research article rapid enzymatic method for pectin methyl...

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2013 Article ID 854763 7 pageshttpdxdoiorg1011552013854763

Research ArticleRapid Enzymatic Method for Pectin MethylEsters Determination

Lucyna Awkawska-Andrinopoulou1 Efstathios G Vasiliou1 Dimitrios G Georgakopoulos2

Constantinos P Yialouris3 and Constantinos A Georgiou1

1 Chemistry Agricultural University of Athens Iera Odos 75 11855 Athens Greece2 Agricultural Microbiology Agricultural University of Athens Iera Odos 75 11855 Athens Greece3 Informatics Laboratory Agricultural University of Athens Iera Odos 75 11855 Athens Greece

Correspondence should be addressed to Constantinos A Georgiou cagauagr

Received 4 October 2013 Revised 3 December 2013 Accepted 4 December 2013

Academic Editor Jianxiu Du

Copyright copy 2013 Lucyna Łękawska-Andrinopoulou et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Pectin is a natural polysaccharide used in food and pharma industries Pectin degree of methylation is an important parameterhaving significant influence onpectin applications A rapid fully automated kinetic flowmethod for determination of pectinmethylesters has been developed The method is based on a lab-made analyzer using the reverse flow-injectionstopped flow principleMethanol is released from pectin by pectin methylesterase in the first mixing coil Enzyme working solution is injected furtherdownstream and it is mixed with pectinpectin methylesterase stream in the second mixing coil Methanol is oxidized by alcoholoxidase releasing formaldehyde and hydrogen peroxide This reaction is coupled to horse radish peroxidase catalyzed reactionwhich gives the colored product 4-N-(p-benzoquinoneimine)-antipyrine Reaction rate is proportional to methanol concentrationand it is followed using Ocean Optics USB 2000+ spectrophotometer The analyzer is fully regulated by a lab written LabVIEWprogramThe detection limit was 147mM with an analysis rate of 7 samples hminus1 A paired 119905-test with results from manual methodshowed that the automated method results are equivalent to the manual method at the 95 confidence interval The developedmethod is rapid and sustainable and it is the first application of flow analysis in pectin analysis

1 Introduction

Pectin a natural polysaccharide present in the cell walls ofhigher plants is an important compound for food andpharma industries The importance of the compound isrelated to its unique properties and the fact that it is biode-gradable The main raw materials from which commercialpectin is extracted are agricultural by-products that is citruspeel and apple pomace The major applications of pectin infood products rely on its gelling and stabilizing propertiesPectin is commonly used for production of foodstuffs likejams jellies and marmalades as a gelling agent but also inacidified milk drinks beverages or confections as stabilizer[1] The diversity of pectin applications and its beneficialhealth effects [2ndash4] are the reasons why this compound hasreceived a lot of interest during recent years

The gelling properties of pectin are a consequence of itsstructure Pectin is primarily composed of 120572-(1-4)-linked D-galacturonic acid molecules some of them being esterifiedwithmethanol Degree of pectinmethylation (DM) is definedas the percentage of methyl esterified carboxyl groups DM50 is the borderline between low-methoxyl (LM) and high-methoxyl (HM) pectins DM influences the conditions thatshould be applied for pectin to form a gel High-methoxylpectins gel in the presence of sufficient amount of sugar andacid Low-methoxyl pectins require divalent cations to forma gel structure The ability of LM pectins to gel without sugarmakes them especially suitable for dietetic that is low sugarfoodstuffs [1] The degree of methylation is related to settingtimes of pectins [5] Based on the setting times the mostsuitable pectin application can be chosen For example rapidsetting pectins are perfectly suited for jams and marmalades

2 Journal of Analytical Methods in Chemistry

when settling or flotation of fruit pieces has to be avoidedIn contrast slow setting pectins are especially useful forproduction of jellies when additional time for elimination ofair bubbles before gelling is needed [1]

Pectins are used in the pharmaceutical industry in drugdelivery systems [6] Their applications greatly depend onthe degree of methylation for example LM pectins are usedin nasal preparations due to their ability to form gels in thepresence of calcium cations [7] DM also influences the drugrelease time from pectin-based matrix tablets when calciumformulations are used [8] The application of pectin dependsgreatly on its degree of methylation This is the reason whyrapid and reliable methods for fast quantification of pectinmethyl esters are of major interest and importance

