Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

HPLC method for mouse creatinine measurements in a diabetes type 1 model


  • Please log in to reply
17 replies to this topic

#1 esbena

esbena

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 11 July 2013 - 03:02 AM

Hi all.

I need some with some calculations.

I´m currently developing a method for measuring creatinine in mice with HPLC. For this are using a Agilent Zorbax SRC300, 2.1mm x 50 mm, 5u strong cation column and a 5mM sodium acetate pH 4.1 solvent.

We emply a 2-fold creatinine standard (128 uM - 0.5 uM) to interpolate the unknown serum creatinine concentrations from.

Samples are prepped with AcN (Acetonitrile) to precipitate proteins before running onto the column.

We use:

100 ul AcN
15 ul serum

spin 10000 rpm, 10 min

remove supernatant: 90 ul to new epp.tube for SpeedVac (try samples)

Resuspend tried creatinine in 15 ul HPLC solvent

Load 3 ul to each run in HPLC.

Average creatinine levels are 18.29 umol/L (0.207 mg/dl)

I get around 7.395 uM when interpolating in my graph!!

Is there something I´m missing ?? dilution factor? How do I get uM to umol/L

Thanks

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,704 posts
123
Excellent

Posted 11 July 2013 - 04:05 AM

you are drying only ~78% of your sample. do you account for that in your calculations.

have you tried spiking the samples to determine recovery?

uM is umol/L
talent does what it can
genius does what it must
i do what i get paid to do

#3 esbena

esbena

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 12 July 2013 - 02:34 AM

No.. I have not yet tried spiking, but it is a good idea.

Thanks.

Although, I don´t think it will make up for at difference I see, that I may loose some creatinine in the process.

I´m hoping, but...

Could it be my standard curve that I use for interpolating ? It has an r squared at around 0.97...

Also, the samples are prepped as described in the first post, but the standards are not... I know this is an error, and I have to do this, but could this make up for the lower measured amounts?

Is there anything else I should try??

Thanks very much

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,704 posts
123
Excellent

Posted 12 July 2013 - 03:51 AM

r2=0.97 is not too bad for your standard curve.

not treating your standards as you treat your samples can make a significant difference but you may not be able to treat them in the exact same way unless you have serum that is completely deficient in creatinine. try treating them as similarly as possible.

spiking samples with creatinine will give you an idea about recovery.

you can also spike samples and standards with something else so that you can normalize them.
talent does what it can
genius does what it must
i do what i get paid to do

#5 esbena

esbena

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 23 July 2013 - 10:44 PM

Thanks all.

But the acetonitrile is not suppose to precipitate creatinine, but only the proteins in the solution.

However, I have just tried running prepped and non-prepped samples in the same run to investigate this difference.

I used std. samples ranging from 128 - 64 - 32 - 16 - 8 - 4 - 2 - 1 - 0.5 uM.

And... I do not see a significant difference between the prep. vs. non-prep. in both the elution peak or the area under the curve after integration.

However... I do sometimes (every time actually) see inconsistencies in the dilutions.

Normally I see an area under the curve for the 128 uM at 120000 μV*sec and thus around 60000 for 64 uM and 30000 for 32 uM.

But sometimes, and especially at the lower conc. I observe significant higher area under the curve (2 - 1 - 0.5 uM)

And also sometimes for the 64 - 32 - 16 uM concentrations.

What is up with this? What is going on here? I don´t get it!!

Especially with the non-prep. samples where I just load a simple dilution of a stock solution.

Please help with this....

Thanks

#6 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,704 posts
123
Excellent

Posted 24 July 2013 - 03:59 AM

how is your baseline?

what are your peak heights? width?

have you adjusted the integration start and stop so that they are consistent?

can you show a typical chromatogram?
talent does what it can
genius does what it must
i do what i get paid to do

#7 esbena

esbena

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 29 July 2013 - 12:32 AM

IMG_0302.jpg

 

My baseline is 0.0000 AU (+/- 0.00005)

 

my peak heights varies with the creatinine concentrations of course, but for 128 uM it is 0.0075 AU in one run to 0.0060 AU i another.

 

The integration start and stop is adjusted so that they are consistent yes.

 

I will attach a typical chromatogram  

 



#8 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,704 posts
123
Excellent

Posted 29 July 2013 - 04:12 AM

the baseline looks very smooth and the peak looks mostly symmetrical (tailing off towards the end as is typical in hplc). i'm guessing that this is a standard and not a serum sample.

