Simultaneous analysis of tramadol, metoprolol and their metabolites in human plasma and urine by high performance liquid chromatography

Cytochrome P4502D6 (CYP2D6) is an important cytochrome P450 enzyme which catalyzes the metabolism of more than 80 drugs.1 Many of these drugs have a narrow therapeutic index and the combined use of substrates may result in significant pharmacokinetic interactions, leading to substrate accumulation and deleterious drug effects. Since the discovery of the debrisoquine/sparteine oxidation polymorphism in the 1970s, numerous studies have investigated its status in various ethnic groups. Currently more than 70 different alleles have been described.2 The gene frequency of individual alleles of CYP2D6 shows a marked interethnic difference. For Chinese population, the most common alleles of the CYP2D6 gene are CYP2D6*1, CYP2D6*2 and CYP2D6*10.3 Thus, it is very important to study the relationship between drug interactions and the genetic polymorphism of CYP2D6. Tramadol hydrochloride is a synthetic μ-receptor agonist for the relief of moderate to severe acute and chronic pain.4 Tramadol is metabolized in the liver by the isoenzyme cytochrome P4502D6 (CYP2D6) mainly to O-desmethyltramadol (M1) and mono-N-desmethyltramadol, of which only the former is pharmacologically active.5 Several studies indicated that the pharmacokinetics of tramadol was affected by the genetic polymorphism of CYP2D6.6,7 Metoprolol is a selective β1-adrenoceptor antagonist used in treatment of cardiovascular disorders such as hypertension and coronary heart disease. Approximately 70% to 80% of an oral metoprolol dose is metabolized by CYP2D6 in the liver. The α-hydroxylation reaction which makes the formation of α-hydroxymetoprolol (OH-met) from metoprolol in the liver was mediated almost exclusively by CYP2D6 and so it was used to probe CYP2D6 activity.8,9 Because of its good efficacy and low addictiveness, tramadol was administered to more and more patients to relieve pain such as headache or stomachache, which makes it possible to coadministrate tramadol and metoprolol. The genetic polymorphism of CYP2D6 may influence the interaction of tramadol and metoprolol because these two drugs are substrates of CYP2D6. This communication describes a bioanalytical method including a simple liquid-liquid extraction procedure of plasma and urine samples and subsequent simultaneous determination of tramadol and its primary metabolite O-desmethyltramadol and metoprolol and its metabolite α-hydroxymetoprolol. The validated analytical method has been applied to a pharmacogenomic study, which investigated the relationship between two genetic polymorphisms of CYP2D6 and interaction of tramadol and metoprolol. METHODS Chemicals and reagents Tramadol, O-desmethyltramadol standards (purity: 99.8%) and tramadol hydrochloride tablets (50 mg) were a gift from Four-ring Pharmaceutic Co., Ltd, Beijing, China. Metoprolol standard (purity: 99.6%) and metoprolol tartrate tablets were provided by Astra, Beijing, China. OH-met (purity: 99.8%) was presented by Professor Zhou, Zhongnan University, China. Bisoprolol (purity: 99.5%) (internal standard) was purchased from NICPBP, Beijing, China. Acetonitrile was of high performance liquid chromatography (HPLC) grade from Fisher, New Jersey, USA. Other reagents were of analytical grade, obtained from Beijing Chemical Reagents Company, Beijing, China. Ultrahigh quality water (Milli-Q apparatus, Millipore Simplicity 185, USA) was used for chromatography. Apparatus and chromatographic conditions The apparatus used for the HPLC analysis was an Agilent 1100 series with a fluorescence detector [excitation wavelength (λ ex) 216 nm/ emission wavelength (λ em) 312 nm] (Agilent Technologies, USA). Data acquisition and analysis were performed using the ChemStation Rev. A 10.01. Separation was carried out at room temperature by a zorbax RP-select B column (Agilent Technologies, USA) with particle size of 5 μm (150 mm × 4.6 mm I.D.). The injection volume was 40 μl (for plasma sample) or 20 μl (for urine sample). The mobile phase was a mixture of 0.05 mol/L KH2PO4 (adjusted to pH 3.5 with phosphoric acid) - acetonitrile (90:10, v : v). The flow rate was 1 ml/min. Sample preparation Frozen plasma and urine samples were thawed at room temperature and vortexed prior to use. Samples (0.5 ml) were spiked with the internal standard (50 μl of 3 μg/ml bisoprolol