Pectin methyl esters can be hydrolyzed either by alka-line hydrolysis or enzymatically by pectin methylesteraseReleased methanol can be quantified by means of GC [9 10]and HPLC [11] or with alcohol oxidase [12] When alcoholoxidase is used the methanol content can be related toformaldehyde [13] or hydrogen peroxide concentration [14]We present here a new automated flow injection-stopped flowmethod for determination of pectin methyl esters A uniqueaspect of thismethod is that reagents are injected into flowingsample stream This approach called reverse flow injection(rFI) comes alongwith a variety of advantages minimizationof reagent consumption being the most common reason forchoosing rFImode rFI is of interest when expensive reagentslike enzymes are used and also when the sample is in abun-dance for example when water is being analysed MoreoverrFI offers increased sensitivity The sample is not injectedand therefore there is no significant dispersion of the sampleimproving sensitivity of the measurement The limitations ofrFI are related to sample throughput as it might be necessaryto stop the system in order to change the sample or even toemploy washing solution between the measurements Never-theless since the introduction of rFI in 1978 the number ofpublications using this approach is constantly increasing [15]

Flow methods are a well-established branch of analyticalchemistry and their applications in quality assurance of foodcompounds [16] or pharmaceuticals [17] offer a rapid and sus-tainable alternative to manual methods Flow injection sys-tems are automated providing rapid procedures of increasedprecision by minimizing human intervention and errorsThe testing environment is identical for all samples furtherincreasing precision Through flow methods sustainability isbrought into analytical work The method presented here isa rapid and sustainable way for analysis of crucial parameterof pectins with low reagent and sample consumption as wellas minimal waste generation The use of enzymes minimizesamount of chemicals used in the analytical work Enzymesare a natural biodegradable and environmentally friendlyalternative to chemicals The method is based on an easilyconstructed manifold which can be assembled from readilyavailable commercial parts

Flow-injection systems for methanol determination havebeen reported in the literature [18ndash23] However the use offlow-injection for pectin analysis remains unexplored Theapplication of flow-injection methods for pectin analysis

offers novel green technologies which are in agreement withcurrent sustainability trends

2 Materials and Methods

21 Reagents Enzyme working solution (EWS) is used in theflow and comparison method 12 UmLminus1 alcohol oxidase(AOX) (EC 11313) 46UmLminus1 horseradish peroxidase(HRP) (EC 11117) 75mM phenol and 25mM 4-amino-antipyrine in 01M phosphate buffer Enzymes were pur-chased from SigmaAldrich All other reagents were of analyt-ical grade EWS used in the kinetic study contained 1UmLminus1AOX and 40UmLminus1 HRP A commercial preparation of arecombinant Aspergillus oryzae pectin methylesterase (PME)(EC 31111) (Novoshape Novozymes Bagsvaerd Denmark)was a generous gift from Dr Hans Sejr Olsen This prepara-tion was filtered before use

22 Standards and Samples A stock solution of 025 g 100mLminus1 pectin equivalent to 72mM methanol concentrationwas prepared in deionized water using HM citrus pectin of695 degree of esterification Dilutions 002 005 009 012014 018 022 and 025 g 100mLminus1 corresponding to 049 1426 34 40 52 63 and 72mM methanol were preparedfrom the stock solution Methanol concentrations calcula-tions were based on degree of esterification and galacturonicacid contentmeasurementsMethanol standards of 025 049074 099 124 148 and 173mM for comparison methodwere prepared in deionized water and stored at minus20∘C Thestandards were thawed just before measurement Methanolstandards used for the kinetic study were prepared in 01Mphosphate buffer pH 75

Pectins were donated by CP Kelco ApS (Lille SkensvedDenmark) and by Dr Karen Marie Soslashndergaard DuPontNHIB Denmark ApS

23 Kinetic Study A kinetic study of the influence of temper-ature and pH on the EWS was performed The effect of dif-ferent AOX activities ranging from 05UmLminus1 to 25UmLminus1was examined Stability of the EWS upon 3months storage inthe fridge was studied 05mL of MeOH standard was placedin the quartz cuvette of Jasco V-550 spectrophotometerand 25mL EWS was added The time course measurementprogram was started immediately to monitor reaction rate at505 nm for 2-3minutes Reagent blanks were subtracted fromthe analytical signal when necessary

24 Comparison Method For comparison method pectinsaponification and neutralization were performed asdescribed by Klavons and Bennett (1986) [12] The absorb-ance was measured exactly 10 minutes after EWS additionEWS reagent and sample blank were measured and sub-tracted

25 Flow Injection Analyzer The flow injection analyzer isdepicted in Figure 1 Pectin is mixed in the first mixing coilwith pectin methylesterase First mixing coil was fixed at

Journal of Analytical Methods in Chemistry 3

Pump function

Pump

Injection valve

Pectin1mLmin

Pectinesterase1mLmin

Run RunStop RunStop

Load Load

Mixing coil100 cm

Enzymesolution

Mixing coil10 cm

DetectorWaste

Inject

Figure 1 Manifold scheme and timing sequence of the analyzer

100 cm as this is sufficient length for methanol creationMethanol is released as a result of action of the enzyme onmethyl esterified groups of pectin (1)

Pectin-COOCH3

+H2

OPectin methylesterase997888997888997888997888997888997888997888997888997888997888997888997888997888997888997888rarr

