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Critical Reviews in Analytical Chemistry

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Disaccharides Determination: A Review ofAnalytical Methods

Marta Pokrzywnicka & Robert Koncki

To cite this article: Marta Pokrzywnicka & Robert Koncki (2018) Disaccharides Determination:A Review of Analytical Methods, Critical Reviews in Analytical Chemistry, 48:3, 186-213, DOI:10.1080/10408347.2017.1391683

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Disaccharides Determination: A Review of Analytical Methods

Marta Pokrzywnicka and Robert Koncki

Department of Chemistry, University of Warsaw, Warsaw, Poland

ABSTRACTDisaccharides are determined mainly for dietetic purposes, hence the most analyses are carried out forfood and drink samples. Its content can also be used to profile groceries in order to identify the origin andquality of the products. They also can be an indicator of the rate of metabolism as well as for the control ofsome technological and biotechnological processes. Unfortunately most of technological analysis areperformed with nonselective polarimetry methods. Sugars due to specific physicochemical properties ofcompounds are difficult to determine with classical analytical techniques. The most commondisaccharides are composed of several types of monomers connected by a different configuration of theglycosidic bond, therefore, there are subject of the same characteristic reactions. This often enforces theneed for pre-separation of sample components. Therefore, nowadays the most popular analyticalmethodologies for disaccharides determination are based on chromatographic and electrophoretictechniques. An alternative is enzymes application that allow both selective recognition of target analyteand its conversion to easy detected product, allowing detection by relatively simple conventionalanalytical methods. Another approach is the use of advanced chemometric methodologies for computingof data obtained from some spectroscopic techniques. This article is a review of the recent analyticalliterature devoted to non-selective and selective methods for disaccharide determination in real samples.

KEYWORDSChromatography;Disaccharides;Electrophoresis; Enzymaticmethods; Spectroscopy

1. Introduction

From a huge group of sugars, disaccharides are the most popu-lar analytes especially in food industry and agriculture. Amongthem the most popular are sucrose, maltose and lactose(Fig. 1), which were the main analytes reported in 85% of publi-cations cited in this review. It is no surprise that the most oftenanalysed disaccharide is sucrose (68%). In the 19th century thetable sugar became strategic resource with special dotation forproduction and quality control improvement. For many yearsit was recognized as the best source of energy, nowadays it is adietician’s nightmare. WHO considers it as one of the majoritysource of obesity and dental caries and strongly recommendreduction of intake.[1] Therefore sucrose is most often deter-mined in food products, especially soft drinks[2–14] andsweets.[13–17] Because of its plant origin, sucrose is also deter-mined in fruits and vegetables[17–26] (with particular emphasison sugar cane[27] and sugar beet[28–31]) and other plant mate-rial,[32–38] where it is an indicator of regular plant growth.[36]

There are also some examples of sucrose assays in urine[39–41]

and blood plasma[40–43] with special application to blood-brainbarrier permeability investigations.[43]

Similar concern as target analyte share lactose (37%) andmaltose (32%). Lactose, characteristic for mammals milk, isdetermined exactly in milk[44–50] as well as in cheeses,[51]

yogurts[52] and different dairy products.[53] Considering thatabout 50–75% of human population (data from different

sources[51,54]) suffer from lactose intolerance, not surprisingnumber of publications reported new methods for lactose assay.Finally, maltose occurs mostly in cereals grains. Except cerealproduct[55,56] and starch hydrolates[57,58] it is often determinedin alcoholic drinks[59] especially in beer[60–63] where togetherwith glucose it is a marker of progress of fermentation processas well as a marker of quality of final product. Of course notonly these three disaccharides are targets of analytical interest.There are also reported several methods for determination oftrehalose,[34,64–68] lactulose,[69–73] isomaltose,[56,74] cellobi-ose,[68,75–78] xylobiose,[75,79] mellibiose[78,80] and more.

Selective methods of disaccharides determination involvemany analytical challenges. Most of known disaccharides are iso-mers, with the same molecular formula, molecular weight andalmost the same functional group. Only small structural changessuch as the inversion of groups at a single chiral carbon atom or achange in the position of the carbonyl group, decide about differ-ent sweetness, solubility, and chemical reactivity. This diversity isclear even for monosaccharides, for example when simple mono-saccharides like glucose, galactose and fructose are compared. Incase of disaccharides it becomesmore complicated. Disaccharidesare compounds with acetal bond between anomeric carbon ofone monosaccharide molecule and any hydroxyl group of secondmonosaccharide, so more isomer variations appear. In Figure 2the structures of ten different disaccharides are presented. Each ofthem is built of two glucose units only and differ by anomeric

CONTACT Marta Pokrzywnicka [email protected] Department of Chemistry, University of Warsaw, Pasteura 1, Warsaw, 02-093, Poland.Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/batc.© 2018 Taylor & Francis Group, LLC

CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY2018, VOL. 48, NO. 3, 186–213https://doi.org/10.1080/10408347.2017.1391683

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form and order of bounded carbon atom in second molecule.Eight of presented compounds are reducing sugars and theyundergo the same characteristic assays based on Fehling’s,[81]

Smogy-Nelson[82] or Summer[83] methods. Other differences aretaste (for example different sweetness rate or bitter in case of gen-tiobiose[84]) and susceptibility to digestives enzymes. What isobvious, because of different configuration of stereogenic carbonseach of them have different specific rotation angle. These proper-ties could be a base for selective determination method, unfortu-nately effect of the light beam refraction is additive, and thereforesuch approach is useless in case of quantitative analysis of sugarmixtures. Nerveless, polarimetry together with other non-selec-tive techniques such as refractometry and hydrometry are cur-rently used in industrial analysis.[85]

2. Non-selective analytical methods

Similarity in disaccharides structures and properties causesmost of analytical methods non-selective. Therefore in manycases information about disaccharide content, especially in

food and beverages, is limited to total sugar content – theparameter which presents the total concentration disaccharidesand related monosaccharides in sample. This parameter pro-vides satisfactory information from nutritional point of view.In case of these assays there is no necessary to apply any selec-tive techniques. For such purposes, several methods based onnon-selective chemical reactions or physical properties of sugarsolutions have been developed.

2.1. Chemical methods

For estimation of total sugar content often phenol-sulphuricacid assay[86] is applied. This method is useful for determina-tion of reducing and non-reducing sugars in complex samples,also in the presence of salts and proteins residues. The assay isbased on measure of colour of aromatic complex absorbance at490 nm. The method was developed in the middle of XX cen-tury but is still applied because of its simplicity and availabilityof reagents. Also 3,4-dimethylphenol forming colour adductsexhibiting absorption maximum at 510 nm wavelength can be

Figure 1. Structures of three common disaccharides: sucrose, maltose and lactose.

Figure 2. Examples of disaccharides composed of two glucose units.

CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 187

applied for total sugar determination.[87] However because ofdifferent absorptivity of individual sugars, this method can besuccessfully applied only under specific conditions.

Inmany cases almost all sugars presented in sample have reduc-ing properties caused mainly by monosaccharides and reducingdisaccharides. There is a lot of methods for reducing sugars deter-mination and some of them are presented in Table 1. Predomi-nantly these are modifications of classic Fehling’s methoddeveloped in the middle of XIX century[81] based on the reductionof cupric ion to cuprous oxide. The main differences are in thetitrant and titrand. That could be dissimilarities in reaction condi-tions like in Loof–Schorl[88] and Ofner’s[89] methods or exchangethe role of sample and titrant as in case of Lane and Eynonsmethod,[88] where Fehlings solution with known cupric concentra-tion are titrated with sugar solution. Copper based reaction can bealso applied for spectrophotometric detection. Cuprous ions canreact with neocuproine to produce a yellow complex that stronglyabsorb the light of 457 nm wavelength. It is obvious that the deter-mination methods based on reducing properties of sugars arestrongly influenced by other reductans (almost the same methodsare applied for estimation of total antioxidant capacity assays)

Methods based on ferricyanide reduction also have somevariation. Except iodometric Mohr method[92] or simple photo-metric measurements of decay of ferricyanide absorption at480 nm wavelength[92,93] also formation of Prussian Blue, after

addition of ferric nitrate can be applied allowing observation ofabsorption increase at 690 nm wavelength.[35] Under flow anal-ysis condition, thanks to following measurements of hexacya-noferrate obtained after reduction by sugars from non-hydrolysed and hydrolysed sample, quite selective determina-tion of sucrose was possible in beet juices and syrups.[31]

Also benzoic acid derivatives containing nitrogen as 3,5-dinitro-salycilic acid (DNS)[83,91] and p-hydroxybenzoic acid hydrazide(PAHBAH)[94–96] can be reduced by sugars and form colouredproducts that absorb in visible range at 540 and 410 nm wave-length, respectively. An interesting case ismethylamine. Its reactionwith reducing sugars leading to formation product, which colourdepends on sugar structure.With monosaccharides it forms yellowproduct that absorb at 400 nm, whereas with oligosaccharides (dis-accharides and trisaccharides) violet- carmine adduct is formedthat absorbs light of 540 nm wavelength.[97] This way the discrimi-nation and determination of chosen saccharide groups is possibleas well as estimation of total reducing disaccharide content only ifoligosaccharides composed of higher number of monosaccharidesunits are not present.

