Recovery issues for API in presence of excipients in UPLC

<p>I have a situation where technical input from Waters or UPLC forum will be much appreciated. Essentially for a drug substance solution in buffer and methanol, we get 100% recovery in HPLC. We also get ~ 100% recovery with same solution in UPLC. Next, the drug substance is spiked with the excipients for linearity check and provided 100% recovery in HPLC. The same spiked solution in UPLC under identical condition as DS analysis now gives 95-96% recovery.</p><p>The mobile phase is isocratic : Water: Methanol: TFA (47:53:0.1), wash solvents are water, methanol and TFA. The sample solvent is ammonium acetate buffer pH 5.0: methanol. PLNO mode 5 uL loop and 2 uL injection.</p><p>Any clues why the UPLC recovery drops for the DS+excipient solution?. </p>


  • As an addendum to the above problem, syringe draw speed change did not have any effect on the UPLC recovery. The rUPLC ecovery however goes up when the DS +excipient solution is stored in refrigerator for few days.

  • sazimi

    There could be 2-3 possibilities, first of all, freshly prepared samples & solvents are always recommended, i don't know your sample chemistry but some samples need cold environment (like sample prep in ice),

    Did you try full mode instead of PLNO....? inject 1uL... as full mode is more precise...

    I suspect your wash solvents are not ok in comparison with sample solvent.... try 50% MeOH weak & 80% MeOH strong wash... that your wash solvent is highly acidic (TFA 0.1%) where diluent pH is 5.0

    & also try sample temp. 8C with column temp. 35C.... as you said your recovery is good after refrigeration

    ~ Salman

  • Salman,

    Thank you for the suggestions. Actually various combinations of wash solvents were tried but did not improve the recovery of DS in UPLC in presence of excipients. PLNO mode gave 100% recovery for DS. I could check if full loop mode may improve recovery for tablets.

    Cooling the autosampler was also investigated, unfortunately that did not work. The temp was reduced to 7oC, column had to be maintained at 40oC for selectivity. of API from impurities and deg products.

    Hopefully some other suggestions will also be posted.

  • lizh


    I know that your mobile mobile phase is isocratic : (Water: Methanol: TFA (47:53:0.1)), and that your wash solvents are water, methanol and TFA. Can you tell us the exact proportions and composition of your wash solvents for the SW and the WW. You mention you sample diluent, what else do you do for sample preparation in this case.(Sample solvent is ammonium acetate buffer pH 5.0: methanol).

    Further can you let us know what the excipients are (ie. is this a simple sugar matrix or do we have something much more complex like a transdermal patch).

    If everything is usually OK in a regular assay, it is a combination of acid conditions for the dissolution media and/or the interaction of the excipients with the system that we need to unravel.

    Many TX