trends in analytical chemistry, vol. 10, no. 8,199l 261

(Editors), Parallel Problem Solving from Nature, Springer- Verlag, Berlin, 1991, p. 90.

8 F.P. Zscheile, H.C. Murray, G.A. Baker and R.G. Ped- dicord, Anal. Chem., 34(13) (1962) 1776.

9 D.L. Massart, B.G.M. Vandeginste, S.N. Deming, Y. Michotte and L. Kaufman, Chemometrics; a textbook, El- sevier, Amsterdam, 1988.

10 J.J. Grefenstette, IEEE Transact. Syst. Man Cybern., 16(l) (1986) 122.

Dr. C.B. Lucasius and Prof. G. Kateman are at the Laboratory for Analytical Chemistry, Faculty of Science, Katholieke Univer- siteit Nijmegen, Toernooiveld I, 6525 ED Nijmegen, Nether- lands.

The use of membrane electrodes for the determination of inorganic species in pharmaceutical analysis

Vasile V. Coqofre? Bucharest, Romania

Membrane electrodes find wide application for the deter- mination of various inorganic species (cations, anions or simple molecules) in pharmaceutical analysis. In this review their high sensitivity and selectivity are discussed and advantages of the technique presented.

Introduction The development and application of novel elec-

trochemical sensors, including selective membrane electrodes, remains a growth area in analytical chemistry lW3. Current themes include the design of drug-sensitive sensors and the application of com- mercially available membrane electrodes to monitor certain drugs in pure form, in complex pharmaceuti- cal formulations, and in biological materials.

Membrane electrode techniques offer advantages of simplicity, rapidity and accuracy over more estab- lished pharmaceutical analysis methods 4-6, which although accurate, are in some cases lengthy and difficult. For example, with membrane electrode, de- termination often takes less than 15 minutes, the procedure can be directly applied to drug determina- tions in pharmaceutical preparations without prior separation, and frequently the excipients do not con- tribute to the electrode response and less clean-up is required. This makes the technique suitable for de- termination of single pharmaceutical units (e.g., tablet, capsule, suppository), so that variation in preparation can be followed if desired.

General principles A phase that separates two others and prevents

mass transfer between them but still allows passage with varying degrees of restriction of one or more

species contained in the external phases may be de- fined as a membrane (1, in Fig. 1). Such a phase, when used as an electrode in an electrochemical cell, constitutes a membrane electrode. The behaviour of the membrane electrode will be determined by the properties of the membrane, which may be a solid, or a liquid containing ionized of ionizable groups.

Selective membrane electrodes may be broadly classified according to the physical state of the elec- troactive substance that forms the membrane: l Selective electrodes with solid membranes. The

3

\

2 4

-- id- --

I- - -- - ---_

- -- _- -- 7 5 - -_ - - -- -- -

Fig. 1. Schematic representation of membrane electrode cell as- sembly: 1 = membrane; 2 = potentiometer; 3 = internal refer- ence electrode; 4 = external reference electrode; 5 = sample so- lution; 6 = internal filling solution.

262 trends in analytical chemistry, vol. 10, no. 8,1991

membrane may be homogeneous, such as a monocrystal, a sparingly soluble ionic crystalline substance, or a glass - which is considered to be a solid because of the immobility of the ionic com- ponents. Alternatively, the membrane may be heterogeneous, incorporating the electroactive substance within an inert matrix.

l Selective electrodes with liquid membranes. The electrode membrane is represented by an organic liquid immiscible with water. The organic liquid contains a charged electroactive substance that offers “sites” for ion exchange between membrane and solution. The membrane is responsive, and may be selective for the exchangeable ions. The above classification is useful for theoretical

considerations. However, selective membrane elec- trodes should not be classified according to mem- brane homogeneity or heterogeneity as these terms refer to composition, not to operation.

The cell assembly is shown schematically in Fig. 1. The ion selective membrane, is the basic component of the electrochemical cell and separates two electro- lyte solutions with differing ionic activities. The potential difference between the two sides of the membrane is measured potentiometrically through the internal and external reference electrodes in the internal and external filling solutions, respectively. The membrane is usually held in a compact unit con- taining the internal reference electrode and the inter- nal filling solution, which constitutes the selective membrane electrode. In some cases an internal filling solution is omitted and electrical contact is made by connecting a wire to the inner face of the membrane. The use of selective membrane electrodes relies on the determination of membrane potentials that represent the electrical potentials occurring across membranes when they separate two electrolyte solu- tions. These potentials cannot be measured directly, but their changes can be deduced from the e.m.f. values for complete electrochemical cells as shown in Fig. 1.

