HPLC method for rapid determination of acetylator phenotype by measuring urinary caffeine metabolites

I. Klebovich, Pentti Arvela, Olavi Pelkonen

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

A validated reversed-phase high-performance liquid chromatographic (RP-HPLC) method is developed for the selective and rapid determination of two major metabolites of caffeine, namely 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (MX) from human urine. HPLC separation is achieved by means of a Supersphere-60 RP-Select B (4 μm) analytical column using a non-linear gradient elution programme of 70-95% solvent B (2.5% acetic acid-methanol, 60:40, v/v) in solvent A (water-acetonitrile, 80:20, v/v). A selective UV detection method is used for determination of AFMU, MX and internal standard with readings at 284, 268 and 248 nm, respectively. Urine samples are prepared for measurement by a simple chloroform-diethyl ether (80:20, v/v) extraction. The assay is validated with respect to linearity, sensitivity, accuracy, precision and system suitability. All validation parameters are found to be within the required limits. The limit of detection of AFMU and MX is found to be 50 ng/200 μl urine. Calibration curves show good linearity between 0.1 and 5 μg/200 μl urine concentration range for both metabolites. The assay is sufficiently sensitive and rapid (4.5 min chromatographic run) to be applied for routine monitoring of change in AFMU/MX molar ratio, indicating acetylation phenotype and change of caffeine metabolism in clinical cocktail studies.

Original languageEnglish
Pages (from-to)1017-1021
Number of pages5
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume11
Issue number10
DOIs
Publication statusPublished - 1993

Fingerprint

Metabolites
Caffeine
High Pressure Liquid Chromatography
Urine
Phenotype
Assays
Acetylation
Chloroform
Metabolism
Acetic Acid
Ether
Calibration
Methanol
Limit of Detection
Reading
1-methylxanthine
5-acetylamino-6-formylamino-3-methyluracil
Water
Monitoring
Liquids

Keywords

  • acetylation phenotype.
  • caffeine metabolites
  • Liquid chromatography
  • urine

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science

Cite this

HPLC method for rapid determination of acetylator phenotype by measuring urinary caffeine metabolites. / Klebovich, I.; Arvela, Pentti; Pelkonen, Olavi.

In: Journal of Pharmaceutical and Biomedical Analysis, Vol. 11, No. 10, 1993, p. 1017-1021.

Research output: Contribution to journalArticle

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