Separation, Identification, Quantification, and Method Validation of Anthocyanins in Botanical Supplement Raw Materials by HPLC and HPLC−MS

A method has been established and validated for identification and quantification of individual, as well as total, anthocyanins by HPLC and LC/ES−MS in botanical raw materials used in the herbal supplement industry. The anthocyanins were separated and identified on the basis of their respective M+ (cation) using LC/ES−MS. Separated anthocyanins were individually calculated against one commercially available anthocyanin external standard (cyanidin-3-glucoside chloride) and expressed as its equivalents. Amounts of each anthocyanin calculated as external standard equivalent were then multiplied by a molecular-weight correction factor to afford their specific quantities. Experimental procedures and use of a molecular-weight correction factors are substantiated and validated using Balaton tart cherry and elderberry as templates. Cyanidin-3-glucoside chloride has been widely used in the botanical industry to calculate total anthocyanins. In our studies on tart cherry and elderberry, its use as external standard followed by use of molecular-weight correction factors should provide relatively accurate results for total anthocyanins, because of the presence of cyanidin as their major anthocyanidin backbone. The method proposed here is simple and has a direct sample preparation procedure without any solid-phase extraction. It enables selection and use of commercially available anthocyanins as external standards for quantification of specific anthocyanins in the sample matrix irrespective of their commercial availability as analytical standards. It can be used as a template and applied for similar quantification in several anthocyanin-containing raw materials for routine quality control procedures, thus providing consistency in analytical testing of botanical raw materials used for manufacturing efficacious and true-to-the-label nutritional supplements. Keywords: Anthocyanins; ES−MS; Balaton tart cherry; Prunus cerasus L.; elderberry; Sambucus nigra L.; bilberry; Vaccinium myrtillus L.; chokeberry; Aronia melanocarpa Elliot; HPLC; LC−MS; botanical supplements; raw materials