Generally, for a given inner electrode system the membrane potential, E, is given by:

where R is the universal gas constant; T the absolute temperature; F the Faraday constant; a, the ion ac- tivities in the sample solution (monovalent ions); and k r: the selectivity coefficient.

Membrane electrodes in pharmaceutical analy- sis

Four types of membrane electrodes find applica- tion for pharmaceutical analysis:

l Primary electrodes containing crystalline mem- branes prepared from either a single compound (e.g., Ag,S) or a homogeneous mixture of spar- ingly soluble compounds (e.g., AgX/Ag,S ; X = halogen). Most are commercially available and their characteristics and performance are good;

l Primary electrodes containing non-crystalline membranes; glass membrane electrodes (e.g., H+ , Na+) and electrodes with membranes containing a mobile carrier. For the latter the electroactive material is dissolved in either a hydrophobic poly- mer (e.g., plasticized PVC) or a hydrophobic li- quid solvent (e.g., nitrobenzene). Few electrodes in this category are commercially available (e.g., BF; , Ca2+, K+) and most of them are thus lab- oratory-made;

l Gas-sensing electrodes (e.g., NH,, CO,). Most of these are based on a sensitized pH electrode and are commercially available.

l Bio-selective electrodes. Based on enzyme- substrate reactions, they are laboratory-made and very selective for the particular substrate. Most were created when stable and reliable potentiomet- ric sensors for NH,, CO,, H,S became commer- cially available.

Determination of various drug-type substances by membrane electrodes

Ag+/S*- membrane electrode

Sulphonamides, R-SO,-NH-R’ The response of a silver sulphide membrane elec-

trode to sulphonamide drug was investigated for sul- phonamides forming soluble complexes with silver (sulphacetamide, acetazolamide, furosemide and hydrochlorothiazide)’ and for those precipitating sil- ver ions (e.g., sulphathiazole, sulphadimethox- ine) . 7-9 When a silver sulphide membrane electrode is used in solutions free of silver ions, but containing a ligand that forms relatively weak complexes with silver, the ligands do not dissolve the membrane but may instead react with adsorbed silver ions’,“. The equation describing the response of a silver sulphide electrode to a ligand is given by:

t2)

where aAg+ is the silver ion activity due to grain- boundary and adsorbed silver ions, /3, is the appar- ent formation constant of the silver complex formed (p is the coordination number), L is the total ligand activity and -rAg+ the activity coefficient of Ag+.

trends in analyticalchemistry, vol. 10, no. 8,199l

Plots of the measured potentials against expected response were generally obtained within 2 minutes and steady potentials were established within 5 minutes, except for sulpha~etamide, which required about 10 minutes. The slope of the linear portion of the calibration curve and the linear range depend on the respective sulphonamide.

The silver sulphide membrane electrode proved useful for drug analysis in pharmaceutical prepara- tions by both potentiometric titrations and direct potentiometry.

T~i~barbit~rates, NaOyMy S

pd H II

0

In the presence of thiobarbiturate ions (SJ- disubstituted thiobarbiturates are present in two predominant forms in alkaline solution) the silver sulphide membrane electrode responds according to:

where S is the change of the electrode potential, which should in this case be 59.1 mV/decade of con- centration taking into account a stoichiometry of thiobarbiturate-silver (1: 1) and ~~~i~b~~bi~~*~~~ is the ac- tivity of the thiobarbiturate ions. The linear ranges of the calibration curves were 10-3-10-5 M both for thiopental (R, = C,H,; R,= i-C,H& and inactin (R, = C,H,; R, = i-C,H,,) in 0.1 A4 sodium hydrox- ide solution”. Both direct potentiometry and poten- tiometric titration in aqueous solutions with lop2 M silver nitrate were used for quantitative determina- tions of thiobarbiturate drugs.

COOH

Cephalosporins (antibiotic agents) may be deter- mined by a potentiometric method using a silver sul- phide membrane electrode and 10T2 M lead(I1) ni- trate solution as titrant. The method is based on alkaline degradation of the respective cephalosporin and conversion of the resulting sulphide into lead(I1) sulphide . l2 Various cephalosporins were shown to give different but reproducible yields of sulphide. The degradation of cephalosporins (at a concentra- tion of 5 - 10S3 A4) were carried out in 1 M NaOH solution containing 2% ascorbic acid, at lOO”C, for

a duration of 30-60 minutes. Aliquots of the degrad- ed solutions were titrated in 1 M NaOH solution medium containing 2% ascorbic acid and 10% (v/v) glycerol. Lead nitrate was used instead of silver nitrate for the titration of sulphide, because there is less adsorption of sulphide ion on lead than on silver and this gives more accurate results.

