Simultaneous and Sensitive HPLC Determination of Mono- and Disaccharides, Uronic Acids, and Amino...

134 Acta hydrochim. hydrobiol. 31 (2003) 2, 134–144

© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Simultaneous and Sensitive HPLC Determinationof Mono- and Disaccharides, Uronic Acids, andAmino Sugars after Derivatization by ReductiveAmination*

Klaus Fischera,Marion Wachta,Axel Meyera

a Analytische und ÖkologischeChemie, FB VI –Geographie/Geowissenschaften,Universität Trier,Universitätsring 15,54286 Trier, Germany

* Paper presented in parts as aposter at the annual meeting ofthe Water Chemical Society – aDivision of the GermanChemical Society(Wasserchemische Gesellschaft– Fachgruppe in derGesellschaft DeutscherChemiker), Eichstätt/Altmühltal,May 2002.

Correspondence: K. Fischer, E-mail: [email protected]

The reductive amination of low-molecular-weight saccharides, uronic acids, and amino sug-ars, followed by a separation of the derivates by means of ion-pair chromatography or RP-HPLC, offers an interesting alternative to HPAEC-PAD for the environmental analysis ofthese compounds. Under this aspect various potential amination reagents, i.e., p-amino-benzoic acid (p-AMBA), p-AMBA propyl ester, 1-aminopyrene, 2-(2-aminophenyl)indole,and 4-aminoazobenzene, were tested with regard to the formation of derivates and to thechromatographic properties of the formed derivates.p-AMBA, p-AMBA propyl ester and 4-aminoazobenzene proved to be especially suited, be-cause they facilitate the amination of all carbohydrate reference components together with acomplete separation and sensible detection (detection limits < 0.5 mg/L) of the derivates.Mainly the following elution sequence was ascertained: amino sugars (hexosamines) / di-saccharide(s) / monosaccharides (hexoses) / hexuronic acid(s) / N-acetyl-D-glucosamine.Detection limits down to 0.1 µmol/L were realized using p-AMBA as reagent, facilitating thedetermination of the target compounds in landfill leachates and lysimeter percolates. Apply-ing the p-AMBA propyl ester for derivatization, chromatographic interferences with weaklyretained derivates and the coelution of the reagent with its galactosamine derivate can beavoided, since the ester elutes after its derivates unlike p-AMBA itself.

Simultane und sensitive HPLC-Bestimmung von Mono- und Disacchariden,Uronsäuren und Aminozuckern nach Derivatisierung mittels reduktiverAminierungDie reduktive Aminierung von niedermolekularen Sacchariden, Uronsäuren und Aminozu-ckern, kombiniert mit ionenpaarchromatographischer oder RP-HPLC-Trennung der Deriva-te, eröffnet eine interessante Alternative in der Umweltanalytik dieser Verbindungen gegen-über der HPAEC-PAD. Vor diesem Hintergrund wurden einige potentielle Aminierungsrea-gentien, u.a. p-Aminobenzoesäure (p-AMBA), der p-AMBA-Propylester, 1-Aminopyren,2-(2-Aminophenyl)-indol und 4-Aminoazobenzol, hinsichtlich der Derivatbildung und derchromatographischen Eigenschaften der Derivate getestet.Dabei erwiesen sich p-AMBA, ihr Propylester und 4-Aminoazobenzol als besonders geeig-net, da mit ihnen eine Derivatisierung sämtlicher Kohlenhydratreferenzkomponenten sowieeine vollständige chromatographische Auftrennung der Derivate und ihre empfindliche De-tektion (NWG < 0.5 mg/L) möglich ist. In der Regel wurde folgende Elutionssequenz erhal-ten: Aminozucker (Hexosamine) / Disaccharide / Monosaccharide (Hexosen) / Hexuronsäu-re(n) / N-Acetyl-D-glucosamin. Mit p-AMBA wurden Nachweisgrenzen von bis zu 0.1 µmol/Lerzielt, was die Bestimmung der Zielkomponenten in Deponiesickerwässern und Lysimeter-perkolaten erlaubte. Bei Verwendung des p-AMBA-Propylesters, der im Unterschied zur p-AMBA nach den Derivaten eluiert, können Interferenzen mit wenig retardierten Derivatenund die Coelution mit Galactosamin vermieden werden.

