High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid-liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-microm Nova-Pak C18 column (150x3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2-10 microg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1-50 microg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 microg/l for plasma and vitreous humour using a 1-ml sample and was 1 microg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100-102% for plasma, 91-106% for aqueous humour and 96-111% for vitreous humour. The between-day recovery (n=9) was 90-114% for plasma, 83-115% for aqueous humour and 90-105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7-7.0% and 8.1-13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.