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

A simple and rapid reversed-phase HPLC/UV procedure for the examination of creatine and creatinine on a Thermo Scientific Hypercarb (porous graphitic carbon) column is described herein. Under reversed-phase, isocratic conditions, excellent separation of these polar analytes is achieved within five minutes and the chromatographic data exhibit excellent precision. Introduction Creatine [2-(methylguanidino) ethanoic acid] is a naturally occurring nitrogenous substance which is synthesized predominantly in the liver and kidneys and stored primarily in skeletal muscle as the high energy molecule phosphocreatine. Supplements of creatine are used sometimes by athletes and bodybuilders who wish to increase their muscular mass. During its critical role in cellular metabolism, phosphocreatine is converted eventually to creatinine [2-amino-1-methyl-5H-imidazol-4-one] which is subsequently excreted in the urine. Thus, measurement of the rate of clearance of creatinine (via the glomerular filtration rate) can be used as an indicator of renal dysfunction. Despite widespread utilisation of the non-specific Jaffe reaction [1,2] and enzymatic methods [3,4] for the determination of serum and urinary creatinine, chromatographic techniques (including ion-pair chromatography [5], ion-exchange chromatography [6] and, in particular, liquid chromatography-tandem mass spectrometry procedures [7,8]) continue to grow in popularity. As a result of their high polarity, creatine and creatinine are unretained on traditional alkyl-bonded (e.g., C18) silicas. Moreover, the practical limitations inherent to both ion-pair and ion-exchange approaches have provided an impetus for the adoption of alternative chromatographic procedures (e.g., HILIC) and the development of new stationary phases (e.g., polar embedded and perfluorinated) that allow alternative mechanisms of interaction in addition to those dispersive interactions which are typically associated with alkyl-immobilised phases. HypercarbTM is a chromatographic support comprised of 100 % porous graphitic carbon. Hypercarb behaves as a strongly retentive alkyl-bonded silica for hydrophobic analytes, but its retentivity and selectivity towards polar and structurally-related compounds (e.g., diastereomers and geometric isomers) is substantially different [9]. The purpose of this investigation is to illustrate the effectiveness of Hypercarb for the retention and separation of the polar molecules creatine and creatinine under reversed-phase conditions. 2 Experimental Details Consumables Part Number Fisher Scientific water (HPLC gradient grade) W/0106/17 Fisher Scientific acetonitrile (HPLC grade) A/0626/17 Trifluoroacetic acid (TFA, HPLC grade, 99+ %) T/3258/PB05 Fisher Scientific Finnpipette F2 pipettor kit (10 μL 100 μL, 100 μL 1000 μL, 1 ml 10 mL) PMP-020-220F Fisher Scientific Finntip pipette tips, 10 μL PMP-107-110W Fisher Scientific Finntip pipette tips, 200 μL PMP-107-600F Fisher Scientific Finntip pipette tips, 1000 μL PMP-103-206K Fisher Scientific Finntip pipette tips, 10 mL PMP-107-040R Thermo Scientific borosilicate glass vials 60180-600 (2 mL, 12 mm x 32 mm) with 8 mm black screw cap fitted with a silicone/PTFE seal Creatine monohydrate (> 99 %) supplied by Fluka Analytical Creatinine (anhydrous, > 99 %) supplied by Sigma-Aldrich Sample Preparation Analytical standards Primary analytical standards of both creatine and creatinine were prepared separately in water at a concentration of 1000 μg/mL. Thereafter, a mixed working standard was prepared in mobile phase (96.95:3:0.05 (v/v) H2O/MeCN/TFA) by combining 5 parts of creatine primary standard, 1 part creatinine primary standard and 94 parts of mobile phase. The concentrations of creatine and creatinine were 50 μg/mL and 10 μg/mL respectively. Separation Conditions Part Number Instrumentation: Thermo Scientific HPLC system equipped with a photodiode array (PDA) detector Column: Thermo Scientific Hypercarb 35005-102130 5 μm, 100 mm x 2.1 mm Mobile phase: H 2 O/MeCN/TFA (96.95:3:0.05 v/v) Flow rate: 0.2 mL/min Column temperature: 30 °C Autosampler temperature: 20 °C Detection: UV at 216 nm Injection volume: 5 μL Syringe flush: mobile phase