Measurement of Dextromethorphan and Dextrorphan in Human Urine using a Novel Isocratic Liquid Chromatography – Tandem Mass Spectrometry Method. Denise A McKeown, Terry D Lee, David W Holt and Atholl Johnston. Analytical Unit, St George’s Hospital Medical School, London, UK. CALIBRATION CURVES REPRESENTATIVE CHROMATOGRAMS CH INTRODUCTION CH3 3 Figure 4 represents a typical chromatogram for a urine N N Figure 2 and Figure 3 show the calibration curves for both dextromethorphan and dextrorphan urine calibrators calibrator. The retention times of dextromethorphan, The analysis of dextromethorphan and its metabolites; respectively. The assay range for dextromethorphan and dextrorphan and ethylmorphine are approximately 4.4, 4.3 dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan was 0 to 5060ng/mL and 0 to 4950ng/mL for dextrorphan. and 4.7 minutes respectively. (Figure 1), has been encountered in both clinical and forensic settings. However this poster will focus on dextromethorphan H C O HO D e x t r o m e t h o r p h a n ( 4 .4 m in s ) and dextrorphan. 3 Dextromethorphan Dextromethorphan has therapeutic properties as an antitussive Dextromethorphan Dextrorphan 3 0 0 0 0 2 5 0 0 0 7 ) drug and is available without prescription as a component of s H p 2 0 0 0 0 c H ( oo 6 ii y tt t cough remedies. The drug can be orally administered and is i 1 5 0 0 0 s N aa N 5 n e RR t widely available, allowing it to be exploited in research as a 1 0 0 0 0 n I aa 4 1-2 ee probe drug for oxidative phenotyping. A large number of rr 5 0 0 0 AA 3 kk 0 drugs are metabolised in the body by the Cytochrome P450 aa 2 0 1 2 3 4 5 6 ee (CYP) enzymes, which are highly polymorphic. CYP3A4, PP 1 C o n c e n t ra t i o n (n g / m L ) CYP2C19 and CYP2D6 are a few examples of these enzymes HO 0 3 H3C O D e x t r o r p h a n (4 .3 m in s ) involved in the biotransformations. Dextromethorphan is 0 1000 2000 3000 4000 5000 3-methoxymorphinan 3-hydroxymorphinan metabolised to the 0-demethylated metabolite, dextrorphan, via Concentration (ng/mL) 1 4 0 0 0 o 1 2 0 0 0 i CYP2D6 enzyme pathway. The ratio of dextromethorphan to t Figure 1: Structures of Dextromethorphan and its metabolites a 1 0 0 0 0 R dextrorphan reflects the CYP2D6 enzyme activity and can be a e 8 0 0 0 Figure 2: Dextromethorphan calibration curve. Regression linear r A used to classify an individual as either a poor or extensive k 6 0 0 0 through zero, weighting (1/(x*x)), y = 0.00125x, r = 0.9989. a e 4 0 0 0 1 EXPERIMENTAL P metaboliser. 2 0 0 0 Dextromethorphan and dextrorphan can be routinely detected D extrorphan 0 0 1 2 3 4 5 6 and quantitated by gas chromatography-mass spectrometry Extraction C o n c e n t ra t i o n (n g / m L ) (GC/MS) in our forensic toxicology service, and has been Internal standard (ethylmorphine, 2.5mg/L, 100µL), sodium 4.0 3.5 E th y l M o r p h in e (4 .7 m in s ) present in several post mortem cases. This type of abuse is not hydroxide (100µL, 1M) and methyl tertiary butyl ether (2mL) oo ii 3.0 tt unexpected as a desperate individual will try to use any legal or aa 7 0 0 0 were added to each urine calibrator, control or human RR 2.5 aa 6 0 0 0 ee illegal drug that is readily available to them. The recreational ) sample (100µL). The tubes were capped, mixed (approx 5 rr 2.0 s 5 0 0 0 AA p c ( kk abuse of dextromethorphan (DXM) is also increasing. Its use is minutes) and then centrifuged (3500rpm, 5 minutes). The 1.5 4 0 0 0 y aa t i ee s 1.0 n 3 0 0 0 PP characterised in two ways; firstly as a drug knowingly being upper organic layer from each was transferred to a tube e t n consumed and secondly being sold as ecstasy. Countries containing 0.1% formic acid (500µL). After mixing (approx. 