Monosaccharide compositional analysis of marine polysaccharides by hydrophilic interaction liquid chromatography-tandem mass spectrometry

ORIGINAL PAPER

Monosaccharide compositional analysis of marinepolysaccharides by hydrophilic interaction liquidchromatography-tandem mass spectrometry

Qiuju Gao & Ulrika Nilsson & Leopold L. Ilag &

Caroline Leck

Received: 5 October 2010 /Revised: 16 December 2010 /Accepted: 21 December 2010 /Published online: 11 January 2011# Springer-Verlag 2011

Abstract A simple and sensitive method was developedusing hydrophilic interaction liquid chromatography cou-pled to tandem mass spectrometry for determination ofmonosaccharides liberated from marine polysaccharides byacidic hydrolysis. Optimal separation of diastereomericmonosaccharides including hexoses, pentoses, and deoxy-hexoses was achieved using an aminopropyl bondedcolumn with mobile phase containing ternary solvents(acetonitrile/methanol/water) in conjunction with MS/MSin SRM mode. Mechanisms for fragmentation of deproto-nated monosaccharides with regard to cross-ring cleavagewere proposed. Matrix effects from coeluting interferenceswere observed and isotopic-labeled internal standard wasused to compensate for the signal suppression. The methoddemonstrated excellent instrumental limits of detection(LOD), ranging from 0.7 to 4.2 pg. Method LODs rangefrom 0.9 to 5.1 nM. The proposed method was applied tothe analysis of polysaccharides in seawater collected fromthe open leads of the central Arctic Ocean in the summer of2008.

Keywords Hydrophilic interaction liquid chromatography .

Tandem mass spectrometry .Monosaccharides .

Polysaccharides . Marine microcolloids . Marine gels

Introduction

The radiative or reflective (albedo) properties of clouds(particularly marine stratocumulus in mid to high latitudes)are strongly dependent on the number concentration ofairborne water-soluble particles known as cloud condensa-tion nuclei (CCN) [1]. However, a critical element inimproving our ability to assess the potential role of marinebiogenic CCN in climate forcing is related to theuncertainty in explaining the maintenance of the numberof CCN over remote marine areas. Blanchard et al. [2, 3]have long advocated that a significant proportion of theremote oceanic CCN is derived from bubble bursting. Thebubbles result from entrainment of air induced by windstress at the air–water interface. In this process, bubblesscavenge sea salt, debris, and high molecular weightsoluble organic surface-active compounds as they risethrough the water prior to their injection into the atmo-sphere. There is recently a substantial body of evidencesuggesting that the microcolloids and their gels (amorphousgel-like material), called exopolymer secretions or micro-gels [4], derived from microbes as a result of extracellularexcretion, grazing, and cell-lysis, can be transferred bybubble bursting from the sea-air interface into the atmo-sphere and play a potential role in cloud formation [5–9].

Marine microcolloids with high molecular weight(HMW) ranging from 1 to 200 kDa, comprise a major partof the organic matter in the ocean, of which about 30∼50%is composed of polysaccharides with a minor fraction ofassociated protein and lipids [10, 11]. Characterization ofthe molecular composition of marine polysaccharides isprimarily accomplished by the study of monosaccharidesreleased by acidic hydrolysis. Techniques commonly usedfor monosaccharide determination include gas chromatog-raphy with flame ionization detector [12] and high

Q. Gao (*) : C. LeckDepartment of Meteorology, Stockholm University,10691 Stockholm, Swedene-mail: [email protected]

U. Nilsson : L. L. IlagDepartment of Analytical Chemistry, Stockholm University,10691 Stockholm, Sweden

Anal Bioanal Chem (2011) 399:2517–2529DOI 10.1007/s00216-010-4638-z

performance anion exchange liquid chromatography withpulsed amperometric detection (HPAEC-PAD) [11], both ofwhich lack sufficient selectivity and consequently it unableto overcome the interferences from complex matrix. Liquidchromatography coupled to tandem mass spectrometry (LC/MS/MS), as a highly selective and sensitive technique, ispotentially advantageous and has recently obtained wide-spread interest. However, the traditional reversed phaseseparation technique is limited in its capability to retainhighly polar and hydrophilic saccharides with numeroushydroxyl groups. For carbohydrates in various matrices,there are recently some successful LC/MS applications withhydrophilic interaction liquid chromatography (HILIC)reported (i.e., food or biological fluids) employing adductformation between carbohydrates and positive or negativeions (i.e., Cs+, I−, HCOO−, and CH3COO

