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HPLC calibration problem


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#1 nube

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Posted 14 November 2010 - 09:39 PM

Hi guys,

I have just started doing HPLC, and am trying to find the concentration of drug in plasma after dosing. I am confused about the calibration curve I have made. For my highest calibrator, I used 50uL of a 100ug/mL solution and added it to 100uL blank plasma (so, 150uL in total. 100uL plasma was used for calibration standards as my actual samples are 100uL). I was doing this to get a 50ug/mL calibration sample. However, in retrospect I now think that this calibrator is 33.3ug/mL as the 50uL of 100ug/mL was diluted into a total volume of 150uL (50uL working solution, and 100uL plasma). The samples then undergo SPE, and are dehydrated before being reconstituted and put through the LC machine.

My lab partner told me this was the correct way to do it as the extra 50uL volume is irrelevant as it is dehydrated before undergoing HPLC. However, in my mind I need to have the same concentration of drug in my calibrator as I do in my actual plasma samples. So I think the concentration of my calibrator prior to SPE is what is important, not the total content of drug.

Can anybody confirm which way is the correct way to do this??

Thanks very much!

#2 nightingale

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Posted 15 November 2010 - 11:57 AM

Hi guys,

I have just started doing HPLC, and am trying to find the concentration of drug in plasma after dosing. I am confused about the calibration curve I have made. For my highest calibrator, I used 50uL of a 100ug/mL solution and added it to 100uL blank plasma (so, 150uL in total. 100uL plasma was used for calibration standards as my actual samples are 100uL). I was doing this to get a 50ug/mL calibration sample. However, in retrospect I now think that this calibrator is 33.3ug/mL as the 50uL of 100ug/mL was diluted into a total volume of 150uL (50uL working solution, and 100uL plasma). The samples then undergo SPE, and are dehydrated before being reconstituted and put through the LC machine.

My lab partner told me this was the correct way to do it as the extra 50uL volume is irrelevant as it is dehydrated before undergoing HPLC. However, in my mind I need to have the same concentration of drug in my calibrator as I do in my actual plasma samples. So I think the concentration of my calibrator prior to SPE is what is important, not the total content of drug.

Can anybody confirm which way is the correct way to do this??

Thanks very much!


Hello,
ok ... let me summarize what have you done :
you are spiking a plasma sample with different concentrations of a certain drug, in order to do a calibration curve right ?
well, after that ... things seemed a bit messy,
the data available :

C1 V1 = C2 V2

C1 = the conc. of the drug ( stock )
V1= how much you will take from the stock in order to reach a certain conc.
C2 = the concentration wished to reach
V2 = the total volume of both the sample and the drug.

100ug/ml x ???? = 50ug/ml x 150ul

V1 = 75 ul must be taken from your drug with the 100ug/ml concentration + 75 ul from the plasma in order to reach a concentration of 50ug/ml of your drug in this total volume ( 150 ul ).


if your question was about :
what matters more, the concentration of the working solution of the drug before undergoing SPE, OR
the concentration of the drug, after reconstitution ( being dehydrated and a solvent added to before applying to HPLC )
the answer is :
you must pay attention to both ...
it matters ...

" The more you learn, the more you realize how little you know ... "

#3 nube

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Posted 15 November 2010 - 05:24 PM


Hi guys,

I have just started doing HPLC, and am trying to find the concentration of drug in plasma after dosing. I am confused about the calibration curve I have made. For my highest calibrator, I used 50uL of a 100ug/mL solution and added it to 100uL blank plasma (so, 150uL in total. 100uL plasma was used for calibration standards as my actual samples are 100uL). I was doing this to get a 50ug/mL calibration sample. However, in retrospect I now think that this calibrator is 33.3ug/mL as the 50uL of 100ug/mL was diluted into a total volume of 150uL (50uL working solution, and 100uL plasma). The samples then undergo SPE, and are dehydrated before being reconstituted and put through the LC machine.

My lab partner told me this was the correct way to do it as the extra 50uL volume is irrelevant as it is dehydrated before undergoing HPLC. However, in my mind I need to have the same concentration of drug in my calibrator as I do in my actual plasma samples. So I think the concentration of my calibrator prior to SPE is what is important, not the total content of drug.

Can anybody confirm which way is the correct way to do this??

Thanks very much!


