A Method for Determining 4‐Aminopyridine in Plasma: Pharmacokinetics in Anaesthetized Guinea Pigs after Intravenous Administration

An HPLC assay has been developed to measure 4-aminopyridine (4-AP) in guinea pig plasma. For the assay, all plasma samples (50μL) were microfiltered following the addition of an internal standard (3,4-diaminopyridine). Filtrates (10μL) were directly injected into a spherical silica column (100×2.1 mm; 5μm); detection was achieved at 266 nm. Standard curves had correlation coefficients ranging from 0.9923 to 0.9992 and coefficients of variation expressed as a percentage (%CV) of below 8%. Precision was expressed as between-day and within-day variability of five test sample concentrations. Between-day %CV ranged from 4.0 to 6.5%. Within-day %CV ranged from 3.6 to 6.9%. Accuracy was assessed by examining expected within-day test sample concentrations against calculated concentrations; per cent errors were all below 10%. Stability studies demonstrated %CV below 5% after repeated freezing. The method was employed to study the pharmacokinetics of 4-AP after intravenous administration to anaesthetized guinea pigs. Serial blood samples (150μL) were collected at predetermined time intervals up to 4 h post-4-AP (2 mg/kg, i.v.) administration. 4-AP demonstrated a biexponential decline in the plasma-concentration curve as a function of time indicating a two compartment model for this drug. Selected mean pharmacokinetic parameter estimates were α-half-life, 0.37 min; β-half-life (biological half-life) for terminal slope, 109 min; and volume of distribution at steady state, 1036.18 mL/kg. 4-AP was found to rapidly and extensively partition into a peripheral tissue compartment and demonstrated a relatively long biological half-life. The findings from the current pharmacokinetic experiments support the pharmacology of 4-AP in its role for reversing saxitoxin (STX)- and tetrodotoxin (TTX)-induced diaphragmatic failure in terms of onset of action and duration of effect in anaesthetized guinea pigs.