−) [13–16], mostlyby means of postcolumn techniques. The postcolumnaddition makes the instrument setup more complex andthe instability of the systems makes it unsuitable for routinequantification. The presence of undesired clusters of multiadduct ions further complicates the interpretation of themass spectrum. The insufficient sensitivity of those meth-ods outlined above limits the application for trace levels ofsaccharides in environmental samples with complex matrix(i.e., seawater and atmosphere). Furthermore, none of theLC/MS/MS methods has addressed the difficulties associ-ated with simultaneous separation and quantification of alldiastereometric monosaccharides naturally occurring in themarine environment including pentoses, hexoses, anddeoxy-saccharides.

The objective of this paper is to present a sensitive andsimple method for the determination of monosaccharides inenvironmental samples by HILIC/MS/MS without requir-ing ion adduct formation and postcolumn addition. Themonosaccharides selected for the initial phase of this studyare three pairs of diastereomers including hexoses (glucoseand galactose), deoxyhexoses (fucose and rhamnose), andpentoses (xylose and arabinose). The applicability of thedeveloped method was demonstrated by analysis ofseawater samples collected from the open leads of thecentral Arctic Ocean in summer.

Experimental

Chemicals and materials

D-Arabinose, L-fucose, D-galactose, D-glucose, L-rhamnosemonohydrate, D-xylose, D-glucose 13C6, trifluoroacetic acid(TFA), HPLC grade acetonitrile (ACN), and methanol(MeOH) were purchased from Sigma-Aldrich (St. Louis,MO, USA). Ultrapure water (18.2 MΩ cm) was obtainedfrom a Millipore (Billerica, MA, USA) water purification

system equipped with a multi-wavelength UV lamp and anorganic acid polishing cartridge. Solid-phase extraction(SPE) cartridges were Bond Elut AccuCAT (mixed-mode,SCX, and SAX; 200 mg and 3 ml) from Varian Inc (HarborCity, CA, USA).

Standards and sample preparation

Individual stock solutions of each monosaccharide andinternal standard (IS) D-glucose 13C6 were prepared at aconcentration of 1 mg mL−1 with water. Working standardsolutions were prepared by appropriate dilution of the stocksolutions with ACN/H2O (80:20 v/v). External calibrationstandard solutions were prepared at concentrations from 10to 1,000 ng mL−1. Each external standard solutioncontained IS at a concentration of 200 ng mL−1. Allsolutions were stored at 5 °C.

Seawater samples (triplicate collection, 10 L each)collected in the Arctic pack ice area was used to isolatemicrocolloidal fraction (5 to 220 kDa) based on the methodfrom Gao et al. [17]. Twenty milliliters of desalted retentatewas obtained by ultrafiltration onboard for subsequentanalysis. An aliquot of 1 mL of the desalted retentate wasplaced in 1.5 mL Eppendorf vials and was vacuum dried(Eppendorf concentrator 5310, Hamburg, Germany). Theresidue was dissolved by 2×1 mL of 4 M TFA. Thesolutions were transferred into hydrolysis tubes and werehydrolyzed at 100 °C for 2 h. Excessive TFA was removedby vacuum evaporation and the residue was reconstitutedwith 1 mL of water. Further cleanup was accomplished bySPE. The SPE cartridge was preconditioned by 3 ml ofmethanol followed by 3 ml of ultrapure water. The samplesolution was loaded onto the cartridge and the eluent wasdirectly collected with 2×3 mL of water. The eluate wasevaporated to dryness under vacuum and then reconstitutedwith 1 mL (or greater if response was too high) of ACN/H2O (80:20 v/v) for LC/MS/MS analysis. An amount of200 ng of IS was added to the reconstituted solution. Forevaluation of matrix effects, the vacuum-dried residue wasreconstituted to 20 mL with ACN/H2O (80:20 v/v) and wasused to prepare matrix-containing standard solution (videinfra). Blanks were prepared by filtering 10 L of ultrapurewater and thereafter treated in the same way as for theseawater samples described above.

