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Analytical Methods for the Determination ofChlorhexidine: A ReviewFlávia Angélica Másquio Fiorentino a , Marcos Antonio Corrêa b & Hérida Regina NunesSalgado a ba Programa de Pós-graduação em Ciências Farmacêuticas, Faculdade de CiênciasFarmacêuticas de Araraquara, UNESP, Araraquara, SP, Brazilb Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas deAraraquara, UNESP, Araraquara, SP, BrazilVersion of record first published: 06 May 2010.

To cite this article: Flávia Angélica Másquio Fiorentino , Marcos Antonio Corrêa & Hérida Regina Nunes Salgado (2010):Analytical Methods for the Determination of Chlorhexidine: A Review, Critical Reviews in Analytical Chemistry, 40:2, 89-101

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Critical Reviews in Analytical Chemistry, 40:89–101, 2010Copyright c© Taylor and Francis Group, LLCISSN: 1040-8347 print / 1547-6510 onlineDOI: 10.1080/10408340903232020

Analytical Methods for the Determinationof Chlorhexidine: A Review

Flavia Angelica Masquio Fiorentino,1 Marcos Antonio Correa,2 and HeridaRegina Nunes Salgado1,2

1Programa de Pos-graduacao em Ciencias Farmaceuticas, Faculdade de Ciencias Farmaceuticas deAraraquara, UNESP, Araraquara, SP, Brazil2Departamento de Farmacos e Medicamentos, Faculdade de Ciencias Farmaceuticas de Araraquara,UNESP, Araraquara, SP, Brazil

Chlorhexidine is a type of antiseptic belonging to biguanidic group and it is large, used indentistry, human, and veterinary medicine because it is active against Gram-positive andGram-negative microorganisms. It can be incorporated in several types of preparations andfor this reason are necessary effective analytical procedures for quality control and pharma-codynamic and pharmacokinetic studies. Chlorhexidine can be analyzed by many types ofassays, however the HPLC is the most used method. In this review, various methods to analyzechlorhexidine including spectrometry, chromatography, colorimetric reaction, solid phase ex-traction, and capillary electrophoresis are presented and the application of these methods forthe determination of chlorhexidine in biological fluids and formulations are discussed.

Keywords review, chlorhexidine, methods of analysis.

INTRODUCTIONGeneral Aspects of Chlorhexidine

Antiseptics are products which destroy microorganisms orinhibit their reproduction or metabolism (1). The antisepticsdamage coagulation protoplasmatic or denaturation of proteins,cell lyses by structural change of the cell membrane, decreaseof surface tension, inhibition of essential enzymes and they areused just to prevent infections because they destroy microor-ganisms in external surface and in the skin (2, 3). The use ofantiseptics started in 1847 (4) and chlorhexidine, a type of an-tiseptic, was synthesized in the 1940s and commercialized in1954 (5).

Chlorhexidine is an excellent cationic antiseptic belonging tobiguanidic group, chemically known as 1,1′–hexamethylenebis{5–(p-chlorophenyl) biguanide} (Figure 1) (6, 7). It has a va-riety of salts, as digluconate (Figure 2), acetate (Figure 3), glu-conate, and hydrochloride (Figure 4) (7, 8).

Chlorhexidine has many types of uses because is possibleincorporate it into many types of products, for example, in soaps,

Address correspondence to Prof. Herida Regina Nunes Salgado,Programa de Pos-graduacao em Ciencias Farmaceuticas, Faculdade deCiencias Farmaceuticas de Araraquara, UNESP, Rodovia Araraquara-Jau, km 1, CEP 14801-902, Araraquara, SP, Brazil; E-mail: sal-gadoh@fcfar.unesp.br

oral rinse, mouthwash, solution of irrigation, gel, spray, and gum(9) and it has an extensive safety record, strong binding potentialthat results in effectiveness and low cost (10).

