HPLC method for mouse creatinine measurements in a diabetes type 1 model - (Jul/11/2013 )

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. 

esbena on Tue Jul 30 08:39:23 2013 said:

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

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?

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.

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...  

This is what I interpolate from the standard curve:

~ 0.05 mg/dL

and this is a published average:

~ 0.205 mg/dL 

This is what I get when I interpolate from my standard curve (given above)

0.05 mg/dL

(but is this just the concentration in the injected 3 ul of the 25 ul in the vail? How do I relate this to the published average concentration in serum??

and this is the published average for the same strain of mice, and approx. same age:

0.205 mg/dL

concentration is the same no matter how much you inject. actual mass injected depends on concentration and volume of the injection.

the only thing i can think of for your differences (other than possibly abnormal mice) is interference by the baseline and/or neighbor peaks.