articol edta

Analytica Chimica Acta 535 (2005) 57–63

EDTA determination in pharmaceutical formulations andcanned foods based on ion chromatography with suppressed

conductimetric detection

A.A. Krokidis, N.C. Megoulas, M.A. Koupparis∗

Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece

Received 21 October 2004; received in revised form 6 December 2004; accepted 6 December 2004Available online 19 January 2005

Abstract

A novel direct method for the determination of EDTA was developed and validated based on ion chromatography with suppressed con-ductimetric detection and anion exchange column (Dionex AS-14, 4 mm× 250 mm). Depending on coexisting substances, suitable eluentsare 10 mM carbonate buffer/pH 11.0 or 10.5 (t = 5.5 and 9.4 min, respectively), and 120 mM borate buffer/pH 8.5 (t = 16.2 min).F( ions( ference ofp etal cations( solutions,s %).©

K

1

ciwaut[tdfat

eddress-

nd tohela-andningantsion, be-eenn ofnr theces-

c

0d

R,EDTA R,EDTA

or 10 mM carbonate buffer/pH 11.0 and isocratic flow rate of 1.0 ml min−1, a linear calibration curve was obtained from 2.7 to 100�g ml−1

r > 0.998), with LOD 0.87�g ml−1 and %RSD 1.5 (5�g ml−1, n= 9). Good resolution was achieved from commonly coexisting anchloride, metabisulphite, ascorbate and citrate), and other aminopolycarboxylic acids (EGTA, NTA and DTPA). The potential interharmaceutical substances (caffeine, phenytoin, nembutal, tolbutamide, dicumarol, acetylsulphisoxazole and paracetamol) and mCa2+, Cu2+ and Fe3+) was also examined. The ion chromatographic method was applied for the assay of EDTA in contact lens careynthetic injection drug solutions, canned mushrooms and mayonnaise, with simple treatment and good recovery (range 74–1082004 Elsevier B.V. All rights reserved.

eywords: EDTA; Ion chromatography; Suppressed conductimetric detection; Pharmaceuticals; Foods

. Introduction

Ethylenediaminetetraacetic acid (EDTA) is a powerfulhelating agent, forming stable complexes with most metalons. Due to its ability to sequester metal ions, EDTA isidely used in medicine, chemical industry, food technology,griculture and pharmaceutical technology. In medicine, it issed for the treatment of lead poisoning[1] and in agricul-

ure to enhance the delivery of metal micronutrients in plants2]. Its industrial applications include pulp, paper, metal andextile production[3]. EDTA, in its disodium salt or calciumisodium salt form, is frequently added in pharmaceutical

ormulations and foods, because of its stability, compatibilitynd low toxicity. In pharmaceutical formulations it enhances

he action of preservatives and antibacterials and stabilizes the

∗ Corresponding author. Tel.: +30 210 7274559; fax: +30 210 7274750.E-mail address:[email protected] (M.A. Koupparis).

action of antioxidants[4,5]. In foods (e.g. canned or picklvegetables, canned mushrooms, mayonnaise and saladings) EDTA is added to prevent deteriorative changes, apreserve color, odor and flavor. Its action is based on the ction of metal ions, which catalyze oxidation reactions,the inactivation of enzymes that cause enzymatic brow[6,7]. At the same time, usage of EDTA or other sequestris strictly regulated by U.S. Food and Drug Administrator relevant agencies. In the field of analytical chemistrysides its use in complexometric titrations, EDTA has breported to be a very useful ligand for the complexatiometals, which enables their chromatographic separatio[8].

