Sensitive and Selective Reversed-Phase High Performance Liquid Chromatographic-UV Spectrophotometric Determination of Dextromethorphan and its CYP2D6 Mediated Metabolite, Dextrorphan in Human Urine

Purpose: To develop a simple, sensitive and selective method for the determination of dextromethorphan and its metabolite, dextrophan in human urine using reversed-phase high performance liquid chromatography with UV-spectrophotometric detection (RP-HPLC-UV). Methods: Pre-column sample clean-up was carried out by liquid-liquid extraction of the analytes with chloroform: isopropanol (70:30) solution after alkalization of 1000 µL sample and spiking of internal standard, morphine. The samples were chromatographed in a reversed-phase (C-18) ultra sphere silica (5µm particle size and 250 x 4.6 mm I.D). The mobile phase consisted of methanol: acetonitrile: 0.5% w/v ammonium acetate (10:10:80) adjusted to pH 2.8 with orthophosphoric acid and pumped through the column at 1ml/min flow rate. The analytical method was validated for accuracy and precision as well as the recovery of the analytes, dextromethorphan and its metabolite, dextrophan over the concentration range of 0.20 to 5.0µg/ml. Results: The standard curves were linear over the concentration range of 0.2 to 5.0µg/ml for dextromethorphan and dextrorphan. The regression coefficients (R 2 ) of the analytes were >0.99. The method was reproducible with coefficient of variation for the analytes being < 10 %. Dextromethorphan was well resolved from its metabolite, dextrorphan and the internal standard, morphine. The limits of detection of dextromethorphan and dextrorphan were 50ng/ml and the recoveries and accuracies were greater than 85 and 90 %, respectively. Conclusion: The analytical assay method exhibits good precision and selectivity and it was applied to the analysis of dextromethorphan and dextrorphan in urine for the assessment of CYP2D6 activity.

3-MEM is further metabolized to 3-hydroxymorphinan (3-OHM) by CYP2D6.DP and 3-OHM are then eliminated via the kidneys after glucuronidation [2,3].There are four phenotypic subpopulations that define the rate of drug metabolism by CYP2D6: individuals with poor (PM), intermediate (IM), extensive (EM), and ultrarapid metabolizers (UM) [4].DMP is a well established probe drug used to assess CYP2D6 function because it is relatively safe and readily available as an "over-the counter" medication, with only mild adverse effects [5,6].CYP2D6 activity is predicted using the metabolic ratio (MR) from molar concentration of DMP/DP [7,8].
Various methods have been described in literature for the determination of DMP and DP in urine, which include high-performance liquid chromatography (HPLC) with fluorescence detection [9-13], HPLC with ultraviolet detection [14], and gas chromatography with flame ionization [15], and mass spectrometric detection [16].Specific quantitation was obtained in most of these methods but assay sensitivity was inadequate for the determination of the metabolites.Also, the lower limit of quantification of DMP was insufficient for the accurate determination of the DMP/DP ratio.There were several interferences from endogenous peaks under the chromatographic conditions specified in some of these methods thereby complicating the quantification of DMP or DP.Additionally, the sample preparation procedures described in these methods generally suffered from low analyte recoveries.Again, the assay selectivity in the presence of other metabolites was not mentioned and the selectivity of the method in urine samples originating from different subjects was not evaluated [17].All these may adversely affect the determination of DMP, DP and other metabolites.Although the effectiveness of HPLC-MS technique is very accurate, the equipment is rather costly.This study therefore aims to develop a simple, cost-effective and sensitive HPLC-UV method for the simultaneous quantitation of DMP and DP in urine samples in resource-limited settings.

Preparation of standard solutions
Stock solutions of 100µg/ml standard DMP and DP were prepared by dissolving 10 mg of each separately in 100 ml of distilled water.The stock solutions were serially diluted with water to give working standard concentrations of 0.2, 0.5, 1.0, 2.0, 4.0, and 5µg/ml for both DMP and DP.A stock solution of 1 mg/ml for the internal standard, morphine sulphate, was also prepared in water.