Cu2+ membrane electrode

Guanidine derivatives, R-y-f-NH-f-NH,

R’ NH NH

Various guanidine derivatives (biguanides, Big), which are used as hypoglicaemic agents, may be potentiometrically determined with a copper(I1) membrane electrode13, a determination based on the formation of fCu(Big),]X, complexes by the reac- tion between copper amine complexes and biguanides. An excess of copper(I1) amine precipi- tates [Cu(Big),]X,; the excess copper(I1) may be sub- sequently determined by a potentiometric titration with 5 * low2 M EDTA using a copper(I1) mem- brane electrode as indicator electrode. Fig. 2 shows the potentiometric titration curve of an ammoniac mixture of [Cu(Big),]X, and excess copper(I1) sul- phate.

The method has the advantage that the residual impurities from the synthesis of the biguanide, e.g., dicyandiamide, n-alkyl-guanidine, amines, etc., do not interfere with the determination. The selectivity

0 1 2 3 4 5 5x&l EUTA,mt

Fig. 2. Potenfiometric titration curve of an ammoniac mixture of [Cu(Big)JXz and copper(U) sulphate using EDTA as titrant.

264 fret& in analytical chemistry, vol. IO, no. 8, I991

of the method allows monitoring of the formation of the biguanide directly in the reaction medium.13.

Cystaphos, H d-k ‘DH

2 P\ & ONa 0

The quantitative determination of cystaphos (a compound used for the prevention and treatment of radiation sickness) with a copper(I1) membrane elec- trode is based on the reaction:

H,N-CH,-CH,-SPO,HNa + HOH s

H2N-CH,-CH2-SH + NaH,PO, (4)

Because the S-P bond is very labile in acidic medi- um, the reaction takes place quantitatively to cystea- mine and ortophosphate in less than 1 minute at 100°C and pH 2-3 (adjusted with 1 MT perchloric acid). The cysteamine produced was stable for 15 to 20 minuteG4. The copper(I1) membrane electrode gives a good response to cysteamine solution. The electrode function is stable and reproducible over a wide concentration range (10-2-10-6 M) and the response times permit use of the electrode for direct potentiometry. The electrode slope is - 58 mV/de- cade, in good agreement with 59.1 mV/decade for the redox interaction at the membrane-solution in- terface according to:

2Cu2+ + 4H,N(CH,),SH - 2H,N(CH,),SCu

+ H,N(CH,),SS(CH2),NH2 + 4H+ (5) Drugs containing a carboxyamide group A simple potentiometric method for drugs con-

taining a carboxyamide group (e.g., enthenzamide, nicotinamide, pyrazinamide, salicylamide) is based on refluxing the sample in 20% HCl for 20 minutes, hydrolizing carboxyamide and liberating an equiva- lent amount of ~monium ion.

Copper(H) oxidises cysteamine to cystamine with the formation of Cu+H,NCH,CH,S- .

CO, membrane electrode

Gentamicin and related antibiotics A potentiometric method using a CO, membrane

electrode has been developed for microbiological as- say of antibiotics such as gentamicin, neomycin, streptomycin and tetracycline15,‘6. The method is based upon the antibiotic inhibition of carbon diox- ide production by a suspension of Escherichia co/i, which is directly measured with the potentiometric gas sensor after an incubation period. The optimum conditions for bioassays of the above antibiotics were pH 7.8; T = 37°C; time of incubation 120 minutes; stock E. coli cell concentration of 4.5 - lo8 cells/ml and a stock nutrient solution of 2.4 g/100 ml nutrient broth. The linear ranges of the log

(dose-response) curves were 0.017-3.3, 0.17-3.3, 0.3-16.7 and 33-167 pg/ml for gentamicin, neomy- cin, streptomycin and tetracycline hydrochloride, respectively.

Digoxin A potentiometric enzyme immunoassay technique

utilizing polystyrene beads in conjunction with a gas sensing membrane electrode has been described by Keating and Rechnitz 17. The technique was illustrat- ed with the measurement of digoxin (secondary glycoside from Digitalis lavata with cardiotonic ef- fect) via competitive inhibition of antidigoxin- horseradish peroxidase conjugate activity. The rate of CO, generation from the peroxidase-pyrogallol (the hydrogen donor) reaction in the presence of antibody-labelled horseradish peroxidase enzyme- with competition between the free digoxin to be de- termined and polystyrene-bead immobilized digoxin -was monitored.

