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Bull. Pharm. Sci., Assiut University, Vol. 35, Part 2, 2012, pp. 199-214

Bulletin of Pharmaceutical SciencesAssiut University

ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــReceived in 15/10/2012 & Accepted in 17/12/2012

*Corresponding author: Gamal A. Saleh, E-mail: [email protected]

BATCH AND FLOW INJECTION POTENTIOMETRIC MONITORINGOF CEFTRIAXONE SODIUM IN PHARMACEUTICAL PREPARA-TIONS USING TWO NOVEL MEMBRANE SENSORS

Gamal A. Saleh1*, Ibrahim H. A. Badr2, Sayed M. Derayea3 and Deena A. M. Nour El-Deen3

1Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut University,Assiut, Egypt

2Department of Chemistry, Faculty of Science, Ain Shams University, Cairo, Egypt3Department of Analytical Chemistry, Faculty of Pharmacy, Minia University, Minia, Egypt

Two novel potentiometric sensors are prepared, characterized and successfully used forstatic and continuous determination of ceftriaxone sodium (CRXN). Both sensors are based onthe use of plasticized PVC matrix membranes incorporating tetradodecylmethyl ammoniumbromide (TDMAB), ortridodecylmethyl ammonium chloride (TDMAC) ion-exchangers and usedfor quantitative determination of CRXN at concentration level down to 29 µM using bothsensors with a good accuracy. Both sensors offer the advantages of fast response, reasonableselectivity, elimination of drug pre-treatment or separation steps, low cost and possibleinterfacings with computerized and automated systems. The use of plasticized membraneelectrodes were used for continuous monitoring of CRXN offers the advantages of simpledesign, ease of construction and possible applications to small volumes of drug solutions withlittle manipulation and without pre-treatment. Both detectors display a wide dynamicmeasurement range of the drug under continuous mode of operation with a flow rate of 2.0ml.min-1 and used for quantitative determination of CRXN. The developed sensors were utilizedin static continuous modes successfully for the determination of CRXN in pure powders and indosage forms. It is worth noting that the developed membrane electrodes exhibited goodselectivity toward CRXN over other cephalosporins such as; cefradine, ceftazidime, cefadroxil,cefaclor and cefoperazone, as well as other additives found in the pharmaceutical preparationssuch as; glucose, fructose and maltose.

INTRODUCTION

Cephalosporins are the second majorgroup of β-lactam class of antibiotics, isolatedfrom Cephalosporium species or preparedsemi-synthetically. They are antibiotics withbroad spectrum on antimicrobial properties.They have an added advantage over the firstknown group of this class of antibiotics,penicillin, that penicillin allergic patients canbe treated with these antibiotics;cephalosporins. Their antibacterial andpharmacokinetic properties have widetherapeutic use1. The broad spectrum ofactivity and favourable safety profile make thecephalosporins one of the most widely

prescribed class of antimicrobials. The earliergeneration cephalosporins are commonly usedfor community-acquired infections, while thelater generation agents, with their betterspectrum of activity against Gram-negativebacteria make them useful for hospital-acquiredinfections or complicated community-acquiredinfections2. CRXN (Fig. 1) is a parenteral thirdgeneration cephalosporins (M.W. 661.6). Itdisplays a broad spectrum of activity againstGram-negative and Gram-positive pathogens2.

Due to the extensive use of cephalosporinsin our market and the great importance of them,many reports have been suggested forquantification and stability assessment of them.These methods include spectrophotometry3-11

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Gamal A. Saleh, et al.

200

spectrofluorimetry12-15, TLC16&17, HPTLC18-20

and HPLC21-26. Most of these methods,however, utilize expensive instrumentation,suffer from lack of selectivity, involve carefulcontrol of the reaction conditions orderivatization reactions, and require time-consuming pre-treatment steps which affecttheir usefulness for routine analysis.

In view of the above factors, apotentiometric method was considered, beingcheaper, faster and sometimes more efficientthan HPLC. Very little work was found in theliterature for the electrochemical determinationof the investigated drug27&28.

Application of potentiometric sensors inthe field of pharmaceutical and biomedicalanalysis has been advocated. The approachprovides simple, fast, and selective techniquefor determination of various drugs29-34.However, as far as the available literature isconcerned, very little records were knownabout the use of this technique for the analysisof one member of the cephalosporins namely;cefuroxime35&36.

The objective of the present study is toconstruct potentiometric membrane sensors forstatic and hydrodynamic, flow injectionanalysis (FIA) of CRXN in pure powder and itspharmaceutical preparations. The developedsensors incorporate ion-exchangers; TDMACor TDMAB embedded in plasticized PVCmatrix membranes. Performance characteristicsof both sensors reveal low detection limit, goodsensitivity and selectivity, fast response, longlife span and application for accuratedetermination of CRXN in pharmaceuticalpreparations under static and hydrodynamic(FIA) modes of operation.

