Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectrometry
© Ibáñez and Bauer; licensee BioMed Central Ltd. 2014
Received: 18 July 2014
Accepted: 22 September 2014
Published: 8 October 2014
For the development of lignocellulosic biofuels a common strategy to release hemicellulosic sugars and enhance the enzymatic digestibility of cellulose is the heat pretreatment of biomass with dilute acid. During this process, fermentation inhibitors such as 5-hydroxymethylfurfural, furfural, phenolics, and organic acids are formed and released into the so-called hydrolysate. The phenolic inhibitors have been studied fairly extensively, but fewer studies have focused on the analysis of the organic acids profile. For this purpose, a simple and fast liquid chromatography/mass spectrometry (LC/MS) method for the analysis of organic acids in the hydrolysate has been developed using an ion exchange column based on a polystyrene-divinylbenzene polymer frequently used in biofuel research. The application of the LC/MS method to a hydrolysate from Miscanthus has been evaluated.
The presented LC/MS method involving only simple sample preparation (filtration and dilution) and external calibration for the analysis of 24 organic acids present in dilute acid pretreated biomass hydrolysate is fast (12 min) and reasonably sensitive despite the small injection volume of 2 μL used. The lower limit of quantification ranged from 0.2 μg/mL to 2.9 μg/mL and the limit of detection from 0.03 μg/mL to 0.7 μg/mL. Analyte recoveries obtained from a spiked hydrolysate were in the range of 70 to 130% of the theoretical yield, except for glyoxylic acid, malic acid, and malonic acid, which showed a higher response due to signal enhancement. Relative standard deviations for the organic acids ranged from 0.4 to 9.2% (average 3.6%) for the intra-day experiment and from 2.1 to 22.8% (average 8.9%) for the inter-day (three-day) experiment.
We have shown that the analysis of the profile of 24 organic acids present in biomass hydrolysate can be achieved by a simple LC/MS method applying external calibration and minimal sample preparation. The organic acids eluted within only 12 min by isocratic elution, enabling high sample throughput. Repeatability (precision and accuracy) and recovery were sufficiently accurate for most of the organic acids tested, making the method suitable for their fast determination in hydrolysate. We envision that this method can be further expanded to a larger number of organic acids, including phenolic acids such as p-coumaric acid and ferulic acid and other molecules depending on the researchers’ needs.
KeywordsOrganic acids Dilute acid pretreatment Inhibitors Liquid chromatography Mass spectrometry Ion exchange chromatography Biomass
In the quest for renewable and sustainable energy, lignocellulosic biomass, such as herbaceous plants and hardwoods and softwoods, has been shown to be a promising feedstock for the production of second generation biofuels . Lignocellulosic biomass essentially consists of the polysaccharides cellulose and hemicellulose and the aromatic macromolecule lignin. These compounds are present in the plant cell wall as a three-dimensional network giving the plant structure, stability, and resistance.
Pretreatment of the biomass is necessary in order to overcome this recalcitrance and facilitate degradation of polymeric structures -. In particular, the pretreatment methods aim to improve the conversion efficiency of the plant cell wall polysaccharides into fermentable monosaccharides by reducing the cellulose crystallinity or by simply splitting the carbohydrates and lignin for separate downstream processing technologies.
Various pretreatment methods have been developed for this purpose, comprising alkaline, acidic, or oxidative conditions (for a review see -). Dilute acid pretreatment is the most common pretreatment method and results in an almost complete solubilization of hemicellulose and a high enzymatic digestibility of the cellulose in the pretreated biomass. The acidic conditions and the higher temperature applied during this process also lead to degradation of the released monosaccharides and the lignin polymer . These degradation products comprise compounds such as phenolics, furans, and organic acids which are inhibitory to fermenting microorganisms ,.
Whereas the phenolic inhibitors have been studied fairly extensively (see, for example, ,), fewer studies have focused on the analysis of organic acids present in hydrolysate -. The predominant organic acids found in the hydrolysate after dilute acid (and other) pretreatment are acetic acid (released from acetate groups of hemicellulose and lignin) and levulinic and formic acid (both mainly derived from sugar degradation) ,. Besides these, other organic acids are also observed, although in lower concentrations ,. However, these compounds add up to the overall organic acid loading and can even contribute to synergistic toxic effects. A variety of analytical techniques have been developed for the measurement of organic acids, predominantly involving chromatography and capillary electrophoresis ,. Although gas chromatography methods exist ,, liquid chromatography (LC) is the preferred technique, since it does not require derivatization. Many different stationary phases have been tested for this purpose, including reversed-phase ,,,-, normal phase -, and ion exchange -. If available, liquid chromatography coupled to a mass spectrometer results in specific detection of individual organic acids and unambiguous compound confirmation in contrast to refractive index (RI), ultraviolet (UV), or electrochemical detection. This is especially advantageous when the analysis has to be performed on samples with a complex matrix including potentially interfering compounds such as those found in hydrolysates. Only a few studies exist that apply mass spectrometry (MS) and also cover methodical approaches including basic method validation steps for the analysis of organic acids in pretreatment hydrolysates ,,. Chen et al. used reversed-phase chromatography and UV detection for the analysis of both aliphatic and phenolic acids and aldehydes after an organic solvent (methyl tertiary butyl ether) extraction step . The method was further revised by a combination of UV and triple quadrupole MS detection to improve the specificity of the analysis . This method was applied by Du et al. for the measurement of both aliphatic and phenolic acids and aldehydes after a variety of pretreatments and also by Chundawat et al. for the analysis of decomposition products formed by ammonia fiber expansion and dilute acid pretreatments. A single quadrupole MS method for formic acid and acetic acid was reported by Davies et al..
