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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.

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Hervé Billy , Nicolas Audonnet, Elodie Mazé and Alex Hundt

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Herv billy nicolas audonnet elodie maz and alex hundt

250mL 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 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 [1]:

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.

Pharmacokinitics [1]:

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 [2]. The method was applied to the analysis of human plasma samples collected during a bioequivalence study.





Mass spectrometry

  • Agilent 1100 binary pump

  • Column : Lichrospher RP60 Select B 125 x 4mm, 5mm

  • Temperature : 25°C

  • Flow rate : 0.5 mL/min

  • Mobile phase in isocratic mode :

  • Methanol/Ammonium acetate 5mM (75/25) + 0.1% formic acid

  • Retention time:

  • Tramadol : ~3 min

  • Paracetamol : ~2.5 min

  • CTC Analysis PAL System

  • Temperature : 4°C

  • Injection volume : 20mL

  • Wash solvent 1 : Methanol/Water 80/20

  • Wash solvent 1 : Methanol

  • Sciex API3000 operating in MRM mode

  • TIS positive mode (350°C)

  • MRM transitions: (Dwell Time : 200 ms)

Validation results for Tramadol and Paracetamol

  • The extraction process was extensively optimized for both selectivity and specificity, and no endogenous peaks or interference are present under the same MRM transition at retention time of the products.

  • The chosen internal standards, Tramadol-d6 and Paracetamol-d4, were found to compensate very well for variations in extraction and analysis.

  • There is no carryover effect observed for either analytes or internal standards after injection of several samples at high concentration.

  • There is no effective matrix effect observed for either analytes used with their respective internal standards.

  • Overall recovery were between 85 and 91% for Tramadol (92% for Tramadol-d6) and between 76 and 80% for Paracetamol (70% for Paracetamol –d4).

  • Stability of Extract (SOE) : 120 hours for extract samples stored at +4°C.

  • Stability in matrix (SIM): 24 hours in plasma stored at room temperature in polypropylene tubes.

  • Freeze/thaw Stability (FTS) : 3 cycles at -20°C in polypropylene tubes.

  • Long TermStability (LTS) : 54 days in plasmastored at -20°Cin polypropylene tubes.

Representative chromatograms



Example of mean results of a bioequivalence study

Double Blank plasma sample

Spiked plasma sample : STD 01 (LLOQ)

Spiked plasma sample : STD 08 (ULOQ)

Calibration curves


  • A highly sensitive, selective and specific method for the simultaneous determination of Tramadol and Paracetamol in human plasmahas been developed and fully validated with excellent accuracy and reproducibility.

  • This method was validated for the range from 1 ng/mL up to 200 ng/mL for Tramadol and from 50 ng/mL up to 10000 ng/mL for Paracetamol.

  • This analytical method was successfully used for the simultaneousanalysis of Tramadol and Paracetamol in human plasma samples collected on K3 EDTA during a bioequivalence study [3].

  • For this bioequivalence study after 37.5/325mg oral doses of Tramadol and Paracetamol respectively, Cmax concentrations at approximately 120 ng/mL of Tramadol and 3500 ng/mL of Paracetamol (based on mean results) were reached after approximately 2h and 1h respectively.


We would like to thank all of the SYNEXEL personnel who contributed to the success of the development and the validation of this assay.


[1] HTTP///WWW.zaldiar.com/ZAL/en_EN/pdf/saldiar_core_product_profile.pdf

[2] U.S. Department of Health and Human Services, Food and Drug Administration : Guidance for Industry, Bioanalytical Method Validation, May 2001

[3] SYNEXEL Study code BW0189A : LC-MS/MS Determination of Tramadol and Paracetamol in human plasma samples collected during the study PAT/2009/472

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