Fig. 1: Chemical structure of ceftriaxone sodium(CRXN).

EXPERIMENTAL

ApparatusAll membrane potential measurements

were made at 25±1°C using data acquisitionsystem, eight-channel electrode-computer

interface (Nico-2000 Ltd., London, UK)controlled by Nico-2000 software. An OrionRoss pH electrode (Model 80-02) was used forpH adjustment. A double-junction Ag/AgClelectrode (Orion Model 90-02-00) with anOrion (90-02-02) internal filling solution wasused as the reference electrode. The outercompartment of the reference electrode wasfilled with 1.0 M lithium acetate. Thecomponents of the FIA system were similar tothose used previously37. The flow injectionanalysis (FIA) system manifold (Fig. 2)consisted of a two channel Ismatech MS-REGLO model, peristaltic pump polyethylenetubing (0.71 i.d.) and an Omni fit injectionvalve (Omni fit, Cambridge UK) sample loopof 150 µl volume, the potential signals wererecorded using a high impedance dataacquisition 8-channel box connected to a PCthrough the interface Nico-2000 (Nico-2000Ltd) and Nico-2000 software. SpectronicTM,GenesysTM, ultraviolet-visible spectrophoto-meter (Milton Roy Co., USA) with matched 1cm quartz cells connected to an IBM computerloaded with the WinspecTM applicationsoftware.

Materials and reagents• All solvents used were of analytical-reagent

grade.• High molecular weight polyvinyl chloride

(PVC) was obtained from Poly sciences(Warrington, PA). Dichloro-tin(IV)-tetraphenylporphorin (Sn TPP-Cl2), copper-octaethylporphorin (Cu OEP), zinc-octa-ethylporphorin (Zn OEP), tridodecylmethylammonium chloride (TDMAC), tetra-dodecylmethyl ammonium bromide(TDMAB), bis(2-ethylhexyl) sebacate(DOS), benzyltributyl ammonium bromide(BTBAB), o-nitropheynloctylether (o-NPOE), ethylenebis-triphenylphosphinebromide (EBTPPB) and 2-fluorophenyl-2-nitrophenylether (FPNPE) were obtainedfrom Fluka (Ronkonkoma NY).Tetrahydrofuran (THF) was purchased fromFisher (Fairlawn, NJ). Tris-(hydroxymethyl)-aminomethane (TRIS) was obtained fromSigma (St. Louis, MO). Dichloro-bis(triphenyl-phosphine)palladium(II) (PdTPP-Cl2) was obtained from Aldrich(Milwaukee, WI). The sodium salts ofthiocyanate and sulfate were obtained from

COONa− +

201

Fig. 2: Manifold for the FIA set up used for the determination of CRXN.

Matheson (Cincinnati, OH). Sodium salts ofiodide and nitrate were purchased from J.T.Baker (Philipsburg, NJ). Sodium salt ofacetate was obtained from Sigma (St. Louis,MO). Sodium chloride was obtained fromFisher Scientific (Cincinnati, OH).Ceftriaxone sodium and ceftazidime-pentahydrate (T3A Pharma Group, Assiut,Egypt). Cefaclor monohydrate and cefradineanhydrous (Sigma Chemical Co., St. Louis,USA), cefadroxil monohydrate (AmounPharmaceutical Industries Co., APIC, Cairo,Egypt) and cefoperazone sodium (Pfizer Co.,Egypt) were obtained as gifts and were usedas supplied. Pharmaceutical formulationscontaining the studied drugs were purchasedfrom the local market.

• Acetate buffer was prepared by titrating 50mM solution of the acid form withconcentrated sodium hydroxide to a pH-value of 5.5±0.01. Phosphate buffer wasprepared by titrating 50 mM solution of theacid form with concentrated sodiumhydroxide to a pH-value of 7.0±0.01 andTris buffer was prepared by titrating 50 mMof the base form with concentratedhydrochloric acid to a pH-value of 7.4±0.01.All standard solutions and buffers wereprepared with doubly distilled water.

Preparation of standard solutionsA stock solution (10-2 M) of CRXN at pH

5.5 was prepared by dissolving 66.2 mg ofCRXN in 10 ml acetate buffer pH 5.5. Dilutesolutions of CRXN were freshly prepared bydiluting the stock solution with acetate buffer

(pH 5.5). All experiments were performedusing doubly distilled water. The stock andworking standard solutions were freshlyprepared.

Sensors constructionTable 1 shows the composition of

membranes used in this study and formulatedwith different ion-exchangers, differentionophores and solvent mediators. Differentcompositions were prepared aiming for theselection of the best sensing system that wouldprovide the bestperformance characteristics.