Pectin-COOminus +H+ + CH3

OH(1)

CH3

OH +O2

Alcohol oxidase997888997888997888997888997888997888997888997888997888997888997888rarr HCHO +H

2

O2

(2)

2H2

O2

+ phenol + 4-AAPHorseradish peroxidase997888997888997888997888997888997888997888997888997888997888997888997888997888997888997888997888rarr

4-N-(p-benzoquinoneimine)-antipyrine + 4H2

O(3)

EWS is injected further downstreamMethanol is oxidized byAOX releasing formaldehyde andhydrogen peroxide (2)Thisreaction is coupled toHRP catalyzed reactionwhich gives thecolored product 4-N-(p-benzoquinoneimine)-antipyrine (3)The reaction rate is proportional to methanol concentrationand it is followed usingOceanOpticsUSB 2000+ spectropho-tometer

The analyzer is managed by a lab written program devel-oped in LabVIEW which provides for data acquisition fromthe detector and control of the pump and injection valveTheprogram also controls the timing of the analyzer as presentedin Figure 1 displays raw data in the screen and stores onthe hard disc Dedicated functions have been developed forcontrolling the load and inject positions of the Vici Valcoinjection valve and the stop and run positions of the GilsonMinipuls 3 peristaltic pump Program runs on a personalcomputer incorporating the PCI-1760U Advantech cardmdashused for controlling the peristaltic pump and the injectionvalve Data acquisition from the spectrophotometer modulewas through the USB port controlled through LabVIEWmodule developed

One minute is allowed for (1) to proceed then EWS isinjected Nine seconds are allowed for the reaction mixtureto reach the flow cell Then the pump stops for five minutesto allow monitoring of (3) If needed the operator has thechoice to flush the system between successive measurementsPTFE tubing 08mm id was used

26 Treatment of Data The LabVIEW program acquires thewhole spectrum 189ndash1037 nm every second This creates atime-dependant data matrix A separate lab written programwas used for extracting data at 505 nmTheprogramwas builtusing Visual Basic and gets input from the file created by theLabVIEW program

Reaction rates were calculated by regression analysisusing the significant part of the data for each measurement

3 Results and Discussion

A kinetic study of the reactions preceded the developmentof the manifold To assure that the measured reaction rateis proportional to methanol concentration the methanoloxidation reaction (2) must be the rate limiting step Onthe other hand the HRP catalyzed reaction (3) shouldbe very fast The influence of AOX concentration on thereaction curves was investigated for the methanol concen-tration as expected from the HM sim025 g 100mLminus1 pectinsample When AOX activities higher than 15UmLminus1 areused methanol is consumed within the monitoring timeand linearity is lost towards the end of the measurementIn contrast low AOX activities such as 05UmLminus1 resultin lower reaction rates as shown in Figure 2 The activity of1 UmLminus1 was chosen as a tradeoff between high analyticalsignal and linearity

Robustness of the method strongly depends on the pHand temperature that affect enzyme activity Figure 3 showsthe effect of the EWS temperature in the range 20ndash30∘C Thereaction rate increases only 002mA sminus1∘Cminus1 This proves thatroom temperature is appropriate for the automated methodInvestigating the most optimal pH for the EWSwas of specialimportance since enzymes in the EWS have different optimalpH AOX 75 [24] and HRP 6ndash65 [25] Figure 4 shows thatpH changes of the enzyme working solution in the 65ndash75 range do not have any influence on the reaction rateTo summarize the influence of temperature and pH on thereaction proved to beminor and the robustness of themethodwas confirmed A long term stability study showed 195 and30 loss of EWS activity after 48 and 99 days respectively


0200400600800

10001200140016001800

0 50 100 150 200

Time (s)

a b c

d

e

A(m

A)

Figure 2 Alcohol oxidase (AOX) concentration influenceon the analytical signal-reaction rate a AOX concentration25UmLminus1(88 plusmn 02) times 119905 (s) + (198 plusmn 25) 119903 = 099 b AOXconcentration 2UmLminus1 (80 plusmn 01) times 119905 (s) + (13plusmn 1) times 10 119903 = 0997c AOX concentration 15 UmLminus1 (714 plusmn 005) times 119905 (s) + (89 plusmn 5)119903 = 09991 d AOX concentration 1 UmLminus1 (612 plusmn 002) times119905(s) + (79 plusmn 2) 119903 = 09997 and e AOX concentration05UmLminus1 (312 plusmn 001) times 119905(s) + (43 plusmn 1) 119903 = 09998 Concentrationof the methanol standard 76mM Absorbance presented inmilliabsorbance (mA)

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8 9

b

a

c

ΔAΔtminus

1(m

A sminus

1)

CMeOH (mM)