It is used in veterinary medicine as a disinfectant for cleansingwounds, skin, instruments, and equipment (11) and for treatmentof dermatitis and piodermitis (12). In clinical practice, it isused for antisepsis of skin, hands, and mucous membranes andis currently recommended for treatment of wounds and burns(13, 14) and for preparation of patients in procedures (15). Indentistry it is used as a mouth antiseptic, for prevention ofdental plaque formation and treatment of gingivitis, to eliminateoral pathogens, as an endodontic irrigant, and as an intracanalmedication (11, 16).

The use of chlorhexidine has been suggested in spermicidesto prevent the transmission of the HIV virus since it does notbreak the vaginal epithelium, has in vitro activity against thevirus, and may prevent contraception because it spreads in thecervical mucus at concentrations of about 1 mg/mL and preventsthe entry of sperm (17). Also, WHO recommends chlorhexidinefor cleansing umbilical cords because it can markedly reducethe risk of omphalitis (10). Stevens and coworkers (18), Haand Cheung (19), and Abad-Villar and collaborators (20)describe the use of chlorhexidine to preserve ophthalmicsolution and to disinfect contact lenses because of its lowtoxicity.

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FIG. 1. Chemical structure of chlorhexidine. CAS: 55-56-1.

Chlorhexidine is active against Gram-positive bacteria andless effective for Gram-negative bacteria, fungi, and speciesof Proteus; it has activity only for certain types of envelopedviruses, including hepatitis virus, herpes simplex, HIV, cy-tomegalovirus, influenza, and respiratory syncytial virus. Formycobacteria, chlorhexidine presents minimal activity; againstendospores and cysts of protozoan the activity is nil (3, 8, 9,17, 21). The anti-microbial activity against spores is much dis-cussed. Some authors say it has low activity (21, 22), whileothers say it does not (8, 11, 23). Martindale (17) and Bambaceand coworkers (24) complement the chlorhexidine is effectiveagainst spores only in high temperatures.

Chlorhexidine acts on the cell membrane causing disrup-tion and its consequent loss of intracellular material, respiratoryinhibition, and cytoplasmic coagulation (9, 25). The links inthe cell membrane probably occur between the positive chargeof chlorhexidine with the negative charge of carboxyl groupsavailable for proteoglycans and the phosphate groups of te-icoic and lipoteicoic acids in bacterial inner wall. Regardingthe metabolism, chlorhexidine inhibits the enzyme complex re-sponsible for the incorporation of glycosis in the bacterial cell(5). The action of chlorhexidine is inhibited by nicotinic acid,but it the exact mechanism of this inhibition is not known; it isbelieved to occur by blocking the receptors of chlorhexidine byacid (26).

Side effects are significant discoloration of the teeth, restora-tions, and back of the tongue, flaking, and oral sensitivity; thereis also a bitter taste and interference with the taste for a few hoursafter the mouthwash (27). When it is associated with soap, itcan cause local or systemic toxicity; change in the normal floraof the skin predisposes it to the colonization by Gram-negativebacteria (28). Contact with strong chlorhexidine solution in eyesand sensitive tissue can cause irritation, when ingested, it can

FIG. 2. Chemical structure of chlorhexidine digluconate. CAS:18472-51-0.

FIG. 3. Chemical structure of chlorhexidine acetate. CAS: 56-95-1

cause gastrointestinal irritation, vomiting, and dizziness, whilesystemic toxicity is rare (17, 21).

ANALYTICAL METHODS FOR DETERMINATION OFCHLORHEXIDINE

Development and validation of analytical methods is very im-portant for the pharmaceutical industry to guarantee the qualityof the commercialized products. Several techniques have beendeveloped for the determination of chlorhexidine and the offi-cial method for salts of chlorhexidine described in the literatureis an aqueous titration using 0.1 M perchloric acid (6, 29) andhigh performance liquid chromatography (HPLC) (7). For irri-gation solution and mouthwash with chlorhexidine, HPLC is thedescribed method in the European and British Pharmacopoeias(6, 29). The United States Pharmacopoeia (7) describes HPLCto analyze oral rinse with chlorhexidine gluconate. All theseofficial compendiums describe HPLC to analyze related sub-stances. The United States Pharmacopoeia (7) describes HPLCto analyze p-chloroaniline and the European and British Phar-macopoeias (6, 29) describe a colorimetric method. For analysisof chlorhexidine and its salts, gas chromatography (GC), (30)and HPLC; are the methods presented in the literature (31) to an-alyze impurities and related substances, catalytic oxidizer andHPLC (18), GC (32), and HPLC (33) are the methods men-tioned. The parameters used in these analyses are presented inTables 1 and 2.