The digoxin-BSA ratio in the digoxin-BSA con- jugate was 2O:l (the polystyrene beads were coated with digoxin-BSA by physical adsorption). The opti- mum conditions for the enzymatic reaction in the presence of antibody-HRP conjugate were substrate (H,O,) concentration of 3 * lop3 M; a pyrogallol con- centration of 3.2. 10m3 M, pH 6 (phosphate buffer),

The method gives a very sensitive assay for digoxin in the nanogram range and offers faster analysis times in comparison with previous methods using plastic beads.

NH, membrane electrode

After alkalinization, the converted ammonia is de- termined by an ammonia membrane electrodeI us- ing the appropriate calibration curve (linearity was observed over the range 2. 1O-5-1O-2 M). The method was successfully applied for the determina- tion of nicotinamide in pharmaceutical preparations such as capsules and tablets.

Drugs containing a carbothionamide group Both ethionamide and prothionamide (tuberculo-

static agents) contain a carbothionamide group which decomposes to ammonium chloride, hydrogen sulphide and the respective carboxylic acid on heat- ing with hydrochloric acid, thus:

trends inanalyticalchemistry, vol. 10, no. 8,1991 265

2 e R- COOH + NH&I + H,S 2H,O (6)

It was possible to use an ammonia membrane elec- trode to determine converted ammonia from the am- monium chloride”. The decomposition of ethiona- mide and prothionamide (W3 mol for each) was performed by heating on reflux with 20% HCI solu- tion for 1 h. After cooling and alkalinization with NaOH solution (pH > 1 l), the sample concentration was determined from the calibration graph, which was linear for a drug concentration range of 10-2-5+ low5 M. The method was applied with good results for the determination of pure drug substances as well as for the tablets.

Conclusions The membrane electrode technique offers the ana-

lytical chemist the means for quick, precise and selec- tive assay of complex pharmaceuticals.

References 1 R.L. Solsky, Anal. Chem., 62 (1390) 21R. 2 J. Koryta, Anaf. Chim. Acta, 233 (1990) 1. 3 G. J. Moody and J.D.R. Thomas, Selective Electrode Rev.,

12 (1990) 261.

V.V. Coaofre!, Membrane Electrodes in Drug Substances Analysis, Pergamon Press, Oxford, 1982. V.V. Coaofret and R.P. Buck, Selective Electrode Rev., 6 (1984) 59. Z. Zong-Rang and V.V. Coaofret, Selective Electrode Rev., 12 (1990) 35. F. Malecki and R. Staroscik, Anal. Chim. Acta, 139 (1982) 353. MS. Ionescu, S. Cilianu, A.A. Bunaciu and V.V. Coaofret, Talanta, 28 (1981) 383. G.E. Baiulescu, G. Kandemir, MS. Ionescu and C. Cristes- cu, Talanta, 32 (1985) 295.

10 P.C.K. Tseng and W.F. Gutknecht, Anal. Chem., 47 (1975) 2316.

11 V.V. CoSofret and A.A. Bunaciu, Anal. L&t., 12 (1979) 617. 12 A.G. Fogg, M.A. Abdalla and H.P. Henriques, Analyst, 107

(1982) 449. 13 G.E. Baiulescu, V.V. Co9ofret and F.G. Cocu, Talanta, 23

(1976) 329. 14 M.S. Ionescu, V.V. Coaofret, T. Panaitescu and M. Costes-

cu, AnaLLett., 13 (1980) 715. 15 D.L. Simpson and R.K. Kobos, Anal. Lea., 15 (1982) 1345. 16 D.L. Simpson and R.K. Kobos, Anal. Chem., 55 (1983) 1974. 17 M.Y. Keating and GA. Rechnitz, Anal. Lett., 18 (1985) 1. 18 S. Tagami and M. Fujita, J. Pharm. Sci., 71 (1982) 523. 19 S. Tagami and H. Maeda, J. Pharm. Sci., 72 (1983) 988.

Dr. Vasile V. Copofre/ is senior researcher at the Institute of chemical and Pharmaceutical Research Bucharest, 74351 SOS Vi- tan 112, Bucharest - 3, Romania. At present he is visiting profes- sor at the Department of Chemistry, University of North Caroli- na, Chapel Hill, NC, USA.

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