Figure 3 Calibration data obtained at different temperatures a30∘C Δ119860Δ119905minus1 (mAsminus1) = (066 plusmn 0003) times 119862 (mM) + (03 plusmn 01)119903 = 0997 b 25∘C Δ119860Δ119905minus1 (mAsminus1) = (058 plusmn 002) times 119862 (mM) +(001 plusmn 009) 119903 = 0998 and c 20∘C Δ119860Δ119905minus1 (mAsminus1) = (048 plusmn002) times 119862 (mM) + (001 plusmn 001) 119903 = 0997

31 Flow Method Optimization Univariate optimization wasperformed for the following parameters (i) PME commercialpreparation dilution (ii) total flow rate (iii) injection volume(iv) preincubation time the time allowed for methanolproduction (1) and (v) time allowed for the reaction mixtureto reach the flow cell that is the second run time in Figure 1Following parameter ranges were selected for the univariateoptimization total flow rate from 1 to 4mLminminus1 injectionvolume from 70 to 250120583L preincubation time from 2 to 60 sand second run time from 3 to 11 s The ranges were selectedbased on the limitations of fluidic systems

The effect of dilution of the PME preparation was testedby diluting filtered Novoshape solution in deionized water

0

05

1

15

2

25

3

35

4

0 2 4 6 8 10

a b

c

CMeOH (mM)

ΔAΔtminus

1(m

A sminus

1)

Figure 4 PH influence on the calibration curve a pH 70Δ119860Δ119905

minus1

(mAsminus1) = (044 plusmn 002) times 119862 (mM) + (02 plusmn 01) 119903 = 0999b pH 75 Δ119860Δ119905minus1 (mAsminus1) = (0433 plusmn 0009) times 119862 (mM) + (015 plusmn005) 119903 = 09998 and c pH 65 Δ119860Δ119905minus1(mAsminus1) = (044 plusmn 002) times119862(mM) + (00 plusmn 01) 119903 = 09991

Table 1 Effect of pectin methylesterase commercial preparationactivity on the analytical signal

Pectinesterase dilution (vv) Ratio signalblank1 3 141 5 161 7 241 10 311 20 1131 60 2761 100 352

up to 1 100 High PME activities increase the blank thatreaches sample signal This is probably due to glycerolcontained in Novoshape as a stabilizer AOX is not specifictowards methanol and although it has higher affinity towardsmethanol it probably also oxidizes glycerol This is furthersupported by the observation that the signal of the blankwas increasing along Novoshape concentration To minimizereagent blank higher Novoshape dilutions were assessed Itshould be noted that as expected lower PME activities resultin lower analytical signalThat is although dilutions 1 60 and1 100 show high signal to blank ratios (Table 1) they wererejected on the basis of 12 and 22 lower net analytical signalthan for 1 20 dilution which was finally selected

The range of total flow rate considered for optimizationwas 1ndash4mLminminus1 A total flow rate of 2mLminminus1 resultedin the best mixing of reagents giving the highest analyticalsignal Figure 5 shows that signal increases along the increaseof injected EWS volume in the range 70ndash123 120583L 123 120583L waschosen as further increase up to 250120583L does not affectsignificantly the analytical signal

Preincubation time is related to PME action influencingmethanol production in the first mixing coil Table 2 presentsthe effect of preincubation time on reaction rate in the rangeof 2 to 60 s Although further increase of preincubation timeis expected to result in increased signal 60 s were chosen


005

115

225

335

445

0 50 100 150 200 250 300 Injection volume (120583L)

ΔAΔtminus

1(m

A sminus

1)

Figure 5 Injection volume effect on the reaction rate using pectinstandard corresponding to 57mMmethanol

Table 2 Preincubation time effect on the reaction rate using pectinstandard corresponding to 57mMmethyl esters

Preincubation time (s) Reaction rate plusmn SE (mA sminus1)20 24 plusmn 0250 38 plusmn 01100 49 plusmn 01200 56 plusmn 01300 64 plusmn 01600 69 plusmn 01

as a tradeoff between analytical run time and sensitivityTo shorten development time 10 s preincubation time wasused throughout optimization A second run time used forallowing the reaction mixture to reach the flow cell (depictedin Figure 1) was varied from 3 to 11 s The analytical signalincreases along time the maximum being observed at 9 sand then it decreases Interestingly reagent blank observedduring the kinetic study was fully eliminated

Mixing coil placement after the injection valve facilitatesmixing of EWS with pectinPME stream To assure that thereaction mixture is rapidly transported for monitoring inthe flow cell coil length should be the smallest possibleIt should be noted that coils dampen peristaltic pumppulsations resulting in increased precision of flow analyticalmethods Therefore and also due to the fact that (3) proceedsimmediately a very short mixing coil that is 10 cm wasselected

