Food Chemistry 220 (2017) 198–207

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Food Chemistry

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Analytical Methods

Improvement of simultaneous determination of neutralmonosaccharides and uronic acids by gas chromatography

http://dx.doi.org/10.1016/j.foodchem.2016.10.0080308-8146/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: xinlu@nwsuaf.edu.cn (X. Lü).

Xin Wang, Lihui Zhang, Jingli Wu, Weiqi Xu, Xiaoqin Wang, Xin Lü ⇑College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi Province 712100, China

a r t i c l e i n f o

Article history:Received 8 June 2016Received in revised form 27 September2016Accepted 1 October 2016Available online 4 October 2016

Keywords:DerivatizationGas chromatographyMonosaccharide compositionn-PropylaminePolysaccharideNeutral monosaccharideUronic acid

a b s t r a c t

Although pre-column derivatization with n-propylamine and acetic anhydride combined with the gaschromatograph was a useful method for the analysis of monosaccharide composition, failure often occursbecause of lack of detail information on the mechanism as well as the operating key point in derivatiza-tion process. In this study, the key points in the derivatization (lactonization time, the amount of n-propylamine and acetic anhydride) were investigated and optimized to improve the method. Underthe optimal conditions, the derivatives of seven neutral monosaccharides and two uronic acids weresimultaneouly obtained, after which they were well separated and detected by GC. It was also found thatall derivatives of monosaccharides were stable even stored for 20 days. The linearity, sensitivity, preci-sion, reproducibility and recovery rate of the improved method were evaluated. Thereafter, five polysac-charide samples from different sources were analyzed to validate the improved method.

� 2016 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, many polysaccharides were separated, purifiedand characterized from different sources (Li et al., 2015; Lorenz,Erasmy, Akil, & Saake, 2016; Wang et al., 2015; Wang, Zhao, Pu,& Luan, 2016; Zeng, Zhang, Zhang, Cui, & Sun, 2015) for their bio-logical activities and function properties, such as antitumor,antioxidant, anti-inflammatory, immunomodulatory activities(Huang, Li, Wan, Zhang, & Yan, 2015; Mao et al., 2015; Yang &Zhang, 2009). They can also be used in food and pharmaceuticalindustry for the special rheological, thermal and gel properties(Patova, Golovchenko, & Ovodov, 2014; Tao et al., 2015; Weiet al., 2015). Therefore, the structure-activity relationship of thepolysaccharides attracted more and more attention, in whichmonosaccharide composition analysis is one of the most importantbasic steps to investigate the complex structure of polysaccharides.However, there are four challenges in monosaccharide composi-tion analysis: (a). Separation: It is difficult to simultaneously sepa-rate monosaccharides because of their similar structure, andespecially many of them are epimers of each other (such as glu-cose, mannose and galactose) (Zhang, Khan, Nunez, Chess, &Szabo, 2012). (b). Detection: Most monosaccharides lack charge,

UV absorbing and/or fluorophore groups; furthermore, it is alsodifficult to gasify monosaccharide (Guttman, 1997). Consequently,the ultraviolet detector (UV), flame ionization detector (FID) andfluorescence detector (FD) which usually be used in gas chro-matography (GC), high performance liquid chromatography (HPLC)and other chromatography techniques cannot be directly appliedto monosaccharide’s detection. (c). Derivatization of uronic acids:As an important component of acidic polysaccharides, uronic acidwidely exists in nature, and which are much more difficult to bederived than neutral monosaccharides for the more complex andstable structure (Lehrfeld, 1987). Although carbazole-sulphuricacid (Dische & Rothschild, 1967) and meta-hydroxydiphenyl col-orimetric methods (Blumenkrantz & Asboe-Hansen, 1973) can beapplied to analyze uronic acids, the determination is susceptibleto the interference of neutral monosaccharide in quantification.(d). Simultaneously determination: It is indeed hard to obtainand detect the derivatives of neutral monosaccharides and uronicacids simultaneously because of their different structureproperties.

