Identification and quantitation of potential fermentation inhibitors in biomass pretreatment hydrolysates using high performance liquid chromatography in combination with ultraviolet detection and tandem mass spectrometry.
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Sharma, Lekh Nath.
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Conversion of lignocellulosic biomass to ethanol involves pretreatment of biomass materials to increase the accessibility of carbohydrates in lignocellulose for enzymatic hydrolysis prior to ethanol fermentation. The pretreatment hydrolysate contains not only cellulose and fermentable sugars but also a wide variety of degradation products, such as aliphatic and aromatic acids, aromatic aldehydes, and phenols. These degradation products exert an inhibitory effect on downstream microbial processes, reducing the overall efficiency for bioconversion of lignocellulosic materials to ethanol. Therefore, development of a reliable quantitative analysis method for individual degradation products is critical in order to advance a more fundamental understanding of lignocellulose pretreatment as well as subsequent microbial processes. Various analytical techniques have been applied to analyze degradation products in hydrolysates, such as gas chromatography-mass spectrometry (GC-MS), GC-flame ionization detection (GC-FID), and liquid chromatography (LC) with ultraviolet (UV) or refractive index (RI) detection. Difficulties in derivatizing samples of unknown composition for GC methods and incomplete analyte resolution in LC experiments have caused researchers to typically employ LC methods targeting a limited group of analytes (e.g., a single analyte class) in quantitative work. To address these limitations, we have developed an improved high performance liquid chromatography (HPLC) method utilizing photodiode array (PDA) and tandem mass spectrometry (MS/MS) detection. The novel HPLC-PDA-MS/MS method enables simultaneous identification and quantitation of 40 degradation products in hydrolysate samples in a single, 60-minute run. Because a unique MS/MS transition is monitored for 37 of 40 target analytes, this approach essentially alleviates resolution limitations of our previous HPLC-UV approach. Upon successful development of the method, it was further applied to analyze various biomass pretreatment samples. Even though coeluting components do not interfere with the detection of analytes using LC-ESI-MS/MS, they can significantly influence the ionization efficiency of analytes at the ionization source. Thus, a series of experiments was conducted to evaluate the influence of matrix interference on quantitation of degradation products. A simplified matrix-spiking approach was investigated and validated for the quantitation of degradation products using LC-ESI-MS/MS methods, which eliminates the necessity of employing labor intensive, multipoint standard addition experiments to compensate for matrix interferences during quantitative analysis.