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I would like to know the opinion of yours about the Matrix Effect.
If this effect is present only for the internal standard, what will
be the influence in the bioequivalence study?
Regards,
Kelen Soares
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Dear Kelen Soares,
This depends on whether the matrix effect (1) enhances or (2) suppresses
ionization of the internal standard, (3) whether the enhancement/
suppression
is a constant effect (improbable) or whether it fluctuates (highly
probable), and (4) whether the matrix effect is a factor only in the
clinical standards or whether it is consistently present, whatever the
matrix source may be.
In short, there is no simple answer except to ensure that your method is
free of interfering effects for the internal standard.
Ian M. Davis, M.S.
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Matrix effect is caused by the ions and other substances present in the
sample. If it is plasma the anticoagulants added to the sample also
cause
suppression (mostly) or enhancement. The amount of suppression varies
but
for some analytes you can manipulate assay conditions to avoid matrix
effect. For example, ESI is more affected than APCI.
The matrix ions may not interfere with the internal standard but might
suppress the analyte signal. If this happens you see less
concentration in
the sample than actually present. In samples with low concentration of
analyte matrix effect could be significant enough to go below LLOQ.
Chandrani Gunaratna
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Dear Kelen Soares,
In theory, matrix interference may be present in non-MS based assays,
however, the majority of assays being developed today are MS based,
and pertains to HPLC-MS/MS based bioanalytical methods.
Matrix effect may change the instrument's response either increased
(enhancement) or decreased (Suppression) for a given concentration of
analyte in the presence of other sample components.
Absolute matrix effect- The ratio of the instrument response of a
standard spiked after extraction into an extract of control matrix
(B) to that of a neat standard injected directly (A).
Matrix Effect (%) = B/A x 100
Relative Matrix Effect- Variation of the absolute matrix effect
between different lots (sources) of matrix.
Standards are not typically prepared in the same lot of matrix as its
composition may vary over the sampling time course due to Food
intake, diurnal variation of endogenous components, co-administered
medications.
In many instances variation of ionization of internal standard may
cause the suppression of analyte signal, which results in showing the
less concentration of analyte in sample rather than actually present in.
A properly chosen internal standard may significantly reduce relative
matrix effects.
Matrix effects "should be investigated" to ensure that they do not
compromise the precision, selectivity, and sensitivity of assays
before initiating the bioanalysis of BE study.
Regards
Shravan Kumar.Eduru.
Dr.Reddy's laboratories Ltd.
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Matrix effects are nasty because they are elusive - you have to
measure it to know whether you have it or not. The easiest way to
measure it is to spike the unknown samples with a known amount of
standard and see if you are getting the right difference (before and
after the spiking). For example, if your unknown sample gives you 500
ng/ml, you spike it with another 500 ng/ml, and after the re-run you
only get 750 ng/ml instead of the expected 1000 ng/ml, means that you
have your analyte suppressed by a factor of 2. This does not solve
the problem yet, but at least you know you have it, and to what
extent. To solve it you can do many things, but my first choice is
always to try and co-elute your internal standard with the analyte,
because there is a very good chance that the internal standard will
get suppressed to the same extent as the analyte, and the calculated
concentration will be correct. You verify it using the method
described above.
Andrew Volosov, PhD
Inotek Pharmaceuticals
Thanks and regards,
Andrew
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Andrew: There are actually at least two experiments. The first
examination of a matrix effect involves a comparison of analytes from
buffer or extraction media on one hand and the biological matrix on the
other. The second exercise examines the distribution of recoveries
among samples of the biological matrix from unique individual donors.
The latter should be controlled as much as possible with regard to
variables such as sex (balance the number equally to minimize bias),
fasting duration, over the counter meds,prescription drugs, street
drugs, disease states, time of day, etc. Unfortunately not many of
these
can be controlled and when dealing with human donors we depend upon the
kindenss of strangers. I have even run across a situation where the
anticoagulant, heparin, would bind to and mask the analyte of interest.
Unless you compare with a defined media-buffer- you might not be aware
of that kind of matrix effect. Besides binding or aggregation there is
also the contribution of enzymes which catabolize the analyte.
The internal standard is always a good choice especially if it is only a
deuterated version of the analyte. If not, you may want to incubate
analyte and internal standard to determine if the matrix affects both
equally.
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
609-228-0243
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