for plasma samples or 10 μg/ml bisoprolol for urine samples). Then, 200 μl 0.1 mol/L NaOH was added, and after vortexing for 2 minutes, 6 ml of redistilled ethyl ethanoate was added. The tube was capped, vortexed for another 2 minutes and centrifuged at 2000 × g for 10 minutes. The supernatant was transferred to another clean tube and 100 μl 0.1 mol/L H2SO4 added. The tube was capped, vortexed for 2 minutes and centrifuged at 2000×g for 10 minutes. Aqueous layer (40 μl for plasma sample or 20 μl for urine sample) was injected into the chromatographic system. Calibration Referential mixtures of tramadol, M1, metoprolol and OH-met were prepared by diluting appropriate volumes of stock solution with drug free human plasma to achieve the following concentrations: 12.5, 25, 50, 100, 200, 400, 600, 800 ng/ml for tramadol; 5, 10, 20, 40, 80, 160, 240, 320 ng/ml for M1; 10, 20, 40, 80, 100, 200, 300, 400 ng/ml for metoprolol and 5, 10, 15, 30, 60, 120, 240, 360 ng/ml for OH-met. Three individual samples were prepared at each calibration level. Analogous mixtures of tramadol, M1, metoprolol and OH-met were prepared by diluting appropriate volumes of stock solution with drug free human urine to achieve concentrations: 25, 50, 100, 200, 400, 1000, 2000, 4000 ng/ml for tramadol; 10, 20, 100, 200, 400, 800, 1600, 3200 ng/ml for M1; 10, 20, 100, 200, 400, 1000, 2000, 4000 ng/ml for metoprolol and 10, 50, 150, 300, 600, 1200, 2400, 3600 ng/ml for OH-met. Three individual samples were prepared at each calibration level. The extraction procedure was the same as described in the above section. Validation The method was validated by analysis of human plasma or urine quality control samples prepared at four concentrations spanning the calibration range. Quality control samples were prepared by spiking drug free human plasma with the different working standard solutions of tramadol, M1, metoprolol and OH-met. The concentrations of each analyte in these solutions were 25, 200, 600 ng/ml for tramadol; 10, 80, 240 ng/ml for M1; 20, 100, 300 ng/ml for metoprolol and 10, 60, 240 ng/ml for OH-met. Quality control samples were prepared by spiking drug free human urine with the different working standard solution of tramadol, M1, metoprolol and OH-met. The concentrations of each analyte in these solutions were 50, 400, 2000 ng/ml for tramadol; 20, 200, 1600 ng/ml for M1; 20, 200, 2000 ng/ml for metoprolol and 50, 300, 2400 ng/ml for OH-met. Assay performance of the analytical method was assessed by all of the following criteria: selectivity, linearity, precision, limit of quantitation (LOQ), recovery and stability. Preliminary test for pharmacogenomic study From Chinese healthy volunteers who have been genotyped in our previous study, two volunteers (One is CYP2D6*1/*1 and the other is CYP2D6*10/*10) participated in this preliminary, pharmacogenomic study. The protocol was approved by the ethics committee of Chinese People's Liberation Army General Hospital. The study was carried out in accordance with Good Clinical Practice guidelines. Written informed consent was obtained from all volunteers. After an overnight fast, the two subjects took 100 mg tramadol hydrochloride and 100 mg metoprolol tartrate with 200 ml water. Two standardized meals were then provided at 4 and 10 hours after drug administration. Venous blood samples (5 ml) were collected in heparinized tubes from a forearm vein of each subject immediately before and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24 and 32 hours after drug administration. Plasma was separated and kept frozen at -20°C until analysed. RESULTS Specificity Average retention times for tramadol, M1, metoprolol, OH-met and the internal standard were 28.80, 8.20, 31.06 and 4.80 minutes, respectively. There were no interfering peaks from the plasma or urine matrix in the chromatographic region. A typical chromatogram of plasma sample and urine sample after administration of metoprolol tartrate and tramadol hydrochloride was presented in Fig.Fig.: Chromatogram of (A) plasma sample and (B) urine sample from a subject after administration of 100 mg metoprolol tartrate and 100 mg tramadol hydrochloride.Linearity The linearity of the calibration curve was tested and evaluated [y=ax+b, where x is the concentration of tramadol, M1, metoprolol and OH-met and y is the corresponding peak area ratio of each analyte to bisoprolol (I.S.)]