Liquid chromatography/mass spectrometry

LC separations were performed on an Accela system(Thermo Fisher Scientific, San Jose, CA, USA) using anaminopropyl-silica column (150×2.1 mm, 5 μm, ZorbaxNH2) from Agilent Technologies (Santa Clara, CA, USA)equipped with a 4×2 mm guard column (Agilent) at roomtemperature. Samples were injected with Thermo Accela

2518 Q. Gao et al.

auto-sampler (Thermo Fisher Scientific). The mobile phasewas composed of acetonitrile, methanol and water(70:10:20, v/v). The flow rate was 400 μL min−1. Injectionvolume was 5 μL.

The LC system was coupled to a triple-quadrupole massspectrometer (TSQ Vantage, Thermo Fisher Scientific)equipped with heated electrospray ionization (H-ESI)probe. The ionization interface was operated in the negativemode. The mass spectrometer was tuned and calibratedusing solution containing polytyrosine-1,3,6 by directinfusion at flow rate of 5 μL min−1. Operating parameterswere fine tuned for maximum intensity of the deprotonatedprecursor ions (M−H)− in full scan mode (m/z 50–500) bydirect infusion of standard solution (2 ng mL−1) with amixing tee union at a flow rate of 10 μL min−1 for analyteand 400 μL min−1 for mobile phase (ACN/H2O, 80:20 inv/v). Typical optimized conditions are shown in Table 1.Argon was used as collision gas. Data were acquired andprocessed using Xcalibur 2.0 software package (ThermoFisher Scientific). Quantification was undertaken in select-ed reaction monitoring (SRM) mode with the ion transitionsoutlined in Table 2 together with optimized collision energyand S-lens voltages for each transition.

Evaluation of matrix effects

Matrix effects were investigated by construction of calibrationcurves using matrix-containing solution (matrix-adapted)and matrix-free mobile phase. The preparation of matrix-containing solution was described in “Standards and samplepreparation.” Each calibration curve contained six concen-tration levels. The slopes were compared statistically using aStudent’s t test. The pooled standard deviation (sp) of theslopes between the two set of calibration curves wascalculated and the computed t value was compared with acritical t value (tc) tabulated at 95% confidence level todetermine if the matrix effect is significant.

Method validation

The linearity was evaluated from standard solutions intriplicate at six concentrations ranging from 10 to1,000 ng mL−1. The square of the correlation coefficients(r2) of the calibration curves were determined by linearregression. The calibration curve was assumed linear whenr2 was greater than 0.99. The instrumental limit of detection(LOD) and the limit of quantification (LOQ) wereestimated for a signal-to-noise ratio of 3 and 10, respec-tively. Precision was expressed in terms of relative standarddeviation (%RSD) in quintuplicate at two different concen-tration levels (40 and 400 ng mL−1). Method accuracy wasestimated by means of SPE recovery experiments at levelsof 40 and 400 ng mL−1.

Results

Mass spectrometric method setup

Electrospray ionization (ESI) of monosaccharides in nega-tive ion mode was chosen in the present study sincemonosaccharides in positive ion mode gives rise to multipleadducts formation of (M+Na)+ or (M–H+2Na)+ with theubiquitous Na+ from glassware. Furthermore, adducts withalkali metals are often difficult to fragment efficiently byuse of collision induced dissociation. Fragmentation pat-terns of each precursor ion (M–H)− was investigated.Typical MS/MS product ion spectra of hexose, pentose, and

Table 1 Working parameters of H-ESI-MS/MS

Spray voltage (v) −3,500Capillary temperature (°C) 300

Vaporizer temperature (°C) 250

Sheath gas pressure (arbitrary unit) 40

Auxiliary gas pressure (arbitrary unit) 5

Ion sweep gas pressure (arbitrary unit) 0

Declustering voltage (v) 10

Collision gas pressure (mTorr) 0.5

Mass resolution (Da)a

Q1 0.7

Q3 0.7

aMass resolution at full width at half maximum

Table 2 SRM specifics for all analytes of interest and the optimumconditions

Monosaccharide SRMtransition

CE(eV)

