Development of high performance liquid chromatography method for analysis of tetracaine for a tape stripping pharmacokinetic study: a comparison with capillary zone electrophoresis (CE)

Faisal Al-Otaibi1, Essam Ghazaly1, Arthure Tucker1,3, David Perrett2, Atholl Johnston1. 1Clinical Pharmacology, William Harvey research Institute Barts & The London, School of Medicine and Dentistry, London, United Kingdom, 2BioAnalysis, William Harvey Research Institute Barts & The London, School of Medicine and Dentistry, London, United Kingdom, 3Clinical Vascular & Microvascular Unit, Dept of Clinical Physics, St Bartholomew’s Hospital, West Smithfield, London, United Kingdom.

Topically applied tetracaine is a local anaesthetic used for numb skin prior to venipuncture. A reversed phase high performance liquid chromatography (HPLC) method for the analysis of tetracaine was compared with a short end direction (reversed voltage) format capillary zone electrophoresis (CE) method1. Both were developed and validated for the separation and quantification of tetracaine in skin samples removed by “tape stripping” as a part of pharmacokinetic study.

The extraction of tetracaine from tape samples was achieved using 100% methanol, which was then diluted to 50% with water before injection. Tetracaine and internal standard, procaine, were separated, on a reverse phase Luna PFP(2), 3μm, 150mm × 4.6mm column at ambient temperature using isocratic elution with KH2PO4 buffer (pH 2.5) and methanol (35:65,v:v). The flow rate was set at 1mL/min, with UV detection at 312nm. The CE separation was performed in 485mm (400mm to window) × 50μm internal diameter fused silica capillary using a background electrolyte of 0.1M phosphoric acid–Tris at pH 2.5 maintained at -25kV with UV detection at 312nm on a HP3D CE system. Injection was hydrodynamic at -500mBar.sec.

The migration/retention time of procaine and tetracaine was 1.25 and 1.36min, respectively for CE, and 2.6 and 3.8min, for HPLC. The limit of quantification for tetracaine for CE and HPLC was 0.5 and 0.3μg, respectively (signal to noise ratio greater than 3). The calibration lines for CE and HPLC were linear for 25, 50, 200, 400, 600, 800, 1000, 1200μg and 0.3, 1, 3, 10, 30, 100, 300,1000μg, respectively and both had r2 values greater than 0.99. The CV% for both within and between assay imprecision and the percentage of inaccuracy for the quality control samples including lower and upper limits of quantitation for HPLC were < 6% and < 10%, respectively, and for CE were < 12.1% and < 11%, respectively. The absolute mean recovery of tetracaine in HPLC was > 92%, and in CE was > 97%. Compared to CE, the mean percentage error and the absolute mean percentage error was 0.62 and 6.29. The methods have been applied in a number of pharmacokinetic studies.

The accuracy and selectivity of the two methods allowed the measurement of tetracaine in samples obtained from a skin tape stripping study in healthy subjects. Although the imprecision is higher, the lower cost and volume of electrolyte to use and faster running time make CE preferable over HPLC in this kind of study.

1. Al-otaibi, F., Tucker, A. T., Johnston, A. & Perrett, D. 2009. Rapid analysis of tetracaine for a tape stripping pharmacokinetic study using short-end capillary electrophoresis. Biomed Chromatogr, 23, 488-91.