In biological fluids, chlorhexidine is determined by severalmethods. For studies or the retention of chlorhexidine in themouth is determined by radiolabelled chlorhexidine (14C) (34),by direct UV spectroscopy (35), by fluorometry (36), by HPLC(37–39), and by solid phase microextraction (40) in saliva; by

FIG. 4. Chemical structure of chlorhexidine hydrochloride.CAS: 3697-42-5.

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ANALYTICAL METHODS FOR CHX, REVIEW 91

TABLE 1Parameters described in the literature to analyze chlorhexidine and its salts.

Reference Method λ(nm) Mobile phase or solvent Column Linearity Sample

USP (2008) HPLC 239 Solution A: sodiumphosphate buffer pH3.0 with triethylaminein water, mixing withacetonitrile SolutionB: acetonitrile

L1 Gluconate

EP (2005) Titration Perchloric acid SaltsEP (2005) Titration Perchloric acid SaltsSiefert et al. (1975) Gas chromatography 63Ni detector Supelco 50–200 ppm AcetateDoub et al. (1996) Gradient HPLC 230 Phase A: ammonium

acetate pH 5.0 PhaseB: acetonitrile

Nucleosil C18 10 µg/mL to10 mg/mL

Gluconate

HPLC in urine (41, 42), in urine and serum, (43–45) and in serum(46), by liquid chromatography-electrospray ionization massspectrometry (LC-ESI-MS) in hemolyzed blood (47), and de-termination of the elimination lifetime of chlorhexidine residuesin milk of cow (25). The parameters used in these analyses arepresented in Table 3.

In pharmaceutical products with chlorhexidine, HPLC is themethod most used to analyze this antiseptic (7, 11, 19, 48–55); solid-phase extraction (SPE) with UV spectrophotometry(56), gas-liquid chromatography (57), liquid chromatography(6, 29), capillary electrophoresis (CE) (20, 58), flow injectionextraction-spectrophotometry (59, 60); and voltametry (DPV)(61) also are used to assess it. The parameters used in theseanalyses are presented in Table 4.

Chlorhexidine has been incorporated in products to have con-trolled release delivery in the skin and in the mouth to prevent thebiofilm formation and for the treatment of periodontal disease.HPLC is the method frequently used to analyze the liberation ofchlorhexidine (62–67); however, spectrophotometry also can beused (68). The parameters used in these analyses are presentedin Table 5.

The percutaneous absorption of chlorhexidine digluconatesolution was assessed by reverse phase adsorption chromatog-raphy (69). The parameters used in this analyze is presented inTable 6.

DISCUSSION AND CONCLUSIONThere are many methods described to analyze chlorhexidine,

its degradation products, and impurities in the literature. How-ever, HPLC is the most used. The physico-chemical propertiesof chlorhexidine indicate that HPLC with UV detection shouldbe the analytical technique of choice (41). The method related inthe European and British Pharmacopoeias (6, 29), colorimetricreaction, is loss-sensitive and accurate; the methods proposedby Revelle and coworkers (33) and by Doub et al. (31) only

separate and identify the impurities of chlorhexidine, but do notquantity them.

The spectroscopic method proposed by Jensen andChristensen (35) to analyze chlorhexidine in biological fluidsis no, specific for chlorhexidine because recovery compoundsof saliva, but is a simple method and easy to carry out. How-ever, fluorescence described by Vries and coworkers (36) is aneasy and exact method to determine chlorhexidine in aqueoussolution and in saliva. The method proposed by Bosnevoll andcoworkers (34) assesses the quantity of chlorhexidine retainedin the mouth 24 hours after the use of 0.2% chlorhexidine diglu-conate aqueous solution.

