Precise simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples following their successive i.p. administration

- The new HPLC method that allows simultaneous quantification of methadone and cocaine in rat serum and brain samples.
- The method is highly sensitive in comparison to other reported methods.
- It can be employed to analyze methadone and cocaine samples following drug - drug interactions pharmacokinetic studies conducted in rats to investigate the effect of methadone on cocaine PK.
- The method also can be potentially applied to human biological serum samples to monitor compliance to methadone maintenance therapy and to detect possible cocaine - methadone co-abuse.

A sensitive high-performance liquid chromatography (HPLC) assay with dual UV detection has been developed and validated for the simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples. Liquid-liquid extraction using hexanes was applied for samples extraction with Levo-Tetrahydropalmatine (L-THP) as the internal standard. Chromatographic separation of the analytes was achieved on a reversed-phase Waters Symmetry® C18 column (150 mm × 4.6 mm, 5 μm). A gradient elution was employed with a mobile phase consisting of 5 mM potassium phosphate containing 0.1% triethylamine (pH = 6.5) (A) and acetonitrile (B) with a flow rate of 1 mL/min. UV detection was employed at 215 nm and 235 nm for the determination of methadone and cocaine, respectively. The calibration curves were linear over the range of 0.05-10 μg/mL for both methadone and cocaine. The assay was validated according to FDA guidelines for bioanalytical method validation and results were satisfactory and met FDA criteria. Inter-day accuracy values of serum and brain samples ranged from 96.97 to 105.59% while intra-day accuracy values ranged from 91.49 to 111.92%. Stability assays showed that both methadone and cocaine were stable during sample storage, preparation, and analytical procedures. The method was successfully used to analyze biological samples obtained from a drug- drug interaction pharmacokinetics (PK) study conducted in rats to investigate the effect of methadone on cocaine PK. Our method not only can be used for bioanalysis of samples obtained from rats but also can potentially be applied to human biological serum samples to monitor compliance to methadone maintenance therapy (MMT) and to detect possible cocaine-methadone co-abuse.