Development and validation of a gradient reversed-phase high-performance liquid chromatographic assay for...

A gradient reversed-phase HPLC method for potency determination of N-0923 (10 mg) from a transdermal delivery system (TDS), was developed and validated with single point calibration using internal standard quantitation. N-0923 and the internal standard, N-0434, are eluted from a reversed-phase C18 column using a gradient which contains 0.1 M triethylamine-0.04 M citrate buffer, pH 5.9, water, and acetonitrile with UV detection at 272 nm. N-0923 is isolated from the transdermal delivery system by extraction with n-heptane followed by extraction of the resulting organic phase with 0.1 M citric acid containing the internal standard. The method was free from matrix interferences in both untreated and forced degraded placebo delivery systems. Acceptable linearity and quantitative recovery from spiked placebo delivery systems over the range 50-150% of nominal label claim were demonstrated. Within-day assay precision from individual samples of active transdermal delivery systems (n = 10) was 5.6% R.S.D. The detection limit was at least 0.1 microgram/ml which is equivalent to 0.05% of the working standard concentration. Replicate injection precision at this level was 0.08% R.S.D. (n = 4). Analysis of thermally stressed active and placebo delivery systems with this HPLC method and photodiode-array detection showed that the chromatography was stability-indicating as demonstrated by the absence of measurable interferences from principal degradation products of either the n-0923 or the delivery system excipients.