Some reactions of reducing sugars allow to distinguish aldo-ses and ketoses. For example Seliwanoff’s test is characteristicreaction of ketohexoses, which in acidic solutions are convertedinto hydroxymethylfurfural forms that react with resorcinol togive red complexes with strong, characteristic absorption peaksat 398 and 480 nm wavelength. This qualitative test has beensuccessfully applied for quantitative photometric determinationof lactulose in pharmaceutical samples.[98] Hydroxymethylfur-fural can react also with cysteine hydrochloride–tryptophanreagent producing pink chromophore with maximum absorp-tion at 518 nm. This reaction also has been applied for determi-nation of lactulose in pharmaceutical products.[99]

So-called Raybin test based on reaction of sugar with 5-diaz-ouracil gives positive results (bluish precipitate) for compoundscontaining 1,2-glycosidic bond such as sucrose, raffinose or sta-chyose, while palantinose and melezitose also containing thisbond type yielded a reddish-brown precipitate. This test hasbeen adapted for photometric determination of sucrosetogether with rafinose in honeys samples.[100]

Reducing sugars in alkaline solution tautomerize to form eno-diols. These compounds can react with zirconyl chloride to formfluorescence derivatives. Tautomerisation to enodiols is muchfaster for ketoses, so under adequate conditions (lower reactiontemperature and shorter time) selective fructose determination canbe achieved. Similar fluorescent complexes with zirconyle chlorideare formed by fructose-based disaccharides, so this method is alsouseful for fluorometric determination of sucrose.[7]

Finally, in case of reducing sugars determination it is worthto mention about qualitative osazone test.[101] At high tempera-tures reducing sugars react with phenyl hydrazyne to form yel-low crystals called osazones. For each reducing sugar crystalswith different structure, precipitation time and melting pointare formed. Comparison of those parameters is useful for sac-charides differentiation.

2.2. Physical methods

Physical techniques developed for investigations of liquidslike polarimetry, hydrometry and refractometry also belong to

Table 1. Methods for reducing sugars determination. Table 1. Examples of non-selective chemical methods for determination of reducing sugars.

Method Reaction Detection Source

Lane and Eynon’smethod*

sugars reduce cupric ionsto cuprous oxide

end point titrationindicated bymethylene blue

[88]

Ofner’s method* sugars reduce cupric ionsto cuprous oxideexcess of cupric ionsare reduced bypotassium iodide

iodine titrated by sodiumthiosulphate withstarch as indicator

[89]

Loof -Schorlmethod*

sugars reduce cupric ionsto cuprous oxideexcess of cupric ionsare reduced bypotassium iodide

iodine titrated by sodiumthiosulphate withstarch as indicator

[88]

Knight and Allenmethod*


residual cupric ions aretitrated with EDTAwith murexide asindicator

[88]

Munson andWalkermethod


cuprous oxide are driedand weight

[89]

Smogyi- Nelsonmethod

sugars reduce cupric ionsto cuprous oxidecuprous oxide reducedmolybdic acid tomolybdenum blue

photometricdetermination ofmolybdenum blue at500 nm.

[82,86]

Sumner’s method sugars reduce 3,5-dinitrosalycilic acid to3-amino-5-nitrosalicylic acid

photometricdetermination of 3-amino-5-nitrosalicylicacid at 540 nm.

[83,91]

Hagedorn-Jensenmethod

sugars reduceferricyanide toferrocyanide

iodometricallydetermination offerricyanide by theMohr method

[92]

photometricdetermination offerricyanide at480 nm.

�official methods recommended by ICUMSA.

188 M. POKRZYWNICKA AND R. KONCKI

non-selective methods of disaccharides determination. How-ever, there are not applied to total sugar determination becausecontributions of each solute (sugar) in total liquid density,refraction index and light beam polarization are individual andnot simply additive as in case of chemical stoichiometric reac-tions reported above. Those techniques are widely applied insamples where influence for refractive index is expected onlyfrom single disaccharide.[9,15] However, due to measurementsimplicity and non-expensive equipment, from over 100 years,these methods are still widely used for routine analysis in sugarindustry. There are also so called Saccharimeters the speciallydesigned polarimeters and refractometers with scale recalcu-lated from angle of rotation or refractive index to sucrose con-centration. Analysis is very simple because proper values aretabulated.

Obviously, density, rotation angle and refractive index dependon temperature. Additionally, polarization and refractive indexdepend also on wavelength and therefore the measurements haveto be performed under precisely specified conditions: temperatureof 20�C (293K), and wavelength of the D line of sodium(589.3 nm) as standard and symbolized by nD. It should be empha-sized that accuracy of such measurements is based on assumptionof substance purity. Although the result of analysis is true only ifsingle sugar is present in sample, for fast routine assays suchmethod is sufficient. These techniques could be applied to analysisof complex samples after sugars separation. However, in the analyt-ical practice only refractometry has found wider application asdetection technique in liquid chromatography. Recently[15], thetechniques based on refractive index difference were applied fordetermination of sucrose content. In case of candy floss analysisretro-reflected beam interference based on refractive index detec-tion has been applied For cola drinks analysis instead of conven-tional refractometer measurements were carried in photometricflow cell. They were based on detection of light deflected by Schlie-ren effect formed due to refractive index gradient.[9] The result ofmeasurements were presented using Brix degrees.

Brix (�Bx) and related scales (Balling �Bg, Plato �P) arewidely applied for sugar liquids characterization. One Brix,Balling and Plato degree is percentage by weight of sucrose inpure water solution. Difference between these three indices isin reference temperature and closeness of measurements values(3,5 and 6 decimal places for �Bx, �Bg, and �P, respectively).Nowadays the most popular and the most used is the Brixindex. Plato index is sometimes applied in brewing industry.Generally these units are closely connected with concentrationof sucrose in pure water but sometimes the industry uses theseunits somewhat loosely to refer to any sweet solids in a prod-uct.[102] In case of sugars other than sucrose Brix is called the“apparent Brix” and is always a relative value. The Brix indexcan be determinated both by hydrometry and refractometry.Specially designed devices have scale both in [kg/m3]/ [g/cm3]or nD and Brix. However, in some cases values obtained byhydrometer and refractometers can differ each other, especiallyin case of “apparent Brix” measurements and samples withcomplex matrix. For example analysis of orange juice samplesrequired special correction because of sample acidity.[103]

An interesting issue is industrial analysis of sucrose. Nowadaysall commonly accepted methods for table sugar assay are codifiedby ICUMSA (International Commission for Uniform Methods of

Sugar Analysis). ICUMSA Methods Book.[104] contains descriptionof official methods recommended for quality control in sugarindustry, both for sucrose and impurities (lead, arsenic, copper,iron, etc.) concentrations determination as well as tabulated valuesof specific rotation, refractive index and density of sucrose solu-tions. This book contains also regulations concerning some selec-tive determinations based on chromatographic and enzymaticmethods, reported in the next paragraphs of this review.

3. Selective analytical methods

Taking into account rather high content of sugars in real sam-ples and practically unlimited accessibility of these samples(mainly food and agriculture products), very low detection lim-its and wide determination ranges are not extremely importantparameters of modern methods developed for analysis ofsugar-containing products. A crucial analytical factor of thesemethods is a selectivity allowing detection of particular saccha-ride in the presence of complex matrix of sample additionallycontaining various other very similar sugars. Currently thereare three main trends for the development of methods for selec-tive determination of disaccharides. The most popular andeffective are various separation techniques like chromatographyand electrophoresis. The second direction is the development ofbioselective methods based on almost specific recognition ofanalyte by enzymes and its conversion into easily detectedproduct. A third relatively new direction, also dedicated forsample analysis without analyte separation, is the application ofadvanced spectroscopic methods (IR, Raman) combined withuse of sophisticated chemometric tools.

3.1. Separation-based methods

Nowadays the most dynamically developed and widely reported inthe analytical literature separative technique for disaccharidesdetermination is liquid chromatography. Some recent papersdescribing application of various chromatographic methods fordisaccharide determination are cited in Table 2. High PerformanceLiquid Chromatography (HPLC) is the official technique for rou-tine sugars analysis recommended by AOAC International (Associ-ation of Official Analytical Chemists).[86] HPLC gives bothqualitative (identification of the carbohydrate) and, with peak inte-gration, quantitative information. The analysis is rapid, applicableto samples with a wide range of sugar concentrations, precise andaccurate and do not required derivatization of carbohydrates.

Liquid chromatography for disaccharide separations exploit dif-ferences in polarity (HPLC normal and reversedphase, HILIC,HTLC) or electrical charge (IEC: cation, anion exchange and ionexclusion) of target molecules. The most popular chromatographicmode is HLPC with normal-phase configuration. In this modecommonly applied stationary phase is silica gel with amino groups,whereas acetonitrile–water (40–95% acetonitrile) is used as amobile phase. The elution order is monosaccharides, disaccharidesand finally higher oligosaccharides. The gradient elution allows toavoid interferences caused by sugar alcohols (mannitol can coelutewith maltose and lactose while inositol with sucrose). In reversedphase HPLC the hydrophobic stationary phase is silica gel withadded alkyl chains, for example C18 column.[42] Thismode of sepa-ration has been used for separation of mono- di- and trisaccharides


Table2.

ApplicationofLiqu

idchromatograph

yford

isaccharides

determ

ination.