Since the paper chromatography was firstly used to investigatemonosaccharide composition at the 1940s (Partridge, 1946), manymonosaccharide composition analysis methods were developedbased on pre-column derivatization, such as GC/GC–MS(Bonaduce et al., 2007; Cai et al., 2015), HPLC (Zhang et al., 2013)and capillary electrophoresis (CE) (Hu, Wang, Yang, & Zhao,2014), and some of which can simultaneously determine the

X. Wang et al. / Food Chemistry 220 (2017) 198–207 199

neutral monosaccharides and uronic acids. There are also anothermethods developed recently which can investigate monosaccha-rides directly without derivatization, for instances, HPLC-MS(Ghfar et al., 2015), high-performance anion-exchange chromatog-raphy with pulsed amperometric detection (HPAEC-PDA) (Xieet al., 2013), subcritical fluid chromatography (Salvador,Herbreteau, Lafosse, & Dreux, 1997), fourier transform infraredspectroscopy (FTIR) (Plata, Koch, Wechselberger, Herwig, & Lendl,2013), and nuclear magnetic resonance (NMR) (de Souza,Rietkerk, Selin, & Lankhorst, 2013).

Although HPLC-MS, HPAEC-PDA, FTIR and NMR methods do notrequire derivatization process, all of them need special instru-ments or detectors (Que & Novotny, 2003; Zhang et al., 2013).Moreover, the accuracy and precision of FTIR and NMR cannotmeet the qualitative and quantitative requirements of monosac-charide composition analysis (Coimbra, Barros, Barros, Rutledge,& Delgadillo, 1998; de Souza et al., 2013). Therefore, the conven-tional chromatography instruments including HPLC and GC aremore suitable for many researchers to determine the monosaccha-ride composition of polysaccharides. Compared with HPLC, theaccuracy, precision, sensitivity and efficiency of GC analysis are rel-atively higher, which makes GC analysis more popular.

Jacob Lehrfeld firstly developed GC method in 1980s for simulta-neously determining neutral monosaccharides and uronic acids(Lehrfeld, 1987), which has been applied in many studies(Makarova, Patova, Shakhmatov, Kuznetsov, & Ovodov, 2013;Peng et al., 2012; Shakhmatov, Toukach, Michailowa, &Makarova, 2014). In the described method, the monosaccharideswere converted to their corresponding alditol acetates and N-propylaldonamide acetates which can be separated and detectedby GC (Lehrfeld, 1987). It is generally known that the derivatizationprocess is the crucial step for the analysis, however, lack of detailedinformation on the principle, specific instruction and optimal con-ditions of derivatization sometimes lead to the failure of the anal-ysis process. Moreover, the linearity, sensitivity, precision,reproducibility and recovery rate of the method should be supple-mented to our knowledge.

Therefore, three key steps in the derivatization process,including lactonization time, the amount of n-propylamine andacetic anhydride were investigated and optimized in this study.Furthermore, the derivatization was discussed in detail, includingthe chemical transformations undergone by the neutral monosac-charides and uronic acids in each reaction step. The validation ofthe improved method was investigated on the aspects of linear-ity, sensitivity, recovery, precision and reproducibility. At thesame time, the stability of the derivatives of monosaccharideswas also investigated. Further, five polysaccharide samplesobtained from different sources were used to verify the applica-tion of the method.

2. Materials and methods

2.1. Materials and reagents

Standard monosaccharides (L-rhamnose, D-arabinose, L-fucose,

D-xylose, D-mannose, D-glucose, D-galactose, L-glucuronic acid, L-galacturonic acid) were purchased from Sigma Chemical Co. (St.Louis, MO, USA). Sodium borohydride (NaBH4), cation-exchangeresin (Amberlite 732) and trifluoroacetic acid (TFA) were pur-chased from Shanghai Aladdin biochemical technology Co. (Shang-hai, China). Cation-exchange resin was pretreated according to themanufacturer instructions. All other chemicals and solvents wereanalytical grade unless otherwise specified.

The apple pomace pectic polysaccharide (APP-P), GrassleafSweelflag Rhizone polysaccharide (GSR-P), Agrimonia Pilosa Ledeb

polysaccharide (APL-P), lactic acid bacteria exopolysaccharides(EPS) and pretreatment effluent of biomass (PEB-P) were usedto verify the application of the improved method in this paper.APP-P was obtained by the method described in our previousstudy (Wang & Lü, 2014). GSR-P and APL-P were isolated byhot-water extraction and ethanol precipitation. ESP was isolatedfrom the culture medium of Lactobacillus plantarum KX041 bycentrifugation and ethanol precipitation. PEB-P was collectedfrom the pretreatment of switchgrass by subcritical water(200, 0 min). After pretreatment, the extracting solution wasfiltered for the analysis of monosaccharide composition whichindicates the hydrolysis of lignocellulose in the pretreatmentprocess.

