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Dear Pharm PK members,
Here, again I have a query regarding irinotecan, topotecan and SN-38
HPLC-UV method development for plasma samples. Topotecan is used as
an internal standard.
There are two issues with irinotecan and topotecan, whereas SN-38
behaves normally:
1) For calculating recovery of the extraction method: Irinotecan and
topotecan peaks show more area when spiked in plasma as compared to
when spiked in aqueous solutions (MeOH, acetonitrile, buffer pH 3.0)
and injected. Plasma processing is done by cold precipitation method
using MeOH:Acetonitrile (50:50 or 80:20).
2) Plasma linearity is coming out very well but this is not the case
with aqueous linearity.
Waiting for the feedback.
Thanks in advance
Regards
Tripta
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The following message was posted to: PharmPK
Dear Tripta,
The problem with your compounds is due to their aqueous instability I
guess. I hope following reference will be useful for you.
Li WY, Koda RT., Am J Health Syst Pharm. 2002 Mar 15;59(6):539-44
Please also check for the co eluents in case of plasma samples.
With Regards.
Suresh
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The following message was posted to: PharmPK
Hi Tripta,
In your solution linearity, I guess, the solvent mixture (MeOH,
acetonitrile, buffer pH 3.0) that you have used in place of plasma is
your mobile phase. While this approach seems to work with mobile phases
that are predominantly aqueous, it may pose issues with mobile phases
that are predominantly organic in nature, especially when you are using
precipitation method and using 100% organic solvent (which in your case
is MeOH:Acetonitrile (50:50 or 80:20)) to achieve that. In case of your
solution linearity, doing so would lead to a greater percent of organic
solvent in the injection solvent than that present in the mobile phase
which is not good for reverse phase chromatography. The reason that you
don't have any issue with plasma linearity is that plasma samples being
aqueous in nature might be changing the composition of injection solvent
to suit the mobile phase used.
Therefore, in cases where the mobile phase is predominantly organic, it
is always advisable to make sure that the injection solvent has greater
percent of aqueous than in the mobile phase to avoid peak broadening
which may some time (e.g. if the volume injected is high) lead to peak
splitting more so with peaks eluting late. As it is not possible to
spike your analyte into 100% aqueous buffer (to mimic plasma spiking)
owing to potential precipitation/adsorption issues, I advise you to
continue to spike into the current solvent mixture (MeOH, acetonitrile,
buffer pH 3.0) that you are using but use a mixture of aqueous and
organic solvent (aqueous percentage should be higher than that of the
mobile phase) instead of pure organic as the precipitation solvent for
your solution linearity. Hope this should fix your problem.
Kasiram.
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