Chromatograph

icseparatio

n

Detectio

ncolumn

elution

Determined

disaccharid

esOtherdeterm

ined

substance

Sample

Reference

Normal-Phase

HPLC

ELSD

Zorbax

RX-SIL(m

odified

byethylene

diam

ine)

isocratic

acetonitrile-water

(72%

:28%

)with

0.03%

ethylene

diam

ineand0.05%

ammoniumhydroxide

sucrose,maltose,lactose

glucose,fructose,raffinose

Drin

ks(app

lejuicepineapple

juiceorange

juicegrapewine

liquor)

[10]

ELSD

carbohydratecolumn

Isocratic

acetonitrile–w

ater

(70%

:30%

)sucrose,maltose,lactose

glucose,fructose,galactose

raffinose

fruits,vegetables,grains,seeds

andleaves

[32]

ELSD

Phenom

enex

Luna

5uNH2100A

isocratic

acetonitrile-water

(82.5%

:17.5%

)sucrose

glucose,fructose,sorbitol

fruits(peach,app

lewatermelon,

cherry)

[23]

ELSD

SpherisorbNH2

gradient

from

81%acetonitrile

/19%

waterto

75%

acetonitrile

/25%

waterover

40min

maltose

glucose,fructose

beer

[62]

ELSD

PrevailCarbohydrateES

isocratic

acetonitrile-water

(80%

:20%

)lactose,lactulose


synthetic

samples

[105]

YMCPack

Polyam

ine

Zorbax

Carbohydrate

Analysis

UnisonUK-Am

inoHT

ELSD

andC-CA

DNH2-Krom

asil

isocratic

acetonitrile–w

ater

(70%

:30%


glucose,fructose

maltotriose

sauces,syrup

s,jellies,glazes,

hone

anddairy

products

[13]

CAD

ShodexAsahipak

NH2P-50E4

gradient

from

90%acetonitrile

/10%

waterto

77%

acetonitrile

/23%

waterover

22minandkept

constant

until40

min

sucrose,maltose

glucose,fructose,erythritol,

xylitol,sorbitol,mannitol,

maltitol

drinks

(juices,nectarsand

syrups)

[106]

RID

Tracer

carbohydratescolumn

isocratic

acetonitrile–w

ater

(75%

:25%

)sucrose,lactose,lactulose


milk-based

form

ulae

[69]

RID

SupelcosilLC-NH2

isocratic

acetonitrile–w

ater

(80%

:20%

)trehalose

—Selaginella

lepidophyllaplant

[67]

RID

Zorbax

Carbohydate

aminopropyl

isocratic

acetonitrile–w

ater

(82%

:18%


glucose,fructose,m

annose,

sorbito

l,xylitol

wine,juices,honey,dairy

products,biscuits

[14]

RID

UltraAminoColumn

isocratic

acetonitrile–w

ater

(75%

:25%

)lactose,lactulose

—HeatTreated

Milk

[44]

PinacleIIAm

ino

isocratic

acetonitrile–w

ater

(75%

:25%

)2PinacleIIAm

ino(in

series)

isocratic

acetonitrile–w

ater

(75%

:25%

)RID

Zorbax

Carbohydate

aminopropyl

isocratic

acetonitrile–w

ater

(82%

:18%


glucose,fructose,m

annose,

sorbito

l,xylitol

wine,juices,honey,dairy

products,biscuits

[14]

RID

UltraAminoColumn

isocratic

acetonitrile–w

ater

(75%

:25%

)lactose,lactulose

—heattreatedmilk

[44]

PinacleIIAm

ino

isocratic

acetonitrile–w

ater

(75%

:25%

)2PinacleIIAm

ino(in

series)

isocratic

acetonitrile–w

ater

(75%

:25%

)RID


isocratic

acetonitrile–w

ater

(75%

:25%

)lactose,lactulose

—conservedmilk

[45]

RID

Agilent

Zorbax

Carbohydrate

isocratic

acetonitrile–w

ater

(80%

:20%

)sucrose

glucose,fructose,sorbitol,

organicacids

grapefruitpu

lps

[24]

RID

SpherisorbAm

ino

isocratic

acetonitrile–w

ater

(70%

:30%

)lactulose

–lactose-free

milk

[107]


ESIM

S/MS


isocratic

acetonitrile–

ammoniumform

ate

(70%

:30%

)

lactose,lactulose

ESI/M

SAcqu

ityBEHam

ide

gradient

from

95%ofacetonitrile/5

%0.1%

ammoniainwaterisocratic

for

2min70%ofacetonitrile/3

0%0.1%

ammoniainwaterfor2

minand60%of

acetonitrile/4

0%0.1%

ammoniain

waterfor2

min

sucrose

glucose,fructose,kestose,

nystose

datesfruits

[22] MS/MS

Acqu

ityBEHam

ide

isocratic

acetonitrile–0.1M

ammoniumhydroxidein

water(80%

:20%

)

sucrose

—ratp

lasm

a,blood,andbrain

homogenate

[43]

MS/MS

Acqu

ityBEHam

ide

gradient

from

75%of

0.1%

ammoniumhydroxidein

acetonitrile/2

5%0.1%

ammoniumhydroxidein

waterto

67.5%of0.1%

ammoniumhydroxidein

acetonitrile/3

2.5%

0.1%

ammoniumhydroxidein

waterfor1

0min,to62.5%of

0.1%

ammoniumhydroxide

inacetonitrile/37.5%0.1%

ammoniumhydroxidein

waterfor2

minand75%of

0.1%

ammoniumhydroxide

inacetonitrile/25%0.1%

ammoniumhydroxidein

waterfor0

.1minthen

isocratic

for6

min

isom

altose

pannose,isom

altotriose

milk

powder

[74]

MS/MS

ZIC-

HILIC

gradient

from

75%acetonitrile

/25%

5mMofam

monium

acetateinwater

to40%

acetonitrile

/60%

5mMof

ammoniumacetateinwater

in10

min

sucrose,lactulose

raffinose,m

annitol

urine

[39]

AscentisSi

gradient

from

80%acetonitrile

/20%

5mMofam

monium

acetateinwater

to65%

acetonitrile

/35%

5mMof

ammoniumacetatein

waterin6min

SupelcocilLC-NH2

gradient

from

75%acetonitrile

0.05%form

icacid/25%

H2O

0.05%form

icacidto

40%

acetonitrile

0.05%form

icacid/60%

H2O

0.05%

form

icacidin6min

(Continuedon

nextpage)


Table2.

(Continued)

Chromatograph

icseparatio

n

Detectio

ncolumn

elution

Determined

disaccharid

esOtherdeterm

ined

substance

Sample

Reference

chem

iluminescence

Krom

asilNH2

isocratic

acetonitrile–w

ater

(70%

:30%

)lactose

glucose,fructose,xylose,

arabinose

grapefruitsextracts

[108]

Reversed

-Phase

HPLC

DAD

andRID

Aminex

HPX-87H

isocratic

3mMsulfuric

acid

lactose

glucose,fructose,organicacids

goat’smilk

yogu

rts

[52]

MS

Alltech

C18

gradient

from

90%0.1%

acetic

acidinwater/1

0%acetonitrile

with

0.1%

acetic

acidto

100%

0.1%

aceticacid

inwater

over8min,isocratic

for3

min,to0.1%

aceticacid

inwater/10%acetonitrile

with

0.1%

aceticover3min

andisocraticallyat90%0.1%

aceticacidinwater/10%

acetonitrile

with

0.1%

acetic

for3

min,for

atotalrun

time

of17

min

sucrose

—equine

serum

[42]

Hydroph

ilicInteractionCh

romatog

raph

y[HILIC]

QTO

FMS

XamideColumn

gradient

5%,10%

or20%of

100mMam

moniumform

ate

(pH3.2)in:0%acetonitrile/

100%

water

to80%

acetonitrile/2

0%waterfor

60min

lowmolecularweigh

theparindisaccharid

eheparin

—[109]

ESI/TOFM

S/MS

Zorbax

NH2am

inopropylsilica

gradient

from

88%8mM

ammoniumform

atein

acetonitrile/1

2%8mM

ammoniumform

ateinwater

isocratic

for1

0minto

80%

8mMam

moniumform

atein

acetonitrile/2

0%8mM

ammoniumform

ateinwater

for8

min,to75%8mM

ammoniumform

atein

acetonitrile/2

5%8mM

ammoniumform

ateinwater

for4

min,to70%8mM

ammoniumform

atein

acetonitrile/3

0%8mM

ammoniumform

ateinwater

for5

min,and

isocratic

for

2min

sucrose,mellibiose

glucose,fructose,galactose,

raffinose,m

anninotriose,

stachyose,verbascose

crud

eandprocessedRadix

Rehm

anniae

[80]

ELSD

Acclaim

Trinity

P2isocratic

acetonitrile–100

mM

ammoniumform

iatebu

ffer

pH3.65

(80%

:20)

lactulose

—milk

[72]


HighTemperature

Liqu

idCh

romatog

raph

y[HTLC]

ELSD

Hypercarb

isocratic

water

sucrose,maltose,lactose


milk,orang

eandmandarin

efruits

[110]

Ion-Exchange

Chromatog

raph

y[HPA

EC](anionexchange)

PAD

CarboPac

PA1

gradient

from

16mMsodium

hydroxideto

250mMsodium

hydroxidefor6

0min

maltose,lactose,trehalose,cellobiose

glucose,galactose,rafinose,

ribose,rham

nose,arabinose,

ethanolsorbitol,glycerol,

arabito

l,erythrito

l

yeastculturesandferm

entatio

nbroths

[76]

CarboPac

MA1

isocratic

480mMsodium

hydroxide

PAD

CarboPac

PA10

isocratic

5mMsodium

hydroxide

sucrose

glucose,fructose

wastewaterfrom

thebeverage

indu

stry

[66]

