Highly sensitive UHPLC–MS/MS method for the simultaneous estimation of propafenone and its metabolites 5-hydroxypropafenone and N-depropylpropafenone on human dried blood spots technique and application to a pharmacokinetic study

Abstract A highly sensitive, rapid and selective UHPLC–MS/MS method has been developed and validated for quantification of the propafenone (PF), 5-hydroxypropafenone (5-OHPF) and N -depropylpropafenone ( N -DPF) on human dried blood spot (DBS). The assay procedure involves a solid–liquid extraction of PF, 5-OHPF and N -DPF and amlodipine (internal standard, I.S.) from dried human DBS cards using water and acetonitrile. The chromatographic resolution was achieved on a BEH C 18 column using a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile with 0.1% formic acid at flow rate of 0.6 mL/min. The UHPLC–MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Total run time of analysis was 1.1 min and elution of PF, 5-OHPF, N -DPF and I.S. occurred at 0.69, 0.6, 0.68 and 0.73 min, respectively. A detailed method validation was performed as per the regulatory guidelines and the standard curves found to be linear in the range of 5.11–1000 ng/mL for PF and 5-OHPF and 0.51–100 ng/mL for N -DPF with a correlation coefficient of ≥0.99 for all the drugs. The intra- and inter-day accuracies were in the range of 95.6–107 and 93.5–103; 93.4–106 and 96.3–107 and 87.9–103 and 96.5–102%, for PF, 5-OHPF and N -DPF, respectively. The intra- and inter-day precisions were in the range of 2.50–5.52 and 3.38–5.18; 2.16–6.34 and 3.23–4.94 and 2.63–7.55 and 1.56–10.2%, for PF, 5-OHPF and N -DPF, respectively. The validated assay method was successfully applied to a pharmacokinetic study in humans. The key pharmacokinetic parameters AUC 0-∞ and C max were 6057 ± 1526, 2002 ± 515 and 525 ± 202 ng*h/mL and 653 ± 183, 295 ± 37.5 and 68.4 ± 13.6 ng/mL for PF, 5-OHPF and N -DPF, respectively.