 

you'll note the little blip near the end of the integrated portion of the peak. the tailing and blip will add area to the peak. this may be the cause of the deviation from ideal of your standard curve. a possible remedy for this may be to start and stop integration a little above the baseline, at points where you eliminate the tail (not sure how well this will work with real samples).

 

6mAU (.0060AU) and 7.5mAU can be considered nearly identical (depending on the sensitivity of the detector, the stability of the baseline, the purity of the sample, etc).

 

do you run samples and standards in triplicate and take an average for each point and sample? if so, is there significant deviation from the average?

 

can you show a plot of a typical standard curve? it's possible that it isn't truly linear, at least not early.

 

have you attempted a standard curve with spiked serum?


talent does what it can
genius does what it must
i do what i get paid to do

#9 esbena

esbena

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 29 July 2013 - 04:53 AM

I run the samples in duplicates, and there are fine (largest SD is 6%, and the smallest at 0.16%

 

Spiked serum??

 

Does you mean a serum sample spiked with a given std. concentration?

 

Two attached pictures of std.curves (I still don´t know if I´m suppose to use linear regression or non-linear regression... 

 

Skærmbillede 2013-07-29 kl. 14.29.36.png

Skærmbillede 2013-07-29 kl. 14.31.40.png

Skærmbillede 2013-07-29 kl. 14.38.35.png

 

Thanks



#10 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,704 posts
123
Excellent

Posted 29 July 2013 - 05:45 AM

yes, that's what i mean by spiked serum. although i would recommend the entire standard curve be done with spiked serum (use the "0" concentration as a subtractive blank) so that you can account for the influence of neighboring peaks. it may also account for the recovery after precipitation (unless spiked after precipitation, you could do it both ways to determine recovery efficiency).

 

how reproducible are those concentration points in the standard curve? if they are then it would appear to be at least bi-modal and not necessarily linear.


talent does what it can
genius does what it must
i do what i get paid to do

#11 esbena

esbena

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 30 July 2013 - 12:39 AM

I don´t really use a standard serum to make the standard curve, but a simple dilution of creatinine in solvent. But I see your point in using a serum spiked with the different standards. This is great help.

 

But, would´nt I need a standard serum already low in creatinine? 

 

 

The concentration points in the standard curve are very reproducible. 



#12 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,704 posts
123
Excellent

Posted 30 July 2013 - 03:37 AM

But, would´nt I need a standard serum already low in creatinine?
not necessarily. that's why i said you also need a zero point as a subtractive blank. just ensure that you use the same batch of serum for all of the standards.

talent does what it can
genius does what it must
i do what i get paid to do

#13 esbena

esbena

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 05 August 2013 - 02:42 AM

Can you please help with another thing?

 

I think I´m messing up in my post-calculations... When I interpolate a unknown serum sample onto my standard curve, I get an underestimation as compared to a publicized value.

 

We both using the same strain of mice, and approx. same age. They measure 0.207 mg/dL and I measure 0.05 mg/dL (~ factor of x4 lower)!

 

Am I doing something wrong in my post-calculation?

 

This is what I do:

 

100 ul AcN
  25 ul serum

 

vortex

 

15 min at -20 degrees

spin 10000 rpm, 10 min, at 4 degrees

remove supernatant: 100 ul to new epp.tube for SpeedVac (dry samples)

Resuspend dried creatinine in 25 ul HPLC solvent 

Load 3 ul to each run in HPLC.

 

My standard curve is in umol/L, and I just convert these values to mg/dL.

 

But is my concentration of creatinine just related to the original 25 ul? Do I have a factor dilution somewhere I´m forgetting?



#14 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,704 posts
123
Excellent

Posted 05 August 2013 - 02:27 PM

what is your calculation?

 

i see that you use 80% of the total to dry. if you want to make it the equivalent of the starting material then you would have to resuspend to 20 ul.

 

but this may only account for part of the difference.


talent does what it can
genius does what it must
i do what i get paid to do

#15 esbena

esbena

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 06 August 2013 - 10:20 PM

Hmm... I don´t really have any calculations. 

 

I just interpolate my unknowns using the standard curve, as depicted above, and use the given value. I mean.. The relation between the standard samples and the unknown serum samples are the same since they have been treated equal.

 

But I want to relate the interpolated values from the unknown samples to my initial serum to give a real concentration

 

Yes, I use 100 ul of the 125 ul to speedvac (80%), so that I don´t get any precipitate, but this is also done with the standard samples, so would this have anything to do with final serum concentration?

 

I would like to get a concentration in either umol/L or mg/dL in my initial serum, but I can´t figure out the calculations...  






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.