A 2.0 mg portion of TDMAC or TDMABion-exchanger (corresponding to 1.0 wt%) wasthoroughly mixed with 132.0 mg of o-NPOE(corresponding to 66.0 wt%), 66.0 mg of PVC(corresponding to 33.0 wt%) and 2.0 ml THFin a glass ring placed on a glass plate coveredwith filter paper and left to stand overnight toallow slow evaporation of THF at roomtemperature38. The master membrane wassectioned with a cork borer (7 mm diameter)and glued to a PVC tubing (~3 cm length, ~8mmi.d.) using THF. A solution of 1 mM NaClplus 1 mM CRXN in 50 mM acetate buffer, pH5.5, was used as the internal filling solution.Ag/AgCl internal reference electrode (1.0 mmdiameter) was immersed in the internalreference solution. Prior to use, membraneelectrodes were conditioned for ~24 hrs in asolution having the same composition as theinternal filling solution and were stored in the 1mM NaCl solution when not in use. Allpotentiometric measurements were made atambient temperature.


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Table 1: Composition and response characteristics of metallo-porphyrins and ion-exchangers basedmembrane electrodes for CRXN determination.

PVC Membranesensor

PlasticizerElectroactive

speciesAdditive Slope

Linear range(M)

E1 o-NPOE TDMAB - -28.84 2.9x10-5 – 1x10-2

E2 DOS TDMAB - -28.63 2.9x10-5 – 1x10-2

E3 FPNPE TDMAB - - -

E4 o-NPOE TDMAC - -25.71 2.9x10-5 – 1x10-2

E5 DOS TDMAC - -22.39 2.9x10-5 – 1x10-2

E6 FPNPE TDMAC - - -

E7 o-NPOE BTBAB - - -

E8 o-NPOE EBTPPB - - -

E9 o-NPOE Pd TPP-Cl2 - -10.65 1x10-4 – 1x10-2

E10 o-NPOE Sn TPP-Cl2 - -16.21 1x10-4 – 1x10-2

E11 o-NPOE Cu OEPTDMAB

(50 mol %)-22.15 1x10-4 – 1x10-2

E12 o-NPOE Zn OEPTDMAB

(50 mol %)-23.27 1x10-4 – 1x10-2

The sensors were calibrated under staticmode of operation by transferring differentaliquots of 1x10-2 − 1x10-3 M aqueous solutionof CRXN to 10 ml beaker containing 10.0 mlof acetate buffer, pH 5.5. The sensors wereimmersed in the solution in conjunction with adouble junction Ag/AgCl reference electrode.The potential readings were recorded afterstabilization to ±0.2 mV and the EMF wasplotted as a function of logarithm CRXNconcentration.

To investigate the influence of dielectricconstants of plasticizerson the performance ofthe developed sensors, different plasticizerswere used (o-NPOE ε = 24; FPNPE ε = 50; andDOS ε = 3.88) and used in membranepreparation to select the solvent mediatorwhich exhibits the best workingcharacteristics39 (ε is the dielectric constant ofthe plasticizer).

Different buffers were tested in theanalysis using the proposed electrodes such asacetate buffer (pH 5.5), phosphate buffer (pH7.0) and Tris buffer (pH 7.4) to select thebuffer system that exhibits the bestperformance characteristics.

Tubular detector constructionTubular sensors were constructed as

described previously37. Coating solutions wereprepared by mixing 2.0 mg portion of TDMACor TDMAB ion-exchanger, 132.0 mg of o-NPOE, 66.0 mg of PVC and 2.0 ml THF. Thissolution was deposited 3-4 times using adropper directly into a hole sectioned in thetube. The tubular sensor was inserted into theflow injection system as schematically shownin figure 2. A 50 mM acetate buffer (pH 5.5)was used as a carrier solution at a flow rate of 2ml.min-1.

The detector was calibrated at 25°C underhydrodynamic mode of operation by injectionof CRXN samples through a valve loop of 150μl in the carrier stream. After a steady-state, thebaseline was reached; the potential signalswere recorded using a high-impedance dataacquisition 8-channel box connected to a PC asmentioned above. An Orion Ag/AgCl doublejunction reference electrode was placed in aPetri-dish downstream from the indicatorsensor just before the solution went to waste.

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Determination of CRXN in pharmaceuticalpreparations

CRXN is found in the market in the formof vials only. The available pharmaceuticalpreparations of this drug were analysed suchas; Cefotrix® 0.25 gm, Cefotrix® 0.5 gm,Cefotrix® 1 gm, Cefaxone® 0.25 gm,Cefaxone® 0.5 gm, Cefaxone® 1 gm andCeftriaxone® 1 gm; for intravenous andintramuscular injections. An accuratelyweighed amount of powder equivalent to 66.2mg of CRXN was transferred into a 10-mLvolumetric flask, dissolved in about 5 mL of 50mM acetate buffer, pH 5.5, diluted to the markwith the same buffer, mixed well and filteredthrough a double filter paper; the first portionof the filtrate was rejected. Further dilutionswith the same buffer were made and then thegeneral procedure was followed.