S-Lens(v)

Scantime (s)

Scanwidth(m/z)

Xyl 149.06>59 15 22 0.2 0.020149.06>89 5 22

Ara 149.06>59 15 22 0.2 0.020149.06>89 5 22

Rha 163.06>59 15 25 0.2 0.020163.06>103 10 25

Fuc 163.06>59 15 25 0.2 0.020163.06>103 10 25

Glu 179.06>59 15 28 0.2 0.020179.06>89 5 28

Gal 179.06>59 15 28 0.2 0.020179.06>89 5 28

13C6-Glu 185.06>61 15 28 0.2 0.020185.06>92 5 28

Xyl D-xylose, Ara D-arabinose, Rha L-rhamnose monohydrate, Fuc L-fucose, Glu D-glucose, Gal D-galactose

Monosaccharide compositional analysis of marine polysaccharides 2519

deoxy-hexose are shown in Fig. 1. Monosaccharidesselected in this study showed similar MS/MS fragmentationpathways with relatively abundant product ions at 59, 71,89, and 119m/z. Deoxyhexoses yield an additional distinctfragment at m/z 103. A major fragmentation pathway forsaccharides is believed to be cross-ring cleavage and neutrallosses [18–20]. The product ions at m/z 119, 103, 89, and59 can be explained by the neutral losses corresponding tounits such as (CH2O)2, (CH2O)3, (CH2O)4, or CH3CHO-

CHOH. Ions at m/z 71 are produced by the loss of H2Ofrom the fragment dissociated at C3–C4. A typical frag-mentation pathway of monosaccharides is proposed inScheme 1. A considerable degree of fragmentation wasobserved when collision energy (CE) was set below 25 eVand collision gas pressure below 1 mTorr. Optimized CEfor each ion and each transition is shown in Table 1.

Offset voltage applied to S-lens (an implementation ofstacked rings), ion optics located closest to atmospheric

Rel

ativ

e A

bund

ance

Rel

ativ

e A

bund

ance

a

b

Fig. 1 MS/MS product ionspectra of (M−H)− of monosac-charides at CE of 10 eV a Xyl,b Rha, c Glu, and d IS

2520 Q. Gao et al.

pressure ionization source, was evaluated by loop injectionin MS/MS mode to obtain a maximum intensity of signal.The optimum value of S-lens voltages varies slightly witheach compound (Table 2), but are all below the values

(55∼70 V) obtained by automatic tuning with polytyrosine-1,3,6. About 100-fold or greater increase in terms ofabsolute ion counts was observed for all analytes of interestversus the auto-tuned settings.

Rel

ativ

e A

bund

ance

Rel

ativ

e A

bund

ance

c

d

Fig. 1 (continued)


Fragmentation of monosaccharides can also be inducedby elevating the temperature of the heated capillary transfertube, which assists in desolvation of emitted droplets.Increase of capillary temperature from 200 to 300 °Cyielded no significant signal reduction or improvement.Decreased sensitivity was observed when capillary temper-ature was lower than 200 °C probably due to insufficientdesolvation. When the capillary temperature was increasedto 320 °C or higher, fragment ions (i.e., m/z 59, 89, or 119)were observed in the mass spectrum suggesting a heat-induced decomposition. A dramatic reduction of signalintensity was also found when the temperature of auxiliarygas (controlled by the vaporizer in H-ESI probe) was higherthan 270 °C. The performance of H-ESI is compounddependent and caution has to be taken in application of

labile compounds to avoid quantitative artifacts introducedthermally.