PAD

CarboPac

PA10

isocratic

50mMpotassium

hydroxide

sucrose,lactose

glucose,fructose

chocolate

[16]

PAD

CarboPac

PA10

gradient

87.5mMsodium

hydroxideisocratic

for

10min,to500mMsodium

hydroxidefor0

.1min,

isocratic

for7

min

maltose,isomaltose

glucose,rib

ose

bloodserum

[111]

PAD

CarboPac

PA20

isocratic

8mMsodium

hydroxide

lactose

glucose,galactose

naturally

“lactosefree”hard

cheese

[51]

PAD

CarboPac

PA20

isocratic

water

sucrose


arabinose,mannose,

rham

nose,m

annitol

greencoffeebean

[33]

PAD

AminoPac

PA10

gradient

from

17.5mMsodium

hydroxideto

25mMsodium

hydroxidefor2

min,to

37.5mMsodium

hydroxide

for3

min,to90

mMsodium

hydroxidefor6

min,2

min

isocratic,to100mMsodium

hydroxidefor2

min,to

175mMsodium

hydroxide

for5

min,to175mMsodium

hydroxideand3mMsodium

acetatefor0

.1min,to

175mMsodium

hydroxide

and7mMsodium

acetatefor

8min,to175mMsodium

hydroxideand10

mM

sodium

acetatefor5

min,to

175mMsodium

hydroxide

and20

mMsodium

acetate

for7

min,to75

mMsodium

hydroxideand60

mM

sodium

acetatefor5

min,

10minisocratic

maltose,isomaltose

glucose,fructose,arabinose,

maltotriose,isomaltotriose,

panose,m

altotertose,

aminoacids

ricewines

[59]

PAD

CarboPac

PA20

isocratic

1mMsodium

hydroxide

sucrose


mannose,xylose

aqueousextractsand

hydrolysates

ofbiom

ass

[112]

(Continuedon

nextpage)


Table2.

(Continued)

Chromatograph

icseparatio

n

Detectio

ncolumn

elution

Determined

disaccharid

esOtherdeterm

ined

substance

Sample

Reference

PAD

CarboPac

PA200

gradient

0.1M

sodium

hydroxide

isocratic

for9

min,to0.1M

sodium

hydroxideand0.04M

sodium

acetatefor1

7min,to

0.1M

sodium

hydroxideand

0.25Msodium

acetatefor

0.1min,isocraticfor1

4min

cellobiose,xylobiose

linearxylo-oligosaccharides

and

cello-o

ligosaccharides

lignocellulosicprocessing

products

[75]

PAD

Ham

ilton

RCX-30

gradient

from

50mMsodium

hydroxide25

mMsodium

acetateto

50mMsodium

hydroxide100mMsodium

acetatefor1

5min,isocratic

for1

0min

maltose,isomaltose

glucose,malto-oligosaccharides

wheatflour

[55]

PAD

CarboPac

PA10

gradient

2mMsodium

hydroxideisocratic

for5

min,

to5mMsodium

hydroxide

for3

min,isocraticfor2

min,

to20

mMsodium

hydroxide

for2

min,isocraticfor3

min,

to30

mMsodium

hydroxide

for1

min,isocraticfor2

min,

to40

mMsodium

hydroxide

for3

min,isocraticfor1

min,

to45

mMsodium

hydroxide

for3

min,isocraticfor2

min

sucrose,lactose,trehalose


ribose,xylose

rawsugar

[65]

ESI/M

SCarboPac

PA20

gradient

3mMpotassium

hydroxideisocratic

for

30min,to80

mMpotassium

hydroxidefor5

min


glucose,fructose,m

annitol,

glucosylglycerol

intracellularextractsof

cyanobacteria

[113]

ELSD

CarboPac

PA1

isocratic

32mMpotassium

hydroxide

sucrose


xylose

drinks

(cola,orange

juice,

watermelon

juice)

[12]

Ion-Exchange

Chromatog

raph

y[HPC

EC](catio

nexchange)

ESI/M

SIOA-1000

9mm

isocratic

20mMform

icacidand

10mMtrichloroacetic

acid

sucrose,lactulose,sucralose

rham

nose,erythritol

urineandbloodplasma

[40]

RID

Carbosep

Corgel87H3

isocratic

5mMsulfuric

acid

sucrose

glucose,fructose,sorbitol

apple’sleafandfruitp

eel

[25]

RID

RNMCarbohydrateNaC

isocratic

water

xylobiose

xylo-oligosaccharides

enzymaticallyhydrolysed

pulp

[79]

RID

SugarP

akI(Ca

2C)

isocratic

water

lactose,lactulose

—heattreatedmilk

[44]

RID

RezexRSO-OligosaccharideIE

(AgC

mode)

isocratic

water

maltose

glucose,fructose

beer

[61]

IEXC

a2C

isocratic

water

Ion-ExclusionCh

romatog

raph

y

RID

Bio-RadAm

inex

HPX

87H

Isocratic

0.005n

phosph

oricacid

sucrose

glucose,fructose,organicacids

fruitjuices

[114]

Abbreviatio

ns:CAD

-Charged

AerosolD

etectio

n;C-CA

D-C

orona-Ch

argedAe

rosolD

etector;DAD

-Diode

ArrayDetector;ESI-Electrospray

Ionizatio

n;ELSD

-EvaporativeLigh

tScatteringDetectio

n;MS-MassSpectrom

etry;M

S/MS-Tan-

demMassSpectrom

etry;PAD

-PulsedAm

perometric

Detectio

n;QTO

FMS-Quadrup

olTime-Of-Flight

MassSpectrom

etry;RID-R

efractiveIndexDetector;TO

FMS-Time-Of-Flight

MassSpectrom

etry.


by groups. A main disadvantage of this technique concerns mono-saccharides separation (short retention time results in elution as asingle unresolved peak). There could be also problem in the pres-ence of anomers that can results in peak doubling and/orbroadening.

For HPLC systems a variety of detectors can be coupled:Refractive Index Detector (RID),[14,24,44,45,67,69] EvaporativeLight Scattering Detectors (ELSD),[10,23,32,62] various MassSpectrometry techniques (tandem MS,[39,43,74] ESI MS[22,115])Charged Aerosol Detectors (CAD)[13,106] or even chemilumi-nescence.[108] A simple and economic RID seems to be the bestsolution for determination of separated sugars although it isless sensitive then other types of detectors. Unfortunately dueto its strongly dependence on solvent, type RID cannot beapplied in case of gradient elution. On the other hand, ELSDand MS required solvent evaporation.

An alternative HPLC mode for separating polar compounds ishydrophilic interaction liquid chromatography (HILIC). In thiscase a polar-hydrophilic stationary phase, characteristic for normalphase mode, is coupled with also polar water-containing, mobilephase characteristic for reversed phase.[116] But unlike in reversed-phase chromatography, gradient elution HILIC begins with a low-polarity organic solvent and elutes polar analytes by increasing thepolar aqueous content.[42,80] Such inversion of elution order allowsshortening of analysis when the target analyte is a single particularsaccharide. Another variation of HPLC is high temperature liquidchromatography (HTLC).[110] In HTLC thanks to raising separa-tion temperature to about 100�C there is a possibility to apply purewater as eluent without elongate time of analysis and degradationin resolution.

Carbohydrate separation can be achieved also by High Per-formance Thin Layer Chromatography (HPTLC).[117] Silica gel60 F254 plates with dropped of 1 mL of sample were developedat room temperature with a mobile phase of acetonitrile: water(8.5:1.5, v/v). Before determination sample was derivatives byaniline diphenylamine o-phosphoric acid. This method wasapplied for determination of maltose and total sugars in insweet potato (Ipomoea batatas L.).

Disaccharides separation based on their electrical charge is alsopossible, because carbohydrates are very weak acids (pKa values>12).[118] In strongly alkaline solutions some carbohydrate hydroxylgroups are ionized allowing sugars separation on anion-exchangecolumns. The employed mobile phases are simple and inexpensivesodium hydroxide[33,51,65,66,76,111,112] or potassium hydrox-ide[16,113,114] solutions, with or without addition of acetatesalt.[55,59,75] Also water may be used,[33] but then postcolumn addi-tion of a electrolyte solution is required for obtain adequate detec-tion conditions. This separation mode is very often connected withPulsed Amperometric Detection (PAD). Amperometric detectionhave a lot of advantages. Under specific pH and voltage conditionsonly carbohydrates will undergo the redox reaction. Coupling thismethod with chromatographic separation additionally increasesselectivity of detection. The development of so-called triple-pulsedamperometric detector,[118] solve the problem of electrode poison-ing by accumulation of oxidized products on its surface andallowed electrochemical detection for carbohydrates. The entirecleaning process takes milliseconds and is ongoing throughout therun. Because electrode reaction relies on oxidation of carbohydratehydroxyl and aldehyde groups, this detector is suitable for both

reducing and nonreducing carbohydrates. Also example of applica-tion of ELSD detector coupled with cation exchange separation isdescribed.[114] This type of detector requires evaporation of eluentbefore detection step and therefore the non-volatile potassium saltsin the basic eluent has to be removed by a suppressor.

