IS for creatine/creatinine in blood serum or in urine

[Kazimierz]

Dear chromatographers,

I would like begin determinate creatinine and creatine in newborns
using HPLC and Primesep 200 column.

There are 2 question appeared:

1) creatine/creatinine ratio is pH dependent and what sample preparation
would be prefer - precipitation with acetonitrile or methanol or ultrafiltration.


2) what is source of potential internal standard 4-(2-Aminoethyl)benzene sulfonamide.

sincerely

Kazimierz H. Kozlowski

[Uwe Neue]

What is your matrix? Urine, blood or ground-up newborns?

[SIELC_Tech]

Dear Kazimierz,

I think that the biggest challenge you have is proteins in your samples. You need to do a minimum sample prep to remove proteins. Proteins will irreversible bind to Primesep 200 column reducing the life of your column. You need to use a guard column. My advice is to use a weaker guard in combination with Primesep 200 column. Primesep C guard or even Primesep B2 guard might help you to trap proteins and then flash them during the run into waste. Please see the our newsletter "Rapid HPLC Analysis of Complex Mixtures" It shows a general guard approach for analysis and removal of compounds which retain very long on the column (in your case proteins from blood, urine, etc.). Guard is connected to the switching valve o agilent 1100 system and is being wash while your compound is analyzed:

http://www.sielc.com/Literature_Newsletters.html


Also check our methods for creatine and creatinine:

http://www.sielc.com/compound_112.html


Let me know if you need any help with method development.

Kind regards,

Vlad

[Kazimierz]

Dear Professor Neue,

I need determinate creatine/creanitine in blood-serum as well as in urine for:

a) determination of cretinine clearance when creatine production is very low (for example in newborns, in undernutrition, in inborn errors etc) or in the cases of analytical interferences with routine method (hepathobiliary disorders, hepatitis). Population Age-reference norms for CLcr in infants depending on covariates (age, BSA, creatine levels, immaturity etc) for population pharmacokinetics of ganciclovir, aminoglycosyde antibiotics or cisplatin and other drugs eliminated by kidneys.

b) as services for GC-MS Laboratory working on detection of congenital enzymatic inborn errors in Creatine metabolism.

Dear Dr Sielc,

I do not like to manipulate with samples, or disturbing cretine/creatinine oryginal equillibrium state with acids or buffers - preanalytical errors.

I will try to compare 2 methods of sample prepation: deproteination with acetonitrile/methanol or separation of proteins by ultrafiltration with hydrophilic YMT-1 ultrafiltration membrane (Amicon). HPLC on Varian Prostar with 3th-gradient, autosampler 400 and UV-VIS detector. Kolumns Primasep-200, 3x150 or 250mm + precolumn (problems with mounting with steel fittings because columns are smooted only). TFA not available, but HFB acid available, also TCA acid.

Question for checking: Is creatinine potentialy metabolize to creatine on the column in the mobile phase of acidic pH?

Sincerely

Kazimierz H. Kozlowski

[SIELC_Tech]

Kazimriez,

You can use formic acid and reproduce our method for these compounds on Primesep 200 column. With formic acid you will have mild enough conditions to avoid ring formation. Expect retention on 150 mm around 8-15 minutes for both compounds. Don't use 250 mm column as creatine and creatinine will stay much longer on the column. You will have shorter retention time if you use stronger buffer. Our method was developed on 50 mm column with no ion-pairing reagent. You can also use ammonium acetate or formate at pH-3 (5-20 mmol) (see the link in my previous message)


Regarding the frits: the column is designed for finger tightening, be careful with the metal frits you can easily over tight it and destroy PEEK insert inside the column.

Regards,

Vlad

[Uwe Neue]

Interesting...

Ultrafitration might be your best choice to preserve the equilibrium. With respect to acetonitrile precipitation, you can get some reasonable results with a roughly 4:1 volume of acetonitrile to sample. But you will need a precolumn, no question.

I would also check in a blank experiment, if the addition of acetonitrile changes the equilibrium.

[HW Mueller]

One routine colorimetric estimation of creatine is done via the conversion to creatinine with hot acid. I never heard of an equilibrium between creatinine and creatin at room temp. (no enzymes).