ESI is a suitable interface for MS of polar and thermallylabile compounds. Volatile additives (i.e., ammonium saltsor formic acid) are very often used to deprotonate acidicmolecules in negative ion mode and vice versa for positivemode. However, ESI is more challenging for monosacchar-ides than other bio-molecules, since the neutral moleculesare not easily ionized in the initial solution under mild pHconditions. Gas-phase proton transfer reaction has beensuggested for the formation of the ions observed [21, 22].In the case of the proton transfer mechanism, it has beenshown that the signal response did not increase with thepresence of additives in the mobile phase [23], andprotonated molecules were often observed with alkali

m/z 131

-

m/z 59

m/z 113

-

- 18 Da

Ring opening

- 60 Da

A

- 18 Da

- 18 Da

m/z 71

-

m/z 149

m/z 89

-

-- 90 Da

–

OHO

OHO

OH

––

–

–

–

–

–

–H2O

H2O

H2O

Scheme 1 ESI-MS/MS fragment pattern of deprotonated Xyl (a), Rha (b), and Glu (c)

2522 Q. Gao et al.

mobile phases and deprotonated molecules under acidicconditions, referred to as “wrong-way-round ioniza-tion”[24]. Some reports indicated that additives in mobilephase lead to ion suppression [25]. The effect of additiveson ESI response was examined semi-quantitatively in ourstudy by loop injection in MS/MS mode with LC columnomitted. Two common mobile phase additives includingformic acid (0.1%) and ammonium acetate (5 mM) were

investigated. Neither showed signal enhancement comparedwith the mobile phase with absence of additives. Theaddition of formic acid shows a reduction of about 10% insignal intensity in terms of absolute ion count. Signalreduction was more evident with mobile phase containingammonium acetate—about 30% of decrease was observed.The decreased ESI response is probably arising from thecompetition of proton transfer reaction between analytes and

-

m/z 145

Ring opening

- 60 Da

-CH3CHO

B

- 44 Da

- 30 Da

- 18 Da

- 18 Da

- CH2O

-

m/z 85

m/z 59

m/z 119

-

m/z 71

m/z 103

m/z 163

-

m/z 89

-- 74 Da

- 18 Da

H2O

H2O

H2O

HO

HOHO

O

Scheme 1 (continued)


additives [25]. Deposition of ammonium salts on the ESIinterface is another reason responsible for the decrease insensitivity [20]. Ammonium hydroxyl as alternative additivecommonly used in LC/MS/MS-negative ion mode was notexamined in this study since the presence of ammoniumhydroxyl in mobile phase can deteriorate the silica support ofthe LC column [26]. In fact, ammonium salts or formic acid,

even at low concentrations, have been found to have thiseffect also on an amino-bonded LC column [20].

Optimization of LC conditions

Monosaccharides existing in multiple isomeric formscannot be readily differentiated by MS/MS. Chromato-

-

m/z 161

Ring opening

- 60 Da

C

- CH2O

m/z 143

- 18 Da

- 18 Da

m/z 71

m/z 59

-

m/z 101

-

- 30 Da

-

m/z 119

m/z 179

m/z 89

-

-- 90 Da

- 18 Da

- 18 Da

OHOH

–

OHO

OH

–

–

–

– – –

––

–

H2O

H2O

H2O

H2O

Scheme 1 (continued)

