An Experimental Design Approach to Selecting the Optimum LC Conditions for the Determination of Local Anaesthetics

Abstract

A sensitive high performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of four local anaesthetics: lidocaine, proparacaine, bupivacaine and oxybuprocaine. A full factorial design was used. The chromatographic separation was achieved using a Bondesil C₈ (4.6 × 2.5 mm i.d., particle size 5 μm) analytical column. An optimised mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH = 3.0, 20 mM) (30:70, v/v) at a flow rate of 1.2 mL min⁻¹. Local anaesthetics detection was performed by UV-Vis detector at 220 nm. The retention times for lidocaine, proparacaine, bupivacaine and oxybuprocaine were 5.74, 9.28, 16.84 and 26.26 min, respectively. HPLC-UV-Vis method was linear in the range of 50–5,000 ng mL⁻¹ for lidocaine and proparacaine and 100–5,000 ng mL⁻¹ for bupivacaine and oxybuprocaine. The limit of detection (LOD) was 25 ng mL⁻¹ for lidocaine, proparacaine and 30 ng mL⁻¹ for bupivacaine and oxybuprocaine. The limit of quantification (LOQ) was found to be 50 ng mL⁻¹ for lidocaine, proparacaine and 100 ng mL⁻¹ for bupivacaine, oxybuprocaine. In intra-day and inter-day precision and accuracy analysis, the relative standard deviation was found to be less than 8%.