5 0.5 I 2 0 0 0 1 0 0 0 4 0.0 reporting use in these contexts include America, Korea and minutes) and centrifugation (3500rpm, 5minutes) the top 0 0 1000 2000 3000 4000 5000 Spain5. Its increased use is believed to be related to cost, legal solvent layer was removed and discarded. The aqueous 0 1 2 3 4 5 6 Concentration (ng/m L) T i m e (m in s ) access and wide availability. extracts were then transferred to autosampler vials ready for analysis. The human urine samples were pre-incubated with Figure 4: Representative chromatogram of a urine calibrator; β-glucuronidase for a minimum of 12 hours prior to Figure 3: Dextrorphan calibration curve. Regression linear AIM dextromethorphan (4.4min), dextrorphan (4.3min) and ethylmorphine extraction. through zero, weighting (1/x*x)), y = 0.000701x, r = 0.9984. (4.7min). To develop a liquid chromatography-tandem mass HPLC Conditions and MS Parameters spectrometry method (LC/MS/MS) for the measurement of SUMMARY The HPLC equipment consisted of a Perkin Elmer PE200 dextromethorphan and dextrorphan in human urine. This series autosampler (injection volume, 10µL) and pump. A method was to be used in a clinical study investigating oxidative The aim to develop a method for the measurement of dextromethorphan and dextrorphan in human urine using LC/MS/MS was silica column (Supelcosil LC-Si, 10cm x 4.6mm, 5µm) was phenotyping. Many researchers have measured achieved. A novel isocratic method using a silica column gave good retention of the drug, metabolite and internal standard. An maintained at 50°C in a Schimadzu CTO-10A column oven. dextromethorphan and dextrorphan using high performance isocratic method has advantages over using a gradient method because it does not use as much mobile phase and avoids long The mobile phase, consisting of acetonitrile/de-ionised liquid chromatography (HPLC) with fluorescence, UV or mass equilibration times allowing a much more efficient method for detection. This method will be used in our clinical toxicology water/formic acid (50/50/0.2) was pumped at 1mL/min and spectrometric detection. Published methods commonly use C8 laboratory for clinical studies investigating oxidative phenotyping. The method can also be used for any assays that require the split 10:1 before entering the mass spectrometer. columns either isocratically or with a gradient system in an measurement of these drugs and can be used for different sample matrices such as blood. In the forensic toxicology laboratory A Sciex API2000 triple quadrupole mass spectrometer attempt to reduce the difference in elution times between these we currently use GC/MS for the qualitative and quantitative analysis of dextromethorphan and dextrorphan. However, the equipped with a turbo-ion spray interface maintained at two compounds. Gradient systems are preferred, but this often LC/MS/MS method has the advantage that it uses less sample volume for detection. This will be advantageous when there is 500°C was used for detection. The method was run in results in longer equilibration times and more solvent waste. limited sample which is often the case with post-mortem samples. positive ionisation mode and set to detect the precursor and Here we report a LC/MS/MS method using a novel isocratic product ions of dextromethorphan (m/z: 272.04/212.98), system which elutes dextromethorphan, dextrorphan and the dextrorphan (m/z: 257.99/198.80 and ethylmorphine (m/z: internal standard, ethylmorphine in less than 6 minutes. REFERENCES 314.01/165.08). Total run time of method was 6.0mins. 1. S.S. Vengurlekar et al., J. Pharm. Biomed. Anal. 30 (2002) 113-124 4. H. Chung et al., Ann. N.Y. Acad. Sci. 1025 (2004) 458-464 2. J.M. Hoskins et al., J. Chromatogr. B 696 (1997) 81-87 5. S. Abanades, A.M. Peiro, M. Farre, Med Clin (Barc) 123(8) (2004) 3. S.A. Testino Jr, G. Patonay, J. Pharm. Biomed. Anal. 30 (2003) 305-311 1459-1467.