2.2. The derivatization procedures of neutral monosaccharides anduronic acids

2.2.1. The process of the derivatization pretreatmentPretreatment of samples and derivatization of monosaccharides

were carried out according to the reported studies (Jacob, 1985;Lehrfeld, 1987; Osborn, Lochey, Mosley, & Read, 1999; Penget al., 2012; Rumpel & Dignac, 2006) with some modifications.The whole process was divided into three parts, which were shownin Fig. 1.

Part 1: Hydrolysis of polysaccharide samples.The dried solid polysaccharide sample (10 mg) was accurately

added into a reaction test-tube. The samples were dissolved in10 mL of 2 M TFA and kept at 120 �C for 3 h to hydrolyze thepolysaccharides into monosaccharides. The hydrolysate was cooledto room temperature, and filtered by the membrane filter (0.45 lmpore size). The supernatant was collected and dried by rotary evap-oration (60 �C) to remove the excess TFA for three times by adding2 mL distilled water each time. Thereafter, the monosaccharideswere ready for the further experiments.

10 mL liquid polysaccharide sample was firstly dried by rotaryevaporation (60 �C), followed by the pretreatment process men-tioned earlier for the solid sample.

Part 2: Reduction of aldehyde groups in neutral monosaccha-rides and uronic acids.

Hydrolysis of lactone is an important step before the reduc-tion. Because part of uronic acids existed in lactone form whosealdehyde group could not be reduced by NaBH4, 200 lL of 0.5 MNa2CO3 was added to the samples obtained from the part 1 forthe conversion of lactone into sodium uronate, and the mixturewas held in water bath at 30 �C for 45 min to ensure the completehydrolysis of lactone. Thereafter 50 mg NaBH4 and 2 mL distilledwater were added into the mixture and kept at room temperaturefor 1.5 h to reduce the aldehyde group in neutral monosaccha-rides and uronic acids. The reaction was terminated by adding25% (v/v) acetic acid drop by drop until no bubbles wereobserved.

After the reduction, some residues i.e. sodium ions, borate ionsand excess acetate acid must be removed to avoid the interfer-ence to the next reactions. A rotary evaporator (60 �C) was usedto reduce the volume to approximately 2 mL and remove excessacetate acid. In order to remove sodium ions which significantlyinfluence the lactonization of the aldonic acid obtained from thereduction of uronic acids, the mixture was loaded onto a columnof cation-exchanged resin (9 mL) and eluted with 10 mL water.The eluate was dried using rotary evaporator (60 �C) and borateions which would affect the formation of alditol acetate wereremoved by evaporating methanol from the mixture for 4 times(adding 4 mL methanol each time; transforming borate tomethyl borate completely). Transparent solid would be obtainedafter drying, which indicated that the ions were removedcompletely, but if not, white solid particles would appear after

Fig. 1. Flow chart and mechanism of the simultaneous derivatization of neutral monosaccharides and uronic acids.

200 X. Wang et al. / Food Chemistry 220 (2017) 198–207

evaporation of the mixture. As the aldonic acids cannot react withn-propylamine to form N-propylaldonamide, it was necessary toheat the dried mixture at 85 �C for 4 h to completely convert

the aldonic acids into aldonolactones. Then the samples wereready for the next derivative reactions with n-propylamine andacetate anhydride.

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Part 3: Formation of alditol acetate and N-propylaldonamide.The sample was dissolved by 2 mL pyridine and 0.5 mL n-

propylamine in the test-tube, which was capped and held at55 �C for 0.5 h. The mixture was then dried by a rotary evaporator(60 �C), after which 2 mL pyridine and 2 mL acetate anhydridewere added and kept at 95 �C for 1.5 h for esterification. The mix-ture was then evaporated (80 �C) to remove the excess pyridineand acetate anhydride. CH2Cl2 was added into the mixture to afinal volume of 2 mL, after which the sample was filtered(0.45 lm pore size) before injection.

2.2.2. The optimization of the derivatization conditionsIt was an essential step to convert aldonic acid into aldonolac-

tone, which was obtained by the reduction of uronic acids withsodium borohydride. The lactonization would significantly influ-ence the reaction of the aldonic acid with n-propylamine, thusthe different lactonizaiton times (0, 1, 2, 3, 4, 5 h) were appliedto obtain the optimal parameters for the derivatization.

Acetic anhydride and n-propylamine directly reacted with thereduced products of uronic acids and neutral monosaccharides,which would significantly influence the formation of monosaccha-ride derivatives. Therefore, n-propylamine and acetic anhydridewith a series of amounts (0, 0.5, 1, 1.5, 2, 2.5 mL) were used to opti-mize the two steps in the derivatization process respectively.

