Profiling phenolic glycosides in Populus deltoides and Populus grandidentata by leaf spray ionization tandem mass spectrometry† Dalton T. Snyder, a M. Christina Schilling, ab Cris G. Hochwender bc and Arlen D. Kaufman * ab Phenolic glycosides (PGs) are phytochemicals known to be present at up to 30% of dry plant mass in the Salicaceae family. These compounds are secondary metabolites that play an important role in the interactions between plants and herbivores by functioning as defense chemicals. The use of leaf spray as an ambient ionization source, along with tandem mass spectrometry (MS/MS), is described here as a useful tool for qualitatively determining the presence or absence of a variety of PGs in Populus deltoides (eastern cottonwood) and Populus grandidentata (bigtooth aspen). Sensitivity and selectivity were enhanced by the addition of sodium or potassium ions to the spray solvent. Introduction Leaf spray, 1 a derivative of paper spray, 2,3 is an ambient ioni- zation technique for analysis of leaves and other plant tissues. The method is procedurally similar to paper spray, where solvent and high voltage are applied to a triangular piece of paper (leaf tissue in the case of leaf spray), resulting in a spray of highly charged droplets containing analyte ions. Aer des- olvation, the analyte ions can be separated, fragmented, and detected by a mass spectrometer. Leaf spray ionization requires virtually no sample preparation and can be used to investigate the chemical prole of leaves and other plant tissues in vivo with high throughput and minimal sample disruption. Previously, leaf spray has been used to quickly detect poison ivy allergens in situ. 4 In addition, leaf spray has been used to investigate Stevia leaves in order to identify their primary glycosides and semiquantitatively determine their relative concentrations in leaves of different origins. 5 Oxidation prod- ucts of tulsi leaf extracts due to aging have been detected in vivo by leaf spray mass spectrometry. 6 Herein, the application of leaf spray to the study of phenolic glycosides is presented. Phenolic glycosides (PGs) are phytochemicals known to be produced in the Salicaceae. These secondary compounds function as defense chemicals against herbivores, including both insects and mammals. 7–18 Furthermore, they are found in high concentration among many species, being reported at up to 30% of dry plant mass in the case of Populus tremuloides. 8 In particular, the hydroxycyclohexen-on-oyl (HCH) functional group, present in salicortin, tremulacin, HCH salicortin, and lasiandrin, has been shown to confer considerable toxicity against herbivores. 18 However, some specialist herbivores, including the native willow beetle, preferentially feed on PG- rich tissue in order to obtain PGs, which they chemically cleave and alter to salicylaldehyde to be used as a deterrent against generalist predators. 16,17 Thus, PGs mediate multiple trophic interactions (plant-herbivore and herbivore-predator), and, as a result, PGs are of great ecological interest. Investigation of PGs by current methodology requires several cumbersome and time-consuming steps. During this process, inappropriate handling of the samples can result in degrada- tion of salicortin and other PG esters to salicin via hydrolysis, so care must be taken to preserve the leaves prior to and during analysis. 19 First, the leaves must be dried, either by vacuum- drying, air-drying, or freeze-drying. Next, a solvent system (usually methanol) is used to extract the PGs from the homog- enized leaves. This step must take place in a cold environment to minimize sample degradation. Subsequent analysis can then be performed by chromatographic separation with an appro- priate form of detection (mass spectrometry, molecular absor- bance spectroscopy, etc.). While adequate, this approach suffers from low sample throughput, and the potential for sample degradation and other errors is increased. In the present study, leaves from Populus deltoides (eastern cottonwood) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the PGs in the species. Subsequently, leaf spray ionization tandem mass spectrometry was shown to be an effective high-throughput method for determining which PGs were present in Populus deltoides and Populus grandidentata (bigtooth aspen). a Department of Chemistry, University of Evansville, Evansville, IN 47722, USA. E-mail: ak2@evansville.edu b Environmental Studies Program, University of Evansville, Evansville, IN 47722, USA c Department of Biology, University of Evansville, Evansville, IN 47722, USA † Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ay02639j Cite this: DOI: 10.1039/c4ay02639j Received 5th November 2014 Accepted 8th December 2014 DOI: 10.1039/c4ay02639j www.rsc.org/methods This journal is © The Royal Society of Chemistry 2015 Anal. Methods Analytical Methods PAPER Published on 17 December 2014. Downloaded by Purdue University on 17/12/2014 12:49:51. View Article Online View Journal