Particular attention has to be paid to avoid carry-overbetween samples and to diminish the effect of absorp-tiondesorption processes on the manifold These phenom-ena are particularly exaggerated in case of viscous samplessuch as pectin Readings for the first daily calibration curvediffer significantly from the following First and two sub-sequent analyses of the standard corresponding to 14mMmethanol resulted in 103 plusmn 003 181 plusmn 003 and 179 plusmn003mAsminus1Thiswas solved by allowing 8 standard injectionsbefore actual measurements on startup improving precisionof 10 consecutive injections to 53 RSD

Typical readings acquired during the second stop of thepump illustrating the linearity of the method are shown in

0

100

200

300

400

500

600

700

800

900

20 70 120 170 220 270 320Time (s)

b

c

d e f g

h

a

A(m

A)

Figure 6 Stopped flow injection peaks for pectin calibrationstandards corresponding to following methanol concentrations (a)0mM (b) 049mM (c) 14mM (d) 26mM (e) 34mM (f) 40mM(g) 52mM (h) 63mM and (i) 72mM Absorbance presented inmilliabsorbance (mA)

Figure 6 The presented data are smoothed by substitutingvalues with the mean resulting from the previous 20 andfollowing 20 readingsThe calibration curve was reaction rate(mAsminus1) = minus005691198622 + 08487119862 + 04622 119903 = 0994

Results from the analysis of samples in comparisonto those acquired with the manual method are shown inTable 3 The detection limit calculated as LOD = 33 times119878interceptslope was found to be 147mM The analysis rateof the developed method is 7 samples hminus1 Analysis time isshorter than in manual method where at least 30 minutesare required for saponification To verify that the proposedmethod is equivalent to manual method a paired 119905-test wasperformed resulting in 119905experimental 0357 when 119905theoretical is318 for 3 df at confidence level 95 proving evidence thatthere is no significant difference between the two methodsFor HM pectin the difference between two methods wasinsignificant while for LM it was higher This is probablydue to the lower hydrolysis rate of LM pectins resultingfrom their lower methyl ester content A remedy is to matchsamples with standards using LM pectins for calibrationwhen analyzing LM pectins It is interesting to note that bothreactions catalyzed by PMEandAOX do not come to an endMoreover physicalmixing of the injected EWS cocktail is alsokinetic in nature not coming to the steady state during theanalytical run These are clear differences from the manualmethod giving the opportunity of lower analysis time but alsothe challenge for selecting appropriate calibration standards

In kinetic methods of analysis only the initial fraction ofthe reaction is monitored providing a fast alternative to time-consuming end point methods Analytical parameter is notthe absolute value of the signal but the rate of its changeThiseliminates end point problems due to sample andor reagentblank With kinetic methods of analysis interferences fromslow reactions are insignificantwhile those resulting from fastreactions are eliminated by a short preincubation step whereinterfering species are consumed [26]


Table 3 Comparison between the automated and the manualmethod

Methanol content mMAutomated method Manual method

Sample 1 40 44Sample 2 75 59Sample 3 58 62Sample 4 20 38

In order to increase the analysis rate and decrease thedetection limit the analyzer could be coupled to a more sen-sitive detection technique that is chemiluminescence withwhich concentrations as low as 10 nM can be measured [27]Use of chemiluminescence would allow higher dilution ofsamples and therefore the washing step could be eliminatedand the monitoring time shortened

4 Conclusions

This study reports the development of an automated flowmethod for determination of pectin methyl esters Detectionlimit down to 147mM was achieved at the analysis rate of 7samples hminus1 Paired 119905-test has shown no significant differencebetween results from the developed analyzer and manualoffline methodThe developed method is the first applicationof flow analysis in pectin quality assessment Samples ofincreased viscosity such as olive oil or pectin need specialattention when pumped in flow injection systems Viscoussamples can lead to carry-over effect between the samplesIn order to avoid these problems additional washing stepor saturation of the system prior to measurements mightbe necessary Another solution could involve merging theanalyzer with more sensitive detection method for examplechemiluminescence This would allow using higher dilutionsof the sample introducing therefore lower viscosity Furtherresearch should focus on the development of flow analyzer forgalacturonic acid content which together with the analyzerpresented here would constitute a system of analyzers for thedegree of methyl esterification

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The research leading to these results has received fundingfrom the (European Communityrsquos) Seventh Framework Pro-gram (FP72007ndash2013) under Grant agreement no 238084 asa part of the EU-ITN LEANGREENFOOD network projectMahsaNaghshineh is acknowledged for providing data on thedegree of esterification and galacturonic acid content of thesamples

References

[1] J N BeMiller Carbonate Chemistry for Food Scientists AACCInternational St Paul Minn USA 2007

[2] E G Maxwell N J Belshaw K W Waldron and V J MorrisldquoPectinmdashan emerging new bioactive food polysacchariderdquoTrends in Food Science and Technology vol 24 no 2 pp 64ndash73 2012