2.3. Gas chromatographic analysis

Gas chromatography (Shimadzu 2014C) with a high perfor-mance capillary column, DB-17 (30 m � 0.25 mm ID, 0.25 mmfilm thickness, Agilent), was used to determine the derivatizationproducts of monosaccharides. The temperatures of the injectorand the detector were 250 �C and 280 �C respectively. The injec-tion (1 lL) was normal injection without splitting. The flow rateof carrier gas (N2), air and hydrogen were 1.5 mL/min, 450 mL/min and 60 mL/min, respectively. Two temperature programswere used to analyze the effects of chromatography conditionson the separation of monosaccharide derivatives, which werenamed as TP-A (Temperature Program-A) and TP-B (TemperatureProgram-B).

Briefly, for TP-A, the initial temperature was held at 180 �C for2 min, and increased to 210 �C with a linear gradient in 5 minand held for 2 min. The temperature was then ramped at 0.3 �C/min to 215 �C and held for 20 min, and then increased at 6 �C/min to the final temperature of 240 �C and held for 10 min, whichresulted in a total run time of 60 min.

For TP-B, it was same as TP-A until the temperature increased to215 �C. Then the temperature was raised at 8 �C/min to 240 �C andheld for 17 min, resulting in a total analysis time of 45 min.

2.4. Validation of the method

Linearity, sensitivity, precision, reproducibility and recoveryrate were evaluated to validate the analytical method (Xieet al., 2013). The linearity of each monosaccharide was evaluatedby using a series of standard solutions. The calibrations curveswere conducted based on peak area versus concentrations ofstandard monosaccharides. The limit of detection (LOD) wasdetermined as the gradational standard monosaccharides concen-tration at which the signal-to-noise ratio was 3:1. Intra- andinter-day variations were chosen to determine the precision ofthe method and expressed by relative standard deviation (RSD).The intra-day precision was evaluated by performing determina-tion with the interval of 2 h in the same day and inter-dayprecision was evaluated by performing determination for 10 dayswith an interval of 2 days. For the recovery determination, thestandard monosaccharides were added into polysaccharide

sample (APP-P) and then pretreated under the optimizedderivatization conditions. The reproducibility was examined by4 repeated determinations of the monosaccharides hydrolyzedfrom five samples (Wu, Jiang, Lu, Yu, & Wu, 2014). In addition,the stability of the monosaccharide derivatives was evaluatedby determination of same standard monosaccharides mixturefor 20 days with an interval of 10 days.

2.5. Statistical analysis

All experiments were carried out in triplicate. The relativestandard deviation (RSD) and the mean values were calculatedusing Microsoft Excel 2013 software.

3. Result and discussion

3.1. Optimization of the derivatization conditions

Although lactonization time, the addition of n-propylamine andacetic anhydride are the essential steps in monosaccharidesderivatization, no report was found to provide detail informationabout their roles in the derivatization as well as their optimalparameters.

3.1.1. Lactonization timeAccording to the derivatization procedure described above,

the mixed monosaccharide standards (7 neutral monosaccha-rides, 1.5 mg/mL; 2 uronic acids, 2 mg/mL) were pretreated withdifferent lactonization time while all other conditions were keptsame (2 mL n-propylamine and 2 mL acetic anhydride). Theeffect of lactonization time on derivatization was summarizedin Fig. 2(A), which indicated that the lactonization time had asignificant effect on the derivatization of uronic acid. Aldonicacid obtained from uronic acid reduction cannot directly reactwith n-propylamine, therefore, it is necessary to convert aldonicacid to aldonolactone. As the reduced products of neutralmonosaccharides, alditols have no carboxyl group, so the deter-mination of neutral monosaccharides was not affected by thelactonization time, which can be proved by the little change ofpeak area with the increase of lactonization time. Some aldonicacids existed in the form of aldonolactone naturally, hence,despite 0 h lactonization pretreatment, part of the aldonic acidsstill can be determined. With the increase of lactonization time,the peak area of uronic acid increased firstly and then reached aplateau at 4 h. Therefore, shorter time resulted in partial conver-sion of the aldonic acid to aldonolactone, and the completeconversion was necessary to be achieved with the reaction atleast 4 h at 85 �C.

