Polarographic determination of chlorhexidine in pharmaceutical preparations

T&znto,

voL 22,pp

100-1004

PergamonPress 1975

Pnnted I” Great Brltam

POLAROGRAPHIC DETERMINATION OF CHLORHEXIDINE IN PHARMACEUTICAL PREPARATIONS EINARJACOBSENand BJ~RN GLYSETH Department of Pharmacy, Umverslty of Oslo, Blmdern, Oslo 3, Norway (Recetved 25 October 1974 Accepted 14 January 1975) Summary-The electroreductlon of chlorhexldme has been studied by d c, a c and pulse polarography Polarograms of the drug recorded from ammomum acetate buffers exhlblt a single well-defined wave The current 1s &tTuszon-controlled and proportIona to the concentration The reduction wave 1s due to an ureverslble &electron reduction of the four > C=NH groups m the molecule to ammo groups The drug IS strongly adsorbed on the electrode surface over a conslderable potential range Hence, the drug can be determined by polarography m the presence of other weaker surfactants often present m pharmaceutical formulations Procedures have been proposed for pulse-polarographlc determination of the drug m antlseptlc cream and liquids The proposed method 1s simple and accurate and does not involve hmeconsummg separation of the drug from msoluble constituents present m the sample

The antrseptrc drug chlorhexrdme [l-l’-hexamethyleneb@-p-chlorophenylbrguamde)] is strongly bactericidal towards a large number of orgamsms and IS mcorporated m several formulatrons, m a range of concentratrons.1-4 Relatrvely large amounts of the drug may be determmed by titration m non-aqueous medium, but smaller amounts are usually determined spectrophotometrrcally ‘e6 However, these methods mvolve trme-consummg separatrons and are mconvement for routine analysrs The present work was carried out m order to study the electroreductron of chlorhexrdme m aqueous solutrons and to investigate the applicatron of pulse polarography to rapid determination of the drug m pharmaceutrcals EXPERIMENTAL chlorhexldme dlacetate was Pharmaceutical-grade obtained from A/S Apothekernes Laboratormm for Speclalpraeparater, Oslo Stock solutions were prepared by dlssolvmg the appropriate amount of the commercial product m distilled water All other chemicals were reagent grade and were used without further purlficatlon Hlbltane antiseptic cream and liquid was obtained from Imperial Chemical Industries Ltd Cheshire. Great Brltam Polarograms (a c and d c ) were recorded with a Metrohm/E 261 R Polarecord connected to a Metrohm E 393 a c modulator An Aa/A&l/saturated KC1 electrode _. _ served as reference electrode and a tungsten electrode was employed as auxllrary electrode The capdlary character1stlc.sof the dropping mercury electrode, measured m 0 IM ammonmm acetate buffer at an applied potential of - 1 58 V and a corrected mercury hetght of 48 2 cm, were m = 1973 mg/sec and t = 3 02 set Dissolved oxygen was removed from the solutions by bubblmg oxygen-free nitrogen through the cell for 10 mm and passing it over the solution during the electrolysis Pulse polarograms were recorded with a PAR polarographlc analyser, model 174, m the dlfferentlal pulse mode Cychc voltammetry experiments were performed with a versatde solid-state Instrument constructed m this laboratory followmg the design of Goolsby and Sawyer ’ A Mosely 7030 AM X-Y recorder was used m conJunctIon I

TM. 22112-E

with the instrument A Metrohm E 410 hanging mercury drop was used as workmg electrode and a platinum co11 served as auxlhary electrode All experiments were performed at 25 f 0 1” RESULTS AND DISCUSSION

