Analysis of Creatine and Creatinine on a Porous Graphitic Carbon Column by HPLC/UV

Importance of the Topic

The quantification of creatine and creatinine is critical for monitoring renal function, assessing muscle metabolism, and evaluating dietary supplements in clinical and sports science applications.

Objectives and Overview

This study presents a rapid and simple reversed-phase HPLC/UV method using a porous graphitic carbon (Hypercarb) column for the efficient separation and detection of creatine and creatinine within five minutes under isocratic conditions.

Methodology and Instrumentation

Sample Preparation:

• Primary standards prepared at 1000 µg/mL in water.
• Mixed working standard in mobile phase (H2O/MeCN/TFA, 96.95:3:0.05 v/v) with 50 µg/mL creatine and 10 µg/mL creatinine.

Chromatographic Conditions:

• HPLC system with photodiode array detector.
• Hypercarb column (5 µm, 100 mm × 2.1 mm).
• Mobile phase: water/acetonitrile/trifluoroacetic acid (96.95:3:0.05 v/v).
• Flow rate: 0.2 mL/min; column temperature: 30°C; autosampler temperature: 20°C.
• Detection at 216 nm; injection volume: 5 µL; total run time: 5 minutes.

Main Results and Discussion

The porous graphitic carbon column demonstrated strong retention of the highly polar analytes through dispersive and polar surface interactions, achieving baseline separation in under five minutes. Repeatability was excellent, with retention factors (k′) of 1.12 (creatine) and 2.44 (creatinine), plate counts exceeding 30 000, tailing factors near 1.4, and resolution of 4.81 (RSD <2%).

Benefits and Practical Application

• Rapid throughput suited for routine clinical and quality control laboratories.
• Simplified isocratic method avoids ion-pair reagents and complex gradients.
• High precision and reproducibility support reliable quantification of polar compounds.

Future Trends and Applications

Further developments may include coupling Hypercarb separations with mass spectrometry for enhanced sensitivity, extending the approach to other polar biomarkers, miniaturized column formats for reduced solvent usage, and integration into automated high-throughput workflows.

Conclusion

The described Hypercarb-based HPLC/UV method delivers a fast, robust, and precise solution for the analysis of creatine and creatinine, offering a compelling alternative to conventional polar compound separations.

Reference

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