For CRXN measurement under staticmode of operation, a 10-ml aliquot of the drugsolution was potentiometrically measured. Thedrug sensor and reference electrode wereimmersed in the solution, and the potentialreadings were recorded after reaching theequilibrium response. The concentration ofCRXN was calculated using a calibrationgraph.

For continuous measurements (FIA), aflow stream of 50 mM acetate buffer of pH 5.5carrier solution was allowed to pass throughthe tubular electrode at a flow rate 2.0 ml.min-1.Successive 150 μl aliquots of the standardCRXN and unknown test sample solutionswere injected into the flowing stream. Thecorresponding potential change was measuredand recorded vs. time. A typical calibrationplot was made and used to determine theconcentration of the unknown samples.

RESULTS AND DISCUSSION

Response characteristics of the developedsensors

Plasticized PVC membrane electrodesformulated with different electro-active species(ion exchangers or metal ion complexes) wereprepared and evaluated. Membranes wereformulated using a casting solution of thecomposition 1:33:66 wt% of electro-activespecies, PVC and plasticizer, respectively.Such polymer-membrane electrodes based oneither ion-exchangers (TDMAB, TDMAC,

BTBAB and EBTPPB) or metal-ion complexes(PdTPP-Cl2, SnTPP-Cl2, CuOEP and Zn OEP)were prepared.

As shown in figure 3, TDMAB andTDMAC based membrane sensors exhibitednear-Nernstian responses (-28.84 and -25.71mV.decade-1) over the concentration ranges of2.9x10-5 – 1x10-2M. However, sensorsincorporating BTBAB or EBTPPB as cationicexchangers did not exhibit any detectableresponses. Cu, Zn, Pd, and Sn based ion-carriers were selected as ionophores based onthe well-known drug interactions with suchmetal ions40-42. However, due to the highhydrophilicity of the drug2 (see the selectivityorder based on ion-exchange mechanismbelow) and its weak binding ability to suchmetal ion complexes, were found to be notsuitable carriers for such drug type. Forinstance, sensors incorporating Cu OEP and ZnOEP based membrane sensors exhibited near-Nernstian responses (-22.15 and -23.27mV.decade-1) over the concentration ranges of1x10-4 – 1x10-2 M. Unfortunately, the obtainedresponses were found to be of the Hofmeistertype and due to the presence of 50% TDMABas an additive in the membrane construction, asindicated by the selectivity order. On the otherhand, membrane electrodes based on Pd TPP-Cl2 and Sn TPP-Cl2 exhibited sub-Nernstianresponses (-10.65 and -16.21 mV.decade-1,respectively) over the concentration ranges of1x10-4 – 1x10-2 M. Therefore, membraneelectrodes based on TDMAB and TDMACwere selected for further investigations.

log [CRXN], M

Fig. 3: Potentiometric response of various PVCmembrane electrodes towards CRXNmeasured in 50 mMacetate buffer, pH 5.5.The ion-exchangers tested were (●)TDMAB, (■) TDMAC, (▲) EBTPPB and(♦) BTBAB.

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F, m

V


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Potentiometric response characteristics ofCRXN selective membrane electrodes werestudied in order to probe the effect of differentbuffers on the potentiometric responses. Trisbuffer (pH 7.4), acetate buffer (pH 5.5) andphosphate buffer (pH 7.0) were tested to selectthe buffer system which gives the bestperformance characteristics. It was found thatno responses were obtained upon using Trisbuffer and phosphate buffer; however acetatebuffer gave near-Nernstian response, so it wasselected for all subsequent work. This may beattributed to that upon using acetate buffer, pH5.5, about 99.9% of CRXN was ionized in theanionic form [CRXN-2] so maximum responsewas obtained at this pH [pKa of CRXN; 2.37(COOH), 3.03 (aminothiazole) and 4.21(hydroxytriazinone)]43.

The dielectric constants of solventmediators much influence the sensorcharacteristics and response. The performancecharacteristics of the proposed membranesensors are greatly affected by the usedplasticizer. Sensors were constructed usingthree different plasticizers; o-NPOE, DOS andFPNPE then the performance characteristicswere investigated (Table 1). It was found thato-NPOE was the best plasticizer whichexhibited the best performance characteristicsproved by giving nearer-Nernstian slopes -28.84 and -25.71 mV.decade-1 for TDMAB andTDMAC based membrane sensors, respectively(Table 1 and Figs. 4&5).

log [CRXN], M

Fig. 4: Potentiometric response of TDMAB basedmembrane electrodes formulated withdifferent solvents mediators; (♦) o-NPOE,(●) DOS and (▲) FPNPE.

log [CRXN], M

Fig. 5: Potentiometric response of TDMAC basedmembrane electrodes formulated withdifferent solvent mediators; (●) o-NPOE,(▲) DOS and (♦) FPNPE.

