UPLC

Hi everyone does anyone has a UPLC developed method for Oxytocin & Chlorobutanol in one go? if Y could you plz load this method.ThxAtif

Answers

  • Did anyone work with this method oxytocin & chlorobutanol on ULPC?

    Atif

  • Hello,

    Do you have an existing HPLC method that you have been successful with? If so, could you please share it with us?

    Thanks,

    --Doug

  • Hi Doug,

    Yes we hav approved method for these two actives on HPLC I will post it shortly...

    Atif

  • Hi Doug,

    First of all accept my apology to reply you late, actually I was on vacations because of our plant shutdown...

    Anyway first there is little correction we have HPLC method for Oxytocin and GC USP method for chlorobutanol. In Oxytocin HPLC method conditions are as follow:

    Mobile Phase A: 0.1M Sodium Phosphate buffer monobasic

    Mobile Phase B: Acetonitrile: Water with 1:1 rartio

    Diluent: 5g chlorobutanol+5ml glacial acetic acid+5g ethyl alcohol+1.1g sodium acetate in 1L.

    Std. Prep:dissolve whole USP vial in 5ml diluent (Conc. 9.2IU/ml)

    sample prep: 5ml sample in 10ml volumetric flast dikute with diluent. (LC: 20IU/ml)

    Chromatographic Conditions:

    Column Temp: RT, wavelenght: 220 nm, flow rate: 1ml, Injection Vol: 100 microlitre, Run Time: 45 min. Column: Novapack C18 4microns 3.9X150mm

    Gradient table:

    time MP-A MP-B

    0 70 30

    20 50 50

    25 70 30

    45 70 30

    SST parameters:

    %RSD: NMT 2.0%

    Resolution b/w Oxytocin & nearest adjacent peak: NLT 1.5

    If you need anything more please don't hesitate to ask.

    Regards,

    Syed Atif

  • I would suggest transferring your HPLC method to UPLC technology using the ACQUITY UPLC Columns Calculator.

    Do you have a copy of the ACQUITY UPLC Columns Calculator? You should have an installed copy on the PC connected to your ACQUITY system. If not, just go to the Overview tab, scroll down a bit and look for the section called 'UPLC Basics.' Click 'HPLC to UPLC Calculator' and download the *.exe file.

    Just use the same LC conditions that you describe in your HPLC method (e.g., mobile phases, temperature, standards, detection wavelength, etc) on your ACQUITY UPLC instrument and change only what the calculator tells you to change. I've attached a file that has some starting conditions based upon the calculator's results. I would suggest using either the HSS T3, BEH C18 or HSS C18 column (2.1 x 50 mm or 2.1 x 100 mm). The eSelectivity chart (located right under the HPLC to UPLC Calculator as described above) suggests HSS T3 is the closest chemistry, but I would just try what you have on hand first before buying anything new.

    Good luck,

    --Doug

  • Thanks for your prompt reply.

    Yes we have that calculator that we have done all this what you attached as a pdf and we are using BEH C18 (2.1 X 50mm) and keeping the all same conditions as we have on HPLC we don't have any problem with Oxytocin peak even we have done almost all validation parameters but we were trying to develop a method for both (Oxytocin & Chlorobutanol) in one run on UPLC that's what I asked but using these conditions we have very bad peak shape and very high tailing for chlorobutanol. So we are working on it but if have some thing about this please just let me know.

    Thanks,

    Syed Atif

  • Dear Atif,

    yes you asked simultaneous determination of oxytocin & cholrobutanol by UPLC, which I know you copied USP method of oxytocin inj. , I have some questions & comments about to develop your method.

    - Did you succeed to develop before, simultaneous method of oxytocin & chlorobutanol by HPLC as USP method of Oxytocin inj. is with chlorobutanol, & oxytocin RS determined with CB

    - If your old HPLC method is only for oxytocin (as you said you are doing CB by GC-FID), then what is the reason,bcuz USP method is for both not for one

    - If you tried your method on UPLC can you load UPLC chromatograms of your method in which you said CB peaks were bad

    - Also provide all other UPLC conditions, like wash solvents, inj. volume, injection modes etc.

    After that I can suggest you how to minimize peak tailing in CB, may be some sample overload there or some pH adjustment requires, as diluents containing CB in high conc.

    Note: for your interest we developed this method isocratically with 100% success, but I don't want to disturb your existing method as you did too much hard work on it, both peaks are possible by UPLC if its true in USP by HPLC.

    ~ Salman

  • Dear Salman,

    First of all make correction in your statement that USP has a mothod for both actives on HPLC no USP has HPLC method only for Oxytocin & GC method for chlorobutanol as I mentioned earlier but bacause now we are in the development phase of this method on UPLC that's why we thought that if its possible to run both actives in one go on UPLC that will save lot of things but before that we had HPLC method for Oxytocin & GC method for CB. As I mentioned that we kept all conditions same except what we got from calculator but the thing is we already have done our validation but we will try to resolve this tailing factor for CB because rightnow we are working with some other method.

    Thanks

    Atif

  • Dear Atif,

    I don't know which USP you have, the method which you copied was from USP2000-2004 etc. & it is clearly written that "RT of oxytocin at 10min & 15-17min for CB" (file attached). Also I checked in all old & our latest USP2008, there are no assay of CB by GC in oxytocin inj. (may be its your company method).

    For your convenience i'm attaching oxytocin inj. assay from USP-2000 & USP-2008, (first method that you followed)

    May be USP-2008 method helping you, mostly similar but somehow easier.

    We did this method 6-7 month before by BEH Shield with slight differences in USP2008 method & every thing was ok plus sharp OT & CB peaks in 1.4 minutes.

    I asked you before load your chromatgram, it will clear either it was 'dewetting', sample overload or anyother cause.

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