The chromatographic methods to analyze chlorhexidine insaliva are laborious, requiring lengthy and complicated ex-traction processes; however, they are more sensitive and spe-cific than other techniques such as colorimetric, titration, flu-orescence, and spectrophotometric methods. The solid-phasemicro extraction allows one to assess several aspects suchas connection and stability of chlorhexidine in the salivaand its pharmacokinetic effects after use of chlorhexidine so-lution. However, it requires many steps for extraction andbecause of this, it is laborious and requires more analysistime.

The method presented by Usui and coworkers (47) is moresensitive and selective than the HPLC-UV method and it alsosupplies more information about the identification of chlorhex-idine and its impurities.

To determine chlorhexidine in urine, HPLC is the most usedmethod. The method described by Wainwright and Cooke (42)allows detecting p-chloroaniline in the urine, but only chlorhex-idine was quantified. The HPLC technique associated with SPE,described by Below and collaborators (45), allows one to de-termine the quantification limit of chlorhexidine in biologicalfluids and in pharmaceutical products and also verify the pres-ence of p-chloroaniline and 1-chloro-4-nitrobenzene, but itsquantification is not exact.

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TABLE 2Parameters described in the literature to analyze impurities and degradation products of chlorhexidine

Reference Method �(nm)Mobile phase

or solvent Column Linearity Product found

USP (2008) HPLC 239 Solution A:sodiumphosphatebuffer pH 3.0withtriethylamine inwater, mixingwithacetonitrileSolution B:acetonitrile

L1 Relatedsubstances andp-chloroaniline

EP (2005), BP(2005)

Gas chromatogra-phy andcolorimetricreaction

Nitrogen OV – 225 4- chloroaniline

EP (2005), BP(2005)

HPLC 254 Sodium octane-sulphonate:glacial aceticacid: water:methanol

Nucleosil C18 Relatedsubstances

Gavlick, Davis(1994)

Gas chromatogra-phy

Helium J&W ScientificDB-1

0.956–10ppm

p-chloroaniline

Revelle et al.(1993)

Isocratic HPLCwith diodearray detector,HPLC-MS*,NMR**

PHLC andHPLC-in: 230RMN: 299.92MHz

HPLC andHPLC-MS:buffer acetatepH 5.0:methanolNMR: D2O ouCD3OD

HPLC and HPLCin Zorbax CN

11 impuritiescalled: B, B1,C, D, D1, D2,E, F, G, G1, H

Stevens et al.(1986)

HPLC-UVdetectorCatalyticoxidizer (CO)

220 H3PO4: sodium 1-heptanesulfonate:CH3CN, pH 2.2

Regis ChemicalC6 Oxygen 300mL/min for CO

1–80µg/mL

p-chloroanilinep-chlorophenylbiguanide andphenyl-biguanide

MS*—mass spectroscopy; NMR**—nuclear ressonance magnetic.

In pharmaceutical preparations, HPLC is the most usedmethod; however, it is necessary to extract the chlorhexidineof the products and in some cases this process is lengty. Themethod described by Miribel and coworkers (57) needed sev-eral steps. Moreover, mass spectroscopy and nuclear resonancemagnetic are carried out to verify the chemical structure ofchlorhexidine. The methods proposed by Gavlick (54) andBauer and coworkers (50) allow the quantification of chlorhexi-dine and chloroaniline. The method proposed by Abad-Villarand coworkers (20) was successful and the detection limitfor chlorhexidine is comparable with the best value reportedfor HPLC with UV-detection; this method is useful in oph-

thalmic drug penetration studies due to its simplicity, sensi-tivity, and limited requirement on sample volume. The othermethod of CE described by Okamoto and coworkers (58) alsowas useful to separate and to quantify seven compounds in anointment.

Calatayud and coworkers (59) described a method that usesthe precipitation of chlorhexidine with thymol blue; this pre-cipitation is evaluated by flow injection. This method was fastand simple. The method proposed by Perez-Ruiz and coworkers(1999) (60) might be applicable for many types of pharmaceu-tical preparations and also has instrumental simplicity whencompared with HPLC.

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98 SALGADO ET AL.