Keywords: Carbohydrates, Ion-pair Chromatography, Reversed-phase Chromatography,Environmental Analysis

Schlagwörter: Kohlenhydrate, Ionenpaarchromatographie, RP-Chromatographie, Umwelt-analytik

DOI 10.1002/aheh.200300484


Reductive Amination of CarbohydratesActa hydrochim. hydrobiol. 31 (2003) 2, 134–144 135

1 Introduction

Carbohydrates represent a small, but ecologically relevantfraction of the natural organic carbon content of surface wa-ters. Because of the fast biological turnover of mono- and di-saccharides and of amino sugars, only a few percent of theaquatic DOC content are apportioned to this class of com-pounds [1]. Nevertheless their analytical determination canbe quite instructive, since the content and the structural diver-sity of low-molecular-weight carbohydrates may be indicativefor their biological sources and for biogeochemical processeswhich regulate the DOC composition and concentration. Thesame applies to uronic acids, i.e., galacturonic and glucuronicacids, which are the monomeric units of several specificpolysaccharides, generated by many aquatic organisms [2].Furthermore carbohydrates are key compounds for the for-mation of humic acids.

Actually high-performance anion-exchange chromatography(HPAEC) hyphenated with pulsed amperometric detection(PAD) is the preferred analytical method for the determinationof low molecular weight carbohydrates in environmental sam-ples [3–5].This analytical technique combines high selectivitywith high sensitivity. Applying this method it is also possible todetermine uronic acids, which are dissociated to a high de-gree above pH 4.0. Consequently the uronic acids have amuch higher affinity for the anion-exchange stationary phasethan the very weakly acidic mono- and disaccharides [6].Therefore the separation of uronic acids on HPAEC resins re-quires eluents exerting elution strengths essentially higherthan used in sugar analysis normally.

The application of HPAEC-PAD to environmental samples isafflicted with several problems. The simultaneous determina-tion of all relevant analyte groups requires gradient elution,but under these conditions, the retention of uronic acids lastsone hour or more [2]. A specific sample pretreatment is nec-essary to remove interfering amino acids [7, 8]. The determi-nation limits of carbohydrates range between 0.1 mg/L and0.5 mg/L typically which is too insensitive for environmentalanalysis.

Besides HPAEC, capillary electrophoresis (CE) in combina-tion with PAD is recognized as a valuable tool for the determi-nation of mono- and oligosaccharides with great potential inthe field of biochemical and pharmaceutical analysis [9]. Toenhance detection sensitivity, derivatization methods apply-ing a number of photometric or fluorometric tags includingseveral amination reagents were developed [10, 11]. Presum-ably due their high matrix sensitivity and their low robustnessthe elaborated methods are not utilized for environmental an-alytical purposes yet.

An interesting alternative for the simultaneous determinationof low-molecular-weight carbohydrates having different struc-

tural properties and acidities is precolumn derivatization fol-lowed by ion-pair or RP-HPLC separation. Derivatizationthrough reductive amination offers some advantages, i.e., un-complicated reaction procedure, transformation of all inter-esting compounds (mono – and disaccharides, amino sug-ars, and uronic acids) and sensible detection of fluorescentderivates. Reductive amination is often applied for the deter-mination of carbohydrates in hydrolysates of oligo- andpolysaccharides and glycoproteins [12–14]. Monosubstitutedaminobenzenes such as 2-aminobenzamide [15, 16], 4-ami-nobenzoic acid esters [17, 18], various aminobenzoic acids[19–21], and 4-aminobenzonitrile [22, 23] were selected asamination reagents preferentially. With the exception of ourown attempts, these reagents were not used for the determi-nation of carbohydrates in environmental samples yet [24].

Our current work deals with the further development of chro-matographic methods based on the application of p-ami-nobenzoic acid and of its alkylesters as derivatization rea-gents [25]. Within a screening test scheme, several other po-tential amination reagents were examined additionally andsome efforts were made to define chromatographic condi-tions suited to separate their carbohydrate derivates.

2 Materials and methods

2.1 Chemicals and stock solutions

The carbohydrates and aldehydes D(+)-lactose monohydrate(Lac), D(+)-xylose (Xyl), D,L-glycerinaldehyde (GA), N-acetyl-D-glucosamine (GlcNAc), D(+)-glucose (Glc), D-glu-curonic acid (GlcUA), and D(+)-galacturonic acid (GalUA)were obtained from Fluka as well as p-aminobenzoic acid (p-AMBA, purity � 99 %), the p-AMBA propyl ester (purity �

99%), 1-aminopyrene (purity � 98%), and tetrabutylammoni-um hydrogen sulfate (TBAHSO4). The amino sugar D(+)-ga-lactosamine (GalN) was purchased from Sigma and servedtogether with the other carbohydrates as reference com-pound. 2-(2-aminophenyl)indole (purity 97%) was obtainedfrom Aldrich, 4-aminoazobenzene (purity 98.9%) fromRiedel-de-Haën.