Chromatographic conditions and equipment
The liquid chromatographic system used was an Agilent 1100 series instrument, (Agilent Technologies, USA) made up of quaternary pumps, a gradient mixer (Agilent, Germany) with a system purge and a variable wavelength (200 -800nm) uv-vis detector model CE1100 (Agilent, Japan) with an 18µL flow cell and detection was done at the wavelength of 278nm.Injection was by a Rheodyne model 7725 valve (Cotati, California, U.S.A.) fitted with a 20 µL loop and an on-line vacuum degasser (Agilent, Japan).The HPLC was connected to a HP computer system and a printer.The column used was a reversedphase (C-18) ultra sphere silica with 5µm particle size and 250 x 4.6 mm I.D (Beckman, USA).The mobile phase consisted of methanol: acetonitrile: 0.5 %w/v ammonium acetate (10:10:80) pumped through the column at a flow rate of 1ml/min.The pH of the mobile phase was adjusted to 2.8 with orthophosphoric acid and the analytical run was performed at ambient temperature.

Preparation of calibration standards and quality control samples for DMP and DP
Blank urine (1 ml) sample was each placed in six different extraction tubes and varying aliquots of the stock solutions (100μg/ml) of DMP and DP were spiked to give concentrations of 0.2, 0.5, 1.0, 2.0, 4.0, and 5µg/ml for calibration standards and 0.2, 0.5, 1.0, 2.0, and 4.0 µg/ml for quality control (QC) samples.20μL of the stock solution of the internal standard, morphine sulphate (1mg/ml), was spiked to each tube.The samples were rendered alkaline with 1M NaOH (0.5 ml) and whirlmixed for 1 min.Chloroform: isopropanol (70:30, 3 ml) solution was added to each of the tubes and whirlmixed for 1min afterwhich the tubes were centrifuged at 2500 rpm for 10 min.The organic layer was transferred into another extraction tube.The extraction process was repeated and the pooled organic extract was evaporated to dryness in a water bath at 40 o C. The residue was reconstituted in 100 μL of 0.1N HCl and whirlmixed before injecting 20 µL onto the HPLC.The peak area ratio was plotted against the concentration of each of the compounds injected.The regression analysis was carried out using Microsoft Excel 2007.Values of the lower limit of quantitation (LLOQ) were calculated according to FDA guidelines for bioanalytical method validation [18] as at least five times the response compared to blank response and a precision of 20 % and accuracy of 80 -120 %.

Determination of precision of the method for DMP and DP
The assay procedure described under the preparation of calibration standards and quality control was repeated five times for QC samples within the same day (intra-day precision) and five times over four days (inter-day precision) both expressed as %RSD values.

Absolute recovery and accuracy for DMP and DP
The recovery was computed by comparing peak areas obtained after extraction of QC samples from urine with peak area results after injecting working standard solutions at the same theoretical concentrations.The accuracy of the measurements was assessed based on replicate analysis of four QC samples for each compound in each run.

Determination of DMP and DP in urine
To 1 ml of urine sample in a centrifuge tube, 20 µL of the internal standard was added and the mixture was incubated for 18 h with 0.5ml of 10, 000 IU β-glucuronidase.After the incubation, DMP and its metabolite DP were extracted under alkaline conditions as previously described.The residue was reconstituted in 100 µL 0.1N HCl and whirlmixed before 20 µL was injected onto the HPLC.The metabolic ratio (MR) was calculated as the molar concentration ratio of dextromethorphan and its O-demethylated metabolite, dextrorphan in a 0 -8 hour cumulated urine collection and was used to estimate CYP2D6 activity.

Statistical analysis
Results were expressed as mean ± SD and RSD (%) for the precision and recovery assays.Statistical analyses were carried out using GraphPad InStat software, version 2, and Microsoft Excel 2007 (Microsoft Corporation, USA) with p < 0.05 as the level of significance.

Chromatographic analysis of DMP and DP
The retention time for DMP, DP and the internal standard MP were 8.7, 5. .These demonstrated that chromatographic peaks of DMP, DP and MP were well resolved with no interferences from endogenous substances in the matrix.
The limit of detection taken as the concentration giving a peak of at least five times the baseline noise was 20 ng/ml for both DMP and DP using 1ml sample, while the value of the lower limit of quantitation (LLOQ) calculated according to the FDA guidelines for bioanalytical method validation [18] was 50 ng/ml for both compounds.