One of the most popular types of liquid chromatography column used in biomass conversion research is a polymer-based matrix of polystyrene-divinylbenzene (for example, BioRad Aminex® HPX-87H, Phenomenex Rezex™-RFQ) ,. This type of column provides good separation of simple sugars (such as glucose and xylose), many organic acids, alcohols (for example, ethanol and n-butanol), and sugar degradation products (such as 5-hydroxymethylfurfural and furfural). It only requires acidified water as the mobile phase, has excellent pH stability, and requires minimal sample preparation. For mass spectrometry coupling, the commonly used sulfuric acid is replaced with the volatile formic or acetic acid ,. With this setup, organic acids have been analyzed -,,, but its application to the analysis of organic acids in hydrolysate has had only very limited study . We therefore evaluated the applicability of measuring organic acids in hydrolysate without any extraction or derivatization steps or the use of internal standards by applying a simple isocratic elution and time-of-flight mass spectrometry detection for high mass-accuracy compound confirmation.
Results and discussion
Relative signal response of the organic acids with the method source parameters chosen (gas temperature 285°C, fragmentor 75 V, and capillary 3,000 V) relative to optimum conditions determined for each organic acid
Relative signal response with method source parameters chosen compared to optimum condition [%]
Calibration range, limit of detection, limit of quantification
Calibration results for each organic acid
y =8187.5x - 7954.7
y =21093x - 5565.6
y =84067x - 16233
y =14419x +1094.2
y =20732x +6991.4
y =15778x +7460.2
y =12550x +6863.1
y =14707x +10512
y =26542x +1315
y =4295.9x +1770.2
y =32279x - 2606.8
y =29560x - 17152
y =12279x +5867.4
y =34174x - 869.82
y =26782x +4284.6
y =18633x +16302
y =23633x +25347
y =33879x - 2761.6
y =34426x - 6184.7
y =31579x +19428
y =79616x +936.42
y =29753x - 3138.7
y =4245.6x +11213
y =4437.7x +2071.3
For some acids, linearity was achieved only in a small range. That was the case for oxalic acid (2.9 to 28.7 μg/mL), glyoxylic acid (1.1 to 19.5 μg/mL), and 2-furoic acid (2.3 to 45 μg/ml) acid. A wider calibration range can be achieved by applying a quadratic calibration equation (data not shown and not further pursued for the purpose of this study). This is an accepted strategy as long as sufficient calibration points are used throughout the measurement range .
Analytical performance characteristics and method application
The evaluation of the method was performed using a dilute acid pretreated biomass hydrolysate containing a complex mixture of innumerable compounds ,. Co-eluting compounds can potentially cause signal suppression or enhancement  and influence the detection and quantification of the organic acids. It is known that dilute acid hydrolysate in general is rich, for example, in monosaccharides and their degradation products as well as acetic acid; these compounds can exceed the concentrations of the other organic acids by a factor of up to 1,000. Their concentrations in the present hydrolysate were 51 mg/mL xylose, 23 mg/mL glucose, 5.8 mg/mL arabinose, 0.9 mg/mL 5-hydroxymethylfurfural (5-HMF), 2.2 mg/mL furfural, and 9.8 mg/mL acetic acid. Whereas the monosaccharides (3.2 to 4.4 min), acetic acid (5.7 min), and 5-HMF (11.5 min) eluted within the 12 min suggested run time, furfural (16.7 min) and potentially other compounds eluted later. Since the method uses isocratic elution applying only one solvent and does not involve any column cleaning steps, later eluting compounds will elute during the next (or later) injection. This is an important fact to consider, because analysis is not only performed on one sample alone but rather on a set of samples that are injected sequentially. Therefore, it was more appropriate to perform a method of evaluation comprising repeatability and recovery/precision using a real hydrolysate matrix. The absolute percentage difference of the values obtained from analyzing the hydrolysate by using a 20 min isocratic LC method (ensuring furfural elution before the next injection) varied from -2.6% to 4.3% compared to the 12 min isocratic LC method (data not shown). Therefore, the longer run time did not improve the accuracy of the results or imply that later eluting compounds did not interfere with the organic acid quantification in the following run when using only a 12-min run time.