In cation exchange liquid chromatography stationary phasesare often resin loaded with one of a variety of metal counterions Ca2C,[44,61] NaC[79] or AgC[61], which react selectively withthe weakly negatively charged hydroxyl groups of sugar mole-cules. The selectivity of this process is controlled with theappropriate choice of resin type and of the metallic speciesbonded to it, as well as by the temperature of column (columnsnormally are operated at elevated temperatures to increase itsefficiency). The mobile phase is typically water. The mechanismof separation is based on the strength of the bonding betweencis-glycols of sugar molecules with the cation loaded on the col-umn. The elution order is related to the number and strengthof cis-glycol complexes formed and takes place in the order ofdecreasing molecular weight.[86]

Ion exclusion chromatography (IEC) also found applicationin disaccharide determination.[114] In this techniques at ionexchange resin the ionic substances are rejected while non-ionic or partially ionized substances are retained and separatedby partition between the liquid inside the resin particles andthe liquid outside the particles. In effect the ionic substancespass quickly through the column. Non-ionic or partially ion-ized substances are held up and eluted more slowly.

Modern liquid chromatography techniques require specializedlaboratory equipment and are often connectedwith high consump-tion of eluent – mostly expensive chemically ultra-pure solvents.Moreover, the most often applicable normal-phase HPLC meth-ods, where organic solvents are applied, stay in contradiction togreen chemistry assumptions. From this point of view Gas Chro-matography (GC) seems to be better solution for disaccharidesdetermination. Without consumption of expensive ultra-purereagents it allows to determine much more analytes in the courseof single run.[119] In the Table 3 some papers from last 20 yearsdevoted to GC determination of disaccharides are collected. Inalmost all cases disaccharides have been determined together withmonosaccharides and sugar alcohols.

Sugars are non-volatile and thermally unstable compounds.Their determination with GC have to be preceded by the deriv-atization process, often complicated and time consuming. Thisderivatization required before separation step is the main prac-tical drawback strongly limiting the development of GC fordisaccharide analytics. For GC sugars determination methyl,acetate, trifluoroacetate and trimethylsilyl derivatives can beapplied.[132] Nowadays the most popular are trimethylsilyls(TMS) and trimethylsilyl oximes (TMSO). Trimethylsilylationof carbohydrates is a simple reaction and because non-volatilereagents or by-products are involved, the complete reactionmixture can be injected directly into the gas chromatograph,avoiding clean-up stages. Trimethylsilyl derivatives of carbohy-drates acquire different chemical properties, molecule increasevolatility and thermal stability and decrease polarity.[133] It isworth to mention that TMSO derivatised disaccharides are iso-meric molecules with monosaccharide ring structure and anopen chain with the oxime group. The only difference is in theposition of the substituents so they can have similar mass


Table3.

ApplicationofGas

Chromatograph

yford

isaccharides

determ

ination.

Chromatograph

icseparatio

n

Detectio

ncolumn

temperature

Determined

disaccharid

esOtherdeterm

ined

substance

Sample

Reference

FID

TR-1capillarycolumn(60m

£0.32

mmi.d.;0.25

mmfilm

of100%

methylpolysiloxane)

80� C

for2

minto

240�Cat5

� C/m

inleftfor5

minto

280�Cat20

� C/m

inleftfor1

5min

sucrose,maltose,lactose,m

elibiose

glucose,fructose,xylose,

mannose,galactose,sorbitol,

myo-inositol

tobacco

[36]

FID

ZB-5(30m

£0.25

mmi.d.;0.25mm

film

of5%

phenylmethylpolysiloxane)

100�Cto

180�Cat4

� C/m

inleftfor

2minto

215�Cat2�C/minto

325�C

at3�C/minleftfor1

0min

sucrose,maltose

trehalose,turanose,

cellobiose,palantinose,isom

altose

glucose,fructose,raffinose,

pann

ose

honeys

[120]

FID

SPB-1(30m£

0.25

mmi.d.;0.25mm

film

ofcrosslinkedmethylsilicone)

170�Cfor1

0minto

215�Cat15

� C/m

into

240�Cat1

� C/m

into

320�Cat

5�C/minleftfor2

0min

sucrose,maltose,a

,a-trehalose,

a,b-trehalose,turanose,cellobiose,

kojibiose,lam

inaribiose,nigerose,

maltulose,trehalulose,palantin

ose,

melibiose,gentio

biose,isom

altose

—honeys

[121]

Rtx-65

TG(25m£

0.25

mmi.d.;

0.1m

mfilm

ofcrossbond35%

dimethyl-65%

diph

enyl

polysiloxane)

200�Cfor2

0minto

270�Cat15

� C/

minto


300�C

at15

� C/m

inleftfor4

0min

FID

OV-101(25m£

0.25

mmi.d.)

180�Cto


290�Cat

10� C

/minleftfor1

5min

sucrose,maltose,lactose,m

altulose


enteralformulations

[122]

FID

CP-SIL5CB(25m£

0.25

mmi.d.;

0.25mmfilm

ofmethylsilicone)

180�Cfor3

6minto

300�Cat10

� C/m

inleftfor5

0min

sucrose

glucose,fructose,m

annose,

galactose,mannitol,

bornesito

l,myo-in

osito

l

coffeeandcoffeesubstitutes

[123]

MS

FID

SPB-1(25m£

0.25

mmi.d.;0.25mm

film


200�Cfor2

0minto

270�Cat15

� C/m

inleftfor4

0min

sucrose

glucose,fructose,inositols

fruitjuices

[124]

MS

FID

fusedsilicacapillarycolumn(7.5m

£0.25

mm)

180�Cfor2

0minto

270�Cat20

� C/m

inleftfor3

0min

sucrose

glucose,fructose,m

yo-in

osito

lorange

juice

[125]

MS

fusedsilicacapillarycolumn(22m£

0.25

mm)

MS

HP-5msUltraInert(15

m£

0.25

mmi.

d.;0.25m

mfilm

of5%

-phenyl)-

methylpolysiloxane)

180�Cfor2

minto

320�Cat7�C/min

leftfor2

min

lactose

—milk,cheese,yogh

urt

[126]

MS

SPB-1(30m£

0.25

mmi.d.;0.25mm

film


270�C

sucrose,maltose,lactose,

a,a-trehalose,turanose,cellobiose,

kojibiose,lam


maltulose,palantin

ose,melibiose,

isom

altose,sophorose,epilactose,

lactulose,leucrose

3-O-b-D-

galactopyranosyl-D-arabinose,

galactobioses,mannobioses

——

[119]

MS

TBR-1(30m£

0.25

mmi.d.;0.25mm

film)

200�Cfor1

5minto

270�Cat15

� C/m

into

290�Cat1�C/minLeftfor3

0min

lactose,a,a-trehalose,cellobiose,

laminaribiose,gentio

biose,

soph

orose

catechin,epicatechin,ethyl-

glucoside,glyceryl

–glucosides

wines

[127]

MS

DP-5MS(30m£

0.25

mm;0.25m)

50� C

to130�Cat30

� C/m

into

300�Cat

10� C/m

insucrose,maltose,m

elibiose

hexoses,hexoltioles,pentose

andpentosealcohols

cerealandpseudo

cerealflour

[128]

MS

DB-5or

HP-5M

S(30m

£0.25

mm;

0.25mmfilm)

50� C

to250�Cover58

min

sucrose,maltose,trehalose,

mannitol,sorbito

lstabilizersform

icrobial

preparations

[129]

MS

Rtx-65TG

(25m

£0.25

mmi.d.;

0.1mm

film

ofCrossbond35%

dimethyl–65%diph

enyl

polysiloxane)

170�Cfor1

0minto

215�Cat15

� C/m

into

240�Cat1

� C/m

into

320�Cat

5�C/minleftfor2

0min

sucrose,maltose,a

,a-trehalose,

a,b-trehalose,turanose,cellobiose,

kojibiose,lam


maltulose,trehalulose,palantin

ose,

melibiose,gentio

biose,isom

altose

raffinose,1-kestose,6-kestose,

pann

ose,erlose,neokestose,

malezito

se,m

altotriose,

isom

altotriose

honeys

[130,131]

Abbreviatio

ns:M

S-MassSpectrom

etry;FID-FlameIonizatio

nDetector.


spectra. Moreover, their retention time could also be similar.That is why coupling of gas chromatography with mass spec-trometry could give foul analytical information about disac-charides contents.[119] For GC disaccharide detection FlameIonization Detectors (FID) and Mass Spectrometers (MS) aremainly applied. MS offer advantages of both qualitative andquantitative information. Depending on the used detector,nitrogen (for FID) or helium (for MS) is applied as carrier gas.

A relatively novel and attractive alternative for LC and GCtechniques is Capillary electrophoresis (CE). CE is an electri-cally driven separation technique with many advantages suchas significant cost-effectiveness, high separation speed and largenumber of theoretical plates. CE requires minimal amounts(only microliters) of buffer, organic solvents and additives. Acommon problem with CE is its low detection sensitivitycaused by extremely low sample injection volume (nanoliters)and small inner diameter of the capillary. On the other hand,CE can be easily coupled with a variety of optical and electro-chemical detectors. CE systems have successfully applied fordisaccharides separation and determination. Some examples ofsuch analytical applications are collected in Table 4. However,none of them is officially recommended analytical method.

