Tramadol is a centrally acting synthetic opioid analgesic and has received special attention due to its abuse potential and unexpected responses induced by CYP2D6 polymorphism. Oral fluid is an advantageous biofluid for drug analysis due to non-invasive sampling and high correlation of drug concentrations with plasma. However, few studies have been performed on distribution of tramadol and its metabolites in oral fluid. In the present study, a linear dual column HPLC-MS/MS method was developed and fully validated for the simultaneous determination of tramadol and its phase I [O-desmethyltramadol (ODMT), N-desmethyltramadol (NDMT) and N,O-didesmethyltramadol (NODMT)] and II metabolites in oral fluid. Furthermore, the distribution of tramadol and its metabolites, in relation to CYP2D6 genetic variations, in oral fluid was investigated following a clinical study including 23 subjects with CYP2D6*wt/*wt, CYP2D6*10/*10 or CYP2D6*5/*5. The validation results of selectivity, matrix effect, linearity, precision and accuracy were satisfactory. Pharmacokinetic parameters, such as C and AUC of tramadol, NDMT and NODMT, in the CYP2D6*10/*10 group were significantly higher than those in the CYP2D6*wt/*wt group. Moreover, the ratios of ODMT/tramadol, NDMT/tramadol and NODMT/NDMT correlated well with the CYP2D6 genotypes. We demonstrated that oral fluid is a promising biofluid for pharmacokinetic evaluation in relation to genetic variations.

The CYP2C19 gene has a high proportion of poor metabolizers (PMs) in Asians, including Koreans, with the CYP2C19*2 and *3 alleles accounting for most of the PMs. CYP2C19*10 is a mutation that differs by 1 bp from CYP2C19*2 and cannot be distinguished by the conventional PCR-RFLP method using SmaI. Thus, the frequency of CYP2C19*10 may appear high in an ethnic group with a high frequency of CYP2C19*2. We classified CYP2C19*2 and *10 using the pyrosequencing method and carried out a study to accurately confirm their frequency. Four hundred and thirty-two healthy Korean subjects were studied. CYP2C19 alleles (CYP2C19*2, *3, *10, and *17) were detected using pyrosequencing assays. The allele frequencies observed here were 63.0% for CYP2C19*1, 26.4% for CYP2C19*2, 9.4% for CYP2C19*3, and 1.3% for CY-P2C19*17. The CYP2C19*10 allele was not detected. The frequencies of the CYP2C19 normal metabolizer, rapid metabolizer, intermediate metabolizer, and PM phenotypes were 40.0%, 1.6%, 45.1%, and 13.2%, respectively. Similar to previous studies, the frequencies of CYP2C19*2 and *3 were relatively high, and the frequency of CYP2C19*17 was low. Finally, in Koreans, the frequency of CYP2C19*10 seems to be extremely low.

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