Results from three replicate studies usingTDMAB and TDMAC based membranesensors exhibited near-Nernstian slopes of -28.84 and -25.71 mV.decade-1 over theconcentration ranges of 2.9x10-5 – 1x10-2 M,with detection limits of 1.17x10-5 and 1.25x10-5

M (7.74 and 8.77 µg.ml-1) for TDMAB andTDMAC based membrane sensors, respectively(Table 2). The response characteristics (e.g.,selectivity, response time, linear range......etc.)of different types of CRXN selectivemembrane electrodes (TDMAB, TDMAC) aresummarized in (Table 2). TDMAB andTDMAC based membrane sensors aresuccessfully formulated for determination ofCRXN and remained suitable for use for about8 weeks (Table 2).

The developed sensor responses aregreatly affected by the pH of the medium. Thepotentiometric responses of TDMAB andTDMAC based membrane sensors wereexamined at different pH values over a pHrange of 2.9-12.4. This was performed inpresence of 10-3 and 10-2 M CRXN. In eachconcentration level, the pH of CRXN solutionwas adjusted using either hydrochloric acid orsodium hydroxide solutions. The potential wasthen recorded after the pH was stabilized. Thevariation in the pH range 4-7 has no significanteffect on the potentiometric responses for bothTDMAB and TDMAC based membranesensors. It was found that the potentials of theprepared membrane sensor considerablydeclined with negative drift at pH values higherthan ~7 then remain constant above pH ~8(Fig. 6). This may be due to due to progressive

∆EM

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∆EM

F, m

V

205

Table 2: Summary of response characteristics of TDMAB and TDMAC based membrane sensorsunder static mode of operation for determination of CRXN.

Parameter TDMAB * TDMAC *Slope, (mV per decade) -28.84 -25.71Working range, M 2.9 x 10-5 – 1.0 x 10-2 2.9 x 10-5 – 1.0 x 10-2

Lower limit of linear range, (M) 2.9 x 10-5 2.9 x 10-5

Response time (t95%)For conc. < 10-3 (sec.)For conc. ≥ 10-3 (sec.)

<30<10

<30<10

Working range, (pH) 4-7 4-7Detection limit, (M) 1.2 x 10-5 1.25x10-5

Life span, (week) 8 8Standard deviation, (%) 1.17 1.42

*Average of five measurements.

pH

Fig. 6: Influence of pH on the potentiometricresponse of TDMAB based membranesensorin presence of: (▲) 10 mM and (♦)1 mM CRXN.

degradation of CRXN at higher pH values andthe hydroxide anion interference. However, atlower pH values, the sensors responses wereseverely influenced by H3O

+ and the decreaseof the ionized/non ionized ratio of CRXN.Based on this, the working pH range for thedeveloped sensors is from pH 4 to 7.

The dynamic response times of theTDMAB and TDMAC based membranesensors were examined by recording thepotential readings at time intervals of 5seconds. The relation between potentialreading and response time was plotted for 29µM to 10 mM CRXN. The time required toattain 95% of the equilibrium by both sensorswas less than 10 s for drug concentration ≥10-3

M. These results indicated that both sensors areamenable for use with automated systems(Fig. 7).

Time, sec.

Fig. 7: Response time of TDMAB based membranesensor towards CRXN measured in acetatebuffer pH 5.5.

Selectivity coefficients ( potCRXNK , ), of

TDMAB and TDMAC based membranesensors towards some interfering ions weredetermined by the separate solutions method(SSM)44. In this method, the selectivitycoefficients of CRXN sensors were evaluatedat a concentration of (1 M) measured in 50 mMacetate buffer pH 5.5 (Figs. 8&9). The

selectivity coefficients (log potCRXNK , ) were

determined with the rearranged Nicolskyequation44:

)log(1log2

121, a

Z

Z

S

EEK pot

CRXN

++

−

=

where, E1 is the potential measured in 1MCRXN, E2 the potential measured in 1 M of theinterfering ion, Z1 and Z2 are the charges of theCRXN and interfering species β, respectivelyand S is slope of the calibration plot.