Various analytical methods have been proposed fodetermination of EDTA in a wide variety of sample matriand reviewed[9]. They include titrimetry[10], spectrophotometry[11], electrochemistry (e.g. polarography[12], dif-ferential pulse polarography[13], catalytic potentiometrititrimetry [14], one-drop square-wave polarography[15],

003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2004.12.011

58 A.A. Krokidis et al. / Analytica Chimica Acta 535 (2005) 57–63

differential pulse anodic stripping voltammetry[16], am-perometry[17]), capillary electrophoresis[18] and chro-matography. Amongst them, gas chromatography and HPLC(reverse-phase ion-pair or ion exchange retention mecha-nism) appear to be the prevailing techniques, despite thefact that EDTA lacks volatility and exhibits low UV/Visabsorptivity. The gas chromatographic methods always in-clude a time-consuming derivatization step, in which EDTAis converted into methyl, ethyl, propyl or butyl esters toobtain volatility [19–21]. Similarly, HPLC methods aremainly based on a pre-column derivatization step in whichcomplexation of EDTA with Cu2+ or Fe3+ is conducted[5,6,22–24], while post-column derivatization for the forma-tion of fluorescent ternary complexes has also been reported[25].

This paper describes a systematic study of the ion chro-matographic behavior of EDTA and the effect of varioussubstances, such as inorganic and organic ions (Fe3+, Cu2+,Ca2+, Cl−, S2O5

2−, ascorbate, citrate), similar aminopoly-carboxylic acids with complexing properties (EGTA, NTA,DTPA) and pharmaceutical substances. Based on this study,a novel ion chromatographic method with suppressed con-ductimetric detection for the direct (without derivatizationstep) determination of EDTA was developed and validated.The method was subsequently used for the determination ofE so-l ods(

2

2

t ont sist-i am-p -1 l-uda thea nsity5 ume1 tiono weree kardm

2

sedw ob-t ).T ula-t yn-

thetic injection drug solutions (containing 500�g ml−1 ofdrug [caffeine, phenytoin, nembutal, tolbutamide, dicumarol,acetylsulphisoxazole, paracetamol] and 100�g ml−1 EDTAin NaCl 0.9%) were also prepared to be analyzed.

2.2.1. EDTA standard solutionsAn EDTA stock solution (equivalent to free acid

500�g ml−1) was prepared by dissolving the appropriateamount of EDTA disodium salt (C10H14N2Na2O8·2H2O,Riedel-de-Haen, extra pure) in water and stored in the re-frigerator (at 4◦C) in a plastic bottle. Working standard solu-tions (in the range of 2.7–100�g ml−1) were daily preparedby appropriate dilution with mobile phase.

2.2.2. Mobile phaseThe selected mobile phases (10 mM aqueous carbonate

buffer with pH 10.5 or 11.0) were prepared by dissolvingsodium hydrogen carbonate in water and adjusting the pHwith a concentrated sodium hydroxide solution. They werestored in the refrigerator in a plastic bottle and renewed every2 weeks. Other examined mobile phases were: (i) 20–32 mMborate aqueous solutions (Na2B4O7·10H2O), without pHadjustment, (ii) 28–150 mM borate buffers of pH 8.5–9.5(H3BO3 + NaOH), and (iii) 7–20 mM carbonate buffers ofp

2

omc t1 thes werefi -p min,u -m tivityr

2tion

d sh-r tedi ent.M ted,fi l oft f or-g lumn( singl be-f xis-t peri-o isr

DTA in pharmaceutical formulations (contact lens careutions and synthetic injection solutions) and canned fomushrooms and mayonnaise).

. Experimental

.1. Instrumentation

Ion chromatographic separations were carried ouhe Dionex DX-100 ion chromatographic system, conng of: a DX-100 high pressure one piston pump, a sle injector equipped with a 25�l loop, an Ion pac AG4 guard (4 mm× 50 mm) and an AS-14 analytical comn (4 mm× 250 mm) (macroporous particles with 9.0�miameter, porous size 100A, total capacity of 78�eq),n ASRS-I micro-membrane suppressor operating inuto suppression recycle mode (selectable current inte0–500 mA), and a conductimetric detector (dead vol.25�l) equipped with a thermistor for the compensaf temperature variations. The chromatographic peakslectronically integrated and recorded with a Hewlett Pacodel 3395 integrator.

.2. Reagents

All chemicals were of analytical reagent grade and uithout further purification. HPLC-grade water was

ained with a Milli-Q water purification system (Milliporehe analyzed canned foods and pharmaceutical form

ions were obtained from local commercial sources. S

H 9.5–11.0.

