Wrong RT on Massrtrak amino acid solution

<p>Hi, </p><p>I'm running the Mass trak amino acid solution on a "officially installed uplc system". After priming the system with the "start up system", I've run a reproductibility sample list. The colum is a new one (lot 1806004097) The problem is that the RT are late (4.75 For AMQ instead of 3.731) at each run and reproductibility is very bad. RT are moving randomly from the beginning to the end of the run. I've changed solvent A and B , the pressure is OK, about 10000 psi, and delta psi 15. I note we had a maintenance with Waters last weak where pistons and joins were changed and pumps were tested . Further more I'ma used to the derivatisation method that I have already practise an HPLC for several years.</p><p>Thanks if anybody can help.</p>


  • lizh


    Here is some general advise. From a previous post to help us further for troubleshooting.

    Many TX


    - 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.

  • Dear Kynurenine,

    We have have running the system for over 18 months, with only a couple of minor problems. The issue you describe may be due to several reasons. The first is temperature. Have you run the column for a number of injections at the same temperature? Secondly you mentioned that the system had been serviced recently. I would perform a static leak test on both pumps to test the plunger seals, check valves and for leaks. This may need to be performed several times to validated any errors that may occur. Have you noticed any small leaks in the system especially near the gradient mixer? We have had a similar problem and it was tracked back to a problem with the seals on pump B resulting in poor RT precision and AMQ RT (4.50 min).


    LC section