(1)

EM

F, m

V

EM

F, m

V


206

log [CRXN], M

Fig. 8: Potentiometric responses of CRXN selectiveelectrodes using TDMAB based membraneelectrodes, measured in 50 mM acetate bufferpH 5.5, of towards various anions, additivesand some cephalosporins: (●) CRXN, (---)Cefadroxil, (◊) Cefoperazone, (….) Cl−, (Δ)SO4

-2, (▲) NO3−, (○) CH3COO−, (*) Cefradine,

(—|—) Cefaclor, (-.-.-.) Ceftazidime, (…♦…)glucose, (□) fructose and (—) maltose.

log [CRXN], M

Fig. 9: Potentiometric responses of CRXN selectiveelectrode using TDMAC based membraneelectrode, measured in 50 mM acetate bufferpH 5.5, towards various anions, additives andsome cephalosporins: (●) CRXN, (---)Cefadroxil, (◊) Cefoperazone, (….) Cl−, (Δ)SO4

-2, (▲) NO3−, (○) CH3COO−, (*) Cefradine,

(—|—) Cefaclor, (-.-.-.) Ceftazidime, (□)glucose, (Δ) fructose and (—) maltose.

It is worth noting that the two membraneelectrodes prepared exhibited good selectivitytoward CRXN over other cephalosporins suchas; cefradine, ceftazidime, cefadroxil, cefaclorand cefoperazone and other additives found inthe pharmaceutical preparations such as;glucose, fructose and maltose. This enables usto use such sensors for determination of CRXNsuccessfully without interference from othercephalosporins and the encountered additives.Moreover, the selectivity of the proposed

electrodes toward other inorganic anionsshowed that the exhibited anionic responsesfollow the classical Hofmeister pattern foranions that is based on anions lipophilicity, inwhich the more lipophilic species are thepreferred ones45. As shown in (Table 3), theselectivity pattern for TDMAB basedmembrane sensor was SCN− > I− >NO3

− >CRXN > cefoperazone > cefadroxil >ceftazidime > cefradine > Cl− > acetate > SO4

-2

> cefaclor. While that for TDMAC basedmembrane sensor was SCN− > I− > NO3

− >CRXN > cefadroxil > ceftazidime > acetate >Cl− > cefaclor > cefradine > SO4

-2 >cefoperazone.

Table 3: Potentiometric selectivity coefficientsof o-NPOE plasticized TDMAC andTDMAB based PVC membranesensors.

log potCRXNK ,Interfering ion,

B TDMAB TDMACCl- ≤-5.51 ≤-5.24

SO4-- ≤-5.75 ≤-5.41NO3- 0.34 0.68

I- 2.74 2.74SCN- 4.45 4.79

CH3COO- ≤-5.55 ≤-5.14Glucose ≤-5.14 ≤-4.89Fructose ≤-5.34 ≤-5.41Maltose ≤-5.62 ≤-5.24

Cefradine® ≤-5.47 ≤-5.38Cefadroxil® ≤-4.10 ≤-4.11Cefaclor® ≤-5.79 ≤-5.27

Ceftazidime® ≤-5.34 ≤-5.01Cefoperazone® ≤-2.88 ≤-5.48

amaximal selectivity limit (≤) was used for ions thatexhibited minimal response towards interferentions44.

Batch monitoring of CRXN inpharmaceutical preparations

The available pharmaceutical dosageforms of CRXN were analysed by the twoproposed potentiometric sensors, as well as byreported method4. The two proposedpotentiometric sensors were appliedsuccessfully for determination of the CRXN inits pharmaceutical dosage forms under staticmode of operation. This finding indicated good

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MF,

mV

207

accuracy and precision in the determination ofthe cited drug in its pharmaceutical dosageforms. The results obtained (Table 4) weresatisfactory compared to those obtained fromthe reported method4. No significant differencewas found by applying t- and F-tests at 95%confidence level indicating good accuracy andprecision. F-test revealed that there was nosignificant difference between the means andvariances of the two sets of results.

Recovery studies were also carried out bystandard addition method under static mode ofoperation. Recovery experiments were made toevaluate the accuracy of the proposed

potentiometric methods on both the pureauthentic and the dosage forms. Differentconcentrations of the authentic drug beingdetermined were added to the samplepreparation which was then analysed for thetotal amount of the drug present. Thedifference between the analytical results of thesamples with and without the added drug gavethe recovery of the amount added drug, (Table5). The results clearly proved the accuracy ofthe proposed method for selectivedetermination of the investigated drug withoutinterference from common excipients.

Table 4: Determination of CRXN in its pharmaceutical formulations by the two proposedpotentiometric sensors under static mode of operation in comparison with the reportedmethod4.