2524 Q. Gao et al.

graphic retention is thus highly desired for their resolution,as well as for minimizing ionization suppression caused bycoeluting matrix. Chromatographic separation of stereo-isomic monosaccharides has been very challenging becauseof their similar retention behavior and their hydrophilicproperties. Some studies showed carbohydrate separationby HILIC on amide bonded column [26], where the isomersbetween glucose and galactose coeluted. Reducing mono-saccharides appeared as double anomeric peaks unless anelevated column temperature (60 °C or greater) wasemployed. Such doublet peaks are undesirable for precisequantitative analysis of monosaccharides. In this work,aminopropyl bonded silica gel column was chosen forchromatographic separation of the isomers. Amino columnis one of the most widely used LC columns in the field ofcarbohydrate study using acetonitrile and water as typicalmobile phase composition. The advantage of aminopropyl-silica over amide-bonded column is the high rate ofmutarotation which prevent the formation of anomericpeaks [27]. The drawback of amino-based column is thataldehyde functional groups in monosaccharides react withamino groups of the stationary phase by carbonyl-aminecondensation yielding a Schiff base which leads todeterioration in column performance on prolonged use[28]. In addition, mobile phase with high water content(>40%) is detrimental to the stability of silica-basedpackings which consequently limits the lifetime of thecolumn[29]. A potential solution to stabilize the aminocolumns by partial replacement of water with methanol inthe acetonitrile/water system was previously reported onseparation of mono-, di-, and tri-saccharides [30]. Glucosewas the only monosaccharide selected in their study and thepotential for separation of diastereomers by the ternaryeluents was not investigated. In this work, the chromato-graphic performance using binary (acetonitrile/water) andternary (acetonitrile/methanol/water) mobile phase systemto separate isomeric monosaccharides was evaluated.Mobile phase with acetonitrile and water at isocratic elution(80:20 in v/v) was chosen initially. Monosaccharides wereeluted in the order of increasing polarity (expressed bythe number of available hydroxyl groups, pentoses<deoxyhexoses<hexoses). The retention decreased whenwater content increased suggesting a normal phase chro-matographic behavior. Peak broadening and poor separationefficiency were observed when acetonitrile content washigh (>85%). For the ternary system with a constantacetonitrile content, reducing methanol content shortenedretention time which was in accordance with the literaturementioned above [30]. Figure 2 shows the retention,represented by the retention factor, k′, of six monosacchar-ides at some binary and ternary conditions. With 20%methanol and 10% water in a ternary system, retentioncapacity is close to those obtained in the binary eluent

containing 20% water. Given the compromise betweenselectivity and sensitivity, mobile phase containing ACN,MeOH, and H2O at 70:10:20 (in v/v) was chosen as theoptimal condition. Figure 3 demonstrates the improvementof chromatographic performance when 10% of water inbinary system was replaced by methanol. It can be seen thatthree pairs of isomers were almost baseline resolved andboth peak shape and signal intensity were improved whenwater was partially replaced with methanol.

Matrix effects

To evaluate if matrix components contribute to ESI signalenhancement or suppression, the standard curves con-structed in matrix-adapted solution were compared withthe external standard line [31]. The results (Table 3) showthat the calibration curves were linear, with r2>0.99, over aconcentration range of 10–1,000 ng mL−1 in all cases. Thelower slopes in the plots of matrix-adapted standardssuggest the occurrence of matrix-induced signal suppres-sion instead of enhancement. Student’s t test (at 95%confidence interval) indicates that the presence of matrixeffects is significant. Use of isotope-labeled IS is consid-ered the most powerful approach to compensate for matrixeffects [31]. Ideally, each analyte should be corrected by itsown isotope-labeled analogue since the matrix effects arevery often compound dependent. One IS, theoretically, isnot adequate for multi-component analysis in the case whenmatrix effects exhibit different patterns for differentanalytes. In this study, the variability in the signalsuppression effect for six monosaccharides, expressed ascoefficients of variation (CV), was considered as a measureof deviation for the absolute matrix effects. An acceptableCV value of 3.5% suggests that the difference in signalsuppression patterns between each monosaccharide isnegligible and therefore it should be sufficient to utilizeone IS for multiple monosaccharides analysis.

0

1

2

3

4

5

6

7

80:0:20 70:0:30 70:10:20 70:20:10

k´

Xyl

Ara

Rha

Fuc

Glu

Gal

Fig. 2 Overview of retention factors (k′) of monosaccharides usingmobile phase with different composition indicated as CAN/MeOH/H2O


Method validation

Linearity, LOD and LOQ, accuracy, and precision of thismethod are summarized in Table 4. Instrumental LODs and

LOQs range from 0.7 to 4.2 pg and 2.2 to 13.8 pg,respectively. Lack of precision, as indicated by %RSD, isbelow 12% for both low and high concentration levels. Theaccuracy of the method, calculated as the percentage of the

