Abstract
Background: Apple pomace represents a low-cost and rich source of bioactive compounds with valuable properties - ursolic acid (UA) and oleanolic acid (OA). Due to the wide range of applications in pharmaceutical and nutraceutical industries, these compounds have a high commercial value, and possessing a suitable analytical method with measurement uncertainty is of great significance and practicability. Aim: The purpose of the present work was to estimate detailed measurement uncertainty for the validated HPLC method combined with the extraction procedure for the determination of UA and OA in apple pomace. Methods: The chromatographic analysis using LC-20AD Prominence Shimadzu System and ultrasound-assisted extraction using the ultrasonic bath DW-5200DTS were performed to obtain and determine UA and OA in apple pomace. The process of measurement uncertainty evaluation was performed by the Ishikawa diagram and a combination of bottom-up and top-down approaches. Results: The content of UA and OA (mg/g) in apple pomace with the value of the expanded uncertainty was calculated, which is 7.06 ± 0.647 mg/g (k=1.96; P=95%) and 4.70 ± 0.422 mg/g (k=1.96; P=95%), respectively. Six sources of all the contributors of uncertainties were observed that affected the measurement. Discussion: The A-type standard uncertainty value was 3 times less than the B-type standard uncertainty for both analytes. The results show that B-type standard uncertainty is a major contributor, and the value of the expanded uncertainty of the validated method will not change from test to test in the same laboratory conditions. Conclusions: The methodology described in this work explains well the details and practical aspects of the hybrid approach using the method validation data and proposes step-by-step instructions to evaluate the measurement uncertainty of the quantitative method.
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