Hello,
ok ... let me summarize what have you done :
you are spiking a plasma sample with different concentrations of a certain drug, in order to do a calibration curve right ?
well, after that ... things seemed a bit messy,
the data available :

C1 V1 = C2 V2

C1 = the conc. of the drug ( stock )
V1= how much you will take from the stock in order to reach a certain conc.
C2 = the concentration wished to reach
V2 = the total volume of both the sample and the drug.

100ug/ml x ???? = 50ug/ml x 150ul

V1 = 75 ul must be taken from your drug with the 100ug/ml concentration + 75 ul from the plasma in order to reach a concentration of 50ug/ml of your drug in this total volume ( 150 ul ).


if your question was about :
what matters more, the concentration of the working solution of the drug before undergoing SPE, OR
the concentration of the drug, after reconstitution ( being dehydrated and a solvent added to before applying to HPLC )
the answer is :
you must pay attention to both ...
it matters ...



#4 nube

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Posted 15 November 2010 - 05:25 PM

Hi there,

Thanks very much for your answer. Can you give me any advice on what to do from here if I don't have enough of the samples to re-run them? The samples have all been run as 100uL samples. Each calibration standard was made using a series of working solutions varying from 100ug/mL down to 0.05ug/mL. 50uL of each was spiked into 100uL blank plasma. And as I said, I was told the excess 50uL of methanol didn't need to be accounted for, but now that I have started writing the method up, I think I need to account for it. So can I count each of the calibration standards as being 'x' ug in 150uL, and compare this to the unknown concentration of my 100uL samples? Eg, my top calibrator would be 33.3ug/mL (using the concentration to compare to my unknown samples?)

I can see this is not ideal, and I am trying to find a way to make it work if possible.

I guess my question is whether or not the 50uL of methanol in which the drug was dissolved to make the working solutions needs to be included in my concentration calculation for the calibrators. My lab partner says it doesn’t, but I think it should be as I am comparing concentration of the calibrator to concentration of the unknown sample. Eg, my lab partner says that adding 50uL of 100ug/mL solution to 100uL of plasma gives a 50ug/mL solution (he doesn’t include the 50uL of methanol in the calculation). But I think it should be 33.3ugmL, as the total volume is now 150uL. Does that make sense?

Thanks again.

#5 nightingale

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Posted 16 November 2010 - 12:22 PM

Hi there,

Thanks very much for your answer. Can you give me any advice on what to do from here if I don't have enough of the samples to re-run them? The samples have all been run as 100uL samples. Each calibration standard was made using a series of working solutions varying from 100ug/mL down to 0.05ug/mL. 50uL of each was spiked into 100uL blank plasma. And as I said, I was told the excess 50uL of methanol didn't need to be accounted for, but now that I have started writing the method up, I think I need to account for it. So can I count each of the calibration standards as being 'x' ug in 150uL, and compare this to the unknown concentration of my 100uL samples? Eg, my top calibrator would be 33.3ug/mL (using the concentration to compare to my unknown samples?)

I can see this is not ideal, and I am trying to find a way to make it work if possible.

I guess my question is whether or not the 50uL of methanol in which the drug was dissolved to make the working solutions needs to be included in my concentration calculation for the calibrators. My lab partner says it doesn’t, but I think it should be as I am comparing concentration of the calibrator to concentration of the unknown sample. Eg, my lab partner says that adding 50uL of 100ug/mL solution to 100uL of plasma gives a 50ug/mL solution (he doesn’t include the 50uL of methanol in the calculation). But I think it should be 33.3ugmL, as the total volume is now 150uL. Does that make sense?

Thanks again.


first : u are most welcome :)
second : hold on ! don't think of repeating anything, till i make sure i have got ur REAL point ;) :)

i have a list of questions :

(1) : ur final volume of standards should have been 100 ul, but unfortunately it is 150 ul since you write, " for my highest calibrator i used 50 ul of 100 ug/ml and added it to 100 ul blank sample.
so, yes ... here the calibrator is : 33.3 ug/ml.

(2) : what do u mean by extra 50 ul ??? or 50 ul MetOH ???
is it the volume of MetOH added to reconstitute ur sample after undergoing SPE ??? or the volume taken from ur drug stock solution ???

if its the volume taken from your stock solution in order to make your working solutions so ...
u should of course count it, as i kindly showed in my first reply ... as it is the volume taken from the stock in order to reach a certain final conc. of ur drug + plasma.

u r welcome again ;)

" The more you learn, the more you realize how little you know ... "

#6 nube

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Posted 16 November 2010 - 03:42 PM


Hi there,

Thanks very much for your answer. Can you give me any advice on what to do from here if I don't have enough of the samples to re-run them? The samples have all been run as 100uL samples. Each calibration standard was made using a series of working solutions varying from 100ug/mL down to 0.05ug/mL. 50uL of each was spiked into 100uL blank plasma. And as I said, I was told the excess 50uL of methanol didn't need to be accounted for, but now that I have started writing the method up, I think I need to account for it. So can I count each of the calibration standards as being 'x' ug in 150uL, and compare this to the unknown concentration of my 100uL samples? Eg, my top calibrator would be 33.3ug/mL (using the concentration to compare to my unknown samples?)