3.1.2. Amount of n-propylamineAs the carboxyl group cannot be esterified by acetic anhy-

dride, aldonic acid generated by uronic acid reduction must beconverted to N-propylaldonamide which can be gasified anddetected by GC. For the purpose to evaluate the influence causedby n-propylamine addition, different amounts of n-propylaminewere added while other conditions did not change, such as theconcentration of monosaccharide standards, lactonization time(4 h), and acetic anhydride (2 mL) described before. The effectof different levels of n-propylamine (0, 0.5, 1, 1.5, 2, 2.5 mL)on the peak area of derivatives was presented in Fig. 2(B). Whenthe n-propylamine was not added (0 mL), the peaks of uronicacids were not found in the chromatogram. With the increaseof the n-propylamine, the peak area of uronic acids increasedto the maximum value at 0.5 mL followed by the stable state.Approximate 8 mg (equal with 0.04 mmol) uronic acid was in

Fig. 2. Effect of lactonization time (A); n-propylamine adding amount (B); acetic anhydride adding amount (C) on derivatization of neutral monosaccharide and uronic acid.

202 X. Wang et al. / Food Chemistry 220 (2017) 198–207

the mixture of standard monosaccharides, it is indicated that0.5 mL (equal with 6.08 mmol) n-propylamine was enough forthe derivatization of uronic acid in polysaccharide samples(10 mg sampling, equal with 0.06 mmol, in general) and theexcess n-propylamine (more than 0.5 mL) would not increase

the peak area of uronic acid. Meanwhile, it was also demon-strated that different levels of n-propylamine addition had noeffect on the determination of neutral monosaccharide. Accord-ingly, 0.5 mL n-propylamine addition was used as the optimalparameter in the derivatization process.

X. Wang et al. / Food Chemistry 220 (2017) 198–207 203

3.1.3. Amount of acetic anhydrideThe acetic anhydride can react with all hydroxyl contained in

the reduced products of neutral monosaccharide and uronic acid,which is the last and the most important reaction in the derivati-zation process. In order to optimize the dosage of acetic anhydride,different levels of acetic anhydride were added. In addition, thelactonization time (4 h), the amount of n-propylamine (0.5 mL)and other conditions were kept same as described previously. Itis shown that the different levels of acetic anhydride have a signif-icant effect on the determination of both neutral monosaccharidesand uronic acids in Fig. 2(C). When the addition of acetic anhydridewas 0 mL, there was no signal for both neutral monosaccharide anduronic acid, which indicated that the acetic anhydride addition wasessential for the derivatization of all monosaccharides. Moreover,with the increase of acetic anhydride addition, the growth rate ofthe peak area was obviously different between neutral monosac-charides and uronic acids. It might be caused by the different num-ber of hydroxyl groups in the reduced products of neutralmonosaccharides and uronic acids. Compared with aldonic acids,more hydroxyl groups for alditols made it more sensitive to thevariation of acetic anhydride addition. With the increasing of theacetic anhydride addition, the biggest peak area of neutralmonosaccharide and uronic acid was obtained at 2 mL additionof acetic anhydride. There were approximately 21 mg (equal with0.12 mmol) neutral monosaccharides and 8 mg uronic acids (equalwith 0.04 mmol) investigated, which meant that 2 mL (equal with21.16 mmol) acetic anhydride could ensure the success of thederivatization of monosaccharides obtained from polysaccharidesample hydrolysis (10 mg sampling in general).

According to the results, lactonization time (4 h), the addition ofN-propylamine (0.5 mL) and acetic anhydride (2 mL) were chosenas the optimal conditions for the derivatization, which could sat-isfy the derivatization for10 mg polysaccharide samples.

3.2. Optimization of chromatographic conditions

The chromatograms of the two temperature programs (TP-A;TP-B) were shown in Supplementary Fig. 1, which indicated thatnine derivatives of monosaccharides were separated successfully(detailed information shown in Fig. 3B). Although the peaks offucose and arabinose did not perform baseline separation com-pletely as other seven monosaccharides, the accuracy of quantita-tive analysis of the two neutral monosaccharides was satisfactory,which would be proved by the results of method validation later.As showed in Supplementary Fig. 1A for TP-A, the peaks of sevenneutral monosaccharides appeared completely when the tempera-ture of the column reached to 215 �C. It was interesting that theseparation of uronic acid derivatives needed a higher column tem-perature (>215 �C). It was also proved that 45 min was enough forthe analysis of 7 neutral monosaccharides and 2 uronic acids by GCwith TP-B which shortened the time of the column temperatureincreasing from 215 �C to 240 �C. There was no significant differ-ence between TP-A and TP-B for neutral monosaccharides separa-tion, which were shown in Supplementary Table 1. Consequently,in the present work, TP-B was chosen for GC analysis in view ofshorter analysis time.

