Chromatography Forum

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 (0.0836 mg/dL) when interpolating in my graph!!

Is there something I´m missing ?? dilution factor? Anything??

Thanks
esbena
technician-in-training


Posts: 9
Joined: Feb 08 2013 2:50 am
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Hi Esbena,

I am sure I don't know what, if anything, you are missing. But, have you tried out creatinine standard(s) at 128 and 0.5 uM levels, and, say, several concentration levels in between these two...placing them through your sample prep routine as if they were samples themselves, to see if there is any loss in creatine due to the sample preparation routine?

Please, see what you think of this idea.

Hello Esbana,

Just an idea: is the creatinine precipitated with the acetonitrile step?

PeterS

Good Morning, All,

As PeterS notes better than I did, this is exactly what I was getting at...see if the acetonitrile precipitation step removes creatinine relative to a "standard prep" without precipitation.

Good Morning, All,

As PeterS notes better than I did, this is exactly what I was getting at...see if the acetonitrile precipitation step removes creatinine relative to a "standard prep" without precipitation.

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

Hi Esbena,

"Supposed To" and "Actually Does" are often two different things...that said, I'm happy to learn that the ACN treatment apparently does not significantly affect the creatinine recovery.

That said...the next step probably aught to be a standard addition study to creatinine-free serum...in precipitating the creatinine, any proteins "crashed out" by the ACN could simply pull out creatinine, just like sticking a hand into a briar bush and finding your arm covered with burrs.

Can't shake the feeling that I've read this before...your column is SCX, 2.1 x 50 mm, and you are using a flow rate of 1.0 mL/min, correct (seems an awfully fast flow rate for a column of these dimensions to me)? That means the column void would be about 120 microliters, and k' = 2 at about 15 seconds...please, at what time does creatinine elute, and if you perform multiple injections of a mid-level standard, what is the %RSD of the peak areas of these injections?

If acetonitrile is a good solvent for creatinine, perhaps a way outside of chromatography would be a good way to check your dilutions...you could remove the column and inject each standard level, record the peak area(s)/heights without a column present...see if that "straightens out" the curve.

Otherwise, I'd think that you didn't have a "true solution" when you attempt to dissolve creatinine in ACN...and perhaps another solvent would be better for the separation.

I'm assuming that your standards are also in ACN, similar to the sample preps?

My average retention time is 3.6 min

I have not yet tried multiple injections of a mid-level standard, but I will.

I make my standard stock solution (128 mM) in the solvent (5 mM sodium acetate pH 4.1), and dilute it down to 128 uM (1ul 128 mM and 1000 ul solvent). From this I make my dilution series: 500 ul 128 uM + 500 ul solvent to give a 64 uM concentration and so on until 0.5 uM

When I prep the standards I take 25 ul and precipitate with 100 ul AcN, and just follow the above-written approach

Thanks

Hi Esbena,

Oh, Okay, I think I see now. Solvent is not a problem at all with the standard prep (and I'm understanding better now), but the way you're making these dilutions, each step-wise from the one before...such as, using a micropipet:

128 uM - 64 uM - 32 uM - 16 uM - 8 uM - 4 uM - 2 uM - 1 uM - 0.5 uM

If, say, the 64 uM prep isn't quite a 1 +1 dilution from the 128 uM, then each subsequent dilution will also be "off" as each dilution relies heavily on the dilution prior to it.

If you set up a dilution scheme from a stock standard concentration level such that all dilutions are made from that single level, chances that multiple levels of standard solutions will be "off" are greatly lessened.

And yes, it's still a good idea to learn the precision of measurement for mid-level standard solution(s).

Please, see what you think.