Extractionless method for the simultaneous high-performance liquid chromatographic determination of urinary caffeine metabolites for N-acetyltransferase 2, cytochrome P450 1A2 and xanthine oxidase activity assessment

Abstract

Urinary metabolic ratios of caffeine are used in humans to assess the enzymatic activities of cytochrome P450 isoenzyme 1A2 (CYP1A2), xanthine oxidase (XO) and for phenotyping individuals for the bimodal N-acetyltransferase 2 (NAT2), all of them involved in the activation or detoxification of various xenobiotic compounds. Most reported analytical procedures for the measurement of the urinary metabolites of caffeine include a liquid–liquid extraction of urine samples prior to their analysis by reversed-phase HPLC. At neutral to basic pH however, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), a metabolite of caffeine, spontaneously decomposes to 5-acetylamino-6-amino-3-methyluracil (AAMU). Since AAMU is not extracted in most organic solvents, the extent of AFMU decomposition cannot be precisely assessed. Although the decomposition reaction can be minimized by immediate acidification of the urine, accurate results can only be obtained when both AAMU and AFMU are monitored, or alternatively, if AAMU is measured after complete transformation of AFMU into AAMU in basic conditions. We report a liquid chromatographic method for the simultaneous quantitative analysis of the five urinary metabolites of caffeine used for the CYP1A2, XO and NAT2 phenotyping studies: AAMU, AFMU, 1-methylxanthine, 1-methyluric acid and 1,7-dimethyluric acid. These metabolites are satisfactory separated from all other known caffeine metabolites as well as endogenous urinary constituents. Sample treatment does not require any liquid–liquid extraction procedure. Urine samples are diluted and centrifuged before being injected (10 μl) onto a YMC-Pack Polyamine II (250×4.6 mm) column. A step-wise gradient elution program is applied using acetonitrile–0.75% (v/v) formic acid: (91:9) at 0 min→(75:25) at 25 min→(65:35) at 35 min→(65:35) at 45 min, followed by a re-equilibration step to the initial solvent composition. The flow-rate is 1.0 ml/min and the separations are monitored by UV absorbance at 260 and 280 nm. The procedure described here represents a substantial improvement over previous methods: a single analysis and a minimal urine sample treatment enables the simultaneous quantitation of five caffeine metabolites, notably AFMU and AAMU, used for the determination of CYP450 1A2, XO and NAT2 enzyme activity. Importantly enough, phenotyping individuals for the bimodal NAT2 is made possible without the uncertainty associated with the deformylation of AFMU, which is likely to happen at all steps prior to the analysis, during sample storage and even in the bladder of the subjects.

Introduction

Calculation of urinary metabolic ratios of caffeine (1,3,7-trimethylxanthine) is commonly used for phenotyping subjects for the bimodal N-acetyltransferase 2 (NAT2), and to assess the activity of cytochrome P450 isoenzyme 1A2 (CYP1A2) and xanthine oxidase (XO). These enzymatic systems are involved in the activation or detoxification of various xenobiotic compounds, including carcinogens [1], [2], which may in turn influence their activity (induction and/or inhibition). Investigating these enzymes is of clinical relevance, since enzyme deficiencies can lead to unusually high plasma concentrations of several drugs due to impaired metabolism, to an increased incidence of side-effects, or conversely, to insufficient therapeutic effect. In environmental, epidemiological and toxicological studies, the determination of the metabolic activity of subjects belonging to a given population (i.e., patients, workers, etc.) may help to evaluate their risk of developing specific exposure-related diseases [3], [4].

Fig. 1 shows the major metabolic pathway of caffeine in healthy human subjects which depends notably on CYP1A2, XO and NAT2 enzymatic systems. In phenotyping studies, the enzyme activities are usually expressed as the urinary molar ratios of the caffeine metabolites (listed in Table 1) with the (AFMU+1X+1U)/17U, 1U/(1X+1U) and AFMU/(AFMU+1U+1X) ratio reflecting CYP1A2, XO and NAT2 activity [5], [6], [7], respectively. Different metabolite ratios have also been proposed [8], [9], [10], notably for CYP1A2 and NAT2 phenotyping, whereby the (AAMU+1X+1U)/17U and AAMU/(AAMU+1U+1X) ratios are used for the calculation, after the basic conversion of AFMU into AAMU. Moreover CYP1A2 activity can also be determined using the paraxanthine/caffeine (17X/137X) ratio albeit in other biological fluids such as plasma and saliva [11].

Most analytical procedures reported for the measurement of the urinary metabolites of caffeine are based on the early work of Grant et al. [12] which include a liquid–liquid extraction of urine samples prior to their analysis by reversed-phase high-performance liquid chromatography (HPLC). At neutral to basic pH however, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), an acetylated caffeine metabolite, spontaneously loses a formyl group and decomposes into 5-acetylamino-6-amino-3-methyluracil (AAMU) (Fig. 1) which is not extracted in most organic solvents, precluding any precise direct assessment of the extent of AFMU decomposition. Although the decomposition reaction can be minimized by acidifying the urine immediately after voiding, complete control and assurance of accurate results is only provided when both AAMU and AFMU are monitored, or alternatively if AAMU is measured after the complete transformation of AFMU in basic conditions. The deformylation decomposition reaction is indeed likely to happen throughout analysis, during the extraction procedure, sample storage, urine collection, and possibly even in the bladder of the subjects [13], [14].

The published procedures reporting the direct quantitation of AAMU require an additional separate analysis, either by HPLC on various chromatographic packings [13], [15], [16], by micellar electrokinetic capillary chromatography (MECC) [17], or by enzyme-linked immunosorbent assay (ELISA) [18]. The only method describing the simultaneous determination of AAMU together with other caffeine metabolites uses a direct injection of urine samples on a C₁₈ reversed-phase column. The reported chromatographic profiles, however, clearly show interferences with endogenous compounds [19].

An important contribution using a normal-phase chromatography has been previously reported by Rodopoulos and Norman [15] but was applied to the assay of AAMU and AFMU only. We therefore propose an improvement of this method enabling the determination of AAMU and AFMU together with three additional metabolites 1X, 1U, and 17U in a single HPLC run with minimal sample preparation. It must be stressed that the proposed sample preparation procedure requires no liquid–liquid extraction step, enhancing phenotyping precision and accuracy by circumventing the poor stability of AFMU and the limited, if any, organic extraction of AAMU from urine.