TABLE 5Parameters described in the literature to analyze the liberation of chlorhexidine in pharmaceutical products

Reference Method λ (nm)Mobile phase

or solvent Column Linearity Sample

Medlicottet al.(1996)

UV spectroscophyHPLC with dualdetector

254 Sodiumcitrate/sodiumhydroxide buffer(for UV)Acetonitrile:glacial aceticacid: sodiumlaurylsulphate(for HPLC)

C18ODS-B Exsil 1 – 60 µg/mL Poly(ε-caprolactone)

Yue et al.(2004)

HPLC withUV-Vis detector

280 Methanol: waterpH 3.0

C18 Nova-Pak Theorical loadingof 10 – 40%

Biodegradablepoly(dl-lacticacid—co—glycolic acid)microspheres bycomplexationwithcyclodextrins

Paler et al.(2004)

RP HPLC 270 Propanol(40%):0.05MNaH2PO4 (40%)and sodiumdodecyl sulphate(0.4%), pH 3.0

C18Joneschromatography

Chlorhexidineacetate in glassionomer cements

Rasimick etal. (2008)

HPLC withUV-Vis detector

288 C18 Waters XBridge

10–150 ppmchlorhexidine

Precipitate ofchlorhexidineand EDTA

Farkas etal. (2007)

UV spectroscopy 255 1–20.0 µg/mL Chlorhexidine andits salts incrystallinestructures

Medlicottet al.(1999)

HPLC with dualdetector

254 Acetronitrile:glacial aceticacid: sodiumlaurylsulphate

Exsil ODS-B C18 2–30 µg/mL Saliva

Giunchediet al.(2002)

RP- HPLC 257 Acetonitrile:buffer(0.1 M disodiumhydrogenphosphate, 0.005M 1-heptanesulfonicacid and 0.05 Mtriethylamine)(35:65

Nucleosil RP-18 Saliva

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ANALYTICAL METHODS FOR CHX, REVIEW 99

TABLE 6Parameters described in the literature to analyze the liberation of chlorhexidine in the skin

Reference Method λ(nm)Mobile phase

or solvent Column Linearity Sample

Lafforgue (1997) RP adsorptionchromatography

254 Acetonitrile: 50 mMacetate buffer(50:50), pH 3.15

0–20 mg/mL Intact andstripped skin

The voltametric method described by Wang and Tsai (2001)(61) to analyze chlorhexidine in pharmaceutical preparationsmight be performed without separation from the fatty constitu-tents and simple extraction procedure, different than the HPLCmethod compared by these authors.

The method presented by Palmer and coworkers (66) wasuseful to demonstrate that chlorhexidine might be incorporate,in glass ionomer cements because it is released into solution.Rasimick and coworkers (67) describe a method that was usefulto quantify chlorhexidine and EDTA in the precipitate formedbetween the association of these products. These results demon-strate that it can be used in dentistry with efficiency. The spectro-scopic method describe by Farkas and coworkers (68) demon-strated that different liquid crystalline structures might be usedto control the release of chlorhexidine and its salts. The authorsconcluded that this method is more simple than HPLC methods.

The methods described by Medlicott and coworkers (63)and Giunchedi and coworkers (64) were also useful to analyzechlorhexidine in the controlled system delivery.

The method described by Lafforgue and coworkers (69) wassimple and important to demonstrate that when the skin is dam-aged the amount of chlorhexidine absorbed and stored is largerthen the skin is intact.

Pharmaceutical products have to obey the law and ensuretheir efficacy without a raise in risk of the life of the consumer.It is necessary for the routine quality control of pharmaceuticalproducts to employ well-characterized and fully validated ana-lytical methods to yield reliable results that can be satisfactorilyinterpreted. The analytical methods are constantly undergoingchanges and improvements, and in many instances, they are atthe cutting edge of the technology (70). This review is importantbecause it presents several methods to analyze chlorhexidine andtheir advantages with technological improvement.

ACKNOWLEDGMENTSThe authors acknowledge to CAPES (Brasılia, Brazil) and

CNPq (Brasılia, Brazil).

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