The other chemicals stemmed from Merck. The solvents(methanol, acetonitrile) were of HPLC quality, all other chemi-cals of “p.A.” or “puriss.” quality. Water was purified by reverseosmosis and then passed through a Millipore Milli-Q unit.

Stock solutions of the reference compounds were preparedby weighing of 50 mg of each in a 10-mL volumetric flask fol-lowed by dissolution in water. These solutions were stored at–20°C and used for further dilutions.


K. Fischer et al.136 Acta hydrochim. hydrobiol. 31 (2003) 2, 134–144

2.2 Apparatus

Chromatographic separations were conducted on a Shimad-zu HPLC system consisting of an autosampler SIL 10A, acontroller SCL-10 AVP, a gradient pump LC-10 ADVP, an on-line degasser GT 104, and a fluorescence detector SPD-10AXL. UV absorption was measured by a dual beam UV detec-tor SPD-10 AVP or by a DAD-detector SPD-M 10 AVP, bothfrom Shimadzu. The column temperature was regulated by aJetstream-Plus thermostat (VDS Optilab). Data acquisitionand processing were accomplished with CLASS VP 5.03 soft-ware (Shimadzu).

The following separation columns, filled with different RP-C18

packings, were used: AQUA 5 C18, 250 mm × 3 mm i.d. (flowrates 0.6 mL/min or 0.5 mL/min), AQUA 3 C18, 100 mm× 3 mm i.d. (flow rate 0.4 mL/min), both from Phenomenex,and Nucleosil 100 C18, 250 mm × 3.5 mm i.d. (Macherey &Nagel; flow rate 0.6 mL/min).The injection volume was 20 µL.Mixtures of phosphate buffer and methanol, whose composi-tion was regulated according to a gradient profile, served aseluents. A pH value of 2.0 or 2.1 was adjusted by adding of85% orthophosphoric acid. Depending on the chromato-graphic method the eluent contained up to 20 mmol/LTBAHSO4 as ion-pair reagent.

2.3 Precolumn derivatization / Screeningprocedure

For the selection of suited derivatization reagents and for thedetermination of adequate derivatization conditions, the fol-lowing specifications were defined and subsequently varieddepending on the achieved results (in parentheses: variationranges): reagent concentration 0.1 mol/L (0.01...0.5 mol/L),prepared in a DMSO/acetic acid solution (70 : 30 v/v); mixingratio sample : reagent solution 1 : 2.5 (e.g. 0.2 mL : 0.5 mL),addition of 10 mg of the reducing agent (NaBH3CN), reactiontemperature 60°C (30...90°C), reaction time 60 min (10...90min). The components were mixed in 2-mL polyethylene vialsand the tightly capped vials were heated on a water bath. Af-ter cooling of the vials the reaction mixture was dissolved in6.3 mL of the eluent.

2.4 Preparation of environmental samples

Several seepages from the sanitary landfill of Trier and efflu-ents from various small lysimeters (fill height approx. 30 cm)were collected. Depending on the test variant, the lysimeterscontained sewage sludge, partially converted into raw soil, amixture of this material with arable soil, a mixture of arable

soil with fresh sewage sludge or arable soil without admix-tures, each of the fillings deposited over a 10 cm sand/gravel-drainage layer.

The samples were filtered through 0.45-µm PTFE filters andsmall amounts of the internal standard glycerinaldehyde (GA)were added. An aliquot (2 mL) of each sample was deriva-tized after addition of 5 mL of the derivatization solution andof 60 mg of NaBH3CN.

3 Results

3.1 Selection of suitable derivatizationconditions

The findings concerning adequate reaction conditions for theprecolumn derivatization of carbohydrates with p-AMBA werecommunicated recently [21]. The method development con-sidered the reaction parameters time, temperature, and rea-gent concentration at given sample:reagent solution ratio.Conditions were searched offering a high reaction yield, shortreaction times, and the possibility to automate the procedureby means of an autosampler. The peak areas (UV or fluores-cence detection) resulting from the subsequent chromato-graphic investigation of the reaction mixtures were chosen asyield criterion. Due to the structural diversity of the analytes, aparameter set providing optimal reaction conditions for allcompounds was not found.

Generally a high reagent excess was needed to achieve asignificant reaction yield. For most of the carbohydrates, a p-AMBA concentration of 0.1 mol/L was sufficient, except forthe (N-acetylated) amino sugars, which required essentiallyhigher reagent concentrations.

