Limits...
Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectrometry.

Ibáñez AB, Bauer S - Biotechnol Biofuels (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Energy Biosciences Institute, University of California, Berkeley, CA 94720 USA.

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Extracted negative ion chromatograms for the deprotonated organic acids [M - H]-based on the theoretical mass-to-charge ratio used for detection and quantification. A standard mixture comprising all 24 organic acids was used. Therefore, extracted ion chromatograms show double peaks for the isobaric pair glucuronic/galacturonic acid and methylmalonic/succinic acid. For these pairs, glucuronic acid and methylmalonic acid eluted before their isobaric counterpart, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4197226&req=5

Fig1: Extracted negative ion chromatograms for the deprotonated organic acids [M - H]-based on the theoretical mass-to-charge ratio used for detection and quantification. A standard mixture comprising all 24 organic acids was used. Therefore, extracted ion chromatograms show double peaks for the isobaric pair glucuronic/galacturonic acid and methylmalonic/succinic acid. For these pairs, glucuronic acid and methylmalonic acid eluted before their isobaric counterpart, respectively.

Mentions: A commercially available ion exclusion column packed with a cation exchange resin based on a polystyrene-divinylbenzene polymer was used for the analysis. This column type has excellent stability at acidic pH and, in our experience, results in very reproducible retention times which are almost unaffected by other matrix components. Since the widely employed non-volatile eluent modifier sulfuric acid is not compatible with mass spectrometry detection, the volatile formic acid was used at a concentration of 0.5% (v/v) [34]. Acetic acid was also used in other works without any advantages in sensitivity in one study [34], although with enhanced sensitivity in another [38]. Lowering the formic acid concentration did not significantly change the retention times, but it can lead to a lower background signal and higher sensitivity [34]. The 0.5% formic acid was kept to ensure appropriate acidity in order to keep the organic acids in their non-ionized state for chromatography. The flow rate of 0.3 mL/min was chosen based on a reasonable compromise of sensitivity and time. A lower flow rate did not result in a better chromatographic separation of the analytes, but it extended the analysis time (data not shown). The mass spectrometer source parameters were varied in the range of 285 to 385°C for the source temperature, 75 to 175 V for the fragmentor voltage, and 3,000 to 4,000 V for the capillary voltage. Despite the aqueous mobile phase, a lower source temperature (285°C), combined with a low fragmentor (75 V) and capillary (3,000 V) voltage, was the best compromise for the detection of the organic acids under study. These settings provided optimum conditions for a larger number of organic acids compared to other settings. As it can be seen in Table 1, this was optimum for 8 acids, and 12 other acids had at least >80% response signal with these source parameters compared to their optimum settings (data not shown). For the remaining 4 acids, the responses were still in the range of 72 to 77%. The negative ion mode resulted in a more intense signal compared to the positive mode, except for acetic and propionic acid (both omitted for the purpose of this study). The internal mass reference ions used during the analysis resulted in a stable mass axis calibration, enabling the measured ions to be kept within the 2 ppm mass accuracy specified by the instrument manufacturer. Figure 1 shows the extracted ion chromatograms (EICs) of a standard mixture of the 24 organic acids analyzed and their theoretical mass-to-charge ratio used for ion extraction. The organic acids selected were chosen based on previous and our own findings in hydrolysate [10,11]. These organic acids were eluted within a narrow retention time window in a comparably short time (<12 min), as observed previously [33]. Good peak separation was achieved based on the combination of chromatographic retention time and accurate mass differences. Exceptions were the two pairs of isobaric compounds glucuronic/galacturonic acid and methylmalonic/succinic acid, which could only be distinguished by their retention time. Although no baseline separation was achieved for the pair glucuronic/galacturonic acid, the results obtained were considered satisfactory. However, the pair methylmalonic/succinic acid was almost baseline separated.Table 1


Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectrometry.

Ibáñez AB, Bauer S - Biotechnol Biofuels (2014)

Extracted negative ion chromatograms for the deprotonated organic acids [M - H]-based on the theoretical mass-to-charge ratio used for detection and quantification. A standard mixture comprising all 24 organic acids was used. Therefore, extracted ion chromatograms show double peaks for the isobaric pair glucuronic/galacturonic acid and methylmalonic/succinic acid. For these pairs, glucuronic acid and methylmalonic acid eluted before their isobaric counterpart, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197226&req=5

Fig1: Extracted negative ion chromatograms for the deprotonated organic acids [M - H]-based on the theoretical mass-to-charge ratio used for detection and quantification. A standard mixture comprising all 24 organic acids was used. Therefore, extracted ion chromatograms show double peaks for the isobaric pair glucuronic/galacturonic acid and methylmalonic/succinic acid. For these pairs, glucuronic acid and methylmalonic acid eluted before their isobaric counterpart, respectively.
Mentions: A commercially available ion exclusion column packed with a cation exchange resin based on a polystyrene-divinylbenzene polymer was used for the analysis. This column type has excellent stability at acidic pH and, in our experience, results in very reproducible retention times which are almost unaffected by other matrix components. Since the widely employed non-volatile eluent modifier sulfuric acid is not compatible with mass spectrometry detection, the volatile formic acid was used at a concentration of 0.5% (v/v) [34]. Acetic acid was also used in other works without any advantages in sensitivity in one study [34], although with enhanced sensitivity in another [38]. Lowering the formic acid concentration did not significantly change the retention times, but it can lead to a lower background signal and higher sensitivity [34]. The 0.5% formic acid was kept to ensure appropriate acidity in order to keep the organic acids in their non-ionized state for chromatography. The flow rate of 0.3 mL/min was chosen based on a reasonable compromise of sensitivity and time. A lower flow rate did not result in a better chromatographic separation of the analytes, but it extended the analysis time (data not shown). The mass spectrometer source parameters were varied in the range of 285 to 385°C for the source temperature, 75 to 175 V for the fragmentor voltage, and 3,000 to 4,000 V for the capillary voltage. Despite the aqueous mobile phase, a lower source temperature (285°C), combined with a low fragmentor (75 V) and capillary (3,000 V) voltage, was the best compromise for the detection of the organic acids under study. These settings provided optimum conditions for a larger number of organic acids compared to other settings. As it can be seen in Table 1, this was optimum for 8 acids, and 12 other acids had at least >80% response signal with these source parameters compared to their optimum settings (data not shown). For the remaining 4 acids, the responses were still in the range of 72 to 77%. The negative ion mode resulted in a more intense signal compared to the positive mode, except for acetic and propionic acid (both omitted for the purpose of this study). The internal mass reference ions used during the analysis resulted in a stable mass axis calibration, enabling the measured ions to be kept within the 2 ppm mass accuracy specified by the instrument manufacturer. Figure 1 shows the extracted ion chromatograms (EICs) of a standard mixture of the 24 organic acids analyzed and their theoretical mass-to-charge ratio used for ion extraction. The organic acids selected were chosen based on previous and our own findings in hydrolysate [10,11]. These organic acids were eluted within a narrow retention time window in a comparably short time (<12 min), as observed previously [33]. Good peak separation was achieved based on the combination of chromatographic retention time and accurate mass differences. Exceptions were the two pairs of isobaric compounds glucuronic/galacturonic acid and methylmalonic/succinic acid, which could only be distinguished by their retention time. Although no baseline separation was achieved for the pair glucuronic/galacturonic acid, the results obtained were considered satisfactory. However, the pair methylmalonic/succinic acid was almost baseline separated.Table 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Energy Biosciences Institute, University of California, Berkeley, CA 94720 USA.

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus