American Journal of Anal yt ical Chemistry, 2011, 2, 422-428
doi:10.4236/ajac.2011.24051 Published Online August 2011 (http://www.SciRP.org/journal/ajac)
Rapid HPLC Method for Determination of
Parachloroaniline in Chlorhexidine Antiseptic Agent in
Mouthrinses, Ophthalmic and Skin Solution
Alain Nicolay, Estelle Wolff, Marie-France Vergnes, Jacques Kaloustian, Henri Portugal
Faculty of Pharmacist, Institut National de la Recherche Agronomique, Marseille, France
Received January 18, 2011; revised February 19, 2011; accepted May 30, 2011
Abstract
We described a simple and rapid method to quantify simultaneously chlorhexidine (CHD) and its major me-
tabolite, para Chloroaniline (pCA) by HPLC with UV detection without the additional need of mobile-phase
amine modifiers or ion-pairing reagents, with good resolution between pCA and CHD, symmetry peak of the
compound and short run time. HPLC-UV analyses were performed using a Dionex® Summit liquid chromato-
graph (Dionex Corp, Sunnyvale, CA, USA). Chromatographic separations were carried out on a Luna® 150
mm×3 mm i.d. column packed with 3 µm CN (cyano) particles (Phenomenex®), guarded by an on-line filter.
Mobile phase consist of methanol:water with sodium chloride with 0.02% of formic acid (55:45). Wavelengths
for pCA and for CHD are 238 and 255 nm respectively. Influence of methanol and of sodium chloride content
in the eluant has been studied. Linearity of CHD is very good, from 0.5 up to 21.2 µg/l while linearity of pCA
is in the range of 0.05 to 10 µg/l with correlation coefficients above 0.999. Resolution between the components
is above 4, asymmetry is about 1.3 and 1.7 for pCA and CHD respectively and the run time is less than 5 min-
utes. This method has been applied to CHD solution of different medical devices. No interference has been re-
ported, and the analysis of direct injection of solution, without any treatment is achieved in less than five min-
utes.In conclusion, we present a validated method for dosage of CHD and its major impurity pCA, known to be
carcinogen, available into medical products or medicinal device for in-vitro diagnostic.
Keywords: Chlorhexidine, Chloroaniline, HPLC
1. Introduction
Chlorhexidine [CHD; 1,1’-hexamethylenebis[5-(4-chlo-
rophenyl) biguanide]] has a wide spectrum of bacteri-
cidal and antiviral activity and is a common ingredient in
various formulations ranging from skin disinfectants in
healthcare products to antiplaque agents in dentistry [1,2].
The presence of two symmetrically positioned basic
chlorophenyl guanide groups attached to a lipophilic
hexamethylene chain (Figure 1) aid in rapid absorption
through the outer bacterial cell wall, causing irreversible
bacterial membrane injury, cytoplasmic leakage, and
enzyme inhibition [3].
This molecule exists as various forms of salts: diacetate,
dihydrochloride, or digluconate, mainly differing by their
solubilizing abilities in aqueous or oily media. CHD di-
gluconate (or gluconate), as most soluble in water or al-
cohol, is the most used form in topical dermatology or
cosmetic preparations. Aqueous solutions of CHD are
most stable within the pH range of 5-8. Above pH 8.0
CHD base is precipitated and in more acid conditions
there is gradual deterioration of activity because the
compound is less stable. Hydrolysis yields p-chloroan-
Cl
NN
NH
N
NH
N
NH
N N
NH
Cl
Cl
NH2
Figure 1. Chemical structure of CHD and pCA.

A. NICOLAY ET AL.423
iline (pCA); the amount is insignificant at room tempera-
ture, but is increased by heating above 100°C, especially
at alkaline pH [4]. CHD diacetate is soluble in alcohol,
glycerol, propylene glycol, polyethylene glycols. Ac-
cording to the manufacturer, 1 part CHD is soluble in 15
parts of 96% ethanol.
This cationic molecule (positively charged species) is
thus generally compatible with other cationic materials,
although compatibility will depend on the nature and
relative concentration of the second cationic species. It is,
however, possible for a reaction to occur between CHD
and the counter-ion (anion) of a cationic molecule which
is negatively charged, resulting in the formation of a less
soluble CHD salt, which then may precipitate. CHD is
incompatible with inorganic anions in all but extremely
dilute solutions. CHD is also incompatible with organic
anions, such as soaps, sodium lauryl sulphate, sodium
carboxymethyl cellulose, alginates, and many pharma-
ceutical dyes. In certain instances, there will be no visi-
ble signs of incompatibility, but the antimicrobial activ-
ity may be significantly reduced because of the CHD
being incorporated into micelles (ionic clusters) [5].
pCA is very toxic if inhaled, swallowed or absorbed
through the skin. It may act as a human carcinogen. It is
readily absorbed through the skin and it may act as a
sensitizer. The lethal dose 50 percent kill is 310 mg·kg–1
and 100 mg·kg–1 for rat and mouse respectively.
Liquid chromatography is the most widely used me-
thod for analysis of CHD [6]; UV detection around 250 nm
is used for quantitative assays [7-9], while for the detec-
tion of impurities, mass spectrometry or photodiode array
detectors are employed [10,11]. Other methods reported
in the literature include fluorometry [12] and direct UV
spectroscopy [13]; both of them have several disadvan-
tages: lack of sensitivity, serious interference.
There are many reports about the determination of
CHD in biological fluids using high performance liquid
chromatography with a UV detector (HPLC-UV) [8,9,
14-18] and gas chromatography mass spectrometry (GC-
-MS) [19-23].
Column used are generally C18 [11,14,15,24], Amide
[11] or phenyl [25]. Furthermore, reversed-phase ion-
-pair system is frequently used.
However, as pCA is the principal product degradation,
and because of his toxicity and to be in line with actual
recommendation for genotoxic impurities [26], it is im-
portant to quantify pCA in CHD solution. Therefore, we
describe hereafter a new very sensible HPLC-UV method
without the need of additional mobile-phase amine modi-
fiers or ion-pairing reagents, with good resolution be-
tween pCA and CHD, symmetry peak of the compo- unds
and short run time. To prove the reliability of this method,
CHD and pCA were analyzed in medical products.
2. Materials and Methods
2.1. Chemicals and Reagents
CHD gluconate solution (20%, with a density of 1.06)
was purchased from Sigma (St Quentin-Fallavier, France)
and stored at 4˚C in the dark. Methanol and formic acid
was of analytical grade from Carlo Erba (Val de Reuil,
France). Other chemicals used were of analytical grade.
Seven pharmaceutical specialities: Prexidine 0.12%®
mouthrinses (Expanscience, France), Septisol® eye drops
(Merck, France), Correctol 0.1%® eye drops (Alcon,
France), Sophtal® eye drops (Alcon, France), Septeal®
for topical application (Pierre Fabre Dermatologie,
France), Chlorhexidine Aqueuse Gilbert à 0.05%® for
topical application (Gilbert, France) and Dacrine® eye
drops (McNeil, France) were purchased in pharmacy.
CHD is used as active substance except in Septisol®,
Correctol 0.1%® and Sophtal® where it is used as ex-
cipient.
2.2. Apparatus and Chromatographic
Conditions
HPLC-UV analyses were performed using a Dionex®
Summit liquid chromatograph (Dionex Corp, Sunnyvale,
CA, USA), equipped with a vacuum solvent degassing
unit, a binary high pressure gradient pump P680, an
ASI100 automated sample injector, an UVD340U vari-
able wavelength UV–VIS detector.
Chromatographic separations were carried out on a
Luna® 150 mm × 3 mm i.d. column packed with 3 µm
CN (cyano) particles (Phenomenex®), guarded by an
on-line filter. Data were collected and analyzed using the
Chromeleon® software v6.50SP4 from Dionex®.
Since it is known that amines tend to form stable salts
with acetic acid, ammonium acetate and phosphate salts,
the mobile phases for chlorhexidine quantitative assay
consisting of methanol:water (55:45, v/v) with sodium
chloride with 0.02% of formic acid (pH = 3) and pumped
at a flow rate of 0.5 ml/min.
Wavelengths for pCA and for CHD are 238 and 255 nm
respectively.
2.3. Sample Preparation
Pharmaceutical specialities were directly injected (20 µl)
into the chromatograph system (without any extraction)
after or not dilution.
2.4. Mobile Phase
Published HPLC assays also suffer from quantitation

A. NICOLAY ET AL. 424
problems caused by irreversible adsorption of CHD onto
the silica-based reversed phase (RP)-HPLC columns [27,
28]. These problems can be overcome by adding NaCl to
the mobile phase [28] and explain the presence of this
compound in our mobile phase. The effect of NaCl can
be explained by the two simultaneously occurring pro-
cesses: the salting out effect, and the electrostatic inter-
actions between polar molecules and salt ions in sample
solution [29].
Chlorhexidine is a dicationic compound with Ka1 =
6.3 × 10−3 and Ka2 = 5.0 × 10−11 and is almost com-
pletely ionized (1+) at pH = 3, pH of our mobile phase.
Because chlorhexidine and some of its degradation
products are strongly basic, they are intensely retained on
most silica-based reversed phase columns [30]; the dif-
ficulty was overcome by using a cyano column and an
acid mobile phase (pH = 3), without the need to use
ion-pairing reagents in the mobile phase. It is known that
chlorhexidine is quite stable at this low pH (some HPLC
methods use pH = 2.5) [9].
2.5. Statistics
Results are presented as the mean  SD and results were
analyzed using Excel® 10, WinStat® v2003.1 and Chr-
omeleon®. A p value < 0.05 was considered as signifi-
cant.
2.6. Validation Criteria
2.6.1 Linearity
Working standards were prepared by dilution in water
and taking account of density for CHD gluconate. Height
reference samples were used for calibration curves of
CHD (0.53 to 21.20 mg/l) and pCA (0.01 to 10.0 ng/ml).
Each determination was done in triplicate. The calibra-
tion factors were calculated according to least-squares
linear regression .
2.6.2 Precision and Accuracy
Precision was determined for both inter- and intra-day
variability. These measurements were made by HPLC
analysis of CHD (5.3 and 12.72 mg/l) and pCA (0.50,
and 2.0 mg/l), on three consecutive days (inter-day varia-
tion or reproducibility) or during the same day for intra-
day variation determination (repeatability).
2.6.3. Limit of Detection (LOD) and Limit of
Quantification (LOQ)
Standard curves were prepared for the two analytes and
the following equation were used to calculate the LOD
and the LOQ for each compound [31]:
LOD = 3.3σ / S; LOQ = 10σ / S
where σ is the standard deviation of the response (esti-
mated from the standard deviation of y-intercepts of re-
gression lines) and S is the slope of the standard curve.
At the LOQ, bias and precision should not exceed
12% [32].
3. Results
To optimise the mobile phase, the influence of concen-
tration of methanol and sodium chloride was first stud-
ied.
4. Discussion
Based on the results presented in Figures 2 and 3 and in
Table 1, the concentration of methanol in the mobile
phase was chosen as 55% in order to have a short run
time (less than 5 minutes) and a good resolution (about
4.1) (Figure 4) between the two components.
There is few influence of sodium chloride concentra-
tion on retention time and peak asymmetry of pCA and
CHD. It is noted that asymmetry for CHD (1.7) and for
pCA (1.3) is very good with this cyano column from
Phenomenex® (Table 2). However, height of CHD de-
crease, when concentration of sodium chloride increase,
while pCA height peak increase too. A concentration of
12‰ of sodium chloride was chosen, the goal is to avoid
an important decrease of CHD and to increase the sensi-
bility of pCA detection, especially because of its toxicity.
Linearity of CHD is very good, from 0.5 up to 21.2 µg/l
(Table 3). This range is in the same order of that de-
scribes by Hebert et al. [7], but with a run time of 22
minutes. Some authors [9,11,14,30] present better sensi-
bility for CHD (LOD is 0.05 mg/l) quantitation but they
don’t quantify pCA. But for Usui [33] which works with
LC/MS, it was difficult to estimate the reliable values at
the concentration range below 0.1 mg/l. Only Below and
Kramer [16] present a dosage method for both pCA and
CHD, with a run time of 20 minutes.
Linearity of pCA is very good (range from 0.05 to
10 mg/l), for a product known to be difficult to be ana-
lysed [9,30] and with LOD and LOQ very low.
This method has been applied to a CHD solution in-
cluded into pharmaceutical specialities (Table 4) (Figure
5). In conclusion, we present a validated method for dos-
age of CHD and its major impurity pCA. No interference
has been reported, and the analysis of direct injection of
solution, without any treatment except an eventually dilu-
tion is achieved in less than five minutes. In all cases,
pCA was less than LOD, and CHD is in the range gener-
ally accepted in pharmaceutical products (95% -

A. NICOLAY ET AL.425
0
1
2
3
4
5
6
30 40 50 60 70 80 90100
k'
% of m eth an ol in mob il e p h as e
pCA
CHD
Figure 2. Influence of the methanol content in the eluant (methanol: 8‰ of sodium solution with 0.02% of formic acid (pH =
3) on the capacity factor of CHD and pCA (Luna CN, 150 × 3.0 mm, 3 µm, 0.5 ml/min); k' (capacity factor) for  pPC and 
CHD.
0.95
0.96
0.97
0.98
0.99
1.00
1.01
1.02
1.03
1.04
468101214161
heig ht normalize d
% of NaCl in mobile phase
8
CHD
pCA
Figure 3. Influence of the sodium chloride content in the eluant (55:45 of methanol: sodium solution with 0.02% of formic
acid (pH = 3) on the variation of height (± SD) normalized by mean of  pPC and  CHD (Luna CN, 150 × 3.0 mm, 3 µm,
0.5 ml/min).

A. NICOLAY ET AL. 426
-10
0
10
20
30
40
50
00.511.522.533.544.55
time (min)
Va lue(mAU)
pCA
CHD
Figure 4. Typical chromatogram of a solution with pCA (1 mg/l) and CHD (5 mg/l).
-20
30
80
130
180
00.511.522.533.544.55
Time (min)
Valu e (m AU)
CHD
Excipients
pCA

Figure 5. Chromatogram of sophtal solution, directly injected. The two first peaks are excipients. pCA is not detected.
Table 1. Intra-day and inter-days precision and accuracy.
Theoretical concentrations intra-day precision (n = 9)inter-day precision (n = 3)
(3 days) RSD
(µg/l) Concentrations found (%)
Substance
(mean  S.D.)
Accuracy (%)
0.50 0.511 ± 0.023 0.90 97.8
pCA 2.0 2.022 ± 0.084 4.16 98.9
5.30 5.203 ± 0.152 2.92 101.9
CHD 12.72 12.90 ± 0.14 0.56 98.6

A. NICOLAY ET AL. 427
Table 2. Influence of sodium chloride c ontent in the eluant (55:45 of methanol:sodium chloride solution with 0.02% of formic
acid (pH = 3)) on the variation (expressed as mean ± standard deviation for a triplicate injection) of retention time (TR) in
minutes, height (H) in mAU and asymmetry (As) of pCA and CHD.
TR CHD H CHD As CHD TR pCA H pCA As pCA
‰ NaCl m SD m SD m SD m SD m SD m SD
6 3.54 0.00 533.6 3.38 1.69 0.01 2.57 0.00 86.8 0.10 1.38 0.06
8 3.48 0.00 525.4 1.96 1.69 0.01 2.56 0.02 89.6 0.25 1.37 0.01
10 3.48 0.00 520.2 0.64 1.70 0.01 2.55 0.00 91.2 0.00 1.34 0.01
12 3.47 0.00 516.7 0.96 1.69 0.01 2.54 0.00 92.1 0.38 1.32 0.01
16 3.48 0.00 506.7 0.80 1.66 0.01 2.53 0.01 93.2 0.25 1.31 0.00
Table 3. Linearity, limits of detection and quatification of standard curvese.
Substance Linear range (mg/l) Calibration equation
(y = area, x = mg/l) Correlation coefficients (r)LOD (mg/l) LOQ (mg/l)
pCA 0.05 – 10 y = 5.5237 x – 0.1299 0.99996 0.0035 0.0105
CHD 0.53 – 21.2 y = 1.6084 x + 1.2123 0.99932 0.15 0.50
Table 4. CHD amounts in pharmaceutical specialities.
CHD theoretical
concentration (mg/l) CHD observed concentration (mg/l) Recovery (%)
Prexidine 0.12% 1200 1174 97.8
Septisol 30* 30.5 101.7
Correctol 0.1% 30* 31.3 104.3
Sophtal 30* 30 100.0
Septeal 0.5% 5000 4707 94.1
Dacrine 100 95.3 95.3
Chlorhexidine Gilbert 0.05%500 517 103.4
* amounts of CHD generally used but not specified when CHD is used as excipients.
105%).
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