The transformation degree of the monosaccharides was al-most independent from temperature within the tested range(30...95°C) at a reaction time of 15 min, whereas the conver-sion of the (N-acetylated) amino sugars increased with in-creasing temperature. A temperature of about 50 °C was opti-mal for the derivatization of the uronic acids.

At a reaction temperature of 95°C, the uronic acids and the(N-acetylated) amino sugars showed an opposite depend-ence of the derivate formation from reaction time.The deriva-tization yield of the amino sugars increased during the wholetime span tested (60 min), whereas yields were highest atshortest contact time (2 min) in the case of the uronic acids.

These results indicate a certain trend in the reactivity of thedifferent structural groups of carbohydrates exposed to ami-nation reagents in the presence of an hydrogen donating



Table 1: Selected reagents for carbohydrate derivatization through reductive amination.

Ausgewählte Reagenzien für die Kohlenhydratderivatisierung mittels reduktiver Aminierung.

Reagent Structure DetectionUV absorbance Fluorescence

1-Aminopyrene λ = 241 nm λex = 241 nmλem = 462 nm

p-Aminobenzoic acid(p-AMBA)

λ = 303 nm λex = 313 nmλem = 358 nm

p-AMBA propyl ester λ = 287 nm λex = 308 nmλem = 356 nm

4-Aminoazobenzene λ = 377 nm(λ = 318 nm,

pH = 2.1)

lowintensity

2-(2-Aminophenyl)-indole

λ = 223 nm λex = 223 nmλem = 397 nm

agent.The uronic acids tend to react relatively fast under mildconditions, but the derivates seem to decompose or to under-go further conversion reactions, at least at high temperature.In contrast to this, the conversion of the amino sugars pro-ceeded slowly and required more vigorous conditions.

With the restriction that a smaller group of reference sub-stances was tested, this general trend holds more or less forthe reaction with other derivatization agents, especially p-aminobenzoic acid propyl ester and 4-aminoazobenzene. Ap-plying the p-AMBA propyl ester, highest derivatization de-grees were achieved for lactose, glucose, and N-acetyl-D-

glucosamine at 90°C/90 min reaction time, applying a 0.1 Mreagent solution. In the case of galacturonic acid, best resultswere attained with a shorter reaction time (20 min) at thesame temperature. Compared with the free p-AMBA acid, thederivatization with its propyl ester needs longer reactiontimes and higher temperatures.

The findings from the derivatization test series applying 4-aminoazobenzene as amination reagent are compiled in Ta-ble 2. It is obvious that in all cases the higher reagent concen-tration was advantageous for the formation of the derivates.At a reagent concentration of 0.1 mol/L, a reaction tempera-



Table 2: Carbohydrate derivatization with 4-aminoazobenzene (4-AAB): Variation of reaction conditions. Separation on Nucleosilcolumn, UV detection (λ = 388 nm). Greatest peak areas marked in bold.

Kohlenhydrat-Derivatisierung mit 4-Aminoazobenzol (4-AAB): Variation der Reaktionsbedingungen.

Carbohydrates GalN Lac Glc GlcNAc GalUAReaction conditions

4-AABmol/L

ϑ°C

tmin

Relative peak area units

60 20 664 1126 3227 n.d. 13280.0190 20 405 1025 1715 497 103990 60 637 1556 1348 365 163490 90 885 1059 2307 623 1841

60 20 503 1695 5747 560 129340.190 20 1384 2528 4302 1046 237690 60 3008 2298 3710 1248 318190 90 3412 2214 3313 1162 3136

n.d.: no peak detected

Table 3: Carbohydrate derivatization with 1-aminopyrene (1-AP):Variation of reaction conditions. Separation on Nucleosil column,fluorescence detection (λex = 241 nm, λem = 462 nm). Greatest peak areas marked in bold.

Kohlenhydrat-Derivatisierung mit 1-Aminopyren (1-AP): Variation der Reaktionsbedingungen.

Carbohydrates GalN Lac Glc GalUA GlcNAcReaction conditions

1-APmol/L

ϑ°C

tmin

Relative peak area units

0.01 60 20 17 206 2775 27 16990 20 47 270 1225 67 38590 60 84 449 1280 120 74690 90 104 505 1238 115 711

0.1 30 90 133 780 3078 192 38660 20 420 1858 5995 2003 99260 90 414 2046 3993 211 78690 20 508 2053 6863 2336 108390 60 1172 2601 8480 5265 194890 90 1054 2676 8838 1341 1937

0.25 30 90 341 1716 3909 273 62760 90 1031 2810 4066 453 80590 90 2606 2597 3894 945 1929

ture of 90°C favoured the transformation of galactosamine, N-acetyl-D-glucosamine, and lactose, whereas the yields of theglucose and of the galacturonic acid derivate especially weresignificantly higher selecting a temperature of 60°C.To achievethe maximum conversion degree a reaction time of 60 min or

90 min is needed for galactosamine and galacturonic acid. Thederivatizationyieldsof lactoseandN-acetyl-D-glucosamineareal-most independent from time within the tested time span, whereasbest yields of the glucose derivate were accomplished witha reac-tion time of 20 min at a reagent concentration of 0.1 mol/L.



