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Hi,
I am trying to develop and validate a LC-MS method for simultaneous
quantitation of four structurally similar analogues. I would like to
know the sample pooling strategies to prepare the calibration curve in
plasma. The procedure I followed was to make individual plasma working
solutions, then take 25ul aliquots each drug in an eppendorf tube
resulting in a pool of 100 ul containing all the four analogues,
followed by protein precipitation using 3 volumes of acetonitrile (The
internal standard is present the acetonitrile used for protein
precipitation). Finally the clear supernatant obtained on
centrifugation was injected in to the LC-MS. The confusion here is
about the range of calibrants? Would it be (a) the concentration range
of the plasma working solutions or (b) the concentration range
obtained after pooling the four 25 ul aliquots. According to me the
it is the latter. Any advice/suggestions on this?
Thanks and regards,
Pradeep B.Lukka
Auckland Cancer Society Research Centre
The University of Auckland
Private Bag 92019
Auckland
New Zealand
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Dear Pradeep,
Your guess is correct. The concentration range obtained after pooling
the
four 25ul aliquots is the right approach. In the same way you have to
take
unknown samples also 100ul for processing.
Comments from the experts are welcomed
Regards
cvsn
Bioserve
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Hi Pradeep,
The calibration range will be calculated on the basis of final dilution.
In your case it will be 25uL in 100uL i.e, 1/4th concentrations of
your plasma working solutions.
Santosh Kumar Manthri
Research Associate
Bioanalytical Research
Clinical Pharmacology Unit
GVK Biosciences Pvt Ltd.
Hyderabad, India
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Dear Pradeep,
You are diluting 25ul to 100 ul. Effective concentration is 4 times
less. So option B is correct. To make life simpler, why don't you make
a standard mix first and then spike, so that you can prepare only one
calibration solution?
Vinayak
Vinayak Nadiger
Manager , Bioanalytical Chemistry
11 Biopolis Way, Helios #08-05
Singapore 138667
E Mail: vnadiger.-a-.combinatorx.com
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Dear Lukka,
Let me know the plasma volume for your method.If your plasma volume is
200 ul then after pooling only 100ul plasma you have to add, In this
case you can take the plasma range working solution( i.e. 25 ul
working solution concentration in 200ul of plasma ). U can prepare
the intermediate working solution by combining all the 4 analogues
provided the calibration range for all four analogues are not varied
drastically.So in 25ul spiking you can have all the four analogues. I
hope i am able to solve your confusion. Better suggestions expected.
thanks,
with regards,
Rashmi ShijuDhirubhai Ambani Life Sciences,Navimumbai- India
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In the pooled standards you have a ULOQ in plasma and an LLOQ in
plasma for
each analyte. That is your working range for each analyte. That is the
only choice. You will want to run each curve independently to
demonstrate
that there is no influence of one curve on the other either in
recovery or
analysis before you run the combined standard.
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DO NOT DO THIS. Your working range is what you spike into plasma,
period. If the assay uses a universal dilution, it is part of the assay
and not used to adjust values. A sample dilution is not considered
unless you want to dilute samples beyond the assay dilution to fit your
curve.
--
Ed O'Connor, Ph.D.
Laboratory Director
Matrix BioAnalytical Laboratories
25 Science Park at Yale
New Haven, CT 06511
Web: www.matrixbioanalytical.com
Email: eoconnor.aaa.matrixbioanalytical.com
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Dear Lukka,
i think what rashmi saying is right, let me simplfy it more , you need
to prepare intermediate solution (means diluted solution of main
working solution) from ur main working solution , and you can club all
four intermediate solution of ur analogue so it will give you one
solution then you can spike this solution to blank plasma
now regarding calulating concentration then no matter how much
acetonitrile you ading and dilutiong it , the actual drug present in
you are sample is the drug which you have spiken in blank plasma so
all steps after that are for extracting ur drug from plasma only so
concentration should be calculated that of combine solution of all
four drug spiked in plasma and not on base of dilition done after that
i hope it will help
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Hello Pradeep,
Calibration curves should be consistent with the sample pooling
strategy that you have proposed. The dilution factors then remain
constant.
If you are using a pooled concentrated stock that contains lets say
100 ug/mL of each compound the you must use a dilution factor
correction in reporting the values.
The plasma spiked with concentrated stock should be quenched in teh
same 3:1 ratio (whether you're ruing 25 ul or 100 uL plasma samples).
Does that help?
Sanjeev Thohan
Director DMPK
Exelixis, Inc
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