HPLC analysis of some bacteriostats in deodorant

J. Soc. Cosmet. Chem., 32, 163-173 (May/June 1981)
HPLC analysis of some bacteriostats in deodorant
sticks and soaps
RAJA G. ACHARI and DAVID CHIN, Research & Development
Center, Bristol- Myers Products Division, 225 Long Avenue, Hillside,
NJ 07207.
Received February 5, 1981.
Synopsis
A stability-indicating HPLC method for the determination of some bacteriostats such as Triclosan
(2,4,4'-trichloro-2'-hydroxydiphenylether) and TCC (3,4,4'-trichlorocarbanilide) is described. The liquid
chromatographic separation is carried out using a •Bondapak Alkylphenyl column and the mobile phase
consisting of 1:1 (V/V) acetonitrile:water. The method had been documented to be precise and accurate
and has been successfully applied in assaying commercially available deodorant sticks and soap samples.
INTRODUCTION
Triclosan (I) (Irgasan DP-300 ©, 2,4,4'-trichloro-2'-hydroxydiphenylether) (Ciba-Geigy,
Greensboro, NC) is a commonly used bacteriostat in deodorant sticks and soaps. TCC
(II) (Monsanto, St. Louis, MO) (3,4,4'-trichlorocarbanilide) is mainly used in deodorant
soaps. Various methods have been reported for the analysis of these bacteriostats in
deodorants, but all of these methods suffer from certain drawbacks. Perfumes, other
UV absorbing substances, or chemical breakdown products of the bacteriostats often
interfere with ultraviolet and colorimetric methods (1,2). The gas chromatographic
procedure of Demars and Yates (3) for the analysis of TCC is cumbersome and
non-specific because the amines which are ultimately analyzed are also the probable
chemical degradation products of TCC. One of the two reported HPLC procedures
requires a gradient elution system (4) and the second requires a radial compression
separation system (5). Both of the above systems are neither suitable nor available in all
laboratories for routine analysis; moreover, neither of the above HPLC methods has
been tested to determine whether it is "stability-indicating" due to the chemical
degradation of the bacteriostats in the finished products.
The present study is aimed at developing a stability-indicating simple isocratic HPLC
method for assaying triclosan in deodorant sticks and TCC in deodorant soaps.

Chromatographic parameters have been provided to assay both triclosan and TCC
should these be present in combination. The chemical structures of triclosan and TCC
are shown below.
163
164 jOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
O Cl
C1 O • Cl C1 NHCNH C1
Cl HO TCC
I II
METHOD
APPARATUS
A modular liquid chromatographic unit consisting of a constant flow solvent delivery
pump (Model 6000A, Waters Assoc., Milford, MA), a continuously variable UV-VIS
spectroflow monitor with a capability of programming to read absorbance of an eluate
at several wavelengths simultaneously, and of obtaining an absorbance spectrum
between desired wavelengths (Model LC-75, Auto Control, Perkin-Elmer Corp.,
Norwalk, CT), a digital recorder (Model 56, Perkin-Elmer Corp.) and an integration
device (Model SP-4000, Spectra-Physics, Santa Clara, CA) to calculate the area under
the curve of the eluates, was used for chromatography.
An alkylphenylsiloxane bonded column (•Bondapak Alkylphenyl, Waters Assoc.) was
used for the separation of the bacteriostats with a mobile phase containing 1:1 (V/V)
acetonitrile:water (HPLC grade, Baker Chemical Co., Phillipsburg, NJ). The bacterio-
stats were monitored at 280 nm.
STANDARD SOLUTIONS
Appropriate amounts of triclosan and TCC were dissolved in methanol (HPLC grade,
Baker Chemical Co.) and the appropriate aliquots of the solutions were further diluted
using the mobile phase or 1:1 (V/V) methanol:water to yield a concentration range of
25 •tg/ml-75 •tg/ml for triclosan, and 6 •tg/ml-24 •tg/ml for TCC, respectively.
SAMPLE PREPARATION
A. Deodorant Sticks

