized sample/standard. Tyrosine is particularly susceptible to variability since this forms a dual adduct. The phenolic adduct needs to be broken down by heating, so if the heating step isn't consistent, this may be the source of the issue.

    - Are all of the amino acids amounts off or just certain ones? Some amino acids (Asp, Glu, Lys, Ala) are sensitive to excess reagent present as well as pH, and will generate smaller yield if conditions are not correct (2-5 fold excess of reagent, pH between 8 and 10). Disproportionate recovery of early to late eluting amino acid peaks in the standard might indicate that there is not enough organic in the derivatized sample, i.e., the late eluting hydrophobic amino acids can come out of solution if the organic concentration is too low. If the organic ratio is incorrect, this could be from an incorrect volume transferred or by evaporation of organic in the vial. The latter should not be a common occurrence if the you are using the total recovery vials with the non-preslit septa, as provided in the solution kit. The ratio should be maintained at 8:2 aqueous:organic.

    - While the use of NVa as an internal standard should improve reproducibility, be sure that you are looking at amounts and not raw areas in that comparison.

    - If there is variability in the analysis of replicate derivatizations, be sure that you are preparing them in a consistent manner. We caution against doing multiple derivatizations at the same time, i.e., we ensure that the sample/standard is mixed with the borate buffer before adding the reagent. We also make sure to mix the vial immediately following the addition of reagent to the vial. The reaction occurs on millisecond timescale, so mixing immediately is important.

    - We would suggest targeting the least abundant amino acid on column to be 1pmol, which should bring it well above the environmental contamination levels typically present in most labs. Also, that should still leave plenty of room at the upper end of the range, as well as have sufficient excess reagent present for the derivatization.

    - A more detailed description of estimating these things can be found in the troubleshooting section of the Rev B manual.

  • Hi!

    I am using AccQTag AAA Reagents, Standard solution is in-house standard adequate to conc's of AA in Solutions for infusion that we produce. Those conc's are within your proposed range. Column and MPhases are according to AccqTag kit. UPLC system is working fine, it is covered by OQ/PQ period. I am not assuming that something is wrong with UPLC system. In meantime, I somehow got the repeatability... I am using waterbath instead of thermoblock kept on 55C, and I think controling of the bath temp during derivatisation could be the critical step. At the end I have repeatability within 5% RSD, but I will keep on mind all the factors you mentioned that could effect accuracy of the method. I have to admit that method shows no roboustness at all, but we will use it anyway...

    Thanks a lot for suggestions.

    Milos Stojanovic
    Senior Associate
    Analytical Support Department
    Quality Control Division
    Hemofarm AD
    tel: + 381 (13) 803 281

  • Hi

    You should expect good reproducibility. Because you mention that some peaks get larger while others get smaller, I suspect derivatization as the source of variability. This is consistent with your observation that better control of the derivatization temperature helps with the reproducibility. The amino acids derivatize at different rates so the specific changes are useful in troubleshooting. Phenylalanine derivatizes most rapidly so it should be most reproducible. At the other extreme, Asp, Glu, Lys, and Ala, roughly in that order, react the slowest. If those four amino acids are more variable, we should suspect incomplete derivatization from one of several causes. The first two things to examine are the height of the AMQ peak and the presence of a peak of mono-derivatized lysine. The AMQ peak should be greater than 1 AU, and the "monoLys" peak, between His and Ser, should be less a couple percent the size of any other peaks. If the AMQ is smaller, it suggests that either not enough reagent was added or that the molar excess of reagent was too small. Both causes should be accompanied by a substantial monoLys. If however, the AMQ is large and there is a substantial monoLys, it suggests that the reagent is partially hydrolyzed, either by absorption of atmospheric moisture or by incomplete mixing when the reagent is added to the sample. We position the tip of the pipet below the surface of the liquid before rapidly expelling the liquid and immediately vortexing the sample.

    If the pattern of irreproducibility doesn't fit the above symptoms, it may reflect evaporation. As evaporation of the derivatized sample occurs, all the peaks are concentrated. However, the acetonitrile evaporates more quickly, so the early eluting, polar peaks get larger and the later eluting peaks come out of solution in the sample vial, tending to give lower peak areas.

    Let me know if these patterns fit your irreproducible results.