[3] L Leclere P van Cutsem and C Michiels ldquoAnti-cancer activi-ties of pH- or heat-modified pectinrdquo Frontiers in Pharmacologyvol 4 article 128 2013

[4] S K Niture and L Refai ldquoPlant pectin a potential source forcancer suppressionrdquoTheAmerican Journal of Pharmacology andToxicology vol 8 no 1 pp 9ndash19 2013

[5] J F Thilbault and M C Ralet ldquoPectins their origin structureand functionsrdquo in Advanced Dietary Fibre Technology B VMcCleary and L Prosky Eds pp 369ndash378 Blackwell ScienceOxford UK 2001

[6] L S Liu M L Fishman and K B Hicks ldquoPectin in controlleddrug deliverymdasha reviewrdquoCellulose vol 14 no 1 pp 15ndash24 2007

[7] G AMorris M S Kok S E Harding and G G Adams ldquoPoly-saccharide drug delivery systems based on pectin and chitosanrdquoBiotechnology and Genetic Engineering Reviews vol 27 pp 257ndash284 2010

[8] S Sungthongjeen P Sriamornsak T Pitaksuteepong A Som-siri and S Puttipipatkhachorn ldquoEffect of degree of esterifica-tion of pectin and calcium amount on drug release from pectin-based matrix tabletsrdquo AAPS PharmSciTech vol 5 no 1 pp 50ndash57 2004

[9] L G Bartolome and J E Hoff ldquoGas chromatographic methodsfor the assay of pectin methylesterase free methanol andmethoxy groups in plant tissuesrdquo Journal of Agricultural andFood Chemistry vol 20 no 2 pp 262ndash266 1972

[10] B J Savary and A Nunez ldquoGas chromatography-mass spec-trometry method for determining the methanol and acetic acidcontents of pectin using headspace solid-phasemicroextractionand stable isotope dilutionrdquo Journal of Chromatography A vol1017 no 1-2 pp 151ndash159 2003

[11] A G J Voragen H A Schols and W Pilnik ldquoDeterminationof the degree of methylation and acetylation of pectins byHPLCrdquo Food Hydrocolloids vol 1 no 1 pp 65ndash70 1986

[12] J A Klavons and R D Bennett ldquoDetermination of methanolusing alcohol oxidase and its application tomethyl ester contentof pectinsrdquo Journal of Agricultural and Food Chemistry vol 34no 4 pp 597ndash599 1986

[13] G E Anthon and D M Barrett ldquoComparison of three colori-metric reagents in the determination of methanol with alcoholoxidase Application to the assay of pectin methylesteraserdquoJournal of Agricultural and Food Chemistry vol 52 no 12 pp3749ndash3753 2004

[14] T J Mangos and M J Haas ldquoEnzymatic determination ofmethanol with alcohol oxidase peroxidase and the chromogen221015840-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and itsapplication to the determination of the methyl ester content ofpectinsrdquo Journal of Agricultural and Food Chemistry vol 44 no10 pp 2977ndash2981 1996

[15] F R Mansour and N D Danielson ldquoReverse flow-injectionanalysisrdquo Trends in Analytical Chemistry vol 40 pp 1ndash14 2012

[16] R Hernandez-Martınez and I Navarro-Blasco ldquoSurvey of totalmercuryand arsenis content in infant cereals marketed in Spainand estimated dietary intakerdquo Food Control vol 30 no 2 pp423ndash432 2013


[17] S M Wabaidur S M Alam S H Lee Z A Alothman and GE Eldesoky ldquoChemiluminescence determination of folic acidby a flow injection analysis assemblyrdquo Spectrochimica Acta Avol 105 pp 412ndash417 2013

[18] J Burfeind B Weigel G Kretzmer K Schugerl A Huwigand F Giffhorn ldquoDetermination of the concentration of higheralcohols with enzyme coupled flow-injection analysis in modelsystemsrdquo Analytica Chimica Acta vol 322 no 3 pp 131ndash1391996

[19] E Forster M Silva M Otto and D Perez-Bendito ldquoEnzymaticdetermination of alcohol mixtures at the nanogram level by thestopped-flow techniquerdquo Analytica Chimica Acta vol 274 no1 pp 109ndash116 1993

[20] CG deMarıa TManzano RDuarte andAAlonso ldquoSelectiveflow-injection determination of methanol using immobilizedenzyme reactorsrdquo Analytica Chimica Acta vol 309 no 1ndash3 pp241ndash250 1995

[21] Y Sekine M Suzuki T Takeuchi E Tamiya and I KarubeldquoSelective flow-injection determination ofmethanol in the pres-ence of ethanol based on a multi-enzyme system with chem-iluminescence detectionrdquo Analytica Chimica Acta vol 280 no2 pp 179ndash184 1993

