PK sampling and analytical methods

For the single‐dose part of the study, blood samples for PK measurements were collected prior to drug administration and at the following times postdose: 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h. For the multiple‐dose part, samples were also collected: (i) within 5 min before the third (day 2), fifth (day 3), seventh (day 4), ninth (day 5), 11th (day 6), 13th (day 7) and 14th (day 7) drug administrations; and (ii) within 5 min before and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 48 and 72 h following the 15th (day 8) administration.

Blood samples were collected and centrifuged (1500 g for 10 min at 4°C) to obtain plasma, which was separated into duplicate tubes and frozen until assayed. Samples from all subjects who received at least one study treatment were assayed. Plasma concentrations of tramadol, M1 and celecoxib were measured using validated high‐performance liquid chromatography with tandem mass spectrometry methods. Sample pretreatment involved the solid phase extraction of tramadol, M1 and celecoxib and their internal standards (propranolol and E‐6087, respectively) from 0.050 ml of human plasma. These compounds were identified and quantified over a theoretical concentration range of 4.00–640.00 ng ml⁻¹ for tramadol, 1.00–160.000 ng ml⁻¹ for M1 and 2.50–1000.00 ng ml⁻¹ for celecoxib. Assay inter‐run precision (coefficient of variation) and accuracy (nominal values) were 8.3% and 102.5%, respectively, for tramadol; 10.1% and 105.2% for M1; and 10.5% and 107.8% for celecoxib. Assay specificity was assessed by employing six independent sources of matrix and verifying for the absence of interference, compared with the respective limit of quantifications at the retention times and mass transitions of analytes and internal standards. Quantitation was carried out using peak area ratios, and back‐calculated concentrations were determined using least squares regression analysis employing a weighted (1/x²) linear regression.