.3. Procedures

The chromatographic elution was performed at roontrolled temperature (22–24◦C) in isocratic mode a.0 ml min−1 flow rate. The selected current intensity foruppressor operation was 300 mA. The eluent solutionsltered through a 0.45�m membrane filter (HVLP, Milliore) before usage. The flow path was rinsed for about 15ntil baseline noise became less then 0.1�S. Other instruental parameters were: air pressure 5 psi and conduc

ange 30�S. Quantitation was based on peak areas.

.3.1. Sample preparationLiquid samples (contact lens care solutions, injec

rug solutions and surrounding liquid of canned muooms) were diluted with the eluent, filtered, and injecnto the chromatographic system without further treatm

ayonnaise samples were mixed with water, mildly healtered and extracted with benzene, for the removahe fatty substances. In the case of coexistence oanic drugs that are adsorbed on the polymeric coe.g. paracetamol), removal of the drug molecules, uiquid–liquid extraction with ethyl acetate is requiredore injection to the chromatograph. In the case of coeence of surfactants (e.g. contact lens care solutions), adical washing of the analytical column with acetonitrileequired.

A.A. Krokidis et al. / Analytica Chimica Acta 535 (2005) 57–63 59

3. Results and discussion

3.1. Selection of mobile phase

EDTA (H4Y) is a weak tetraprotic carboxylic acid.Five EDTA species (H4Y, H3Y−, H2Y2−, HY3− and Y4−,dissociation constants:K1 = 1.02× 10−2, K2 = 2.14× 10−3,K3 = 6.92× 10−7 andK4 = 5.50× 10−11) coexist in aqueoussolutions and their concentration distribution depends on thepH of their environment (mobile phase). The electric chargeof EDTA species strongly influences its selectivity coefficient(retention) but not the conductimetric detector response fac-tor, since electrochemical suppression was occurred beforedetection.

Preliminary experiments were carried out utilizing bo-rate (Na2B4O7·10H2O) eluents in the concentration range of10.0–32.0 mM, without pH adjustment. In all cases, two chro-matographic peaks were recorded for EDTA. The existenceof two peaks in the EDTA chromatograms may be attributedto the formation of EDTA complex with Na+. Despite thelow formation constant of NaY3− complex (logkf = 1.7), therelatively high concentration of Na+ in mobile phase favorsthe complex formation. Therefore, the first chromatographicpeak may be attributed to the NaY3− complex, while thesecond one to the free EDTA species. Increase of borate con-c ntiont

heri s( 0.0( ta se oft db ase ofp iftedt ciesic whilea equalt onec

-i (thec rma-t iono phicp ases( .0.F ratioo k wasf

adst fort hasewo

Fig. 1. Effect of pH of borate mobile phase on the EDTA chromatographicpeaks: (A) pH = 10 (the first peak (tR = 10.6 min) corresponds to NaY3−complex, and the second one (tR = 16.9 min) to free EDTA species), (B)pH = 9.5 (the first peak (tR = 11.6 min) corresponds to NaY3− complex, andthe second one (tR = 18.2 min) to free EDTA species) and (C) pH = 8.5 (thefirst peak was disappeared and only one peak corresponding to HY3− specieswas recorded,tR = 17.4 min).

Calibration curves for EDTA in the range of 5–100�g ml−1 were constructed in order to evaluate the analyti-cal characteristics of various mobile phases.Table 1sum-marizes the chromatographic and analytical characteristicsof each tested mobile phase. As it is shown, an increase ofcarbonate or borate concentration was accompanied by a con-siderable decrease in the retention time of EDTA. The sym-metry of the EDTA peak (asymmetry factor 1.1–1.3) wassuitable for all eluents tested. The resolution from chlorideions (common excipient) in all cases was excellent, whilethe resolution from metabisulphite (S2O5

2−) was better forcarbonate mobile phase (Rs = 1.7) (Fig. 2). Comparing allthe chromatographic and analytical characteristics [analysistime, peak symmetry, resolution between adjacent peaks, sen-sitivity (slope of calibration curve), detectability (detectionlimit) and precision], it was concluded that a 10 mM carbon-ate buffer/pH 11.0 is the optimum eluent (tR = 5.5, LOD =0.87�g ml−1).

entration in mobile phase resulted in decrease of reteime of both peaks.