Table 4: Optimized derivatization conditions.

Optimierte Derivatisierungsbedingungen.

Derivatizationreagent

p-Amino-benzoic acid

(p-AMBA)

p-AMBApropylester

4-Amino-azobenzene

1-Amino-pyrene

2-(2-Amino-phenyl)-indole

Reagent solutionc, mol/L

0.35 0.1 0.1 0.25 0.01

Temperature, °C 60 90 90 90 60Time, min 15 75 60 90 90

Table 3 summarizes data from the derivatization test seriesconducted with 1-aminopyrene.Again for a high conversion de-gree a reagent concentration of at least 0.1 mol/L is necessary.A further increase of the 1-aminopyrene concentration pro-motes the derivatization of galactosamine and lactose, but low-ers the conversion of glucose and galacturonic acid.With a fewexceptions the derivatization yields increased with increasingtemperature. Given a reaction temperature of 90°C and a rea-gent concentration of 0.1 mol/L, best results were obtained forthe derivatization of galactosamine, N-acetyl-D-glucosamine,and galacturonic acid selecting a reaction time of 1 h.At 90 min,a slightly higheramount of theglucose derivatewas generated,but theamountof thegalacturonicacidderivatedroppeddrasti-cally.

Conducting the amination reaction with 2(2-aminophenyl)in-dole, a relatively low surplus amount of the reagent was foundto be sufficient for the formation of aminated carbohydrates.Furthermore a temperature of 60°C and a reaction period of 90min favoured the product formation. Nevertheless the genera-tion of a galactosamine derivate could not be confirmed yet.

The finally selected derivatization conditions are comprised inTable 4.All parameter sets are compromises with regard to thesimultaneous determination of analytes belonging to differentstructural subgroups.

To avoid chromatographic interferences between the derivatesand the excess reagent, several attempts were made to sepa-rate the latter from the derivates prior to the chromatographicrun.The efforts were focussed on the removal of p-aminoben-zoic acid, since this reagent elutes before the analytes underchosen conditions (Fig. 4).

Oneapproachwas toextractp-AMBAby theadditionof organicsolvents to the aqueous reaction solution (volume ratio 1:1).The different solvents reduced the p-AMBA peak area as fol-lows: isooctane: 6%, diethylether 15%, CH2Cl2: 38%, CHCl3:

48%. Since some derivates were partially extracted too, thistreatment marks no progress.

Similar results were obtained testing various cation exchangecartridges. Removal degrees up to 60% were achieved for thereagent, but a loss of derivates occurred also, diminishing thepractical detection sensitivity of the method.

3.2 Chromatographic method development

Thehighlypolarandhydrophilic characterof thecarbohydratesis not essentially reduced after derivatization with p-AMBA.Furthermore the amination reagent introduces a carboxylicgroup into the derivate. Under these conditions a normal RPseparation on strongly hydrophobic stationary phases seemednot to be promising. A satisfying separation could be achievedusing the Phenomenex AQUA column and the ion-pair reagentTBAHSO4 (resulting concentration in the phosphate buffer/methanol eluent: 20 mmol/L).The AQUA column is marked byits reduced silanophilic activity and by its hydrophilic end cap-ping. The reached concentration detection limits range be-tween 20 µg/L and 30 µg/L (0.1...0.2 µmol/L) for fluorescencedetection and between 30 µg/L and 75 µg/L for UV detection at303 nm.

The separation of the p-AMBA propyl ester derivates is possi-ble under similar conditions. Due to the lower polarity and hy-drophilicity of the derivates, a higher methanol and a lowerTBAHSO4 content of the eluent has to be adjusted.

The chromatographic separation resulting under these condi-tions is depicted in Figure 1.The analytes elute with retentiontimes between 7 min and 20 min, followed by the surplus rea-gentandbysomesideordegradationproducts.Comparedwithp-AMBA, the elution of its propyl ester after the derivates is ad-vantageous, especially for the determination of trace concen-trations of carbohydrates.



Fig. 1: Separation of the p-AMBApropyl ester derivates of lactose,glucose, galacturonic acid, N-acetyl-D-glucosamine (5 mg/Leach), and of galactosamine(50 mg/L) on AQUA 5, combinedwith fluorescence detection (λex =308 nm, λem = 356 nm). Chromato-graphic conditions as in Table 5.