3.3. Validation of the GC analysis method

3.3.1. Linearity and limit of detection (LOD)Under the optimized conditions of derivatization and GC analy-

sis, the linear calibration regression equations were established bythe analysis of seven points ranging from 0.025 to 4 mg/mL forneutral monosaccharides and 0.030 to 5 mg/mL for uronic acids.Each point of the calibration plot was repeated three times. Thesummary of calibration curves, linear ranges and limit of detection

for all monosaccharides were listed in Table 1. It was shown thatexcellent linearity between y (peak area) and x (concentration ofmonosaccharide standard) was found in the analysis range of bothneutral monosaccharides and uronic acids. The correlation coeffi-cients (R2) of the calibration curves were at least 0.9982, whichsuggested good linearity within the tested concentration range. Itwas also found that the coefficients of x in each regression equationwere different. Furthermore, the limit of detection (LOD) was cal-culated as the lowest concentration level that was statistically dif-ferent from the blank. LOD of each tested monosaccharide wasobtained by injecting 1 lL of gradational dilutions of mixedmonosaccharide standards sample, and peak height with asignal-to-noise ratio (S/N) of 3 was used to estimate the LOD. Itis given in Table 1 that the LOD values of the nine monosaccharideswere in the range from 0.27 lg/mL to 1.65 lg/mL, which provedthat the sensitivity of the method was satisfactory.

3.3.2. Precision and reproducibilityThe precision of the method was evaluated by measuring the

repeatability which was reflected by the coefficient of variation.Both inter-day and intra-day variability of retention time and peakarea for each monosaccharide were calculated as the relative stan-dard deviation (RSD) by making five repetitive injections of a stan-dard mixture solution under the same optimum conditions. Assummarized in Table 2, the RSD values in intra-day were less than0.31% for the retention time and 3.61% for the peak area, and forthe inter-day, which were less than 0.80% and 4.27% for retentiontime and peak area respectively. These results showed excellentrepeatability of the retention time and the peak area, which indi-cated that the method precision was satisfactory.

The result of reproducibility analysis was summarized in Sup-plementary Table 2 about the analysis of five polysaccharide sam-ples. The RSD values of each monosaccharide were in the range of0.12–2.31%, which suggested that the reproducibility of themethod can satisfy the requirement of qualitative and quantitativedetermination of monosaccharide composition.

3.3.3. AccuracyThe method of standard sample addition was adopted to deter-

mine the recovery of each monosaccharide standards, which can beused to evaluate the accuracy of the method studied in the presentwork. Three replicate experiments were performed under opti-mum conditions and the average recovery rate and relative stan-dard deviation (RSD) of each monosaccharide were determined,which were showed in Table 3. The recoveries of all the ninemonosaccharides ranged from 98.40% to 101.82% and the RSD val-ues were lower than 3.5%. According to the results of the recoverytest, the method was proved to be accurate.

3.3.4. The stability of the monosaccharide derivativesIn order to evaluate the stability of the monosaccharides deriva-

tives, the sample obtained from the derivatization of ninemonosaccharides standard mixture under optimum conditionswas determined three times for 20 days with an interval of10 days. The derivatives were stored at�20 �C. The results summa-rized in Supplementary Table 3 which indicated that the retentiontime of each derivative varied little for the RSD values were lessthan 0.11%. Since the retention time reflects the chemical proper-ties of the derivatives, the results indirectly suggested that thederivatives were stable in 20 days. RSD values of the peak areafor each derivative were more than 10% which was mainly causedby the volatilization of the solvent (CH2Cl2). This can be proved bythe results of the excellent linearity between peak area and storagetime with the R2 more than 0.99, and the similar slope of the linearregression line for all monosaccharide derivatives (the linearregression equation of rhamnose was shown in Supplementary

Table 1The linearity and limit of detection (LOD) of GC-FID method in different monosaccharide references.

