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THE ANALYSIS OF TRAMADOL AND ITS MAJOR METABOLITE O-DESMETHYL
Unit 2, Kelvin Campus, West of Scotland Science Park, Glasgow. G20 0SP TRAMADOL, FROM HUMAN PLASMA USING POSITIVE TURBOIONSPRAY LC-MS/MS
Lindsay Best, Caroline Wood, Jim Hillier
I N T R O D U C T I O N
Tramadol is an analgesic used to treat moderate to moderately severe pain. A specific analytical Back Calculated Concentrations from the Calibration Curve for Tramadol
method has been established and validated for the determination of tramadol and its major Figure 1:
Representative Calibration
Figure 2:
Representative Calibration
metabolite o-desmethyl tramadol in human plasma. The method was validated over the range 5.00 Curve for Tramadol
Concentration of Tramadol (ng/ml)
Curve for O-Desmethyl Tramadol
M E T H O D O L O G Y
Sample Preparation and Extraction - Plasma Samples
Control human plasma samples were fortified with solutions containing tramadol and o-desmethyltramadol. These plasma samples were then extracted by liquid/liquid extraction using methyl tert butyl ether (MTBE). The resultant supernatant was evaporated to dryness followed by reconstitutionin mobile phase.
Back Calculated Concentrations from the Calibration Curve for
O-Desmethyl Tramadol
Sample Analysis
Concentration of O-Desmethyl Tramadol (ng/ml)
METHODOLOGY
Applied Biosystems MDS SCIEX API 3000 TandemMass Spectrometer with the TurboIonSprayTM Source Agilent HP1100 Series Quaternary Pump andDegasser.
20mM Ammonium Acetate/Acetonitrile (50/50; v/v) Intra-Occasion Statistics for Tramadol
Figure 3:
Representative Chromatogram from a Calibration Standard Sample containing
Concentration of Tramadol (ng/ml)
5.00ng/ml of Tramadol and O-Desmethyl Tramadol
V A L I D A T I O N R E S U L T S
Back-calculated concentrations for the plasma calibration samples are shown in Tables 1 and 2.
The calibration curves showed good linearity over the range 1.00 - 500 ng/ml. Typical calibration curves are displayed in Figure 1 and 2. Examples of chromatography from a calibration standardcontaining 1.00 and 500 ng/ml of each analyte are presented in Figures 3 and 4.
Intra-day statistics are presented in Tables 3 and 4.
The inter-day (three occasions) bias for tramadol was 0.8, 0.0, -0.4, -1.0 and -2.2% at 5.00, 12.5, Intra-Occasion Statistics for O-Desmethyl Tramadol
50.0, 400 and 500ng/ml respectively. The inter-batch CV for tramadol at these concentrations was7.3, 4.7, 3.2, 2.8 and 4.0% respectively. The inter-batch (three occasions) bias for o-desmethyl Concentration of O-Desmethyl Tramadol (ng/ml)
Figure 4:
Representative Chromatogram from a Calibration Standard Sample containing
tramadol was 3.8, 0.8, 2.2, -3.3 and -6.2% at 5.00, 12.5, 50.0, 400 and 500ng/ml respectively. The 500ng/ml of Tramadol and O-Desmethyl Tramadol
inter-batch CV for o-desmethyl tramadol at these concentrations was 6.9, 6.2, 5.5, 5.9 and 5.8%respectively. These results demonstrate good accuracy and precision over the range 1.00 to 500 Both analytes were also shown to be stable in plasma for 24hours at room temperature, over 3 freeze/thaw cycles and plasma extracts containing the analytes demonstrated stability for up to 72hours when stored at 4°C±3°C.
C O N C L U S I O N
A sensitive and versatile bioanalytical LC-MS/MS method was developed and validated to determine concentrations of tramadol and o-desmethyl tramadol in human plasma samples.

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