Prehmmary experiments showed that polarograms of chlorhexrdme m acrdrc solutions exhrbrt only a poorly-defined wave, with half-wave potentral close to the reduction wave of the supportmg electrolyte, which 1s not useful for a polarographrc determmatron of the drug Better-defined waves were obtained from slightly alkaline media Experiments showed, though, that the drug IS only slightly soluble m ammoma buffers contammg chloride and that catalytrc waves are obtained with buffers contammg mtrate On the other hand, polarograms recorded for neutral and alkaline ammomum acetate buffers exhibit a single well-defined wave (Fig 1) Hence, 0 1M ammonium acetate/ammonia buffer was chosen as supportmg electrolyte The effect of pH on the reductron wave was mvestrgated by recording d.c polarograms of 0 lmA4 chlorhexrdme m O*lM ammomum acetate buffers of various pH values As mdrcated m Table 1, the hmrtmg current IS independent of pH m the range 6 5-8 3 but decreases of hrgher pH values The steepest wave was obtained at pH values above 9 Moreover, a better separation of the chlorhexrdme wave from that of the supportmg electrolyte was obtamed at higher pH values Consequently, 0 1M ammomum acetate/ 0 1M ammoma buffer at pH 9 3 was used as supportmg electrolyte m the followmg experiments The effect of drop-time was mvestrgated by recordmg polarograms of 0 1mM chlorhextdme m 0 1M ammonmm acetate buffer at varrous heights of the mercury column The constancy of the product zh- ‘I’, where h IS the height of the column after correctron

1002

EINARJACOBSEN and

BJBRN GYLSETH

42-

I

I

-10

E.

1

-08

volts vs

I

I

I

-04

-02

0

I

-06

Ag/AgCl

Fig 2 Electrocaplllary curves of ammomum acetate buffer m the absence (curve A), and m the presence of 25 x 10w5M chlorhexrdme (curve B) and 10e4M chlorhextdme (curve C)

E.

volts vs Ag/AgCl

FIN 1 Polarograms (d c and a c) of 0 1 mM chlorhexrdme m 0 1M ammomum acetate/ammonia buffer pH 9 3

for the “back pressure”, and the temperature coefficlent of 2 1% per degree (over the range 2$45”), mdlcate that the dc current was &ffuslon-controlled The d c polarographlc step was followed by an a c polarographlc wave (Fig 1) The peak potential E,, was about 40 mV more negative that the half-wave potential The appearance of an ac polarographlc wave indicates that a fast electron-transfer step IS mvolved m the electrode reaction On the other hand, the plots of log l/(ld-l) us the potential show two almost straight lmes with slopes of -45 and -28 mV per log unit, respectively, lmplymg that the electron-transfer occurs m two steps and that the overall electrode reaction IS lrreverslble The ac polarogram (Fig 1) also exhlblts a tensammetrlc wave with peak potential - 145 V, mdlcatmg that the drug IS strongly adsorbed on the electrode surface The adsorption was verdied by droptime measurements As shown m Fig 2 the presence of chlorhexldme causes a large decrease m the droptime over a conslderable potential range The drug IS desorbed at - 145 V (Fig 1) Further experiments showed that chlorhexldme IS such a strong surfactant that it may even be used as a maxlmum suppressor Table 1 Effect of pH on dc polarograms of 0 1 mM chlorhextdme dtacetate m 0 1M ammomum acetate/ ammoma buffers PH

z.i> PA

-E,,,> V

-(Ew-Ed

V

m dc polarography The presence of only 00002% chlorhexldme was sufficient to suppress the maxlmum on the wave for 3 mM lead m 0 1M potassrum chlorIde, of 0 5mM mtrazepam m 0 5M sulphurlc acid and of 1mM mcotmamlde m 2M sulphurlc acid The surface activity of chlorhexldme causes a considerable foaming during deaerahon of solutions contammg higher wncentratlons of the drug, but experiments showed that the foaming can be avoided by the presence of a minute amount of n-octyl alcohol The results m Table 2 indicate that the drug can be determmed by dc polarography with a lmear calibration over the concentration range from 5 x 10e6 to 2 x 10v4M which corresponds to Z-125 pg/ ml As the ac polarographlc peak current IS very small (Fig 1) it IS not smtable for a practical determination of small amounts of the drug, but the dlfferentlal pulse polarogram of the drug 1s very well Table 2 Polarographrc data for the reductron of various amounts of chlorhextdme m 0 1M ammonium acetate/ ammoma buffer, pH 9 3 Direct current polarography Cone, mM 0200 0150 0 100 0075 0050 0025 0010 0 005

