Development and Comparison of Analytical Methods for Sugar Quantitation in Corn-to-ethanol Fermentation

The quantitation of various sugar components is a standard procedure during the corn-to-ethanol fermentation process. As starch undergoes enzymatic digestion by α-amylase and glucoamylase enzymes, the simple saccharides produced are metabolized by yeast for production of ethanol and carbon dioxide. Glucose is the primary fermentable sugar produced during starch digestion, though other sugars produced by the breakdown of starch, such as maltose (DP2) and maltotriose (DP3), are also quantified to monitor the progression of fermentation during flask fermentation. The concentration of the sugar analytes with one analytical technique can be difficult as the concentration of these analytes during the fermentation process range over 3 - 4 orders of magnitude. Standard industry methods of quantifying these sugars include the AOAC-approved (996.11) Megazyme® Total Starch Assay or D-Glucose Assay, which utilizes glucose oxidase and peroxidase enzymes (GOPOD) for the indirect quantitation of glucose by UV-Vis detection. This is a simple-to-use UV-Vis method with specificity for glucose; however, the linearity range for glucose is from 100 - 1000 ppm per the manufacturer recommendation. A commonly-practiced method for sugar quantitation in industry is the technique high-performance liquid chromatography with refractive index detection (HPLC-RID). The sensitivity of the RID is in the mg/L range, and analyte identification is based on retention time comparison of standards of target analytes. With complex matrices from corn-to-ethanol fermentation broth, the retention times of the analytes can vary from that of standards leading to misidentification of analytes and improper quantitation. Liquid chromatography mass spectrometry (LC-MS) is selective and sensitive, allowing for the quantitation of lower concentrations over a broad range of concentrations. Full scan mode was utilized to verify the identities of sugar components in fermentation broth samples as defined by HPLC-RID analysis with retention time comparison. As well, selected ion monitoring (SIM) and multiple reaction monitoring (MRM) methods were developed using two triple quadrupole mass spectrometry instruments and utilized in this research for the quantitation of sugar components in corn-to-ethanol fermentation broth samples with success. Several ionization products were identified for glucose (dextrose), maltose, and maltotriose. The ionization products were identified at specific retention times during the chromatographic analysis. Comparing the retention time of standards to that identified in the fermentation broth sample, there was a maximum shift in retention time of 0.298 minutes, showing that retention time matching for standards and samples is not an efficient technique for analyte identification during analysis. The GOPOD analysis, the HPLC-RID method, and the developed LC-MS methods were validated for the quantitation of the glucose, DP2 and DP3. Three biological flask fermentation replicates were analyzed every twelve hours beginning at 14 hours of fermentation (T14) until 62 hours, near the completion of fermentation (T62). The method validation results for glucose quantitation showed that the LC-MS SIM methods of analysis had the lowest limits of quantitation (LOQ) at 2 ppm and 0.3 ppm, and a calibration range of 2.7 - 3 orders of magnitude. The HPLC-RID analysis had a calibration range of 1.5 orders of magnitude with an LOQ of 1500 ppm. The Megazyme® GOPOD analysis had a linear dynamic range (LDR) of 0.9 orders of magnitude with an LOQ of 120 ppm. The analysis for maltose and maltotriose resulted in calibration ranges of 2.0 - 2.3 orders of magnitude for LC-MS MRM and 1.4 - 1.5 for HPLC-RID. The HPLC-RID method was ideal for glucose quantitation as it is present in high concentrations. In contrast, maltose and maltotriose components were found to be present in lower concentrations, such that simultaneous quantitation of the three analytes is difficult during fermentation. The LC-MS methods were the only methods able to successfully quantify the concentration of glucose, DP2, and DP3 in all of the fermentation broth samples collected at various times throughout the corn-to-ethanol fermentation process simultaneously