Separation principles of disaccharides in EC systems is simi-lar to this applied in anion exchange chromatography. Thehigh pH value is required, that is why the most common back-ground electrolyte is 50–75 mM sodium hydroxide. Capillarylength, critical for adequate separation resolution, range from10[11] to 120 cm.[63] Further improvement of separation effec-tivity can be obtained by chemical modifications of active sur-face of used capillaries.[77] There are also described applicationof microchip in place of conventional fused silica capillary.[134]

Disaccharide detection in capillary electrophoresis may seemproblematic. In case of electrochemical detection, because ofhigh voltage applied during separation, detector electrodesshould be somehow separated from capillary. Detector electro-des are placed about 50mm opposite to the capillary outlet.[135]

In case of photometric detection because of lack of chromo-phores for carbohydrates, precolumn derivatization[136] orother transformation[137] is required. There is also possibility toindirect absorbance detection. In this case an ionic chromo-phore is added to background electrolyte for example: 2,6-pyri-dinedicarboxylic acid, maleic acid and phthalic acid,[77] sorbicacid,[138] 1-naphthylacetic acid[63] or 2,6-pyridinedicarboxylicacid.[139] The detector receives a constant signal due to the pres-ence of these substances. The analyte displaces some of theseions, and detector signal decreases during the passage of ananalyte through the detector. Similarly chemiluminescencedetection can be applied with presence of luminol in back-ground electrolyte.[140]

3.2. Bioselective methods

Sugars as natural compounds are participate in many biotransfor-mation processes catalysed by various enzymes. Several biocatalyticpathways of such transformations are useful in the analytical chem-istry of sugars. The enzymes and enzymatic pathways involved inbiorecognition and biodetection for three main disaccharides areshown in Figures 3–5. In almost all cases enzymes involved in sac-charide metabolism are highly selective. Often they exhibit both

substrate and reaction specifity. This specific biorecognition of dis-accharides by respective enzymes can be applied in analyticalchemistry for the development of highly selective methods fordetermination of particular analyte without the need of separationof sugars and other components of samplematrix.

As can be seen from Figures 3–5, in the course of severalenzymatic disaccharides biotransformations various specificco-products, mediators and intermediates are consumed or cre-ated. This way enzymatic biorecognition could be easily cou-pled with various kinds of simple chemical detectors andsensors. A type of applied detector is closely related with classof last enzyme in biotransformation path of target sugar. Areview of detection methods coupled with enzymatic recogni-tion of disaccharides reported in the literature is presented inTable 5. Except amperometry, spectrophotometry, conductom-etry, fluorimetry and chemiluminometry, several detectiontechniques based on potentiometry[50,144] and ion selectivedevices[145–148] are also possible. Light addressable potentiome-try[144] and differential pHmetry[50] are based on acidificationby phosphorylation reaction catalyzed by glucokinase (GK E.C.2.6.1.2) or hexokinase (HK E.C. 2.7.1.1). Ion-Sensitive FieldEffect Transistors (ISFET) are sensitive for hydrogen ions gen-erated during reaction with dehydrogenases (describedexamples concern glucose[147–149] and galactose[148] dehydro-genases), or generated after electrolysis of hydrogen perox-ide.[146] A bit more complicated is situation with ElectrolyteIsolator Semiconductor (EIS). This device sensitive for fluorideions required application of 4-fluoroaniline as HPR mediator.For disaccharides determination also entalpimetric measure-ments of temperature changes during single[27] or multistepenzymatic reactions can be utilized.[150] Examples of applica-tion of Fourier Transform Near Infrared Spectroscopy(FTNIR)[151] or coulometry[152] have been also reported.

Predominantly, the biosensing schemes for disaccharides arebased on sequence of enzymatic conversions of target analyteinto detectable final species (Figs. 3–5) These so-called cascadeenzyme reactions, consisting of at least two even to foursteps,[29,58,145,161] realized in the analytical practise are listed inTable 6. Most of them are based on specific enzymatic hydroly-sis of target disaccharide to respective monosaccharides (thefirst step of enzymatic cascade) and then on their enzymaticallycatalysed oxidation (the second step) allowing detection usingconventional instrumental methods (Tab. 5). The enzymeapplied in the first step defines which disaccharide will be bio-recognized and determined, whereas further enzymes convertintermediate products into final detectable species. For exam-ple, amperometric detection of lactose could be performedusing only two enzymes: b-galactosidase (b-Gal, E.C. 3.2.1.23),glucose oxidase (GOx, E.C. 1.1.3.4).[48] However, in somecases[47,53] horseradish peroxidase (HPR E.C. 1.11.1.7) improv-ing detection of enzymatically generated hydrogen peroxide isalso implemented into biosensing system. Anotherextraordinary applied enzyme is mutarotase (Mut, E.C.5.1.3.3),[3,26,146,153,158,160,165] because most of the hydrolasesdecompose disaccharides into a- glucose, whereas the nextenzymes (glucose oxidase (GOx, E.C. 1.1.3.4) or glucose dehy-drogenases (GDh, E.C. 1.1.1.47)) are specific for b-D-glucose.However, this enzyme is not crucial in these biosensing path-ways, because mutarotation can occurs spontaneously


Table4.

ApplicationofCapillaryElectrophoresisford

isaccharides

determ

ination.

Detectio

ncapillarydimensions

background

electrolyte

voltage

Determined

disaccharid

esOther

determ

ined

substance

Sample

Reference

DAD

indirectdetectionof

maleicacidat210nm

61.5cm

effectiveleng

th138.2mMsodium

hydroxide,

40mMmaleicacid,5

mM1-

tetradecyl--3-

methylim

idazoliumchlorid

e

¡21.7kV

sucrose,lactose,

cellobiose,

xylose,fructose,glucose,

galactose,raffinose,

cellotriose,cellotetraose,

cellopentaose,cellohexaose

biom

ass

[77]

70cm

totallength

22.5mmID

DAD

directdetectionat278

and240nm

71.5cm

effectiveleng

th130mMsodium

hydroxide,36

mM

disodium

phosph

ate

C17kV

xylobiose

xylo-oligosaccharides

enzymaticallyhydrolysed

pulp

[79]

80cm

totallength

50mm

IDDAD

indirectdetectionof

sorbicacidat254nm

8.5cm

effectiveleng

th20

mMsorbicacid,40mMsodium

hydroxide,0.2mMcetyl

trimethylammonium

brom

ide

C25kV

sucrose

glucose,fructose

honey

[138]

60cm

totallength

50mm

IDDAD

270nm

absorbing

interm

ediateform

atted

byph

otooxidatio

n

60cm

totallength

98mMsodium

hydroxide,120mM

sodium

chlorid

e¡1

4kV

sucrose,lactose

glucose,fructose

post-explosion

residu

es,

smokedevice,cough

syrup,applejuice,red

wine

[137]

50mm

ID

DAD

directdetectionat

270nm

60cm

effectiveleng

th130mMsodium

hydroxide,36

mM

disodium

phosph

ate

C10kV

cellobiose,trehalose

fructose,fucose,galactose,

glucose,myo-in

osito

l,mannitol,mannose,

rham

nose,ribose,sorbito

l,xylose

PinotN

oirred

wines

[68]

50cm

totallength

50mm

ID

DAD

indirectdetectionof

1-naph

thylaceticacidat

222nm

120cm

totallength

1mMsolutio

nof1-naph

thylacetic

acidadjusted

topH

12.5with

sodium

hydroxide

C25kV

sucrose,maltose

glucose,fructose,m

altotriose

wort

[63]

75mm

ID

DAD

directdetectionat

280nm

afterp

recolumn

derivatizationwith

p-am

inobenzoicacid

57cm

totallength

20mMsodium

tetraborate

C20kV

maltose

glucose,malto-oligosaccharides

beers,orange

andplum

juices

[136]

75mm

ID

DAD

indirectdetectionof

2,6-pyrid

inedicarboxylic

acidat275nm

104cm

effectiveleng

th112.5

20mM2,6-pyrid

inedicarboxylic

acid,0,5mM

cetyltrimethylammonium

hydroxideadjusted

topH

12.1

with

1Msodium

hydroxide

¡25kV

sucrose,lactose

glucose,fructose

yogu

rt,orang

ejuice,sake

mash,pickledapricot

[139]

cmtotallength

50mm

ID

Chem

iluminescence

65cm

totallength

0.018gluminolin10

mL0.02M

sodium

hydroxidesolutio

nof

10%dimethylsulfoxide

C15kV

sucrose

fructose,rhamnose,cyclodextrin

–[140]

25mm

ID

Cond

uctometric

4cm

effectiveleng

th75

mMsodium

hydroxide

C5kV

sucrose

glucose,fructose,ribose

energy

drinks

[11]

10cm

totallength

10mm

IDAm

perometric

(special

design

edgraphene–

cobaltmicrosphere

hybridpasteelectrodes)

40cm

totallength

75mMsodium

hydroxide

C12kV

sucrose,lactose

glucose,fructose,m

annitol

Honey,m

ilk[141]

25mm

ID

Amperometric

(special

design

edgraphene–

copp

ercomposite

electrodes)

40cm

totallength

75mMsodium

hydroxide

C12kV

sucrose,lactose

glucose,fructose,m

annitol

honey,milk,peach,

banana

[135]

25mm

ID

Amperometric

(nano-NiO

modified

carbon

paste

electrode)

27cm

totallength

50mMsodium

hydroxide

C10kV

sucrose

glucose,fructose,m

annitol

honey

[142]

25mm

ID

Amperometric

40cm

totallength

75mMsodium

hydroxide

C12kV

sucrose

paeoniflorin,paeonoside,

glucose,andfructose

MoutanCortex

[143]

25mm

IDAm

perometric

8cm

totallength

100mMsodium

hydroxide

100V



mannose,xylose

honey

[134]

1mmID

onmicrochip

DAD

-Diode

ArrayDetector.