[22] C Almuzara O Cos M Baeza D Gabriel and F ValeroldquoMethanol determination in Pichia pastoris cultures by flowinjection analysisrdquo Biotechnology Letters vol 24 no 5 pp 413ndash417 2002

[23] X F Yue and Z Q Zhang ldquoSimultaneous determination offormaldehyde and methanol by flow-injection catalytic spec-trophotometryrdquo Journal of Analytical Chemistry vol 62 no 10pp 992ndash996 2007

[24] R Couderc and J Baratti ldquoOxidation of methanol by the yeastPichia pastoris purification and properties of alcohol oxidaserdquoAgricultural and Biological Chemistry vol 44 no 10 pp 2279ndash2289 1980

[25] D Schomberg M Salzmann and D Stephan Enzyme Hand-book 7 EC 11117 1993

[26] A G Vasilarou and C A Georgiou ldquoEnzymatic spectrophoto-metric reaction rate determination of glucose in fruit drinks andcarbonated beverages an analytical chemistry laboratory exper-iment for food science-oriented studentsrdquo Journal of Chem-ical Education vol 77 no 10 pp 1327ndash1329 2000

[27] E G Vasiliou Y M Makarovska I A Pneumatikos et alldquoHydrogen peroxide assessment in exhaled breath conden-sate condensing equipment-rapid flow injection chemilumi-nescencemethodrdquo Journal of the BrazilianChemical Society vol18 no 5 pp 1040ndash1047 2007

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when settling or flotation of fruit pieces has to be avoidedIn contrast slow setting pectins are especially useful forproduction of jellies when additional time for elimination ofair bubbles before gelling is needed [1]

Pectins are used in the pharmaceutical industry in drugdelivery systems [6] Their applications greatly depend onthe degree of methylation for example LM pectins are usedin nasal preparations due to their ability to form gels in thepresence of calcium cations [7] DM also influences the drugrelease time from pectin-based matrix tablets when calciumformulations are used [8] The application of pectin dependsgreatly on its degree of methylation This is the reason whyrapid and reliable methods for fast quantification of pectinmethyl esters are of major interest and importance

Pectin methyl esters can be hydrolyzed either by alka-line hydrolysis or enzymatically by pectin methylesteraseReleased methanol can be quantified by means of GC [9 10]and HPLC [11] or with alcohol oxidase [12] When alcoholoxidase is used the methanol content can be related toformaldehyde [13] or hydrogen peroxide concentration [14]We present here a new automated flow injection-stopped flowmethod for determination of pectin methyl esters A uniqueaspect of thismethod is that reagents are injected into flowingsample stream This approach called reverse flow injection(rFI) comes alongwith a variety of advantages minimizationof reagent consumption being the most common reason forchoosing rFImode rFI is of interest when expensive reagentslike enzymes are used and also when the sample is in abun-dance for example when water is being analysed MoreoverrFI offers increased sensitivity The sample is not injectedand therefore there is no significant dispersion of the sampleimproving sensitivity of the measurement The limitations ofrFI are related to sample throughput as it might be necessaryto stop the system in order to change the sample or even toemploy washing solution between the measurements Never-theless since the introduction of rFI in 1978 the number ofpublications using this approach is constantly increasing [15]

Flow methods are a well-established branch of analyticalchemistry and their applications in quality assurance of foodcompounds [16] or pharmaceuticals [17] offer a rapid and sus-tainable alternative to manual methods Flow injection sys-tems are automated providing rapid procedures of increasedprecision by minimizing human intervention and errorsThe testing environment is identical for all samples furtherincreasing precision Through flow methods sustainability isbrought into analytical work The method presented here isa rapid and sustainable way for analysis of crucial parameterof pectins with low reagent and sample consumption as wellas minimal waste generation The use of enzymes minimizesamount of chemicals used in the analytical work Enzymesare a natural biodegradable and environmentally friendlyalternative to chemicals The method is based on an easilyconstructed manifold which can be assembled from readilyavailable commercial parts

Flow-injection systems for methanol determination havebeen reported in the literature [18ndash23] However the use offlow-injection for pectin analysis remains unexplored Theapplication of flow-injection methods for pectin analysis

offers novel green technologies which are in agreement withcurrent sustainability trends

2 Materials and Methods

21 Reagents Enzyme working solution (EWS) is used in theflow and comparison method 12 UmLminus1 alcohol oxidase(AOX) (EC 11313) 46UmLminus1 horseradish peroxidase(HRP) (EC 11117) 75mM phenol and 25mM 4-amino-antipyrine in 01M phosphate buffer Enzymes were pur-chased from SigmaAldrich All other reagents were of analyt-ical grade EWS used in the kinetic study contained 1UmLminus1AOX and 40UmLminus1 HRP A commercial preparation of arecombinant Aspergillus oryzae pectin methylesterase (PME)(EC 31111) (Novoshape Novozymes Bagsvaerd Denmark)was a generous gift from Dr Hans Sejr Olsen This prepara-tion was filtered before use