The EDTA ion chromatographic behavior was furtnvestigated using borate (H3BO3 + NaOH) mobile phase28–112 mM) with adjusted pH within the range of 8.5–1dominant EDTA species HY3− and Y4−). It was found thadecrease of pH of mobile phase resulted in a decrea

he area of the first peak (NaY3−), which was accompaniey an increase of the area of the second peak. A decreH, along with the decrease of sodium concentration, sh

he protonation equilibrium among the various EDTA spen favor of HY3−, which suppressed the formation of NaY3−omplex. Therefore, the area of the first peak decreased,n increase was observed for the second peak. For pH

o 8.5, the first peak was disappeared, obtaining onlyhromatographic peak (Fig. 1).

In the contrary, for pH above 10.5, the Y4− species domnated, the concentration of sodium in mobile phaseounter cation of hydroxides) increased, and so the foion of NaY3− complex was favored inducing the eliminatf the second and an increase of the first chromatograeak. This effect was observed for carbonate mobile ph7–20 mM) with varying pH within the range of 9.5–11or 10 mM carbonate mobile phase with pH = 10.5, thef the area of the first peak to the area of the second pea

ound to be 25.3.An overall evaluation of the results from this study le

o the conclusion that the ion chromatographic methodhe determination of EDTA should be based on mobile pith pH below 8.5 (borate buffer, dominant species HY3−)r above 10.5 (carbonate buffer, dominant species NaY3−).


Table 1Chromatographic and analytical characteristics of EDTA determination (5–100�g ml−1) for the selection of mobile phase

Mobile phase tR (min) Retentionfactor,k′

Asymmetryfactor

Resolution,REDTA/S2O5

2−Slope of calibrationcurve(×105 �V s ml mg−1)

Correlationcoefficient(n= 5)

Detectionlimit(�g ml−1)

%RSD(n= 3× 3)a

2.3�g ml−1

Carbonate buffer8 mM, pH 10.5 9.4 9.4 1.3 – – – – –10 mM, pH 10.5 6.3 6.0 1.2 1.7 1.64 (±0.03) 0.998 1.2 3.010 mM, pH 11.0 5.5 5.1 1.1 1.4 1.90 (±0.04) 0.998 0.87 1.5

Borate bufferH3BO3 + NaOH, pH 8.5

112 mM 17.0 17.9 1.3 1.0 2.18 (±0.07) 0.997 4.2 1.5120 mM 16.2 17.0 1.3 1.0 2.85 (±0.12) 0.993 0.53 1.5140 mM 12.5 12.9 1.1 1.0 2.55 (±0.02) 0.999 0.22 0.7150 mM 11.8 12.1 1.1 0.9 2.63 (±0.16) 0.996 0.83 2.6

a Three working days (within a week), three injections per day.

3.2. Separation from potential interfering substances

Interferences from substances usually coexisting withEDTA in foods and formulations were studied, using the car-bonate eluent. It was examined, whether ascorbate and citrate,common preservatives for canned foods, affect the chromato-graphic behavior of EDTA. The applied mobile phase was the10 mM carbonate buffer (pH = 10.5). No interference was ob-served for both molecules, since the retention time of citratewas 52.1 min, while EDTA resolution from ascorbate wasRs = 1.2.

Fm[t

Potential interferences from chelating aminocar-boxylic acids [EGTA (ethyleneglycol-bis-(2-aminoethyle-ther)tetraacetic acid), NTA (nitrilotriacetic acid) and DTPA(diethylenetriaminepentaacetic acid)] were also examined.DPTA appeared retention time logger than 30 min (it bearsan extra carboxylate group in comparison to EDTA) and itwas not further investigated. NTA and EGTA were elutedat 6.9 and 7.4 min, respectively, resulting in inadequateresolution from EDTA (tR = 6.3 min). Better separationcan be achieved by reducing the carbonate concentrationin mobile phase. Utilizing an 8.0 mM carbonate eluent(pH = 10.5), the retention time of EDTA was 9.4 min,while the retention time of NTA and EGTA were 12.7 and15.1 min, respectively. Resolution between EDTA and NTAwas 1.2 (Fig. 3). Calibration graph was constructed with

ig. 2. Resolution of EDTA (5�g ml−1) from chloride (100�g ml−1) andetabisulphite (20�g ml−1) with 10.0 mM carbonate eluent (pH = 10.5)

tR = 3.7 min corresponds to chloride,tR = 6.4 min corresponds to EDTA and

R = 8.0 min corresponds to metabisulphite].