I can see this is not ideal, and I am trying to find a way to make it work if possible.

I guess my question is whether or not the 50uL of methanol in which the drug was dissolved to make the working solutions needs to be included in my concentration calculation for the calibrators. My lab partner says it doesn’t, but I think it should be as I am comparing concentration of the calibrator to concentration of the unknown sample. Eg, my lab partner says that adding 50uL of 100ug/mL solution to 100uL of plasma gives a 50ug/mL solution (he doesn’t include the 50uL of methanol in the calculation). But I think it should be 33.3ugmL, as the total volume is now 150uL. Does that make sense?

Thanks again.


first : u are most welcome :)
second : hold on ! don't think of repeating anything, till i make sure i have got ur REAL point ;) :)

i have a list of questions :

(1) : ur final volume of standards should have been 100 ul, but unfortunately it is 150 ul since you write, " for my highest calibrator i used 50 ul of 100 ug/ml and added it to 100 ul blank sample.
so, yes ... here the calibrator is : 33.3 ug/ml.

(2) : what do u mean by extra 50 ul ??? or 50 ul MetOH ???
is it the volume of MetOH added to reconstitute ur sample after undergoing SPE ??? or the volume taken from ur drug stock solution ???

if its the volume taken from your stock solution in order to make your working solutions so ...
u should of course count it, as i kindly showed in my first reply ... as it is the volume taken from the stock in order to reach a certain final conc. of ur drug + plasma.

u r welcome again ;)



#7 nube

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Posted 16 November 2010 - 03:43 PM



Hi there,

Thanks very much for your answer. Can you give me any advice on what to do from here if I don't have enough of the samples to re-run them? The samples have all been run as 100uL samples. Each calibration standard was made using a series of working solutions varying from 100ug/mL down to 0.05ug/mL. 50uL of each was spiked into 100uL blank plasma. And as I said, I was told the excess 50uL of methanol didn't need to be accounted for, but now that I have started writing the method up, I think I need to account for it. So can I count each of the calibration standards as being 'x' ug in 150uL, and compare this to the unknown concentration of my 100uL samples? Eg, my top calibrator would be 33.3ug/mL (using the concentration to compare to my unknown samples?)

I can see this is not ideal, and I am trying to find a way to make it work if possible.

I guess my question is whether or not the 50uL of methanol in which the drug was dissolved to make the working solutions needs to be included in my concentration calculation for the calibrators. My lab partner says it doesn’t, but I think it should be as I am comparing concentration of the calibrator to concentration of the unknown sample. Eg, my lab partner says that adding 50uL of 100ug/mL solution to 100uL of plasma gives a 50ug/mL solution (he doesn’t include the 50uL of methanol in the calculation). But I think it should be 33.3ugmL, as the total volume is now 150uL. Does that make sense?

Thanks again.


first : u are most welcome :)
second : hold on ! don't think of repeating anything, till i make sure i have got ur REAL point ;) :)

i have a list of questions :

(1) : ur final volume of standards should have been 100 ul, but unfortunately it is 150 ul since you write, " for my highest calibrator i used 50 ul of 100 ug/ml and added it to 100 ul blank sample.
so, yes ... here the calibrator is : 33.3 ug/ml.

(2) : what do u mean by extra 50 ul ??? or 50 ul MetOH ???
is it the volume of MetOH added to reconstitute ur sample after undergoing SPE ??? or the volume taken from ur drug stock solution ???

if its the volume taken from your stock solution in order to make your working solutions so ...
u should of course count it, as i kindly showed in my first reply ... as it is the volume taken from the stock in order to reach a certain final conc. of ur drug + plasma.

u r welcome again ;)



#8 nube

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Posted 16 November 2010 - 03:46 PM

Hi there,

Thankyou very very much! I really appreciate your help. The extra 50uL / 50uL Methanol was the volume of the working solution, so you have answered the question for me. I will make sure not to make the same error when validation my next assay!

Thanks again!
:)




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