Development of a Normal Phase HPLC Method for the Separation of Creatinine and Purine Metabolites
Merlin K. L. Bicking, Ph.D.
ACCTA, Inc.
PO Box 25602
St. Paul, MN 55125
Phone: 651-731-3670
Email: mbicking@accta.com

xanthine
mwt = 152
xLogP3 = -0.7

allantoin
mwt = 158
xLogP3 = -2.2

uric acidmwt = 168
xLogP3 = -1.9

http://pubchem.ncbi.nlm.nih.gov/

Some retention, notseparated fromallantoin

No retention* Pentafluorophenylpropyl

B: Acetonitrile

01090

4.0

1090

12.075

25

12.5

75

2512.61090

20.0

1090

Conclusion: No single phase was capable ofproviding adequate retention for all analytes!

• Experiments indicated that Unison UK-Amino (ImtaktUSA), using an ammonium formate buffer (pH ~ 7),
produced retention for all analytes.
• The retention was affected by buffer concentration, withless retention at higher concentrations.
• The retention patterns indicated that the separations
were a combination of Aqueous Normal Phase (ANP)
and anion exchange.
- Retention decreased with an increase in water
concentration.
- Water is a “strong” solvent in this system.

0235

500

3.2265400

4.0210300

5.5290200

Optimized Conditions

235 nm2265 nm

290 nm200

Xanthine

150500 Creatinine
200220240

260

280300

320

340

360

380nm

mAU
800

6040200

Uric Acid

100

810min

700
600
500
400
300

246810121416

CompoundRetention Time, min.

Wavelength, nm

Conc., g/mL

Creatinine

2.66

235

100

Xanthine

3.66

265

10Allantoin

4.79

210100

Uric Acid

10.5

290

1018min

6810min200

046810• Aveka, Inc., St. Paul, MN- HPLC equipment and facilities• College of Veterinary Medicine, University ofMinnesota, St. Paul, MN- Dr. Jody Lulich- Josephine Gnanandarajah- Sara Shepard- Dr. Craig Johnson- Dr. Mike Murtaugh

References

100

2A method has been developed that allows rapid andcomplete separation of the three main purine
metabolites in urine.
• A gradient program with wavelength switching isrequired.
• Real-world samples can be analyzed with no sample
preparation. Only dilution is required. No interferences
are present.

Acknowledgements

DAD1 E, Sig=235,20 Ref=360,80, TT (URINE\URATE_CAT_URINE.D)

Uric AcidAllantoin6• This animal has elevated levels of uric acid, resulting in
the formation of urate stones in the urine.
% Acetonitrile

Xanthine

250No single wavelength will provide
good sensitivity for all analytes(210 nm would work, but too many
interferences would be expected).
Multiple wavelength or wavelengthswitching methods will berequired.

4Urate Cat Urine
210 nm

4Conclusions

DAD1 E, Sig=235,20 Ref=360,80, TT (URINE\PURINESTDS_121808A\STANDARDS-00001.D)
PMP1 , Solvent B

80Norm.

00Allantoin

Absorbance Spectra

50

2100mAU
140150

DAD1 E, Sig=235,20 Ref=360,80, TT (URINE\NORMAL_CAT_URINE.D)
mAU

Wavelength, nm

Unison UK-Amino (3um)

200

100

The normal cat has more allantoin, as allantoin is
the final step in the metabolic pathway and is quite
water soluble. Thus, animals producing moreallantoin do not form stones composed of purine
metabolites. Xanthine is not present.

Time, min.

100

• The four analytes have significantly different
chromatographic characteristics.
- Range of polarity makes column selection difficult• Absorbance spectra are all unique- Single wavelength analysis not practical• Need a single column system with short analysis times
and without the use of ion pairing agents.

Normal Cat Urine

Wavelength Switching120

Analytical Problems• Note: concentrations in urine are high relative to thesensitivity of the method, so actual dilution factors canbe adjusted as needed. However, some pH adjustment
is needed to prevent precipitation of some analytes.

Uric Acid

No retention

A: 50mM Ammonium Formate

Allantoin

Some retention, notseparated from uricacid

Time, min.

mAU

Creatinine

Good retention

DAD1 E, Sig=235,20 Ref=360,80, TT (URINE\XANTHINE_CAT_URINE.D)

Xanthine

Slight retention• This cat, with hereditary xanthinuria, formed xanthinestones in the urinary bladder. Xanthine is found in theurine, and will form xanthine stones as a result.

Uric Acid

Good retention• Collect urine sample• Dilute 1:10 with 50 mM ammonium formate• Filter through 0.45 um filter into autosampler vial
• Inject 5.0 LAllantoin

Uric Acid

No retention
No retention

PFP*• Unison UK-Amino, 150 x 4.6mm (3um), 35 ºC• Flow: 1.5 mL/min• Injection: 5 uL
Gradient Program

Uric Acid

creatinine
mwt = 113
xLogP3 = -1.3

Allantoin

HILIC/Silica

Xanthine Cat Urine

Allantoin

Structures for Creatinine and Purine Metabolites

Creatinine

Reversed
Phase/C18

Sample Preparation

Creatinine

A single column, multi-wavelength method will be shownfor the following analytes: creatinine, xanthine, allantoin,
and uric acid.
This method is currently being used for routine urine
analysis.

•This chart summarizes initial results for initial column
choices.

Optimized Chromatographic Conditions

Creatinine

The determination of creatinine and purine metabolitescan be an important marker for renal function, and theconcentrations of various metabolites are an indicator forcertain disease states. However, this is a difficult
separation problem because of the dramatic differencesin chromatographic behavior of the analytes.
A novel HPLC method was developed that separates andquantifies these compounds of interest on anaminopropyl phase without the use of ion pairing
additives. Important variables that were optimized forthis method include stationary phase selection, sample
solvent composition, mobile phase additives, and pH.

Initial Chromatographic ResultsCreatinine

Abstract

Bryan Evans
Imtakt USA
1511 Walnut St., Ste 310
Philadelphia, PA 19102
Phone: 888-456-HPLC
Email: bevans@imtaktusa.com

min• A Novel Aminopropyl Column with Revolutionary
Aqueous Durability
YAZAWA Itaru, TACHIKAWA Hiroshi, OKAMURA
FumikoImtakt Corporation, Kyoto, Japan
HPLC 2008 – Baltimore, MD, USA