Monosaccharides Retention time (min) Linear range (mg/mL) Regression equationa Correlation coefficient (R2) LODb (lg/mL)

Rhamnose 11.839 0.025–4 y = 52709x + 290.31 0.9985 0.83Fucose 12.349 0.025–4 y = 60516x + 285.11 0.9986 0.71Arabinose 12.559 0.025–4 y = 70817x � 500.69 0.9985 1.06Xylose 13.240 0.025–4 y = 67598x � 642.77 0.9983 1.43Mannose 23.392 0.025–4 y = 62260x + 606.75 0.9986 1.46Glucose 24.095 0.025–4 y = 61517x + 201.76 0.9987 0.49Galactose 24.705 0.025–4 y = 62948x + 114.31 0.9982 0.27Glucuronic acid 36.605 0.030–5 y = 50715x + 507.67 0.9987 1.50Galacturonic acid 38.532 0.030–5 y = 48747x + 536.00 0.9989 1.65

a y and x stand for the peak area and the concentration (mg/mL) of monosaccharide references, respectively.b The detection limits correspond to concentrations giving a signal-to-noise ratio of 3.

Table 2Precision of the retention time and peak area of the tested monosaccharides by GC-FID method.

Monosaccharides Intra-day precision (RSD%, n = 5) Inter-day precision (RSD%, n = 5)

Retention time Peak area Retention time Peak area

Rhamnose 0.12 0.82 0.67 1.89Fucose 0.16 1.18 0.47 3.90Arabinose 0.26 2.63 0.63 3.46Xylose 0.26 3.61 0.73 3.31Mannose 0.24 0.94 0.80 3.60Glucose 0.30 1.87 0.62 3.40Galactose 0.31 1.84 0.50 3.33Glucuronic acid 0.19 0.51 0.30 3.89Galacturonic acid 0.22 0.47 0.24 4.27

Table 3Recoveries of nine monosaccharides with GC-FID method.

Monosaccharides Content/mg Added value/mg Detected value/mg Recovery (%)a RSD (%)

Rhamnose 0.2364 2.00 2.2727 101.82 2.53Fucose 0.0006 2.00 2.0195 100.95 1.27Arabinose 0.1820 2.00 2.1499 98.40 3.48Xylose 0.1205 2.00 2.1145 99.70 2.24Mannose 0.0020 2.00 2.0356 101.68 1.41Glucose 0.1528 2.00 2.1369 99.21 2.62Galactose 0.1419 2.00 2.1578 100.80 1.28Glucuronic acid NDb 2.00 2.0300 101.50 1.10Galacturonic acid 4.1235 2.00 6.1666 101.71 2.94

a Data are means of three experiments.b ND: not detected in the sample.

204 X. Wang et al. / Food Chemistry 220 (2017) 198–207

Fig. 2 as an example). All the derivatives were dissolved in CH2Cl2which was easy to volatilize in the process of transfer and detec-tion. As a consequence, the peak area of monosaccharide deriva-tives had an increasing tendency with the storage time and thenumber of transfers. Therefore, it was indicated that all the ninemonosaccharides derivatives had an excellent stability for 20 days’storage. Thus it was necessary to know the exact volume of thesample before injection. For example, measuring exact volumesample or re-dilute the sample to 2 mL are suggested.

According to above analysis, it can be concluded that linearity,sensitivity, precision, reproducibility and recovery rate of the pro-posed method are good enough for monosaccharides analysis. Thechemical transformations undergone by the neutral monosaccha-rides and uronic acids in each reaction step were discussed indetail. In addition, the good stability of each monosaccharidederivative made it possible to collect a large number of samplefor GC analysis after derivatization.

3.4. Monosaccharide composition analysis of experimental samples

In order to investigate the applicability of the method, fivepolysaccharide samples from diverse sources were selected to

verify the improved method. The standard pretreatment processeswere performed as described in Fig. 1. The monosaccharides con-tents of five samples were calculated and listed in SupplementaryTable 2 and the chromatographic profile of the monosaccharideswere shown in Fig. 3 (A to F respectively). It was clear that deriva-tives of nine monosaccharides could be separated and themonosaccharides of samples were identified by comparing theirretention time with those of the monosaccharide standards(Fig. 3A).