Trennung der p-Aminobenzoe-säure-Propylester-Derivate vonLactose, Glucose, Galacturon-säure, N-Acetyl-D-glucosamin(jeweils 5 mg/L) und Galactosamin(50 mg/L) in Verbindung mit Fluo-reszenz-Detektion (λex = 308 nm,λem = 356 nm).ChromatographischeBedingungen wie in Tabelle 5.

Fig.2:Separation of a carbohydratemixture after derivatization with1-aminopyrene on Nucleosil 100.Chromatographic conditions as inTable 5. Elution sequence of themarked peaks: galactosamine/lac-tose/glucose/galacturonic acid/N-acetyl-D-glucosamine.

Trennung eines Kohlenhydrat-Ge-mischs nach Derivatisierung mit1-Aminopyren an Nucleosil 100.Chromatographische Bedingungenwie inTabelle 5.Elutionsreihenfolgeder markierten Peaks: Galactos-amin/Lactose/Glucose/Galacturon-säure/N-Acetyl-D-Glucosamin.

Above: UV detection, λ = 241 nm.Below:fluorescencedetection,λex =241 nm, λem = 462 nm.

The chromatogram displays an elution order of the derivateswhich seems to be of general importance for the investigatedamination products of the analytes.The elution sequence hex-osamine < disaccharide < monosaccharide (hexose) < uronicacid < N-acetyl hexosamine occurs with derivates of p-AMBA,its alkylesters and of 1-aminopyrene. In the case of 4-amino-azobenene, the positions of the derivates of the uronic acid andof N-acetyl-D-glucosamine are interchanged.

According to our current results further reagents are principallyable to form derivates with carbohydrate compounds repre-senting the various interesting structural subgroups.The sepa-ration of derivates of 1-aminopyrene on a Nucleosil column is il-lustrated in Figure 2.This separation is achieved without an ad-dition of an ion-pair reagent to the eluent. The analytes aremonitored by UV absorbance at 241 nm (Fig. 2, above) and byfluorescence detection (Fig. 2, below). They elute between



Fig. 3: Separation of the 4-aminoazo-benzene derivates of galactosamine, lac-tose, glucose, N-acetyl-D-glucosamine, andgalacturonic acid on Nucleosil 100, com-bined with UV detection (λ = 377 nm). Chro-matographic conditions as in Table 5.

Trennung der 4-Aminoazobenzol-Derivatevon Galactosamin, Lactose, Glucose, N-Acetyl-D-Glucosamin und Galacturonsäurean Nucleosil 100, kombiniert mit UV-Detek-tion (λ = 377 nm). ChromatographischeBedingungen wie in Tabelle 5.

Fig. 4:Determination of galactose (98 µg/L),xylose (100 µg/L), and glucuronic acid (201µg/L) in a landfill leachate after derivatizationwith p-AMBA. Fluorescence detection (λex =313 nm, λem = 358 nm). Chromatographicconditions as in Table 5.

Bestimmung von Galactose (98 µg/L), Xy-lose (100 µg/L) und Glucuronsäure (201µg/L) in einem Deponiesickerwasser nachDerivatisierung mittels p-Aminoben-zoesäure. Fluoreszenzdetektion (λex =313 nm, λem = 358 nm). Chromatogra-phische Bedingungen wie in Tabelle 5.

Table 5: Chromatographic separation conditions.

Chromatographische Trennbedingungen.

Derivatization reagent p-Amino-benzoic acid

(p-AMBA)

p-AMBApropyl ester

2(2-Amino-phenyl)indole

1-Amino-pyrene

4-Amino-azobenzene

Separation column AQUA 5 AQUA 5 AQUA 3 Nucleosil NucleosilEluentcompo-sition

phosphate buffermethanol –gradient, %

100/0in 60 minto 50/50

60/40in 30 minto 25/75

45/5515 min isocrat.,

in 25 minto 0/100

65/35in 35 minto 0/100

50/50in 30 minto 20/80

flow, mL/min 0.5 0.5 0.5 0.6 0.6pH 2.0 2.1 without buffer 2.1 2.1ion-pair reagent TBAHSO4

20 mMTBAHSO4

5 mM / / /

tr, min derivat. reagent 10.1 22.7 18.3 33.8 26.2galactosamine 9.3 7.1 n. d. 25.2 11.8lactose 14.5 14.5 8.4 26.4 14.5glucose 18.1 17.7 32.5 28.7 17.8galacturonic acid 21.1 18.5 n. d. 30.1 20.5N-acetyl-D-galactosamine

25.2 19.9 31.0 31.2 18.7

n. d.: no data (no derivate formation).



