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Hi,
Recently i developed a method for analysis of a compund in human plasma,and got that validated with all the requirements.Now to my surprise the method is not performing as required in Sample analysis(Real condition),?
Even the calibration standards are not meeting the acceptance?
What may be the possible reason?Can anybody explain?
The method is regarding analysis of a morphine category drug, which is resistant to collision induced dissociation, It has to be detected at say 25pg level. the method iam using is simple SPE. the mobile phase is Ammonium Acetate and combination of ACN in gradient mode.
Thanks in Advance
Jacob
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The following message was posted to: PharmPK
Hi Jacob,
You'll have to more specific; e.g. what exactly do you mean by "Even the
calibration standards are not meeting the acceptance"?
You'll have to narrow down the problem, step by step, and when you have
put your finger on the sore spot you figure out what to do about it.
Perhaps your (authentic?) standard has degraded? Your column wants to
retire (...). Your MSD needs a good scrub. Are you using a different
batch of something (e.g. SPE columns, plasma, buffers, etc.)?
Good luck,
Frederik
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The following message was posted to: PharmPK
Dear Jacob,
You may wish to troubleshoot in following order:
1.Ensure that instrument set up is working fine, right from injector to
MS
2.Re-analyse calibrations and QC's
3.If batch fails again, have a re-look at validation data
4.If validation data looks fine, start searching for any differences
between validation condition and actual analysis condition
5.If there are no differences found, you have not developed a robust
method
Here is a paper for your reference:
High-performance liquid chromatographic-electrospray mass spectrometric
determination of morphine and its 3- and 6-glucuronides: application to
pharmacokinetic studies
(R. Pacificia et al ,Journal of Chromatography B Volume 664, Issue 2, 17
February 1995, Pages 329-334)
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Dear Jacob, There may be many reasons but to narrow your hunt, first you should find out the conditional difference between validation and real sample analysis. Check the following points:
* Plasma lot used during validation and real sample analysis is same or not? :- If same then standard should pass if plasma is not the reason
* Matrix effect:- . howz the result during validation with lipemic plasma?
* Column lot and life: Which column you are using. As wn we used cyano column for one of the drug analysis, it needed equillibriation with mobile phase for 2-4 hours.If the time gap between the validation and real sample analysis is more then the same column may need to equillibriate in the same way.
* Solvent lot and make.
* Gradient program
* Standards for real sample analysis are bulk spiked or freshly spiked?
* Check the response at LLOQ and ULOQ during validation and real sample analysis.
thanks,
with regards, Rashmi Shiju
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Dear Jacob,
First you have to check when you formed validation that step are same or not . then you have to re tune compound and that you are getting stable response or not .after this you start with initial stage you are getting satisfactory response for LLOQ level than ok and inject aqueous linearity and then try with spike linearity also you have to check that your blank is clean after that you are not getting spike linearity then try to change matrix lot may be its comes due to matrix affect and once you try spike mqc level by using your study sample predose (0 hr)
Regards
Mahendra sahu
Torrent research centre ahmedabad
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