Brain painkiller reaction rates revealed in real-time

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  • Published: Mar 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Brain painkiller reaction rates revealed in real-time

Need to find levels of tramadol in the brain

Tramadol is a synthetic opioid, used as a painkiller in a wide variety of conditions, since it has less severe side effects and a lower risk of addiction than morphine. However, some side-effects, such as dizziness, nausea, indigestion and a dry mouth, can occur, while withdrawal symptoms can be severe. The pharmacokinetics of tramadol are therefore of great interest. Much work has been carried out on the detection of tramadol and its main metabolite, O-desmethyltramadol (ODT), which is more active than the parent compound, often using LC-MS systems. However, the majority of the work to date has concentrated on investigations of plasma tramadol levels, despite the fact that knowledge of levels in the brain is actually of greater importance.

The researchers from Wuhan attempted to look at the levels of ODT in rat brains, which are often used as models for the human brain in painkiller studies. They attempted to obtain real-time concentrations of cerebrospinal fluid using microdialysis devices.

Microanalysis and LC-MS/MS used to obtain tramadol and ODT concentrations

Cannulas were inserted into the right-lateral ventricle of rat brains under anaesthetic. Later, after administration of tramadol by IP (intraperitoneal) injection, a microdialysis probe, connected to a micro-infusion pump, was inserted through each cannula. The probe was perfused with artificial cerebrospinal fluid. The membrane in the probe allowed diffusion of small molecules from the brain fluid while keeping out larger molecules, such as proteins. Samples of output microdialysis fluid were taken at timed intervals, in order to determine the pharmacokinetics. Samples were stored at -80 °C, which was shown to be cold enough to avoid degradation, prior to thawing and the determination of the tramadol and ODT concentrations by LC-MS/MS.

Before the analysis, known quantities of the cough medicine active ingredient ambroxol were added to thawed samples as an internal standard. HPLC was carried out with an Agilent Zorbax Eclipse XBD-C18 column, with an eluting solvent of 50:50:0.1 acetonitrile:water:formic acid. The flow rate was 0.3 ml/min, while the total run time was only 4 minutes. These conditions proved to be the best out of a number of columns and solvent systems tested.

The mass spectrometer was an Applied Biosystems API 3000 triple quadrupole instrument, with an electrospray (ESI) source in positive ionisation mode. Diagnostic molecular and product ions were noted for tramadol, ODT and the internal standard. The system was run in multiple reaction monitoring (MRM) mode, excluding unwanted peaks and quantifying the compounds of interest by means of precursor to product ion transitions.

The method was shown to have good linearity and selectivity. The limit of quantification was low, at 1 ng/ml for tramadol and 0.5 ng/ml for ODT. Since concentrations of the molecules in native cerebrospinal fluid differ from those found in the artificial cerebrospinal fluid recovered from the microdialysis, calibration of the system was carried out using experiments with spiked fluid, so that actual brain concentrations could be calculated from the analytical results.

The pharmacokinetics results showed that tramadol and ODT appeared in the brain within five minutes of injection. The maximum tramadol level was reached after 21 minutes and the maximum ODT level after 36 minutes. These times are significantly lower than those typically seen after oral administration.

Greater insight into tramadol pharmacokinetics obtained

The combination of microdialysis and LC-MS/MS has helped greater knowledge of the pharmacokinetics of tramadol to be achieved, which should help in devising protocols which maximise pain relief while minimising side-effects. The techniques used could no doubt be applied to other pharmaceuticals that act on the brain.

Related Links

Drug Testing and Analysis, 2017, Early View paper. Liu et al. Intracerebral microdialysis coupled to LC-MS/MS for the determination tramadol and its major pharmacologically active metabolite O-desmethyltramadol in rat brain microdialysates.

Wikipedia, Microdialysis

Wikipedia, Tramadol

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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