For APP-P, there are eight monosaccharides detected by GCexcept glucuronic acid. The molar ratio of rhamnose, fucose, arabi-nose, xylose, mannose, glucose, galactose and galacturonic acidwas found to be 5.53, 0.01, 4.65, 3.08, 0.04, 3.25, 3.02 and 80.42.The molar ratios revealed that the galacturonic acid possibly wasthe backbone component with other seven monosaccharideslinked to it, which was in accordance with the reported study(Wang & Lü, 2014). For GSR-P and APL-P which come from Chineseherbal medicine, there were 3 kinds of monosaccharides detectedfor GSR-P and 4 kinds of monosaccharides in APL-P, and the glu-cose was the dominant component. ESP was produced by Lacto-bacillus plantarum KX041, which was mainly composed ofmannose, glucose, galactose and arabinose. In order to apply the

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Fig. 3. GC-FID chromatography profiles of standard monosaccharides mixture (A), APP-P (B), GSR-P (C), APL-P (D), ESP (E) and PEB-P (F). The peaks in chromatography profile(B) from left to right in order were as follows: (1) Rhamnose; (2) Fucose; (3) Arabinose; (4) Xylose; (5) Mannose; (6) Glucose; (7) Galactose; (8) Glucuronic acid; (9)Galacturonic acid.

X. Wang et al. / Food Chemistry 220 (2017) 198–207 205

206 X. Wang et al. / Food Chemistry 220 (2017) 198–207

proposed method to investigate the monosaccharide compositionof liquid samples, such as fermentation liquor, pretreatment efflu-ent and some sugary fluids, PEB-P which come from the pretreat-ment of switchgrass by subcritical water was used as an examplefor the liquid sample analysis. There were six kinds of monosac-charides successfully separated and detected with the proposedmethod. The dominant monosaccharides were xylose, glucoseand arabinose. As the liquid sample was the residual liquid of thebiomass pretreated by subcritical water, the monosaccharideswere mainly from the hydrolysis of cellulose and hemicellulosein which xylose and glucose cover the most part. In addition,RSD values of each monosaccharide were lower than 5% (calculat-ing from Supplementary Table 2) which proved the good repro-ducibility. Thus, it was confirmed that the optimized GC methodwas applicable to the monosaccharide composition analysis forboth solid and liquid polysaccharides samples.

Besides, the hydrolysis of polysaccharide samples was also cru-cial in monosaccharide composition analysis. Because polysaccha-rides did not perform the same resistant toward acid for thedifferences of glycosidic linkage between the monosaccharides,so the optimal hydrolysis conditions were also different, such asacid concentration, temperatures and hydrolysis times. Sincenumerous polysaccharides exist in nature, it was impossible togive uniform conditions for the hydrolysis process. It is generallysuggested that the polysaccharide sample was hydrolyzed by 2 MTFA at 120 �C for 3 h (Wang et al., 2016; Zhang et al., 2012), whilethe hydrolysis process should be optimized if the polysaccharidesample has high content of uronic acids or side chains (Garna,Mabon, Wathelet, & Paquot, 2004).

Although the improved method can be used for many monosac-charides analysis, it should be paid much attention that it was notsuitable to determine the ketoses and some rare or specialmonosaccharides, such as gulose, talose, allose, apiose, streptose,glucosamine and galactosamine (amino sugar), etc. For example,fructose as ketose can be reduced into glucitol and mannitol inthe derivatization, which make the quantification difficult. Andthe formation of same alditol in the derivatization may also leadingto inaccurate results, for instance, glucose and gulose will be bothtransformed into glucitol. Therefore, it should be noticed that theimproved method cannot solve all problems but it is good enoughto analyze most part of monosaccharides and uronic acids. To ourknowledge, single analytical method for unknown polysaccharidessample is not adequate, thus two or more methods should be com-bined together to exclude the influence of some special monosac-charide in the qualitative and quantitative process. The improvedGC-based method in this study at least can be a reliable and accu-rate method for providing monosaccharides profile of somepolysaccharides based on the common reagents and instruments.

4. Conclusion

The data presented showed that GC-based method is suitedfor the monosaccharide composition analysis because of thegood performance in separation and detection with higheraccuracy and precision. The optimal derivatization parametersand sample pretreatment were obtained and the validation ofthe method was verified. Base on the results, the simultaneousdetermination method for general uronic acids and neutralmonosaccharides assay by GC was improved, which showedgood performance in linearity, precision, accuracy and repro-ducibility, and all the derivatives of monosaccharides werestable during 20-days storage. In addition, it must be stressedthat the proposed method was not ideal for ketoses and somerare or special monosaccharides, such as fructose, gulose, talose,allose, apiose, streptose, glucosamine and galactosamine (aminosugar), etc. To the best of our knowledge, single analytical

methods for the qualitative and quantitative analysis ofunknown polysaccharides composition are not available,therefore two or more methods should be combined togetherfor obtaining reliable results.

Acknowledgements

The author thanks the financial support of Special Fund forAgro-scientific Research in the Public Interest (Grant No.201503135).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2016.10.008.

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