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Abstract: Ruggedness and robustness testing is applied prior to the transfer of a method to various laboratories. It is especially important for official methods that are widely and formally used. Ginsenosides (Rg1, Re, Rf, Rb1, Rg2, Rc and Rd) occurring in Asian and American ginseng, were assayed using high-performance liquid chromatography (HPLC) methods issued by the United States Pharmacopoeia (USP XXVI and XXVII). They have a delicate system suitability to properly execute the method. A robustness test was carried out using an experimental design approach, in order to indicate the suitability of the method in a broad execution range. Seven factors are selected and examined by a fractional factorial design. Twelve responses, involving quantitation-related items, such as recovery of Rg1 and Rb1, and qualitative items, such as resolution (Rs), theoretical plate number (N), relative retention time (RRT) of two critical pairs, are measured. The calculated effects are both graphically and statistically interpreted to identify the significant factors. The robustness tests using fractional factorial design on the assay of ginsenosides provided an effective approach as part of a validation of the method on phytomedicines. It also indicated that methods with different selectivity, of which some are possibly unsuitable, could pass the existing system suitability limits. [Copyright &y& Elsevier]

The work of the ruggedness/robustness evaluation and system suitability tests was oriented to profound understand the practicability of using assay methods issued by United States Pharmacopoeia (USP XXVI and XXVII) for ginsenosides in Asian ginseng and American ginseng. The items chosen for the method validation included quantitative related items such as recovery of Rg(1) and Rb(1), respectively, and qualitative related items such as resolution, theoretical plate number, relative retention time of two critical-band-pairs, Rg(1)/Re and Rb(1) with its neighboring peak, respectively. Totally, 16 column types were used for comparison of different vendors, different packing materials, different size, etc. and five sets of LC systems and two laboratories were involved in comparing the data of both quantitative and qualitative items. The results showed that different packing materials of columns used might significantly alters separation. The column packing material Hypersil afforded the preferable separating for the ginsenosides. No significant difference was observed from the different instrumentations and inter-laboratories. Our results suggest a modification of the system suitability test as given in USP26-NF21 and the latest version of USP27-NF22, which was not suitable for most systems. Using resolutions of Rg(1)/Re and Rb(1) with its neighboring peak as critical parameters for the ginsenosides assay and omitting the relative retention time of both Rg(1)/Re and Rb(1) with its neighboring peak is our suggestion for a more reasonable, yet practicable system suitability. Six typical chromatograms gain from different columns were figured out as well.