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Dear Group,
Our analytical lab has developed an LC/MS method to determination of creatinine in human
urine. They used water as a surrogate matrix for urine. I suspect they did this because
the high levels of endogenous creatinine in urine would prevent develop of an assay with a
reasonable LLOQ. I found some literature supporting this for other urine assays that
measured other analytes that occur naturally in urine.
Yet the FDA bioanalytical guidance recommended "Bioanalytical method validation includes
all of the procedures that demonstrate that a particular method used for quantitative
measurement of analytes in a given biological matrix, such
as blood, plasma, serum, or urine, is reliable and reproducible for the intended use." My
understanding is that the matrix for the method validation should be carried out in urine.
Could anyone comment on whether water or urine should be the matrix for validating the
bioanalytical method of creatinine?
Thanks,
Norman
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Dear Norman,
Regarding use of water as a surrogate matrix for urine in standards
and/or controls:
Since it is impossible (or highly unlikely) to obtain creatinine-free
human urine, I think that your general approach is OK. We used
"synthetic" human cerebrospinal fluid (CSF) in a similar situation where
real human CSF is difficult to obtain and of variable quality, and
received no challenges from regulatory reviewers. In the validation, we
included some authentic CSF samples that had been analyzed before and
after supplementation with additional analyte to show that the amount
added could be accurately measured in a set of samples.
Since urine contains a number of substances other than creatinine, you
might consider supplementing the water with an arbitrary amount of some
of these (protein, urea, etc) in order to match urine properties more
closely. Alternatively (or also with supplemented water), during
validation, add a known amount of creatinine to a representative number
and variety of urine samples (normal, lipemic, colored, containing
blood) and demonstrate that the amount added (total minus amount before
the addition) is accurately and reproducibly determined by your method.
This should be done for 2 or 3 added creatinine concentrations. In
addition to validating water (or the supplemented water) as a surrogate
matrix, this would identify any particular types of urine that your
method cannot analyze. Of course you can also measure urine creatinine
by a different established method as well, to ensure that your LC/MS
method with water or supplemented water as surrogate matrix gives
equivalent results for real samples.
Finally, since urine is readily available, you should consider using
individual or pooled urine for QCs (rather than water), even if you use
the water for standards and blanks.
-Tom
Thomas L. Tarnowski, Ph.D.
Corporate Liaison & Special Tasks Director
Organizing Committee
CACO Pharmaceutical and BioScience Society
ttarnowski1.-at-.aol.com
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The following message was posted to: PharmPK
Dear Norman,
To validate any method you have to consider same media,say urine in your
case.you can screen different lots of urine to get desired detection
level which is acceptable to you.If not you need to process the urine to
remove creatinine without changing the nature of urine.I feel it gives
you some idea.
Regards
Hareendran K Nambiar
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The following message was posted to: PharmPK
Dear,
I suggest you use simulated urine fluid without creatinine. Data on
urine composition should be available in text books . It should be
better than using water as you would account for effect of other
elements in the matrix.
Kind regards
Dr Youssef Hijazi
PKPD Expert
Germany
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Water is rather an ideal and rarefied matrix. Given the complexity of
urine I would not use water unless I could demonstrate absolute recovery
against urine. A surrogate matrix is permitted per guidance in those
cases where actual matrix is difficult to obtain- cerebrospinal fluid
for example. Urine is not difficult to obtain but given the ions and
concentration of the ions and other components- may be very difficult to
work with.
With so many methods out there for creatinine and no real need to push
sensitivity, why choose this LC-MS? If you are looking for accuracy and
precision in estimating GFR why not use inulin or another compound that
is filtered, not metabolized nor re-absorbed? That would remove the
baseline issues you are undoubtedly meeting.
Edward O'Connor
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The following message was posted to: PharmPK
Hi Norman,
Your understanding is right. Method development should be carried out
using urine as matrix. FDA do allow to use surrogate matrix, only when
you prove that sufficient natural matrix is not available.
Vinayak Nadiger
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The following message was posted to: PharmPK
Because the salts can either enhance or reduce ionization and or
detection it would be best to use urine. Measuring creatinine in urine
would or should not require an lloq since you should Never see a sample
w/o creatinine. It would be more appropriate to measure sensitivity in
terms of change from baseline. You could also run an isotope
dilution-stable label-if you want this for GFR you should consider a
different marker insulin like etc
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