Table 6: Mean ± SE of concentrations of short chain carbohydrates in landfill leachates. 3 subsamples of each sample were deriva-tized and each derivatization solution was threefold injected. Leachates of the landfill of Mertesdorf (A.R.T., Trier).

Mittelwerte und Standardabweichungen der Gehalte von kurzkettigen Kohlenhydraten in Deponiesickerwässern.

Carbohydrate Lactose Galactose Xylose Galacturonic acid Glucuronic acidµg/L

Detection limit 30 27.5 20 20 20

Sampling date14.02.2000 n. d. 98 ± 22 100 ± 11 n. d. 201 ± 5516.02.2000 63 ± 11 30 ± 7 42 ± 5 67 ± 20 47 ± 1318.02.2000 97 ± 17 45 ± 10 57 ± 7 73 ± 22 51 ± 14

n. d.: not detectable.

Table 7: Mean ± SE of concentrations of xylose and N-acetyl-D-glucosamine in lysimeter percolates.3 subsamples of each samplewere derivatized and each derivatization solution was threefold injected.

Mittelwerte und Standardabweichungen der Gehalte von Xylose und N-Acetyl-D-glucosamin in Lysimeterperkolaten.

Sample 1 2 3a 4 5 6 7b 8c 9d

Analyte µg/L

Xyl 36 ± 4 54 ± 7 54 ± 7 70 ± 7 71 ± 6 60 ± 6 62 ± 5 38 ± 4 77 ± 10GlcNAc 44 ± 7 n. d. 36 ± 6 29 ± 6 72 ± 6 34 ± 5 38 ± 6 39 ± 5 42 ± 7

a plus 41 µg/L of galactose and 51 µg/L of glucoseb plus 43 µg/L of glucosec plus 126 µg/L of galacturonic acidd plus 117 µg/L of galacturonic acid and 38 µg/L of glucuronic acidn. d.: not detectable

25 min and 31 min, followed by the surplus reagent (33.8 min).According to the chromatographic separation of p-AMBA pro-pylesterderivates the laterelutingcomponentsaresideordeg-radation products of the derivatization reaction mainly.

The separation between galacturonic acid and N-acetyl-D-glu-cosamine is incomplete.Furthermoreapartial peakoverlayoc-cursbetween the latter component andanunknowncompoundif UV absorbance is monitored.This interference does not ap-pear applying fluorescence detection.

As Figure 3 demonstrates, a complete separation of the refer-ence compounds is achievable after derivatization with4-aminoazobenzene.The Nucleosil RP-C18 column was em-ployed as stationary phase. The selected chromatographicconditions facilitateahigh resolutionof all compounds.Thesur-plus reagent elutes after its derivates, approximately 5 min af-ter the latest eluting analyte (galacturonic acid).This excludes

chromatographic interferences between 4-aminoazobenzeneand its derivates.

Adequate conditions for the separation of the carbohydratederivates of 2-(2-aminophenyl)indole could not be found yet.Applying the AQUA 3 column, the retention time difference be-tween lactose (8.3 min) and glucose (32.5 min) was extremelyhigh, but the surplus reagent eluted in between and the resolu-tion between N-acetyl-D-glucosamine and glucose was verypoor. Galacturonic acid coeluted with the reagent. Using theNucleosil column, an identification of the derivates of lactose,galacturonic acid, and glucose was possible, but the latter co-eluted.

Table 5 summarizes the finally selected separation conditionsand the corresponding retention times of the analytes and oftheaminationagents.Toseparate thederivatesofp-aminoben-zoic acid and of its propyl ester, the addition of an ion-pair rea-



gent to the eluent is necessary or at least advantageous. Ex-cept p-AMBA the reagents are more hydrophobic than theirderivates.Therefore they are more strongly retained by the sta-tionary phases than their reaction products. Omitting the datafrom the imperfect determination of the 2-(2-aminophenyl)in-dole derivates, the retention time difference between the firstand the last completely resolved analyte peak was highest inthecaseof thep-AMBApropylesterderivatesand lowestsepa-rating the1-aminopyrenederivates.The fastest separationwasaccomplished with the p-AMBA propyl ester derivates, lasting20 min. But due to the subsequent elution of the surplus rea-gent and of side or degradation products, this result is of limitedpractical value.In contrast to this, the separation of the p-AMBAderivates can be performed with a total run time of 30 min.

