## 1 Introduction
Subsequently, even for relatively simple plant matrices, the use of long chromatographic columns and analysis time of over 15 min, and sometimes over 30 min, are usually required for satisfactory separation [15; 16; 17; 18; 19]. As most of the methods utilize HPLC equipment with ultraviolet (UV) detectors, reduced sensitivity may be also an issue, as triterpenes, in general, exhibit low absorption of ultraviolet light. To address these problems various methods have been applied. Adding of separation facilitating substances, like cyclodext [20; 21], might improve the resolution, as might pre-column derivation [17; 22; 23; 24], which is sometimes used as a solution for low sensitivity. Better limits of detection may be also achieved with detectors other than UV, e.g., evaporative light scattering (ELS) or mass selective (MS) detectors [23; 24; 25; 26]. However, these techniques increase the complication of procedures (additional, time-consuming steps in sample preparation) and  
Figure 1: Molecular structures of unsolic acid (UA) and oleanolic acid (OA). their costs (special chemicals and/or solvents) and, therefore, they might not be suitable for routine, commercial standardisation tests. Over the past decade, there has been tremendous interest in approaches to speed up and increase the resolving power of the chromatographic separation, particularly with the development of columns packed with porous sub-2-\(\upmu\)m particles used in very high pressure conditions (namely UHPLC, for ultra-high performance liquid chromatography). The UHPLC technique is quickly gaining significance in many areas of pharmaceutical industry, including the standardisation of natural products. It takes full advantage of chromatographic principles with superior sensitivity, efficiency and resolution at reduced solvent consumption [27]. Considering these advantages, it might become a suitable alternative to HPLC, especially for analysis of structural isomers. There is, however, only one previous report presenting the use of UHPLC for separation and quantification of OA and UA [28]. Despite the separation of both analytes has been achieved in a relatively short time of 7.2 min, the basic rules of HPLC-to-UPLC method transfer suggest that further improvement might be expected [29]. Optimisation of the (U)HPLC separation is a complex process influenced by numerous factors, such as mobile phase composition, flow rate, column temperature, detector wavelength, etc. The traditional optimisation approach, applied in all previous works for the separation of OA and UA based on trial and errors methodologies, which are hardly efficient, time-consuming, and may not be able to find the optimal conditions [30]. These could be identified by the use of systematic experimental design and statistically-assisted construction of a response function describing relationships between the process variables and their interactions and performance parameters of the chromatographic separation [31]. Thus, the objective of this study was to present the first-time application of multivariate statistical techniques in order to develop a simple, fast, sensitive and fully validated ultra-high performance liquid chromatography-photodiode array (UHPLC-PDA) method suitable for routine standardisation of Ericaceae plant materials in terms of OA and UA content.