Evaluation of the Precision of High-Performance Liquid Chromatography for Wheat Cultivar Identification'

Cereal Chem. 66(2): 112-116 The reproducibility of computer-derived reversed-phase highcomputed peak areas and heights, and percentage peak areas and heights. performance liquid chromatography (RP-HPLC) quantitation parameters Whereas chromatographic resolution was relatively constant over time, following prolonged use of a single commercially available column was prolonged column use significantly retarded peak retention times especially studied. Using a standardized experimental procedure, more than 65 for early eluting components. Results show that without appropriate chromatograms were evaluated based on gliadin extracts from bulknormalization, peak retention times lack sufficient long-term precision in ground meal and composite grinds of four kernels of the bread wheat order to obtain reliable results for cultivar identification and other RPcultivar Neepawa. Statistical results are reported for a set of more than 30 HPLC comparative analysis applications. chromatogram peaks with respect to the precision of retention times, The separation of wheat protein extracts by electrophoretic or chromatographic procedures for computerized wheat cultivar identification and other comparative analysis applications requires precise quantitative parameters to achieve reliable results (Sapirstein and Bushuk 1985a). Previous studies on the use of reversed-phase high-performance liquid chromatography (RPH PLC) for these applications have dealt extensively with optimization of experimental procedures (Bietz et al 1984, Bietz and Cobb 1985, Kruger and Marchylo 1985). These studies sought to improve wheat protein separation. Little attention has been focused on aspects of chromatographic reproducibility, although some reports have addressed short-term effects (Bietz and Cobb 1985, Marchylo et al 1988). This study attempts to evaluate quantitative precision of chromatograms using a standardized experimental procedure, carried out over a two-month period, in order to determine if sufficient long-term analytical precision exists for automated wheat cultivar identification. MATERIALS AND METHODS Materials H PLC grade acetonitrile and ethanol were obtained from Fisher Scientific (Fair Lawn, NJ). Sequanal grade trifluoroacetic acid (TFA) was purchased from Pierce Chemical Co. (Rockford, IL). Water was distilled and then purified with a Millipore Milli-Q system (Mississauga, ON). A certified seed sample of Canadian hard red spring wheat cultivar Neepawa was used for this study. The authenticity and homogeneity of the grain was verified by polyacrylamide gel electrophoresis (Sapirstein and Bushuk 1985a). Apparatus HPLC analyses were performed on a Hewlett-Packard 1090M liquid chromatograph incorporating a DR5 two-solvent reservoir delivery system, auto-injector, auto-sampler, heated column compartment, and diode array detector. Analytical control and quantitation were provided by an HP-310 computer running HP 79994A software for the analytical workstation and HP 79995A operating software. A 55 Mbyte hard-disk was used for system backup and data storage. A Supelcosil LC-308 reversedphase column (5 y m silica particle size, 300A pore size, C8 bonded phase, 25 cm X 4.6 mm i.d.) was used with a Supelguard LC-308 5 cm X 4.6 mm i.d. guard column of the same packing material (Supelco Canada Ltd., Oakville, ON). Peak retention times and 'Presented in part at the AACC 72nd Annual Meeting, Nashville, TN, November 1987. Publication no. 133, Food Science Department. 2Food Science Department, University of Manitoba, Winnipeg, MB, Canada R3T