XylAra

Rha Fuc

Glu

Gal

0

50

100

0

50

100

0

50

100

0

50

100

3.51

3.93

3.47

2.362.79

2.63

2.97

3.54

3.94

3.422.64

3.483.51

IS

a

b

NL: 2.91E4

TIC F: - p ESI SRM ms2 185.060 [60.990-61.010; 91.990-92.010] MS

NL: 3.29E4


NL: 2.25E4


NL: 2.18E4


NL: 2.20E4

TIC MS

0 1 2 3 6 7 8

0

50

100

4 5

0 1 2 3 6 7 8

0

50

1000

50

100

0

50

100

0

50

100

0

50

100NL: 2.63E4

TIC MS

NL: 4.71E4


NL: 3.49E4


NL: 3.36E4


NL: 2.99E4


3.29

2.87 4.853.83 5.30

3.29

3.86

2.87

3.51

4.83

5.33

4.85

4 5

Fuc

Rha

AraXyl

GalGlu

IS

Fig. 3 Reconstructed SRM chromatograms of monosaccharide standard mixture (200 pg of each injected on column) with mobile phasecontaining a ACN and H2O (70:30 in v/v) and b ACN, MeOH, and H2O (70:10:20 in v/v)

2526 Q. Gao et al.

ratio of the determined concentration to the theoreticalconcentration, ranged from 84% to 102%. The methodLODs and LOQs were expressed as molar concentrations ofindividual monosaccharides, resulting in a signal-to-noiseratio at 3 and 10 with an injection volume of 5 μL. Theresults reported in Table 4 imply the potential to detectcarbohydrate in seawater, generally at low nanomolar level,without extensive preconcentration steps. The method washighly specific as no significant signals were observed inthe blanks at the retention times of analytes.

Environmental application

The method was applied for the analysis of monosaccharidecomposition in seawater collected from the central ArcticOcean. The sea water samples studied here were collectedduring an expedition called ASCOS (the Arctic SummerCloud Ocean Study, www.ascos.se) on the Swedishicebreaker Oden to the central Arctic Ocean in the summerof 2008 [32]. Measurements commenced during that part ofthe expedition when the ship was moored to an ice floe at N87°09′, W 10°45′. Two sites were selected for sampling: (1)

ice-covered water beneath ice floe by a through-hull inletsystem at a depth of 8 m and (2) open lead water betweendrift ice taken by bucket at depth of 0.5 m. Standardsolutions for sample quantification underwent the sametreatment as the seawater samples from the hydrolysis step(see “Standards and sample preparation”). The identities ofthe target compounds were verified by comparing retentiontimes and SRM transitions between samples and standardsolutions. With preconcentration of seawater, the LC/MS/MS responses of some analytes obtained from the recon-stituted solution became too high and out of the linear rangeof the calibration curves. Appropriate dilution was madeuntil anticipated response was obtained. It is worth notingthat the preconcentration procedure is necessary in order tocollect colloidal organic matters with desired molecularweights in a representative way. A concentration factorbelow 100 for isolation of colloids from bulk seawatermight retain significant amount of low molecular weightcompounds, leading to an overestimation of HMW organicmatter [33]. Rigorous cleaning and proper handlingprotocol are required in order to obtain unbiased andreliable results [34]. Results are summarized in Table 5.

Table 4 Linearity, limits of detection (LOD) and limits of quantification (LOQ)

Analytes RTa (min) r2 LODb (pg) LOQb (pg) MethodLOD (nM)

MethodLOQ (nM)

%RSD Accuracy (%)

40 ng mL−1 400 ng mL−1 40 ng mL−1 400 ng mL−1

Xyl 3.29 0.9938 0.7 2.2 0.9 3.1 5 5 102 100

Ara 3.86 0.9967 1.5 5.0 2.0 6.7 10 1 86 101

Rha 2.87 0.9973 1.0 3.4 1.2 4.1 10 5 97 99

Fuc 3.51 0.9951 4.2 13.8 5.1 16.8 4 7 90 102

Glu 4.83 0.9970 1.3 4.3 1.4 4.8 12 5 95 96

Gal 5.33 0.9950 1.7 5.7 1.9 6.3 4 9 84 102

Xyl D-xylose, Ara D-arabinose, Rha L-rhamnose monohydrate, Fuc L-fucose, Glu D-glucose, Gal D-galactosea Retention timeb Instrumental LOD and LOQ indicated by the amount of analytes injected on column