Recovery % ± SDa,b

Pharmaceutical product TDMAB basedmembrane sensor

TDMAC basedmembrane sensor

Reportedmethodc

Cefotrix®vialsd

250 mg of ceftriaxonesodium/vial (IV, IM)

100.17 ± 1.07t = 0.68F = 1.38

99.79 ± 1.06t = 0.07F = 1.42

99.72 ± 1.26

Cefotrix®vialsd


99.88 ± 1.64t = 0.46F = 1.77

99.3 ± 1.38t = 0.27F = 1.23

99.51 ± 1.23

Cefotrix®vialsd


98.83 ± 1.40t = 1.11F = 1.54

98.68 ± 1.46t = 1.26F =1.39

99.83 ± 1.72

Cefaxone®vialse

250 mg of ceftriaxonesodium/vial (IM)

98.82 ± 1.15t = 0.43F = 1.38

99.27 ± 1.45t = 0.25F = 2.23

99.08 ± 0.98

Cefaxone ®vialse


99.43 ± 1.09t = 0.64F = 1.11

99.25 ± 1.18t = 0.41F = 1.06

98.25 ± 1.15

Cefaxone ®vialse


100.27 ± 1.33t = 1.70F = 2.35

100.34 ± 1.54t = 1.63F = 3.17

99.17 ± 0.86

Ceftriaxone®vialsf


100.16 ± 1.23t = 1.91F = 1.17

99.63 ± 0.86t = 1.31F = 1.73

98.87 ± 1.12

aEach value is the mean of five determinations.bThe tabulated values at 95% confidence limits are t = 2.78 and F = 6.39, respectively.cReference 4.d T3A Pharma Group, Assiut, Egypt.ePharco Pharmaceuticals, Alexandria, Egypt.fKahira Pharm. & Chem. Ind. Co. under licence from Novartis Pharma S.A.E., Cairo, Egypt.


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Table 5: Standard addition method for the assay of CRXN in its pharmaceutical dosage forms usingTDMAB and TDMAC based membrane sensors under static mode of operation.

TDMAB TDMACPharmaceutical

formulation

Authenticdrug

added(mol.L-1)

Authenticdrug found(mol.L-1)

Recovery(%) ± SDa

Authenticdrug found(mol.L-1)

Recovery(%) ± SDa

Cefotrix® vials2501.0 x 10-5

5.0 x 10-5

7.0 x 10-5

0.95 x 10-5

4.81 x 10-5

6.73 x 10-5

95.0±1.496.2±0.996.1±1.1

0.93 x 10-5

4.74 x 10-5

6.77 x 10-5

93.0±1.594.8±0.696.7±0.9

Cefotrix® vials5001.0 x 10-5

5.0 x 10-5

7.0 x 10-5

0.98 x 10-5

4.86 x 10-5

6.57 x 10-5

98.0±0.597.2±0.993.8±0.8

0.91 x 10-5

4.91 x 10-5

6.72 x 10-5

91.0±1.298.2±0.796.0±0.9

Cefotrix®

vials1000

1.0 x 10-5

5.0 x 10-5

7.0 x 10-5

1.03 x 10-5

4.94 x 10-5

6.68 x 10-5

103.0±1.598.8±0.795.4±0.9

0.96 x 10-5

4.94 x 10-5

6.78 x 10-5

96.0±1.498.8±0.696.8±0.8

Cefaxone®

vials250

1.0 x 10-5

5.0 x 10-5

7.0 x 10-5

0.95 x 10-5

4.88 x 10-5

6.88 x 10-5

95.0±0.797.6±0.698.2±1.2

0.90 x 10-5

4.98 x 10-5

6.89 x 10-5

90.0±1.299.6±0.698.4±0.7

Cefaxone®

vials500

1.0 x 10-5

5.0 x 10-5

7.0 x 10-5

0.96 x 10-5

5.17 x 10-5

6.90 x 10-5

96.0±1.4103.4±0.798.5±0.9

0.97 x 10-5

4.82 x 10-5

6.70 x 10-5

97.0±0.696.4±0.595.7±0.9

Cefaxone®

vials1000

1.0 x 10-5

5.0 x 10-5

7.0 x 10-5

0.89 x 10-5

4.99 x 10-5

6.93 x 10-5

89.0±1.699.8±0.699.0±1.1

0.96 x 10-5

4.82 x 10-5

6.77 x 10-5

96.0±1.196.4±0.796.7±0.8

Ceftriaxone®

vials1000

1.0 x 10-5

5.0 x 10-5

7.0 x 10-5

0.87 x 10-5

5.03 x 10-5

7.32 x 10-5

87.0±1.4100.6±0.8104.6±0.9

0.95 x 10-5

4.93 x 10-5

6.80 x 10-5

95.0±0.898.6±0.597.1±0.5

aAverage of six determination.

Flow injection monitoring (FIA) of CRXN inpharmaceutical preparations

A tubular-type detector incorporatingTDMAB and TDMAC based membranesensors were prepared and used underhydrodynamic mode of operation forcontinuous CRXN quantification. The generalintrinsic response characteristics of the detectorrevealed that the dependency of the peakheight, peak width, and time to recover thebase line depend on the flow rate of the carrierbuffer solution. Successive injection of CRXNstandard solutions through a valve loop of 150µl in the carrier stream with different carrierflow rates (i.e. 1.0-4.0 ml.min-1) revealed thatas the flow rate increased the response peakswidth decreased with a decrease in the peakheight. Consequently, the recommendedoptimal flow rate was chosen to be 2.0ml.min-1. With a flow rates of 1 ml.min-1, thetubular sensor required longer washing time torecover the base line leading to a decrease in

the number of sample outputs. At flow rateshigher than 3.0 ml.min-1, the peak heightdeclined.