216 2 14 2 16 2 14 198 186

1388 1423 1445

1484 1 536 1 586

0 038 0 037 0 036 0 034 0 032 0 030

-E,,z, V

3 65 281 1 84 139 092 0 455 0 188 0 092

1640 1610 1 578 1 564 1544 1531 1 523 1513

‘d/C. wWM 183 187 184 186 184 182 188 184

Dtfferentral pulse polarography (drop ttme 0 5 set, pulse amplitude 50 mV and scan-rate 2 mV/sec) Cone , i7lM

65 72 76 83 90 95

'd,PA

00050 0 OQ25 OOIO 0 00075 0 00050

‘P, PA

-E,. V

1200 0 590 0 243 0 185 0127

1 57 1 58 158 1 57 1 57

$K w@M

240 236 243 247 254

Polarographlc determmatlon of chlorhexldme

1003

defined even at low concentrations and by this pH range 65-8, was I = 115 fl 1 mmole-’ method the concentration range can be extended mg -3/2 sec112 Companson of this value with that of other depolarizers with approximately the same down to 5 x lo-‘M (03 kg/ml) As indicated m Table 2, the half-wave potential 1s molecular size indicates that 8 electrons are involved m the overall electron-transfer reaction The shift of shifted to more negative potentials with increasing the half-wave potential to more negative values with concentration of the drug, whereas the pulse polarographlc peak potential (which was obtamed with a mcreasmg pH (Table 1) suggests that hydrogen ions drop tnne of 05 set) 1s independent of the con- are consumed m the electrode reaction The structure centration This effect 1s probably due to a slow of chlorhexldme 1s adsorptlon of the reduction product on the electrode surface CL NHCNHCNH(CH&NHf”f; Cl Cychc voltammetry experiments were performed at iH AH a hangmg mercury drop electrode Reproducible results were obtained provided that the mercury drop Hence, the reduction wave of chlorhexldme 1s probably due to reduction of the four > C&NH groups was changed between each experiment No anodlc to ammo groups peak resulting from reoxldatlon of the reduction product was observed at any scan-rate or any swltch> C=NH + 2H+ + 2e+ > CH.NH2 mg potential, mdlcatmg that the overall electrode As stated above, the electron-transfer occurs m two reaction 1s lrreverslble As shown m Fig 3, the cathosteps, probably a slow one-electron step followed by dic peak has the characterlstlc symmetrical shape of a fast one-electron step glvmg rise to the a.c wave an adsorption wave If the cychc sweep 1s repeated The four > C%NI-I groups are probably energetically on the same drop the cathodic peak current decreases equivalent and hence they are reduced at the same to a very small value, mdlcatmg that the reduction potential and only one polarographlc wave 1s product IS adsorbed on the electrode surface and that observed the adsorbed4ayer mhlblts further electrode reactlon Further experiments showed that the current function ANALYTICAL APPLICATIONS l,/CV”2 increased with mcreasmg scan-rate and deBecause chlorhexldme 1s reduced at highly negative creasing concentration, confirming that adsorption IS potent&, differential pulse polarography IS the most involved m the electrode reaction * useful method for the determination of the drug m Coulometrlc reductions of chlorhexldme at controlled potential were performed to determme the the presence of other polarographlcally active substances It IS also the most senslttve method number of electrons involved m the overall electronExperiments showed that the antiseptic liquid Hlbltransfer reaction The experiments were carried out m the absence of an m a small electrolysis cell with tane, contammg 5% chlorhexldme dlgluconate, can a mercury pool as working electrode However, these easily be determmed by dlfferentlal pulse polarograexpernnents did not gve reliable results, because the phy by simply dllutmg the sample with ammonmmreduction potential of the drug 1s too close to that acetate/ammonia buffer and recording the polaroof the supportmg electrolyte Hence, the drug could gram The commercial antlseptlc hquld also contams, m addition to chlorhexldme, certain other surfactants not be completely reduced without also reducmg some of the supportmg electrolyte The diffusion cur- which interfere at high concentrations of the hquld rent constant, calculated from the dc current m the Hence, the sample must be diluted with buffer to give a final concentration below 10M4M Recommended procedure