Calibration curves for DMP and DP
The standard curve was a graph of the peak area ratio (y-axis) versus the concentration (x-axis) of each of the compounds and was linear over the concentration range of 0.2 to 5.0 µg/ml for DMP and DP, in urine.The regression equations for the calibration curves for DMP and DP were y = 1.537x -0.115 and y = 0.681x -0.265, respectively with the regression coefficients (R 2 ) being 0.995 and 0.998 for DMP and DP, respectively, which allowed for accurate reading of the concentrations of all the test samples.

Precision of the analytical Method for DMP and DP
The RSD%, which is a measure of the precision ranged from 4.29 to 7.80 % for intra-day run and 1.90 to 5.26 % for inter-day assay for DMP, while RSD% for DP ranged from 1.43 to 7.2 % and Time (Min) 1.85 to 5.23 % for intra-day and inter-day precision, respectively as shown in Table 1.

Recovery and Accuracy of DMP and DP
The recovery of DMP and DP from spiked urine following extraction ranged from 84.8 to 104.8 %, while the accuracy of the analytical method ranged from 92 to 106 % as shown in Table 1.

DISCUSSION
The analytical assay method developed in this study gave a good resolution of dextromethorphan and its main metabolite, dextrorphan.When ten blank urine samples obtained from different individuals were hydrolyzed, cleaned up and injected onto the HPLC to check for endogenous interferences, no endogenous compound was found to interfere with the DMP and DP.Also, few structurally related compounds such as codeine as well as some inhibitors of CYP2D6 like propranolol were assessed to check for possible interferences.None of these drugs interfered with the determination of the analytes.The analytical method therefore gave a good resolution of DMP, DP as well as the internal standard MP, thus facilitating accurate determination of the drug and its metabolite.
The LLOQ for DMP and DP in urine was 50 ng/ml, which made it possible to monitor the unchanged drug and its metabolite in the samples.The method was found to be reproducible, as indicated by values obtained for RSD (< 10 %) for DMP and DP.The recoveries and accuracies for the analytes ranged from 84.8 to 104.8 % and 89 to 110 %, respectively, indicating that the method was quite accurate and sensitive.The stability of QC samples was evaluated for samples stored at −20 o C for 4 months; at room temperature for 24 h; and after three freeze-thaw cycles.Stability of treated samples was also assessed after 24 h [18].In all of the conditions, samples preserved their potency (> 90 %) during the mentioned periods.This method emphasized off-column pretreatment as a necessary panacea for effective extraction and quantitation of DMP and DP in urine.In this study, the choice of chloroform/isopropanol as the extracting solvent resulted in some improved recovery of DMP compared to previous reports [14,19].The analytical method employed in this study is one of the few methods that can detect DMP and its metabolite DP.Besides, the sample preparation procedures described in some previous methods generally suffered from low analyte recoveries [15,20] and in most cases, the selectivity of these methods in the presence of a number of other metabolites of DMP was not clearly established.

CONCLUSION
The HPLC method described is a simple, sensitive and cost-effective assay technique for the determination of dextromethorphan and its CYP2D6-mediated metabolite, dextrorphan, in urine.The main advantage of the method is the use of a simple liquid-liquid extraction process for sample clean-up prior to chromatographic protocol with good analyte recovery.The newly developed method allowed the simultaneous determination of dextromethorphan and its main metabolite, dextrorphan; it is also suitable for separating CYP2D6 phenotypes.
3 and 4.2 min, respectively, as shown in Fig 1.A representative chromatogram of the blank urine sample spiked with 2 µg/ml of standard, DMP and DP and the internal standard (MP) is presented in Fig 2 .The retention times of DMP, DP and MP in the sample chromatograms corresponded to those obtained from the reference compounds by direct injection (Fig 1)

Fig 1 :
Fig 1: HPLC chromatogram following direct injection of test samples containing 2 µg/ml of DMP, DP and internal standard, MP.The peak eluting at 4.2 min represents MP while the peaks at 5.3 and 8.7 mins represents DP and DMP, respectively

Fig 2 :
Fig 2: HPLC Chromatogram of spiked urine containing 2 µg/ml each of DMP, DP and internal standard, MP.The peak eluting at 4.2 min represents MP while the peaks at approximately 5.3 and 8.8 min represent DP and DMP, respectively.