%RSD failed

vas
vas
in Instrumentation and Mass Spectrometry
<p>Hi,</p><p></p><p>During dissolution method validation of mebeverine Hydrochloride standard %RSD came on UPLC 4.5% for five replicate injections.</p><p>Where as in RRLC it is came 0.45%. In two systems used same column and same mobile phase.</p><p>what is problem i don,t understand %RSD came 4.5% in UPLC.</p><p>Please give me suggestions about %RSD failing.</p><p></p><p>Mobile phase: BUFFER : ACN (60:40)</p><p>Strong needle wash : ACN: WATER (90:10) </p><p>Weak needle wash : ACN: WATER (40:60)</p><p></p><p>My doubt is %RSD failed due to incorrect needle washes</p><p></p><p>So please choose correct needle washes.</p><p></p><p>Other wise any System Problem.</p>

Answers

  • stephaneDC
    stephaneDC

    Hi,

    Can you give us more details about your conditions like the type of injection: Full loop, Partial Loop Pressure Assist (PLPA) or Partial Loop with Needle Overfill (PLNO)?

    What is the matrix of your sample, is mebeverine Hydrochloride standard alone in your injected solution?

    Are you using isocratic or a gradient method?

    What's about the retention time of your component? Is there any variation of the retention time?

    I assuming you don't have retention time variation issues. So in this case, you probably experienced peak area variation issues explaining your poor RSD% results which are probably not due to a system problem.

    I'll make hypothesis to help you solving your problem:

    The critical thing is the injection mode:

    -Full loop, take precautions regarding carry over. You may have injected to much and carry over makes the peak area vary from injection to injection.

    -If you are using PLNO, there are several possible causes:

    -You want to inject 1 or 2 microliter with a large loop volume of 20 or 50 microliters that could explain the high variability of your sample. In this case change to a lower loop volume.

    -In PNLO mode, the weak needle wash solvent never comes in contact with your sample (no risk of peak shape issue due to pre diffusion of your sample) so chose the best solvent for a maximum solubility of your standard (in your case according information I've just found, probably better to put 100% H2O maybe with acid to increase the solubility because this component is very soluble in water)

    -If you are using PLPA mode, this could explain the poor repeatability issue you are experienced because this mode is the worst mode for accuracy and precision so change to full loop or PNLO injection mode.

    -If your matrix is viscous, you may adapt the syringe draw rate to a lower value in order to give sufficient time to your sample being aspirate by the syringe with the exact volume you request.

    Here are the few possibilities that could help you solve this problem, I hope that'll help but don't hesitate to give us more information regarding your experimental conditions as mentioned above!

    Best regards,

    Stephane

  • vas
    vas

    Dear Stephane,

    Tanks for your information, My injection mode is PLNO, Loop size is 10µL and injection volume is 3µL.

    Matrix of the sample is mebeverine Hydrochloride standard solution,Prepared with pH6.8 dissolution buffer

    Method is Isocretic, flow is 1.5ml/min

    Retetion time is 1.5min and no variation in retention time.

    Regards

    Vas

  • stephaneDC
    stephaneDC

    OK so in PLNO mode with no variation of retention time, the only explaination I have is what I said earlier:

    I would go for 100% water in your weak needle wash!! Be carrefull, don't put buffer in weak neither strong needle wash, only acid or base are tolerated but not buffer.

    Try this, don't forget to prime your weak needle wash several times, inject 3 or 4 blanks and try again multiple injections on the same vial to see % RSD.

    I've experienced exactly the same problem few months ago and I solved it this way.

    Please let us know about the situation after this!!

    Have a good week-end,

    Cheers.

    Stephane

  • vas
    vas

    Dear Stephen,

    Thanks for your valuable suggestion, I put weak needle wash pH6.8 water (previously pH6.8 adjusted with TEA)

    and injected standard solution 5 times ,%RSD found 0.47. strong needle wash is off.

    so any problem in this case.

    After giving reply i will start the validation.

    so please give me reply as early as possible

    Regards

    Vas

  • stephaneDC
    stephaneDC

    Hi Vas,

    I do not really understand what you want, I assume you solve your %RSD issue by changing the weak needle wash solvent with water ajusted to pH 6.9 with TEA (Tri ethanol amine?)

    I would have choosen ultra pure water alone instead, and leave a strong needle wash active with a mix of water and MeCN (40:60) because when you'll start to inject your unknown matrix you will probably need to rinse as much as possible the needle before each injection from polar and non polar contaminent. Therefore keep strong needle wash.

    Injection repeatability below 0.5% is good (I usually have between 0.1 & 0.3% depending on the application) you can go ahead for the validation!!!

    Good to see that my advices have helped you.

    Please, keep in mind that in PLNO injection mode, you can put whatever solvent you want in strong and weak needle wash because they never get in contact with the injected sample. Just to insure that the needle is totaly rinsed!

    I hope this will answer your question if not, give me a sign!

    Best regards,

    Stephane

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