A pre-column derivatization high-performance liquid chromatography method with diode array detection was developed and validated to determine the total retronecine esters-type hepatotoxic pyrrolizidine alkaloids (RET-HPAs) in herbs. The RET-HPAs reacted with o-chloranil in methanolic solution heated for 3 h, and an oxidative derivative was produced that could be detected at a maximal absorption of 223 nm. The analysis was performed using a C18 column with an isocratic elution of methanol and aqueous 0.01% triethylamine (adjusted to pH 4 with formic acid), and the detection was carried out with DAD at 223 nm. The validation of the method included linearity, sensitivity, recovery and stability. It showed a good linear regression (r(2) > 0.9900) in the range of 2.5-250 microM with a limit of detection (S/N = 3) of 0.5 microM. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.6%. The obtained recoveries for both of the extraction and derivatization process were between 94.6 and 100.7% (n = 3).
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