INTRODUCTION: Hieracium pilosella L. is a medicinal plant encountered in Europe in traditional herbal medicinal products. Caffeoylquinic (ortho-dihydroxycinnamic) acid derivatives are characteristic constituents used as analytical markers in the quality control of the herbal material. Until now, the caffeoylquinic acid derivatives have been assayed using a colorimetric method according to the French Pharmacopoeia. OBJECTIVE: To develop an HPLC-method for quantification of caffeoylquinic acid derivatives in H. pilosella. METHODOLOGY: Samples were prepared by methanol extraction of H. pilosella, dried herb. An HPLC method suitable for analysis was developed and validated. The content of caffeoylquinic acid derivatives was determined and expressed as chlorogenic acid. Individual substances in the samples were identified by analyses of UV-MS/MS spectra and by comparisons with spectra and chromatographic retention times of authentic reference substances. RESULTS: Validation showed that the chromatographic method has good selectivity with no interfering peaks. Sensitivity, linearity, repeatability and accuracy were shown to be adequate. In analyses of several batches of H. pilosella, contents of caffeoylquinic acids ranging from 0.7 to 1.9% were found. Compared to the colorimetric method, this newly developed HPLC method is more specific and results in detection of nominally lower amounts of caffeoylquinic acid derivatives. A new acceptance limit of 1.0% instead of 2.5% caffeoylquinic acid, expressed as chlorogenic acid, for H. pilosella, is proposed when using this HPLC-method. CONCLUSION: A newly developed HPLC method is shown to be appropriate for quantitative determination of caffeoylquinic acid derivatives in H. pilosella.
INTRODUCTION:Hieracium pilosella L. is a medicinal plant encountered in Europe in traditional herbal medicinal products. Caffeoylquinic (ortho-dihydroxycinnamic) acid derivatives are characteristic constituents used as analytical markers in the quality control of the herbal material. Until now, the caffeoylquinic acid derivatives have been assayed using a colorimetric method according to the French Pharmacopoeia. OBJECTIVE: To develop an HPLC-method for quantification of caffeoylquinic acid derivatives in H. pilosella. METHODOLOGY: Samples were prepared by methanol extraction of H. pilosella, dried herb. An HPLC method suitable for analysis was developed and validated. The content of caffeoylquinic acid derivatives was determined and expressed as chlorogenic acid. Individual substances in the samples were identified by analyses of UV-MS/MS spectra and by comparisons with spectra and chromatographic retention times of authentic reference substances. RESULTS: Validation showed that the chromatographic method has good selectivity with no interfering peaks. Sensitivity, linearity, repeatability and accuracy were shown to be adequate. In analyses of several batches of H. pilosella, contents of caffeoylquinic acids ranging from 0.7 to 1.9% were found. Compared to the colorimetric method, this newly developed HPLC method is more specific and results in detection of nominally lower amounts of caffeoylquinic acid derivatives. A new acceptance limit of 1.0% instead of 2.5% caffeoylquinic acid, expressed as chlorogenic acid, for H. pilosella, is proposed when using this HPLC-method. CONCLUSION: A newly developed HPLC method is shown to be appropriate for quantitative determination of caffeoylquinic acid derivatives in H. pilosella.