F n-c .5)[ ndt

ig. 3. Resolution of EDTA from NTA, EGTA and DPA, all at a coentration level of 100�g ml−1, with 8.0 mM carbonate eluent (pH = 10tR = 9.4 min corresponds to EDTA,tR = 12.7 min corresponds to NTA a

= 15.1 min corresponds to EGTA, no peak was recorded for DPA].
R


EDTA standard solutions containing 100�g ml−1 of NTAand EGTA. No significant change was found to be causedby NTA or EGTA in EDTA peak area and in the slope of thecalibration curve (t-test).

Since the scope of our study was mainly the applicationof the IC method for the determination of EDTA in phar-maceutical formulations, seven pharmaceutical substancesof different molecular type (caffeine, phenytoin, nembu-tal, tolbutamide, dicumarol, acetylsulphisoxazole and parac-etamol) were tested for their effect on the EDTA peak. Itwas found that conductimetric detector appear no responsefor these substances, since none of them bear significantelectric charge. However, poor symmetry of EDTA peakwas observed in case of coexistence of paracetamol (N-4-hydroxyphenyl acetamide). Probably paracetamol adsorbedto the polymeric stationary phase reducing the column ca-pacity. It was found that paracetamol can be removed fromthe sample by liquid–liquid extraction with ethyl acetate.The quantity of the extracted paracetramol was monitoredusing the UV absorption spectrum (paracetamol absorp-tion peak at 280 nm). When 500�g ml−1 paracetamol wereadded to a 50�g ml−1 EDTA standard, five sequential ex-tractions with 10 ml ethyl acetate were required for the com-plete removal of the interfering substance. Efforts to removeparacetamol utilizing Sep-pac® (Waters) C8 and C18 car-tu

ns,t froms lentc t, ino salts.IFs l)s -p and1 eaka

thatd ts fort on-a5a

3c

rthera icalf tiond reser-v nlyr witht ased

on liquid–liquid extraction for the elimination of lipophilicsubstances was conducted (according to Section2.3.1). In thecase of coexistence of organic molecules that are adsorbedon the analytical column (e.g. paracetamol), extraction withethyl acetate is required before sample injection. Aqueous10 mM carbonate buffer (pH = 11.0) was the selected mobilephase. A typical chromatogram of the contact lens care solu-tion is shown inFig. 4. A variety of viscosity agents, preser-vatives, surfactants and active drugs were contained in theselected samples. The compositions reported on the productlabels are included inTable 2. Retention times of most excip-ients were examined in order to assure that they do not inter-fere (peak overlapping) with EDTA. However, since some ofthem (e.g. surfactants) were excessively retained by the ana-lytical column (due to strong interactions with the polymericion exchange stationary phase), their elution was not feasi-ble by the selected mobile phase and therefore, a periodicalwashing with acetonitrile was performed.

Fig. 4. Typical chromatogram of contact lens care solution with 10.0 mMcarbonate eluent (pH = 11.0) [tR = 3.3 min corresponds to chloride andtR = 5.2 min corresponds to EDTA].

ridges and the hydrophobic resin Amberlite® XAD-2 werensuccessful.

Since EDTA forms strong complexes with metal catiohe chromatographic determination is expected to suffertrong interference in the presence of divalent or trivaations. Investigation was conducted with borate eluenrder to avoid precipitation of the cations as carbonate

ncreasing amounts of Ca2+ (Kf = 10.7), Cu2+ (Kf = 18.8) ande3+ (Kf = 25.1) were added to a 50�g ml−1 EDTA standardolution. Application of at-test (at 95% confidence levehowed that the presence of Ca2+, Cu2+ and Fe3+ in the samle, at a ratio (EDTA:metal) equal or less than 1:50, 1:10:1, respectively, has no significant effect on the EDTA prea.

