LC–Q-TOF-MS/MS determination of darunavir and its metabolites in rat serum and urine: Application to pharmacokinetics

• LC–Q-TOF/MS method was developed for the determination of darunavir metabolites.
• A simple protein precipitation method was used for extraction of serum samples.
• Urine samples were extracted by solid phase extraction without any matrix effect.
• The developed method was successfully applied to pharmacokinetic studies in rats.

A simple, rapid and reliable liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC–Q-TOF-MS/MS) method was developed and validated for simultaneous determination of darunavir and its metabolites in rat serum and urine. The separation was accomplished on an Agilent RP-18 (250 × 4.6 mm, 5 μm) column using 20 mM ammonium acetate and methanol (40:60, v/v) as a mobile phase at a flow rate of 1.0 mL/min in an isocratic mode. The [M+H]+ ions of darunavir (m/z 548) and metabolites-I (m/z 392) were monitored in positive mode of ionization, while [M−H]− ion of metabolite-II (m/z 172) in negative mode selectively. The matrix effects of rat serum and urine were found to be negligible and the recoveries were 87–93% for all the analytes. The short and long term stability of darunavir and its metabolites was within acceptable limits and the lower limits of quantification were in the range of 3.63–5.24 ng/mL with a linear range of 5–5000 ng/mL in rat serum as well as urine. The method exhibited good intra- and inter-day performance in terms of 2.54–8.92% precision and 0–5% accuracy. The method was successfully applied to a single-dose pharmacokinetic study of darunavir boosted with ritonavir in Wistar rats.

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