Recoveries obtained after spiking 1:10 diluted (1:100 for glucuronic acid, galacturonic acid and glyoxylic acid) hydrolysate with 1 ppm, 5 ppm and 10 ppm
Recovery 1 μg/mL (%)
Recovery 5 μg/mL (%)
Recovery 10 μg/mL (%)
Average concentration of organic acids detected in dilute acid pretreated Miscanthus hydrolysate and relative standard deviation (RSD) of intra-day and inter-day repeatability
mean (μg/ mL)
mean (μg/ mL)
We have shown that the analysis of the profile of 24 organic acids present in dilute acid pretreated biomass hydrolysate can be achieved by a simple LC/MS method applying external calibration and minimal sample preparation comprising only filtration and dilution. Note also that the present method profiles a larger number of non-phenolic acids in the pretreatment hydrolysate than previous studies -,. The 24 organic acids were eluted within only 12 min by isocratic elution, enabling high sample throughput. Repeatability and recovery were sufficiently accurate for most of the organic acids tested, making the method suitable for the fast determination of organic acids in hydrolysate. We envision that this method can be further expanded to a larger number of organic acids including phenolic acids, such as p-coumaric acid and ferulic acid, and other molecules depending on the researchers’ needs.
Methods and materials
LC/MS grade formic acid and water were obtained from Fisher Scientific (Pittsburgh, PA). Organic acids, all 99 +%, were purchased from Sigma-Aldrich (St. Louis, MO).
Hydrolysate was obtained from the National Renewable Energy Laboratory (NREL). Pretreatment conditions were: Miscanthus (around 1 inch size) was incubated with 1.5% (w/w) sulfuric acid at a 25% biomass loading (w/w) at 190°C for approximately 1 min, then the pressure was rapidly released. The liquid phase after filtration is referred to as “hydrolysate”.
Liquid chromatography/mass spectrometry
Compounds were analyzed using a 1200 Series liquid chromatography system (Agilent Technologies, Santa Clara, CA) coupled to a 6520 Accurate-Mass Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with a dual-spray electrospray ionization source. 2 μL aliquots of the diluted samples were injected onto a Phenomenex (Torrance, CA) Rezex™ ROA-Organic Acid H + (8%) (150 mm × 4.6 mm) column equipped with a Phenomenex (Torrance, CA) Carbo-H+ (4 mm × 3 mm) guard column. The compounds were eluted at 55°C with an isocratic flow rate of 0.3 mL/min of 0.5% (v/v) formic acid in water (132.5 mM formic acid in water). The negative ion mode mass spectrometry conditions were: gas temperature =285°C, fragmentor =75 V and capillary =3,000 V, scan range m/z 50 to 1100, 1 scan/s. Internal mass reference ions m/z 112.9856 and m/z 1033.9881 were used to keep the mass axis calibration stable during the analysis.
Sample preparation and analysis
A calibration mixture containing all 24 organic acids studied was prepared in 0.5% formic acid in water at approximately 100 μg/mL of each acid (200 μg/mL for levulinic acid). To determine the linear calibration range, limit of quantification and limit of detection, the calibration solution was serially diluted to 0.01 μg/mL and each concentration level was analyzed five times. The hydrolysate sample was filtered, and 100 μL were diluted with 900 μL 0.5% formic acid in water (100 μL of this dilution were further diluted with 900 μL 0.5% formic acid in water for the determination of glucuronic, galacturonic, and glyoxylic acid). The sample was then analyzed three times with and without spiking of a known standard mixture concentration and run for 12 min in order to determine analyte recovery in the presence of matrix compounds (signal suppression or enhancement). The recovery of the standard spike was calculated as ([measured amount of analyte in spiked hydrolysate] - [measured amount of analyte in unspiked hydrolysate])/[amount of analyte spiked in] × 100%.
For intra-day/inter-day comparison of repeatability, the hydrolysate sample was analyzed three times each on day one and additionally on three different days afterwards. Since trans-aconitic acid, glutaric acid, fumaric acid, 2-hydroxy-2-methylbutyric aid, and adipic acid were below the limit of quantification, the hydrolysate was spiked with about 10 ppm of these compounds.
The extracted ion chromatograms for the individual mass-to-charge ratios were integrated using MassHunter Quantitative Analysis software version B.05.00 (Agilent Technologies). Gaussian peak smoothing was applied with a smoothing function width of 15 and a Gaussian smoothing width of 5.
SB is the academic responsible for funding and supervising the research in addition to coordinating the experimental design and the data analysis and drafting the manuscript. ABI performed the experimental setup and data analysis and drafted the manuscript. Both authors read and approved the final manuscript.
extracted ion chromatogram
liquid chromatography coupled to mass spectrometry
lower limit of quantification
limit of detection
tandem mass spectrometry
relative standard deviation
upper limit of quantification
This work was funded by the Energy Biosciences Institute. Publication made possible by support from the Berkeley Research Impact Initative (BRII) sponsored by the UC Berkeley Library. The hydrolysate was provided by the National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden, CO 80401, a national laboratory of the U.S. Department of Energy managed by the Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy under Contract Number DE-AC36-08GO28308.
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