2.3 LC-MS/MS

The concentrations of naproxen and its major metabolic, 6-O-desmethylnaproxen, were analyzed using an 8040 Triple Quadrupole Mass Spectrometer (Shimadzu, Kyoto, Japan)and piroxicam was used as an IS. Before performing the standard curve for naproxen, 6-O-desmethylnaproxen, esomeprazole, and the IS, the mobile phase was standardized to increase the reliability of the results that will be obtained from saliva samples in the PK studies [7]. As naproxen and esomeprazole ionize in different ways, only the PKs of naproxen and its major metabolite were investigated, as these are the drugs of most interest for pain control.

Initial analysis for drug characterization was performed by LC-MS/MS and separation was performed using a Shim-Pack XR-ODS 75Lx2.0 column and C18 pre-column (Shimadzu, Kyoto, Japan) at 40°C using a mixture of methanol and 10 mM ammonium acetate (70:30, v/v) with an injection flow of 0.3 mL/min. The total analytical run time was 5 min.

The chromatographic effluent was directed to the 8040 Triple Quadrupole Mass Spectrometer (Shimadzu, Kyoto, Japan). Mass spectrometry was performed following the information obtained in the optimization of ions in positive/negative ionization mode using ionizing electrospray, with monitored selection for quantitative analysis. The voltage of the ionization electrospray capillary was 4.5 kV. Source and desolvation temperatures were maintained at 250°C and 350°C, respectively. Nitrogen was used as the mist gas at 3.0 L/min and argon gas as the collision gas at approximately 230 kPa. The cone voltage was defined for each transition and drug molecular ions were fragmented by the variable collision energy. All conditions were achieved by the direct injection of standard drug solutions of naproxen and 6-O-desmethylnaproxen without the separation column, resulting in transitions in which specific father/son ions were employed. The time of analysis for each event was 0.075 ms. The measurements were performed in multiple reaction monitoring mode to quantify the transitions presented in Fig 1. The peak areas for all components were integrated automatically using the LabSolutions software, version 5.97(Shimadzu, Kioto, Japan). The transitions considered for the quantification and qualification of the peaks are shown in Table 1.

^aQuantifier: the most intense peak.

^bQualifier: a second peak (SRM)t has been used as a qualifier for the same analysis.