. The regression equations, determination coefficients and linear range were presented in the table.Table: Standard calibration curves of tramadol, M1, metoprolol and OH-met in plasma and urine samples (n=3)Precision The precision of the assay method was evaluated by determining the inter-day and intra-day relative standard deviation (RSD) of the measured peak area ratios for different concentrations. The RSD were all less than 10% (n=5). Lower limit of quantification (LLOQ) The LLOQ was defined as the lowest concentration in the calibration curve that can be determined with an accuracy of 80%-120% and precision of no more than 20%. In this assay, LLOQ were 12.5, 5, 10 and 5 ng/ml for tramadol, M1, metoprolol and OH-met, respectively. Recovery The absolute recoveries of the four analytes were all more than 77% and the relative recoveries were between 92% and 108% (n=5). Stability A set of standard quality control samples of three different concentrations was prepared from a freshly prepared stock solution of the analytes in drug free plasma and urine and kept frozen at - 20°C. Three samples of each concentration were subjected to three freeze and thaw cycles before analysed. The RSD of the measured concentrations of the four analytes were all less than 10%. Another set of three samples of each concentration were stored in the freezer for one month, thawed and analysed. The RSD were less than 9%. The stability of the extract of processed samples was determined by reinjecting such samples after standing for 24 hours at ambient room temperature. No significant changes in the measured peak area ratios were observed, which suggested that the analytes were stable at room temperature in the extracted samples for at least 24 hours. The mean change in peak area ratios after injection was less than 5% for all analytes. Results of preliminary test The concentrations of tramadol and metoprolol and their metabolites were measured by HPLC. Then the pharmacokinetic parameters were calculated by the DAS 1.0 software. Areas under curve (AUC) of tramadol and metoprolol were higher (3753 ng · ml−1 · h−1 vs 2913 ng · ml−1 · h−1 and 2534 ng · ml−1 · h−1 vs 1325 ng · ml−1 · h−1, respectively) and those of M1 and OH-met were lower (715 ng ·ml−1 ·h−1 vs 1194 ng ·ml−1 ·h−1 and 675 ng ·ml−1 ·h−1 vs 901 ng · ml−1 · h−1, respectively) in subject expressing *10/*10, compared with *1/*1 subject. These data show that the metabolising of tramadol and metoprolol in CYP2D6*10/*10 subject was slower than that in CYP2D6*1/*1 subject. DISCUSSION The methods described for the determination of tramadol and/or its metabolites in biological samples included capillary gas chromatography with a nitrogen selective detector,10 GC-mass spectrophotometry (GC-MS)11 and HPLC with UV12 or fluorescence13 detection. Several methods for assaying metoprolol, with or without metabolites, in biological fluids have been published.14 There has been no simultaneous analysis of these two drugs in the same chromatographic condition reported. Based on the excitation (Ex) and emission (Em) fluorescence spectra of the analytes, tramadol was found to react strongly in the range of 208 nm to 228 nm [max 216 nm (Ex)] and 300 nm to 326 nm [max 308 nm (Em)], and metoprolol in the range of 210 nm to 232 nm [max 224 nm (Ex)] and from 300 nm to 330 nm [max 312 nm (Em)]. To avoid strong reactions of O-desmethyltramadol, OH-met and bisoprolol, Ex 216 nm and Em 312 nm were selected for the fluorescence detection. Acetonitrile: 0.05 mol/L phosphate buffer combinations of 25:75, 20:80; 15:85, 13:87, 11:89, 10:90 demonstrated better resolutions among these five analytes obtained with decreasing organic composition. Here, we chose 10:90 as the proportion of acetonitrile: 0.05 mol/L KH2PO4. The lowest concentration of tramadol and metoprolol can be determined by this method in our preliminary pharmacogenomics study, indicating that this method could meet the requirement of this study. In conclusion, we developed and validated a new bioanalytical method including a simple liquid-liquid extraction of plasma and urine samples and highperformance liquid chromatographic determination and selective separation of tramadol, metoprolol and their metabolites using fluorescence detection. The stability of these four analytes under conditions of storage and handling relevant to the conduct of clinical pharmacokinetic studies was also demonstrated in our study.