The results from this study leads to the conclusionepending on coexisting substances, suitable eluen

he determination of EDTA appears to be 10 mM carbte buffer/pH 10.5 (tR,EDTA = 6.3 min) or pH 11.0 (tR,EDTA =.5 min), 8 mM carbonate buffer/pH 10.5 (tR,EDTA = 9.4 min)nd 120 mM borate buffer/pH 8.5 (tR,EDTA = 6.3 min).

.3. Application to pharmaceutical formulations andanned foods

The proposed ion chromatographic method was fupplied for the determination of EDTA in pharmaceut

ormulations (contact lens care solutions, synthetic injecrug solutions) and canned foods (mayonnaise and pation liquid of mushrooms). For liquid samples the oequired sample treatment was the appropriate dilutionhe mobile phase, while for mayonnaise a procedure b


Table 2Recovery of EDTA from pharmaceutical formulations and canned foods

Sample type/declared excipients EDTA found (%, w/v)(n= 5)

Fortification levela

(�g ml−1)% Recovery % Mean recovery

(±S.D.)

Oxysept 2® Allergan, contact lens care solution [isotonic so-lution containing catalytic neutralizer 0.2% (w/v), thiomersal(disinfectant) 0.001%, w/v]

0.560± 0.007 10 105 99 (±4.3)30 9550 9970 97

Renu® Bausch&Lomb, contact lens care solution [isotonicsolution containing boric acid, sodium borate, DYMED(polyamino-propylbiguanidine) 0.0005% (w/v) and polox-amine 1%, w/v]

0.087± 0.002 20 96 99 (±2.8)30 10250 9870 101

Synthetic drug injection solutions of 500�g ml−1 (caf-feine, phenytoin, nembutal, tolbutamide, dicumarol, acetyl-sulphisoxazole or paracetamol) [containing NaCl 0.9% andEDTA 100�g ml−1]

– – – 97–108

Canned mushrooms [sodium chloride, citric acid, ascorbicacid]

0.095± 0.002 10 74 79 (±4.7)30 7750 8570 80

Mayonnaise [vegetable oils 76%, eggs 8.5%, vinegar, sodiumchloride, sugar, lemon juice, spices]

n.p.b 10 94 93 (±2.5)40 9370 90

100 96

a On diluted sample working solution containing about 30�g ml−1 EDTA; in the case of mayonnaise the solid sample (250 mg) was dispersed in EDTAsolution.

b Not present.

The accuracy of the new method was evaluated performingrecovery experiments by spiking sample working solutions.Recovery ranged from 95 to 105% for pharmaceutical for-mulations and from 74 to 96% for canned foods (Table 2),which revealed sufficient accuracy. Further study of the ma-trix effect on the determination was carried out by dilutionexperiments (determination of EDTA content using a vary-ing dilution factorD (Vinitial /Vfinal) at four different levels).The correlation curves of the concentration found (in the di-luted solution) versusD were linear (r > 0.998) with a slopeequal to the content of the EDTA and a statistically (provenby t-test) zero intercept. Similarly, the correlation curves ofEDTA content found (which corresponds to the initial undi-luted sample) versusD were very linear with statistically(proven byt-test) zero slopes. These results confirmed theabsence of any constant or proportional determinate errordue to matrix (excipients) effect.

4. Conclusions

Ion chromatography with suppressed conductimetric de-tection appears to be an efficient technique for the directdetermination of EDTA in pharmaceutical formulations andcanned foods. This IC-conductimetric method for EDTA hast therH de-t

ersd xes,

this is the first work dealing with an IC method with sup-pressed conductimetric detection for the assay of EDTA andcan also be applied to the assay of other chelating agents withsimilar (polyaminocarboxylate) structure.

Acknowledgements

We gratefully acknowledge support from the Ministry ofIndustry, Energy and Technology, General Secretariat of Re-search and Technology of Greece and the Ministry of Edu-cation (EPEAEK II Program ‘Pythagoras’).

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