(effectively in the presence of phosphate ions[166]). Sometimesadditional enzymes are used in the developed bioanalytical sys-tems for eliminations of interferences. For example speciallydesigned bioreactors with immobilized glucose oxidase (GOx,E.C. 1.1.3.4) and catalase (Cat, E.C. 1.11.1.6) have been appliedfor elimination influences from glucose in the course of sucrosedetermination.[26,165]

There are also some enzymatic paths that allow selectivedetermination only selected disaccharide without theirhydrolysis to respective monosaccharides.[5,27,46,54,58,151,156]

Sucrose phosphorylase (SP E.C. 2.4.1.1) decomposes sucroseto glucose 1-phosphate, a substance which further enzy-matic conversion (Fig. 3) is not interfered by glucose pres-ent in the sample.[5,156] Similar biosensing scheme (Fig. 4)has been developed for maltose using maltose phosphory-lase (MP E.C. 2.4.1.8) in the first biorecognition step.[58]

Lactose can be oxidized by cellobiose dehydrogenase (CDHE.C. 1.1.99.18) and concentration of acceptor are mea-sured.[46,54] However such approach is useful only in case ofsamples that do not contain cellobiose and maltose.[170] Sin-gle enzymatic reaction can be applied also for sucrose deter-mination after hydrolysis with invertase, but in these casesuncommon detection method have to be applied: thermom-etry for measuring of heat produced in the course of bioca-talyzed[27] reaction or subtle changes of Infra-Red spectrabetween substrate and product.[151]

Commercially available photometric assay kits for disaccha-ride determination are based on two steps cascade enzymaticreactions.[107,171–174] These kits contain soluble enzymes, how-ever as can be seen from table 6 a large number of bioanalyticalsystems dedicated for disaccharide determination is based onimmobilized enzymes. They are immobilized in the form of

Figure 3. Analytically useful enzymatic pathways for detection of sucrose.

Figure 4. Analytically useful enzymatic pathways for detection of lactose.


mono- or polyenzymatic bioreactors or as biosensing layersintegrated with respective detectors (biosensors). These biosen-sors[27,46,53,54,57,70,146,148,150,156,157,167,175] and bioreac-tors[3,5,26,64,70,71,155,160,161,165] are often applied in systemsdesigned for flow analysis. Also measurements with freeenzyme are performed under flow analysis conditions,[17,153]

although it is connected with high enzyme consumption. Suchapproach (flow analysis) allows mechanization of multistepanalytical procedure and offers highly reproducible conditionsof biochemical processes (precise control of reaction time, tem-perature, reagent mixing etc.) and detection, what is especiallyimportant in case of reported here biocatalytic analytical meth-ods due to their kinetic, non- stationary character.

Not only enzymes could be applied as biocatalytic mate-rials for selective determination disaccharides. Severalmicroorganisms, natural sources of commercially availableenzyme preparation, were used for development of biosens-ing devices. Yeast cell were successfully used instead ofinvertase[166] and coupled with GOx on surface of biosen-sor. Saccharomyces crevisiae caused fermentation of glucoseobtained from catalysed by lactase lactose hydrolysis andallow determination with carbon dioxide electrode.[176] Con-sumption of oxygen by specially grown mutants of Escheri-chia coli K12 enable to monitoring of sucrose, maltose andlactose.[175] A maltase-displayed bacteria and glucose dehy-drogenase-displayed bacteria were co-immobilized on multi-

Figure 5. Analytically useful enzymatic pathways for detection of maltose.

Table 5. Possible detection methods defined by the last enzyme in biocatalytic path, Table 5 Detection type according to last enzyme type in cascade enzymatic path.

Final enzyme type

Oxidases: GOx,GaOx, PyOx

Dehydrogenases: GDh, GaDh,FDh, G6PDh, CDh Peroxidase: HrP

Detection technique Amperometry oxygen consumption,[17,48,57,153,154]H2O2 detection[3,26,155]

reduction of acceptor[46,54,71,156,157]

oxidation of mediator [47,53]

Conductometry increased in conductivity afterlactone dissociation [158,159]

— —

Spectrophotometry H2O2 chromogenic reactions[160] with G6PDH absorbance of

NADPH (340 nm) [29,58,64,161];with FDH reduction of MTTto MTT formazan (570 nm)[41,162]

product of reaction of 4aminoantrypine and Phenol4 sulphonic acid salt(500 nm) [163]

Fluorimetry — Fluorescence of NADPH(ex- 340 nm; em 460 nm) [5]

reduction of Amplex Red tohigh fluorescent resorufin(ex- 550 nm; em-585 nm)[28]

Chemiluminometry oxidation of luminol by H2O2[164,165]

— —

Abbreviations: CDh- cellobiose dehydrogenase; FDh- fructose dehydrogenase; G6PDh- glucose-6 phosphate dehydrogenase; GDh- glucose dehydrogenase; GaDh- galac-tose dehydrogenase; GOx- glucose oxidase; GaOx- galactose oxdase; HrP- horseradish peroxidase; MTT- 3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide;NADPH- Nicotinamide adenine dinucleotide phosphate; PyOx- pyranose oxidase.


Table6.

Enzymaticdeterm

inationofdisaccharid

es.

Cascadeenzymaticreactio

nsequ

ence

Target

Analyte

1st

enzyme

2nd

enzyme

3rd

enzyme

4th

enzyme

detection

method

immobilizatio

nmethod

Other

determ

ined

sugars

Sample

Reference

immob

ilizedenzyme(biosensor)

sucrose

invertase

mutarotase

glucoseoxidase

horseradish

peroxidase

EIS

cross-linking

with

GA

glucose

—[145]

sucrose

invertase

mutarotase

glucoseoxidase

—Conductometry

cross-linking

with

BSA

andGA

glucose

orange

andapplejuices

[158]

sucrose

invertase

mutarotase

glucoseoxidase

—ISFET

photo-crosslinkable

polymer(PVA

SbQ)

glucose

—[146]

sucrose

invertase

glucoseoxidase

catalase

—Calorim

etry

covalent

attachmentto

activated

with

GA

glucose

—[150]

sucrose

sucroseph

osph

orylase

phosph

ogluco-m

utase

glucose-6ph

osph

ate

dehydrogenase

—Am

perometry

entrappedincarbon

pasteelectrodematrix

—pineapple,peachapple

juice

[156]

sucrose

invertase

fructose

dehydrogenase

——

Amperometry

entrapmentw

itha

dialysismem

brane

fructose,glucose

cond

ensedmilk,

referencematerial

[157]

sucrose

invertase

glucosedehydrogenase

——

ISFET

cross-linking

with

BSA

andGA

glucose

—[147]

sucrose

invertase

glucokinase

——

Ligh

tadd

ressable

potentiometry

cross-linking

with

BSA

andGA

glucose

—[144]

sucrose

invertase


——

ISFET

cross-linking

with

GA

glucose

—[148]

sucrose

invertase

——

—Thermom

etry

cross-linking

with

BSA

andGA

—sugarcanejuice

[27]

lactose

b-galactosidase

glucoseoxidase

horseradishperoxidase

—Am

perometry

entrapmentw

ithin

dialysismem

brane

glucose

chocolate,dairy

samples

[53]

lactose

b-galactosidase

glucoseoxidase


—Am

perometry

cross-linking

with

GA

glucose

milk

[47]

lactose

b-galactosidase

glucoseoxidase


—Am

perometry

—glucose

milk,cheese,yogh

urt

[126]

lactose

b-galactosidase

mutarotase

glucoseoxidase

—Am

perometry

cross-linking

with

GAand

b-cyclodextrin

and

coveredby

nafion

glucose

—[49]

lactose

b-galactosidase

glucoseoxidase

——

Conductometry

cross-linking

with

BSA

andGA

glucose

milk

[159]

lactose

b-galactosidase

glucoseoxidase

——

Amperometry

cross-linking

with

gelatin

eandGA

glucose

milk

[48]

lactose

b-galactosidase

glucoseoxidase

——

Voltammetry

cross-linking

with

GA,

covalent

bond

edwith

polyazetidine

glucose

—[167]

lactose

b-galactosidase


——

ISFET

cross-linking

with

GA

glucose

—[149]

lactose

b-galactosidase

galactosedehydrogenase

——

ISFET

cross-linking

with

GA

galactose

—[148]

lactose

lactase

galactoseoxidase

——

Amperometry

Lang

muir-Blodgetfi

lmof

poly(3-hexyl

thioph

ene)/stearic

acid

galactose

—[168]

lactose

cellobiosedehydrogenase

——

—Am

perometry

physicaladsorptio

n,entrapmentw

itha

dialysismem

brane

—milk

[46,54]

maltose

amylo-

glucosidase

mutarotase

glucoseoxidase

horseradish

peroxidase

EIS

cross-linking

with

GA

glucose

—[54]

maltose

a–g

lucosidase

glucokinase

—Ligh

tadd

ressable

potentiometry

cross-linking

with

BSA

andGA

glucose

—[145]

maltose

a–g

lucosidase


——

ISFET

cross-linking

with

GA

glucose

—[144]

(Continuedon

nextpage)


Table6.