3.3 Environmental analytical applications

Applying p-AMBA as amination agent, first attempts weremade to evaluate the performance of the developed method inthe environmental analysis of the target compounds.This rea-gent was chosen because of the achievable low detection lim-its. As analytical matrices served several leachates from thesanitary landfill of Trier and lysimeter percolates.The sampleswere separated on an AQUA 5 column.The peaks were identi-fied by comparison with the retention times of standard com-pounds and by standard additions.

A typical chromatogram obtained from the analysis of a landfillleachate is shown in Figure 4.The chromatographic window forthe elution of the analytes is marked by the p-AMBA peak andby the glycerinaldehyde signal. Galactose, xylose, andglucuronic acid are detectable in concentrations between0.1 mg/L and 0.2 mg/L. Moreover the chromatogram displayssome further peaks having intensities comparable to those ofthe quantified analytes. It can be assumed that these signalsbelong to carbohydrates too, but their identity is still an openquestion.

Table 6 provides an overview over the carbohydrate contents ofthe sampled landfill leachates.Each sample was divided into 3subsamples and all of them were derivatized individually.Thaneach of the reaction solutions was threefold injected. In totalfive compounds were detected, i.e. the group of the alreadymentioned components, enlarged by lactose and galacturonicacid.The determined concentrations ranged between 30 µg/Land 200 µg/L.

Table 7 compiles the carbohydrate contents of 9 lysimeter per-colates.XyloseandN-acetyl-D-glucosamine (the latterwithex-ceptionofsample2)were found inall samples.Thevariationsofthe concentrations of xylose (single values 36...77 µg/L, mean58 µg/L) and of N-acetyl-D-glucosamine (single values29...72 µg/L,mean42µg/L)were relatively low.Galactose,glu-cose, galacturonic acid, and glucuronic acid were less fre-

quently detected. The concentration of galacturonic acid sur-passed the other compounds significantly.

As theTables 6 and 7 elucidate, the reproducibility of the analyt-ical data is satisfying with respect to the complex sample matrixand to the low analyte concentrations. It has to be emphasizedthat the calculated standard deviations are valid for the wholeanalytical process including the derivatization step. Excludingthe derivatization step, the precision of the determination pro-cedure is 2 to 6 times higher.

On the basis of our current results a few remarks about the dif-ferent composition of the investigated water samples can bemade.N-acetyl-D-glucosamine was found in almost all lysime-ter percolates, but not in the landfill leachates.Contrary lactosewas present in the landfill leachates only.Xylose was detectedin all samples.The carbohydrate composition of the landfill lea-chates was more diverse than the lysimeter percolates.

4 Conclusion

A series of amination reagents, which were previously used incombination with capillary electrophoresis mainly or whichwere not considered for analytical purposes yet, were exam-ined with respect to their suitability for the derivatization andsubsequent chromatographic separation of carbohydrates.Most of them facilitated the reductive amination of mono- anddisaccharides, uronic acids, and amino sugars.In most cases asimultaneous and sensitive determination of the derivates waspossible by means of ion-pair or RP-HPLC, hyphenated withUV or fluorescence detection. Especially the amination rea-gent p-aminobenzoic acid proved very worthwhile for the anal-ysis of various kinds of water samples.The combination of se-lective derivatization and selective fluorescence detection ofthe derivates enables the determination of carbohydrates inhighly loaded waters, i.e., landfill leachates, with very little influ-ence by the matrix components.Under these conditions detec-tion limits of about 0.1 µmol/L are attainable.

Besides p-AMBA several other amination reagents, especiallyp-AMBA propyl ester and 4-aminoazobenzene, bear a certainpotential tobeutilized in theenvironmentalanalysisof carbohy-drates. The propyl ester offers the advantage of an enhancedretardationof thesurplus reagenteluting after thederivates un-der chosen chromatographic conditions.This prevents the co-elution with galactosamine and possible interferences withother early eluting compounds.The separation of the 4-amino-azobenzene derivates is marked by a good resolution of all ref-erence compounds.

In some respects the achieved results are not fully satisfying.One point is the remaining surplus of reagent which gives riseto chromatographic interferences with the analytes in cases



especially where the reagent is eluting before its reaction prod-ucts. Others are the appearance of chromatographic signalspresumably belonging to side products of the derivatization re-action or to decomposition products of the derivates.

Further investigations including an enlarged selection of envi-ronmentally relevant carbohydrates are necessary to evaluatethe analytical capability of the various reagents in greater detailand to refine the derivatization and separation conditions.

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[Received: 13 September 2002; accepted: 22 April 2003]

Simultaneous and Sensitive HPLC Determination of Mono- and Disaccharides, Uronic Acids, and Amino...

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