An HPLC system using a simple liquid-liquid extraction and HPLC with UV detection has been validated to determine tramadol concentration in human plasma. The method developed was selective and linear for concentrations ranging from 10 to 2000 ng/ml with average recovery of 98.63%. The limit of quantitation (LOQ) was 10 ng/ml and the percentage recovery of the internal standard phenacetin was 76.51%. The intra-day accuracy ranged from 87.55 to 105.99% and the inter-day accuracy, 93.44 to 98.43% for tramadol. Good precision (5.32 and 6.67% for intra- and inter-day, respectively) was obtained at LOQ. The method has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.
The aim of the present study is to investigate the influence of the CYP2D6*10 allele on the disposition of tramadol hydrochloride in Malaysian subjects. A single dose of 100 mg tramadol was given intravenously to 30 healthy orthopaedic patients undergoing various elective surgeries. After having obtained written informed consents, patients were genotyped for CYP2D6*10: the most common CYP2D6 allele among Asians by means of allele-specific polymerase chain reaction. The presence of other mutations (CYP2D6*1, *3, *4, *5, *9 and *17) was also investigated. Tramadol was extracted from 1 ml serum with an n-hexane: ethylacetate combination (4:1) after alkalinisation with ammonia (pH 10.6). Serum concentrations were measured by means of high-performance liquid chromatography. The pharmacokinetics of tramadol was studied during the 24 h after the dose. As among other Asians, the allele frequency for CYP2D6*10 among Malaysians was high (0.43). Subjects who were homozygous for CYP2D6*10 had significantly (P=0.046) longer mean serum half-life of tramadol than subjects of the normal or the heterozygous group (Kruskal-Wallis test). When patients were screened for the presence of other alleles, the pharmacokinetic parameter values were better explained. CYP2D6 activity may play a main role in determining tramadol pharmacokinetics. The CYP2D6*10 allele particularly was associated with higher serum levels of tramadol compared with the CYP2D6*1 allele. However, genotyping for CYP2D6*10 alone is not sufficient to explain tramadol disposition.
An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3,500 ng/ml (tramadol) and 50 to 500 ng/ml (M1) with mean recoveries of 94.36 +/- 12.53% and 93.52 +/- 7.88%, respectively. Limit of quantitation (LOQ) was 50 ng/ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra- and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.