(Continued)

Cascadeenzymaticreactio

nsequ

ence

Target

Analyte

1st

enzyme

2nd

enzyme

3rd

enzyme

4th

enzyme

detection

method

immobilizatio

nmethod

Other

determ

ined

sugars

Sample

Reference

maltose

amylo-

glucosidase

glucoseoxidase

——

Cyclicvoltammetry

physicaladsorptio

nglucose

beer

[148]

maltose

amylo-

glucosidase

glucoseoxidase

——

Amperometry

cross-linking

with

BSA

andGA

glucose

starch

hydrolysate

[60]

maltose

a–g

lucosidase

pyranose

oxidase

——

Amperometry

cross-linking

with

chito

san,carbon

nanotube

andGA

glucose,galactose,xylose

beer

[57]

lactulose

b-galactosidase

(inbioreactor)

fructose

dehydrogenase

——

Amperometry

cross-linking

with

BSA

andGA

fructose

milk

[154]

immob

ilizedenzyme(bioreactor)

sucrose

invertase

phosph

oglucose

isom

erase

hexokinase

glucose-6ph

osph

ate

dehydrogenase

Spectrophotometry

covalent

attachmentto

CPGallkylam

inated

with

APTS

and

form

ylated

with

GA

glucose,fructose

synthetic

samples

[161]

sucrose

invertase

mutarotase

glucoseoxidase

catalase

Amperometry

covalent

attachmentto

Amino-Cellulofine

activated

byGA

glucose,fructose

(byFD

H)C

ocacola,kiwi,apple,

banana,m

andarin

[26]

sucrose

invertase

mutarotase

glucoseoxidase(in

solutio

n)catalase

Chem

iluminescence

covalent

attachmentto

CPGallkylam

inated

with

APTS

and

form

ylated

with

GA

glucose

Pepsi,coke,cereal,cake

mix

[165]

sucrose

invertase

mutarotase

glucoseoxidase

—Am

perometry

cross-linking

with

BSA

andGAon

pig’ssm

all

intestine

glucose

fruitjuices

[3]

sucrose

invertase

mutarotase

glucoseoxidase

—Spectrophotometry

covalent

attachmentto

CPGallkylam

inated

with

APTS

and

form

ylated

with

GA

glucose

—[160]

sucrose

invertase

mutarotase

glucoseoxidase

—Am

perometry

cross-linking

tocellulose

mem

branewith

BSA

andGA

glucose

—[155]

sucrose

sucroseph

osph

orylase

phosph

ogluco-m

utase

glucose-6ph

osph

ate

dehydrogenase

—Fluorometry

covalent

attachmentto

CPGallkylam

inated

with

APTS

and

form

ylated

with

GA

—ionicsoftdrink,cola,

orange

juice

[5]

sucrose

invertase

glucoseoxidase

——

Chem

iluminescence

covalent

attachmentto

CPGallkylam

inated

with

APTS

and

form

ylated

with

GA

glucose

—[164]

sucrose

invertase

glucoseoxidase

(biosensor)

——

Amperometry

Glucose

oxidasesand

wich

mem

braneand

invertasebearingsilk

reactor

glucose

—[169]

sucrose

invertase

——

—FT-NIRspectroscopy

covalent

attachmentto

silicon

chipsilanized

with

ATPS

and

form

ylated

with

GA

—softdrinks

[151]

lactose

lactase

glucoseoxidase

——

Chem

iluminescence

covalent

attachmentto

CPGallkylam

inated

with

APTS

and

form

ylated

with

GA

glucose

—[164]


maltose

amylo-

glucosidase

glucoseoxidase

——

Chem

iluminescence

covalent

attachmentto

CPGallkylam

inated

with

APTS

and

form

ylated

with

GA

glucose

—[164]

lactulose

b-galactosidase

(insolutio

n)fructose

dehydrogenase

——

Amperometry

covalent

attachmentto

CPGallkylam

inated

with

APTS

and

form

ylated

with

GA

fructose

milk

[71]

trehalose

trehalase

hexokinase

(insolutio

n)glucose-6ph

osph

ate

dehydrogenase(in

solutio

n)


covalent

attachmentto

Epoxyresin

glucose

ferm

entatio

nbroth

[64]

non-

immob

ilizedenzyme

sucrose

invertase

hexokinase

phosph

oglucose

isom

erase

glucose-6ph

osph

ate

dehydrogenase

Spectrophotometry

—glucose,fructose

beet

root

[29]

sucrose

invertase

mutarotase

glucoseoxidase

—Am

perometry

—glucose

sugarb

eetm

olase

[153]

sucrose

invertase

glucoseoxidase


—Fluorim

etry

—glucose

sugarb

eet

[28]

sucrose

invertase

glucoseoxidase



—glucose

coffeebeans

[163]

sucrose

invertase

fructose

dehydrogenase

——

Spectrophotometry

—fructose

bloodserum,urin

e[41]

sucrose

invertase

glucoseoxidase

——

Amperometry

—glucose

orange

andapplejuice,

icecream,condensed

milk,jellies,greenpea,

corn,w

heat,peanuts

[17]

lactose

b-galactosidase

glucokinase

hexokinase

—DifferentialpH

techniqu

e—

glucose

milk

[50]

lactose

lactase

glucoseoxidase

——

Amperometry

—infant

form

ula

[17]

maltose

maltose

epimerase

maltose

phosph

orylase

phosph

ogluco-m

utase

glucose-6ph

osph

ate

dehydrogenase

Spectrophotometry

——

potato

solublestarch

[58]

maltose

a–g

lucosidase

glucoseoxidase

——

Amperometry

—corn

andmaltsyrup

[17]

maltose

a–g

lucosidase

glucoseoxidase

——

Coulom

etry

—glucose

—[152]

lactulose

b-galactosidase

hexokinase

——

DifferentialpH

techniqu

e—

glucose,fructose

milk

[50]

lactulose

b-galactosidase

fructose

dehydrogenase

——

Spectrophotometry

—fructose

milk

[162]

Abbreviatio

ns:APTS-3-am

inopropyltriethoxysilane;BSA-b

ovineserumalbu

min;CPG

-controlledporous

glass;EIS-electrolyteisolator

semicondu

ctor;FT-NIR-FourierTransform

NearInfraredSpectroscopy;G

A-glutaraldehyde;ISFET-

Ion-Sensitive

FieldEffectTransistor.


walled carbon nanotubes modified glassy carbon electrodeto obtain electrochemical biosensor for maltose and glu-cose.[177] In the course of another investigations[178] therepressor of Escherichia coli lac operon has been engineeredas altered effector for selective recognition of lactulose.

Whereas biorecognition of various sugars using enzymesand biocatalytic materials is widely reported in the analyticalliterature, till now the immunoassays for disaccharide determi-nation are not developed. It is practically impossible to grownan antibody for sucrose due to lack of its immunogenicity.Although maltose and lactose antibodies are available, theiranalytical application is not reported in the literature.

3.3. Spectroscopic methods

Disaccharides, like all organic compounds, have reach characteris-tic spectra in Infra-Red (IR) range of wavelength with specificabsorption bands (table 7). The application of IR spectroscopy fordisaccharides determination required absorbance measurements atmany wavelengths simultaneously and then the use of special mul-tivariate statistical techniques to relate spectral data with the con-centration of the chosen component. Such techniques as multiplelinear regression (MLR) or partial least squares (PLS) regressionequation can predict the concentration of each constituent fromthe absorbance values at selected wavelengths. Because of the struc-tural similarities of sugars the spectra can overlap for each other.The baseline can variate in various apparatus and because of ambi-ent conditions (especially temperature). In case of aqueous solutionthere is high background spectrum of water. Sample surface imper-fections can effect in nonlinear, inhomogeneous and anisotropiclight scattering. In some cases high frequency detector noise canoccur. Obviously, many other compounds present in the samplecan contain similar functional group and thus can interfere.[179]

For IR data processing and interpretation rather sophisticatedchemometric methods are required. In experiments from two[181]

to more than fifty[182] spectra are recorded and averaged. In manycases also further data handling are processed. For smoothing andresolving overlapping peaks first and second derivatives computa-tion[20,182,183] or Savitzky-Golay (SG) filter[8,19,30,73,180,184–186] couldbe applied. Because spectra included very extensive data StandardNormal Variates (SNV),[8,18,30,184,185,187] Principal ComponentAnalysis (PCA)[18,56] or Genetic Algorithm (GA)[187] could be

applied to reduce the dimensionality of the data in order to extractmain and remove insignificant information. GA together withArti-ficial Neural Network (ANN) could also be coupled to define ana-lyte origin when group of samples is compared.[20] To obtaincalibration curves the Partial Least Square Regression (PLS) aremostly applied, however the use of Multiple Linear Regression(MLR)[187] and Principal Component Regression (PCR)[4] is alsoreported in the literature. The PLS and PCR use data reductiontechniques to extract from all extensive data much smaller amountof new variables representative for most of the variability in sam-ples. These newly defined variables can be used to create a calibra-tion curve or develop a regression equation to predict theconcentration of disaccharide in sample. In those methods, it is notnecessary to reduce data dimension, as it is in MLR where only alimited number of wavelengths are used. Examples of applicationof IR for disaccharide determination using various chemometrictools for data processing are presented in table 8. As can be seenfrom this table, chemometrically supported IR spectroscopy is use-ful for analysis of real samples having quite complexmatrix.

It is worth to notice that not only IR spectroscopy can be sup-ported by advanced chemometric methods to estimate disacchar-ides content in sample. There is also example of measurements invisible range with the use of second derivative spectra in combina-tion with PLS regression modelling for determination of sucroseand trehalose in olive leaves.[34] Some electrochemical techniquesmay require statistical data treatment to could be applied for selec-tive disaccharide determination. An Electrochemical ImpedanceSpectroscopy (EIS) was applied for determination of sucrose, glu-cose, fructose and total sugar content in pineapple fruit.[21,189] Afterstandard addition, measurement was performed and ANN techni-ques was applied to predict specific mathematical models for eachone of determined sugars. PLS method was also used to model therelationship between the EISmeasurements and the s

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