A linear relationship between CRXNconcentrations and FIA signals was obtainedover the range 1-10 mM (Fig. 10). The slopeof the calibration plot was near-Nernstian(-23.07±0.32 mV.decade-1) for TDMAB basedPVC membrane sensor. Table 6 shows thegeneral response characteristics of the tubularTDMAB and TDMAC based membranesensors under FIA mode of operation. Theresults obtained for determining CRXN in drugformulations using FIA are shown in table 7.

Validation of the proposed potentiometricmethods for determining CRXN was made bymeasuring the range (R), lower limit ofdetection (LOD), accuracy (recovery), linearity(correlation coefficient) and sensitivity (slope).Results obtained support the application of theproposed potentiometric methods for qualitycontrol assessment of drug formulations.

209

Table 6: Response characteristics of tubular TDMAB and TDMAC based membrane sensors underhydrodynamic (FIA) mode of operation.

Parameter TDMAB TDMAC*Slope, (mV per decade) -23.07 -21.07Correlation coefficient, (r) 0.996 0.994Working range, mM 1 – 10 1 – 10Intercept, (mV) 2.5 18.5Lower limit of detection (mM) 0.5 0.6Optimum flow rate, (ml min-1) 2 2Carrier 50 mM acetate buffer,(pH)

5.5 5.5

* Based on three measurements.

Table 7: Determination of CRXN in its pharmaceutical formulations by the two proposedpotentiometric methods under FIA mode of operation and the reported method4.

Recovery % ± SDa,b

Pharmaceutical product TDMAB basedmembrane sensor

TDMAC basedmembrane sensor

Reportedmethodc

Cefotrix®vialsd


98.83 ± 1.43t = 1.17F = 1.29

100.17 ± 1.98t = 0.44F = 2.49

98.72 ± 1.26

Cefotrix®vialsd


98.86 ± 0.77t = 1.10F = 2.55

100.14 ± 1.78t = 0.74F = 2.09

99.50 ± 1.23

Cefotrix®vialsd


100.65 ± 1.95t = 1.95F = 2.28

99.01 ± 1.56t = 0.87F = 1.22

99.82 ± 1.72

Cefaxone®vialse

250 mg of ceftriaxonesodium/vial (IM)

98.77 ± 1.26t = 0.48F = 1.75

99.14 ± 1.24t = 0.17F = 1.59

99.08 ± 0.98

Cefaxone ®vialse


99.51 ± 1.28t = 0.63F = 2.27

99.61 ± 1.25t = 0.72F = 2.08

98.95 ± 1.15

Cefaxone ®vialse


100.28 ± 1.02t = 2.05F = 1.45

99.80 ± 1.29t = 0.99F = 2.23

99.16 ± 0.86

Ceftriaxone®vialsf


99.65 ± 1.34t = 1.09F = 1.40

99.62 ± 1.35t = 1.03F = 1.51

98.87 ± 1.12

aEach value is the mean of five determinations.bThe tabulated values at 95% confidence limits are t = 2.78 and F = 6.39, respectively.cReference 4.d T3A Pharma Group, Assiut, Egypt.ePharco Pharmaceuticals, Alexandria, Egypt.fKahira Pharm. & Chem. Ind. Co. under licence from Novartis Pharma S.A.E., Cairo, Egypt.


210

Time, s

log [CRXN], M

Fig. 10: Typical (FIA) peaks produced byinjection of CRXN standard solutionsthrough a valve loop of 150 µl in a streamof 50 mM acetate buffer pH 5.5 using theTDMAB based membrane sensor.

ConclusionsTwo potentiometric sensors for CRXN

were prepared, characterized and successfullyused for static and continuous drugdetermination. Sensors based on the use ofplasticized PVC matrix membranesincorporating TDMAB and TDMAC ionexchangers were used for quantitativedetermination of ceftriaxone sodium atconcentration level down to 2.9x10-5 M with agood accuracy. The drug is determined in purepowders and in vials. The sensors offer theadvantages of fast response, reasonableselectivity, elimination of drug pre-treatment orseparation steps, low cost and possibleinterfacings with computerized and automatedsystems. The use of TDMAB and TDMACbased membrane sensors as two detectors forcontinuous monitoring of CRXN offers the

advantages of simple design, ease ofconstruction and possible applications to smallvolumes of drug solutions with littlemanipulation and without pre-treatment. Thedetector displays a wide dynamic measurementrange of the drug under continuous mode ofoperation with a flow rate of 2.0 ml.min-1.

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