RESEARCH PAPER Computer-aided method for identification of major flavone/flavonol glycosides by high-performance liquid chromatography–diode array detection–tandem mass spectrometry (HPLC–DAD–MS/MS) Zhengfang Wang & Longze Lin & James M. Harnly & Peter de B. Harrington & Pei Chen Received: 12 June 2014 /Revised: 4 September 2014 /Accepted: 12 September 2014 # Springer-Verlag Berlin Heidelberg (outside the USA) 2014 Abstract A new computational tool is proposed here for tentatively identifying major (UV quantifiable) flavone/ flavonol glycoside peaks of high performance liquid chro- matogram (HPLC)–diode array detection (DAD)–tandem mass spectrometry (MS/MS) profiles based on a MATLAB- based script implementing an in-house algorithm. The HPLC– DAD–MS/MS profiles of red onion, Chinese lettuce, carrot leaf, and celery seed extracts were analyzed by the proposed computer-aided screening method for identifying possible flavone/flavonol glycoside peaks from the HPLC–UV and MS total ion current (TIC) chromatograms. The number of identified flavone/flavonol glycoside peaks of the HPLC–UV chromatograms is four, four, six, and nine for red onion, Chinese lettuce, carrot leaf, and celery seed, respectively. These results have been validated by human experts. For the batch processing of nine HPLC–DAD–MS/MS profiles of celery seed extract, the entire script execution time was within 15 s while manual calculation of only one HPLC–DAD–MS/ MS profile by a flavonoid expert could take hours. Therefore, this MATLAB-based screening method is able to facilitate the HPLC–DAD–MS/MS analysis of flavone/flavonol glycosides in plants to a large extent. Keywords Flavonol glycoside . Flavone glycoside . HPLC–UV . HPLC–MS . HPLC–MS/MS . MATLAB Introduction Flavonoids are the most universal and diverse secondary metabolites in plants [1]. They function as plant pigments for flower coloration and are involved in ultraviolet (UV) filtration [2]. In the human diet, the flavonoids in food plants may also help reduce the risk of cancer and cardiovascular diseases [3]. Because of the importance of flavonoids to plants and humans, the identification, structural determination, and quantitation of these compounds have become a hot topic in food and plant sciences. However, there are at least eight major subclasses of flavo- noids, which vary from positions of the functional groups to structural arrangement [4]. These eight subclasses include more than 5,000 chemically distinguishable compounds [4]. The large number of flavonoids, their structural diversity, and the unavailability of certified reference materials present a great challenge to the identification and quantification of flavonoids. Recently, high-performance liquid chromatography (HPLC)–diode array detection (DAD)–tandem mass spec- trometry (MS/MS) has become a standard approach for ana- lyzing flavonoids in plants [4]. The negative ion mode is usually used because it is more selective and sensitive for the analysis of flavonoids in plants [5]. A well-developed HPLC–MS/MS method could potentially achieve physical separation and tentative identification of almost all the DAD detectable flavonoids in a complex plant extract (i.e., DAD profiles provide additional information for peak identifica- tion). Although mass spectrometry cannot differentiate Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-8187-8) contains supplementary material, which is available to authorized users. L. Lin : J. M. Harnly : P. Chen (*) Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Services, Department of Agriculture, Beltsville, MD 20705-2350, USA e-mail: pei.chen@ars.usda.gov Z. Wang : P. d. B. Harrington Center for Intelligent Chemical Instrumentation, Clippinger Laboratories, Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701-2979, USA Anal Bioanal Chem DOI 10.1007/s00216-014-8187-8