250 m L plasma. 10µL IS solution (Tramadol-d6 and Paracetamol-D4). Vortex and add 250µL of HCl 0.05N. Rinse with 2x1mL of water. Vortex, centrifuge. Elute with 1mL of Methanol. Load on conditioned Plexa column. Evaporate at 45°C. reconstitute in 200 m L of reconstitution phase.
10µL IS solution (Tramadol-d6 and Paracetamol-D4)
Vortex and add 250µL of HCl 0.05N
Rinse with 2x1mL of water
Elute with 1mL of Methanol
Load on conditioned Plexa column
Evaporate at 45°C
reconstitute in 200mL of reconstitution phase
Inject 20mL on LC/MS/MS system
: Expected Retention Time
Liquid Chromatography-Turboionspray Ionization tandem Mass Spectrometry (LC/TIS-MS/MS) method for the simultaneous determination of Tramadol and Paracetamol in human plasma
Hervé Billy, Nicolas Audonnet, Elodie Mazé and Alex Hundt
SYNEXEL Research International, 144 rue de la Gibauderie 86000 POITIERS, FRANCE
Mechanism of Action :
Tramadol is an opioid analgesic that acts on the central nervous system. Tramadol is pure non selective agonists of the μ, δ, and κ opioid receptors with a higher affinity for the μ receptors. Other mechanisms which contribute to its analgesic effect are inhibition of neuronal reuptake of noradrenaline and enhancement of serotonin release. Tramadol has an antitussive effect. Unlike morphine, a broad range of analgesic doses of tramadol has no respiratory depressant effect. Similarly, the gastro-intestinal motility is not modified. The cardiovascular effects are generally slight. The potency of tramadol is considered to be one-tenth to one-sixth that of morphine.
The precise mechanism of the analgesic properties of paracetamol is unknown and may involve central and peripheral effects.
After a single oral administration of a tramadol/paracetamol (37.5 mg/325 mg) tablet, peak plasma concentrations of 64.3/55.5 ng/ml [(+)-tramadol/(-)-tramadol] and 4.2 μg/ml (paracetamol) are reached after 1.8 h [(+)-tramadol/(-)-tramadol] and 0.9 h (paracetamol) respectively. The mean elimination half-lives t1/2 are 5.1/4.7 h [(+)- tramadol/(-)-tramadol] and 2.5 h (paracetamol).
This poster describes the validation of a specific and sensitive Liquid Chromatography-Turboionspray Ionization tandem Mass Spectrometric (LC/TIS-MS/MS) method for the simultaneous determination of Tramadol and Paracetamol concentration in human plasma using calibrators and quality control samples in human plasma.
A sensitive and specific analytical method for the simultaneous determination of Tramadol and Paracetamol in human plasma was validated with LLOQ of 1 ng/mL for Tramadol and 50 ng/mL for Paracetamol. Specificity, selectivity, linearity, limit of quantification, within-run and between-run precision and bias, extraction efficiency, dilution test, carry-over, matrix effect and stability of Tramadol and Paracetamol under different storage conditions (extract stability, short term stability of the spiked samples maintained at room temperature, freeze / thaw cycles stability, long-term stability at -20°C and stability in solutions) were verified to be within the internationally accepted criteria . The method was applied to the analysis of human plasma samples collected during a bioequivalence study.
Validation results for Tramadol and Paracetamol
Example of mean results of a bioequivalence study
Double Blank plasma sample
Spiked plasma sample : STD 01 (LLOQ)
Spiked plasma sample : STD 08 (ULOQ)
We would like to thank all of the SYNEXEL personnel who contributed to the success of the development and the validation of this assay.
 U.S. Department of Health and Human Services, Food and Drug Administration : Guidance for Industry, Bioanalytical Method Validation, May 2001
 SYNEXEL Study code BW0189A : LC-MS/MS Determination of Tramadol and Paracetamol in human plasma samples collected during the study PAT/2009/472