Transfer an ahquot of the sample, eqmvalent to l-10 mg of chlorhexldme dlgluconate, to a lOO-ml volumetric flask and add 25 ml of 04M ammomum acetate/ammonia buffer (pH 9 3) and one drop of n-octyl alcohol, and dilute to the mark with dlstllled water Shake the flask and transfer a sultable amount to a polarographlc cell Displace dissolved oxygen with pure mtrogen and record a ddferenteal pulse polarogram with drop-time 0 5 set, pulse amphtude 50 mV and scan-rate 2 mV/sec at a startmg potential of - 1 3 V Measure the peak-current and determme the amount of chlorhexldme by the standard addition method

I -I

I 4

I

I

-I E.

I

6

volts vs

I

-I

I

8

Ag/AgCl

Fig 3 Cychc voltammogram of OOSmA4chlorhexldme m ammomum acetate buffer, pH 9 3 Scan-rate 0 05 V/set

The amount of chlorhexldme dlgluconate m Hlbltane antlseptlc liquid (declared amount 5%) was determmed by the procedure above Four determmatlons gave the results 4 98, 4 94, 4 98 and 5 14%

Further experiments showed that chlorhexldme can be determined m antlseptlc cream by almost the same procedure and without any time-consummg separation from the fatty constituents A polarogram of

1004

EINARJACOBSEN and BJ~RN GYLSETH I

I

z

septic cream (declared amount 1 g of chlorhexrdme dlgluconate per 1OOg of cream). Recommended procedure

T

Transfer 0 5 g of antlseptzc cream to a lOO-ml volumetric flask, dilute to the mark with dlstdled water and shake well for a few minutes Transfer 10 00 ml of the suspension to a lOO-ml flask, add 25 ml of 04M ammomum acetate/ ammoma buffer at pH 9 3, one drop of n-octyl alcohol, and dilute to the mark with dlstdled water Transfer a smtable volume to a polarographzc cell and follow the polarographzc procedure above In order to reduce the noise it ISadvantageous to increase the scan-rate to 10 mV/sec

‘PA

?1

Several determmauons of chlorhexldme m 1% Hlbltane antiseptic cream gave the result 1 11-1 13 g of chlorhexldme dlgluconate per 100 g of cream

J 1

-I 3 E,

I

-I 5 volts us

I

-I 7

1

-I 9

Ag /AgCl

Fug 4 Dlfferentlal pulse polarogram of a suspension of Hlbnane anhseptlc cream m 0 1M ammonnzm acetate/ ammoma buffer, pH 9 3 Drop time 0 5 set, pulse amphtude 50 mV and scan-rate 10 mV/sec Hlbltane antiseptic cream suspension m 0 1M ammonmm acetate buffer (pH 9 3) IS given m Fig

4 Experiments showed that the peak current IS proportlonal to the concentration m the range from 3 x 10e6 to 3 x 10-5M. Hence, the suspension must be diluted with buffer to gve a final chlorhexldme concentration of less than 1 &ml before the polarogram IS recorded. On the basis of these experiments the followmg procedure was outlmed for Hlbltane antl-

Acknowledgement-The authors wish to thank cand pharm R GJermundsen and cand pharm V Helm, Pharmaceutical Research Laboratory, A/S Apothekernes Laboratoruzm, Oslo for valuable dlscusszons and for supply of the drugs used m this mvestlgatzon

REFERENCES 1 G E Davies, J Francis, A R Martin, F L Rose and G Swam, Brat J Pharmacol, 1954, 9, 192 2 S R Kohn. L Gershenfeld and M Barr. J Phurm Scz, 1963, ii, 1126 3 M W R Brown, J H S Foster, D A Norton and R M E Richards, Phurm J, 1964, 192, 8 4 R T Yousef, Acta Pharm Sueczca, 1966, 3, 141 5 A Holbrook, J Pharm Pharmacol, 1958, 10, 370 6 M Aokl and Y Kusaka, Yakuzaqaku, 1966, 26, 58 7 A D Goolsby and D T Sawyer, Anal Chem, 1967, 39, 411 , 1967, 39, 1514 8 R H Wopschall and I Sham, zbzd