22 Standards and Samples A stock solution of 025 g 100mLminus1 pectin equivalent to 72mM methanol concentrationwas prepared in deionized water using HM citrus pectin of695 degree of esterification Dilutions 002 005 009 012014 018 022 and 025 g 100mLminus1 corresponding to 049 1426 34 40 52 63 and 72mM methanol were preparedfrom the stock solution Methanol concentrations calcula-tions were based on degree of esterification and galacturonicacid contentmeasurementsMethanol standards of 025 049074 099 124 148 and 173mM for comparison methodwere prepared in deionized water and stored at minus20∘C Thestandards were thawed just before measurement Methanolstandards used for the kinetic study were prepared in 01Mphosphate buffer pH 75

Pectins were donated by CP Kelco ApS (Lille SkensvedDenmark) and by Dr Karen Marie Soslashndergaard DuPontNHIB Denmark ApS

23 Kinetic Study A kinetic study of the influence of temper-ature and pH on the EWS was performed The effect of dif-ferent AOX activities ranging from 05UmLminus1 to 25UmLminus1was examined Stability of the EWS upon 3months storage inthe fridge was studied 05mL of MeOH standard was placedin the quartz cuvette of Jasco V-550 spectrophotometerand 25mL EWS was added The time course measurementprogram was started immediately to monitor reaction rate at505 nm for 2-3minutes Reagent blanks were subtracted fromthe analytical signal when necessary

24 Comparison Method For comparison method pectinsaponification and neutralization were performed asdescribed by Klavons and Bennett (1986) [12] The absorb-ance was measured exactly 10 minutes after EWS additionEWS reagent and sample blank were measured and sub-tracted

25 Flow Injection Analyzer The flow injection analyzer isdepicted in Figure 1 Pectin is mixed in the first mixing coilwith pectin methylesterase First mixing coil was fixed at


Pump function

Pump

Injection valve

Pectin1mLmin


Run RunStop RunStop

Load Load

Mixing coil100 cm

Enzymesolution

Mixing coil10 cm

DetectorWaste

Inject



Pectin-COOCH3

+H2



OH(1)

CH3

OH +O2


2

O2

(2)

2H2

O2



O(3)










0200400600800

10001200140016001800

0 50 100 150 200

Time (s)

a b c

d

e

A(m

A)


0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8 9

b

a

c

ΔAΔtminus

1(m

A sminus

1)

CMeOH (mM)




0

05

1

15

2

25

3

35

4

0 2 4 6 8 10

a b

c

CMeOH (mM)

ΔAΔtminus

1(m

A sminus

1)


minus1








005

115

225

335

445


ΔAΔtminus

1(m

A sminus

1)








0

100

200

300

400

500

600

700

800

900

20 70 120 170 220 270 320Time (s)

b

c

d e f g

h

a

A(m

A)










4 Conclusions




Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Pump function

Pump

Injection valve

Pectin1mLmin


Run RunStop RunStop

Load Load

Mixing coil100 cm

Enzymesolution

Mixing coil10 cm

DetectorWaste

Inject



Pectin-COOCH3

+H2



OH(1)

CH3

OH +O2


2

O2

(2)

2H2

O2



O(3)










0200400600800

10001200140016001800

0 50 100 150 200

Time (s)

a b c

d

e

A(m

A)


0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8 9

b

a

c

ΔAΔtminus

1(m

A sminus

1)

CMeOH (mM)




0

05

1

15

2

25

3

35

4

0 2 4 6 8 10

a b

c

CMeOH (mM)

ΔAΔtminus

1(m

A sminus

1)


minus1








005

115

225

335

445


ΔAΔtminus

1(m

A sminus

1)








0

100

200

300

400

500

600

700

800

900

20 70 120 170 220 270 320Time (s)

b

c

d e f g

h

a

A(m

A)










4 Conclusions




Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0200400600800

10001200140016001800

0 50 100 150 200

Time (s)

a b c

d

e

A(m

A)


0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8 9

b

a

c

ΔAΔtminus

1(m

A sminus

1)

CMeOH (mM)




0

05

1

15

2

25

3

35

4

0 2 4 6 8 10

a b

c

CMeOH (mM)

ΔAΔtminus

1(m

A sminus

1)


minus1








005

115

225

335

445


ΔAΔtminus

1(m

A sminus

1)








0

100

200

300

400

500

600

700

800

900

20 70 120 170 220 270 320Time (s)

b

c

d e f g

h

a

A(m

A)










4 Conclusions




Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


005

115

225

335

445


ΔAΔtminus

1(m

A sminus

1)








0

100

200

300

400

500

600

700

800

900

20 70 120 170 220 270 320Time (s)

b

c

d e f g

h

a

A(m

A)










4 Conclusions




Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






4 Conclusions




Acknowledgments


References






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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