Table 3 Statistical comparison of matrix-free and matrix-adapted calibration curves (n=6)

Analytes Matrix-free standards Matrix-adapted standards Pooled SD Calculated t value Tabulated t (95% confidence) SSE%a

Slope Intercept r2 Slope Intercept r2

Xyl 231 676 0.9997 176 10,152 0.9996 21 3.300 2.776 76

Ara 204 1,639 0.9995 157 1,531 0.9996 19 4.221 2.776 77

Rha 232 −149 0.9999 185 2,105 0.9998 17 4.263 2.776 80

Fuc 198 774 0.9992 147 8,838 0.9989 17 4.414 2.776 74

Glu 274 1,509 0.9989 199 10,295 0.9972 23 3.934 2.776 72

Gal 231 486 0.9989 171 12,647 0.9996 16 5.308 2.776 74

Xyl D-xylose, Ara D-arabinose, Rha L-rhamnose monohydrate, Fuc L-fucose, Glu D-glucose, Gal D-galactosea Percentage of signal suppression/enhancement defined as: SSE% ¼ 100� Slopematrix�adapted=Slopematrix�free


http://www.ascos.se

The RSD values determined from the triplicate samplesranged from 4% to 19%. The concentrations of mono-saccharides in seawater sample from high Arctic wereshown to range from 1.0 to 15.8 nM with higher value atopen lead (55.7 nM in total) than ice-covered seawater(35.6 nM in total). Relative molar percentages (mol%) ofmonosaccharides indicate that the monosaccharide compo-sition was mainly glucose, galactose, fucose and xylose.The contribution of arabinose and rhamnose was low(ranging from 1.8% to 6.3%). The relatively abundantxylose in seawater suggests the existence of biogenicpolymers of phytoplankton origin [35]. The presence offucose is indicative of surface-active polysaccharides andwas suggested to be a subunit of sulphated polysaccharidesfound from varies species of brown algae [36]. Furtherdiscussion on the details and the difference between ice-covered water and open lead water regarding monosaccha-ride profile and abundance is beyond the scope of thispaper.

Conclusions

Analysis of monosaccharides by LC/MS has long beenchallenging because of their high hydrophilicity and lowionization efficiency. HILIC, with high MS compatibility,shows a clear advantages over conventional methods, interms of both sensitivity and selectivity. In this paper, wedemonstrated the HILIC/MS/MS capability for separationand identification of diastereomeric monosaccharides. ESI/MS/MS involving cross-ring cleavage of deprotonatedmonosaccharides, without need of any acidic or basicmobile phase additive, can yield abundant fragment ionsand offer a sufficient sensitivity in SRM mode. It is evidentthat there still is considerable potential to be exploited inthe method development of HILIC/MS for trace detectionof carbohydrate. The proposed method represents the firstmeasurement of neutral monosaccharides naturally occur-ring in marine biopolymers by HILIC /MS/MS withoutderivatization, postcolumn addition or adducts formation.To date, HPAEC-PAD has been the predominant method of

choice for marine carbohydrate research. The application ofthe highly sensitive HILIC/MS/MS, as a promising alter-native to the traditional HPAEC-PAD, makes it possible fordetermine the in situ concentration of marine carbohydrateswith minimal sample pretreatment.

Acknowledgments This work is part of ASCOS (the ArcticSummer Cloud and Ocean Study: www.ascos.se) project. The authorswould like to thank the financial supports from the Swedish ResearchCouncil (VR) and from the Knut and Alice Wallenberg Foundation.

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Concentration(nM)

%RSD Mole (%) Concentration(nM)

%RSD Mole (%)

Xyl 12.9 6 23.2 8.5 4 23.9

Ara 1.0 18 1.8 1.8 17 5.1

Rha 3.5 19 6.3 1.9 17 5.3

Fuc 12.4 6 22.3 9.6 11 27.0

Glu 10.1 11 18.1 8.7 5 24.4

Gal 15.8 16 28.4 5.1 18 14.3

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Monosaccharide compositional analysis of marine polysaccharides by hydrophilic interaction liquid chromatography-tandem mass spectrometry