Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

• Published in: Journal of chromatography. B, Biomedical sciences and applications Vol. 759; no. 2; pp. 209 - 218
• Main Authors: Georga, Kalliopi A, Samanidou, Victoria F, Papadoyannis, Ioannis N
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.08.2001; Elsevier Science
• Subjects: Analysis; Biological and medical sciences; Caffeine; Caffeine - metabolism; Chromatography, High Pressure Liquid - methods; General pharmacology; Humans; Medical sciences; Methyluric acid; Methylxanthine; Pharmacology. Drug treatments; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Uric Acid - blood; Uric Acid - metabolism; Uric Acid - urine; Xanthines - blood; Xanthines - metabolism; Xanthines - urine; Methylxanthine; Methyluric acid; Caffeine; Urine; Validation; Solid phase extraction; Biological fluid; CNS stimulant; Metabolite; Psychotropic; HPLC chromatography; Blood; Reversed phase chromatography; Ultraviolet detector; Xanthine derivatives; Quantitative analysis
• ISSN: 0378-4347; 1387-2273
• DOI: 10.1016/S0378-4347(01)00251-1

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C 4, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm 2. The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day ( n=6) and inter-day calibration ( n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.