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Dear All,
I want to know that whether Post extraction spiking experiment can be used to quantify ion suppression? If so what is the logic in it?
Thanks
Jacob
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The following message was posted to: PharmPK
Dear Jacob,
Post extraction spiking is probably one of the best approaches to evaluate matrix effect, it provides some quantitative measure of its impact (as opposed to e.g. post column infusion, which looks very nice and is interesting qualitatively, but provides very little quantitative information). Adding solution to an extract (often dried down extract) of matrix and comparing to pure solution allows to determine any matrix interference without being confounded by extraction recovery. Indeed any method proficiency issues (comparing pre-spiked QC extract to pure solution) may be defined in terms of extraction recovery and/or matrix effect, so that solutions may be sought in a rational manner. One author that dedicated some effort to the theme is Bogdan Matuszewski, the following article might be of most use to you.
Anal Chem. 2003 Jul 1;75(13):3019-30
Remember though that validation exercises on commercial matrix may not guarantee for potentially interfering components (formulating agents, metabolites ...) present in real samples. The new EMEA Draft guideline on validation of Bioanalytical methods expanded on this theme. Specific experiments may be valuable to evaluate this. A first safeguard during analysis could be to use a stable label internal standard and monitoring its response in all samples (e.g. comparing response in real samples and calibration / QCs, on the lookout for any inconsistencies).
Patrice
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Jacob,
Yes, one way to quantify ion supression is by determing relative recovery= peak area of samples spiked post-extraction/peak area of neat samples. The rationale is that the absolute recovery (peak area of samples spiked pre-textraction/peak area of neat samples) results from losses due extraction efficiency + ion supression. When you spike post-extraction, you bypass the extraction efficiency part because the samples are already extracted. Any loss of signal in that case would be mainly due to matrix effect from the endogenous components that are co-extracted with your analytes.
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Dear Jocab,
First of all you need to clear that what is ion suppression / ion enhancement (Matrix Effect) and recovery and How we can calculate it.
SO Lets prepare 3 equivalent samples-
1) AQ SAMPLE EQUIVALENT TO EXTRACTED ONE
2) EXTRACTED SAMPLE
3) POST SPIKED EXTRACTED BLANK PLASMA
If you compare 2 with 1 that will be your recovery.
and if you compare 2 with 3 , that will be absolute recovery.
and if you compare 3 with 1 that will be matrix effect (ion suppression - ion enhancement ).
As we know matrix effect is due to presence of matrix in plasma that may enhance or decrease drug - and IS response and here we are comparing post spiked sample with aq one which is equivalent to that sample.
we can calculate Matrix Factor as:
MF=(POST SPIKED EXTRACTED BLANK PLASMA - AQ SAMPLE)
ie.
MF=peak response in presence of matrix ions/peak response in absence of matrix ions
Matrix factor may vary for different lots of plasma.
Matrix factor must be close to 1.
while calculation take area ratio of drug and IS
as per guideline you hv to take minimum six different lot of plasma, calculate MF for them and % cv should not be more then 15%.
plz refer some AAPS articles--
http://www.aapsj.org/articles/aapsj0901/aapsj0901011/aapsj0901011.pdf
If the value is not close to unity, but if % CV is with in limit. there is no need to redevelop method.
Is it OK---------------- discussion continued-----------.
ASHISH SAXENA
BIOANALYTICAL RESEARCH
INDIA
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1:2 is absolute recovery, diminution of signal is due to "matrix effect". 3:2 Post extracted sample is the effect of ions on recovery versus the effect of fewer ion on recovery.
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I totally agree with this explanation, but susprisingly the new EMEA draft guidance does not include post extracted spiked samples for matrix effect calculation. Instead, the matrix effect section (4.1.8) says:
"For each analyte and the internal standard, the matrix factor (MF) should be calculated in each lot of matrix, by calculating the ratio of the peak area in the presence of matrix (measured by analysing blank matrix spiked with analyte at a concentration of maximum 3 times the LLOQ after extraction), to the peak area in absence of matrix (pure solution of the analyte). The IS normalised MF should also be calculated by dividing the MF of the analyte by the MF of the IS.
The CV of the IS-normalised MF calculated from the 6 batches of matrix should not be greater than 15 %."
For me, the guideline is mixing concepts, as the ratio of the peak area in the presence of matrix (that obviously will have to be extracted) to the peak area in absence of matrix is RECOVERY.
I would very much appreciate your thoughts about this definition of Matrix effect in the EMEA draft guideline
yazen alnouti
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The following message was posted to: PharmPK
FDA guidaline is correct.
Recovery is the analyte conc. spiked before extraction divided by the analyte conc.spiked after extraction.
Matrix effect is the analyte conc. spiked after extraction divided by the analyte concentration spiked into neat solvent (reconstitution solvent)
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