Approximately 1 g of the deodorant stick was accurately weighed into a 50-ml
volumetric flask. 5 ml of methanol was added and the deodorant was dissolved by
applying gentle heat on a hot plate. 25 ml of 1:1 (V/V) acetontrile:water was added.
The solution gelled. It was warmed until dissolved and diluted to volume with
additional 1:1 acetonitrile:water. The solution was cooled to room temperature and the
volume was adjusted to mark if needed. The solution was mixed thoroughly and
approximately 15 ml of the above solution was transferred into a stoppered centrifuge
tube and placed in a methanol-ice bath for 30 min. The solution was centrifuged while
cold to separate a clear supernatant from a small amount of gel which did not contain
any bacteriostat. The supernatant was transferred to a sample vial and this solution was
used for chromatography.
HPLC ANALYSIS OF BACTERIOSTATS 165
B. Deodorant Soaps
Approximately 1 g of soap was accurately weighed and transferred into a 50-ml
volumetric flask. 10 ml of methanol was added and the soap was dissolved with gentle
heating. The solution was diluted to volume with methanol. 5 ml of the methanolic
solution was transferred to a 100-ml flask and brought to volume with 1:1 (V/V)
methanol:water. The solution was thoroughly mixed and an appropriate amount of the
solution was transferred to a stoppered centrifuge tube. The tube was cooled in a
methanol-ice bath for 30 min and centrifuged while cold and the clear supernatant was
transferred into a sample vial. This solution was used for chromatography.
C. Chemical degradation of bacteriostats for "stability indicating" testing
Several chemical degradation studies of the individual bacteriostats and the deodorant
samples (containing bacteriostats) were carried out. The following tests were
performed: 1) effect of acid; 2) effect of alkali; 3) effect of oxidation; 4) effect of heat;
and 5) effect of UV irradiation.
Triclosan and TCC and the deodorants were dissolved in methanol and to the
methanolic solution acid (HC1), base (NaOH), oxidizing agent (Potassium monoper-
sulphate, Oxone ©) were added, respectively. An aliquot of each of these solutions was
transferred into an ampule and sealed. The ampules were heated at 40øC for 24-48 h.

For UV irradiation the methanolic solution was applied on a clear glass plate and the
solvent was evaporated in vacuum. The process was repeated several times to ensure
that sufficient amounts of the sample had been added onto the glass plate. One half
the glass plate was covered with aluminum foil to serve as a control and the other half
was exposed to UV light. This was accomplished by placing a mercury lamp (Model
//GE, G15T8) about 6 in above the glass plate. In addition, methanolic solutions of
triclosan and TCC in sealed ampules were also exposed to UV light.
QUANTITATION
The calibration standards were injected in duplicate and a regression analysis of the
drug concentration versus the area under the curve was carried out. The concentration
of the bacteriostat was extrapolated from the regression curve and the percent of the
bacteriostat was calculated in the following way:
C
x V x 100 = % Bacteriostat,
SIV
where C = extrapolated concentration of the bacteriostat in mg/ml,
Sir/= sample weight in mg,
V = sample volume.
IDENTIFICATION OF THE BACTERIOSTATS
Initial identification of the bacteriostats was carried out by comparing the retention of
the bacteriostats with those of the authentic reference standards. Further confirmation
166 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
of the identity and purity of the chromatographic peak was effected by the absorbance
ratio measurement technique which is accomplished in the following way. An
authentic bacteriostat standard is chromatographed and the flow of the mobile phase
is stopped approximately at the chromatographic peak maximum. The instrument is
programmed to measure the absorbances of the eluate at pre-selected wavelengths and
the ratios of the absorbances are calculated. Similarly, a sample is chromatographed
and the absorbance ratios of the analytical peak are determined. If the absorbance
ratios of the bacteriostat in the sample compare within _+ 10% with those of the

reference standard, the identity and the purity of the bacteriostat is confirmed.
RESULTS AND DISCUSSION
Under the chromatographic conditions used, triclosan and TCC showed the chroma-
tographic properties listed in Table I.
Table I
Chromatographic Properties of Triclosan and TCC •
Rentention time (sec.): 483 468
k': 6.1 5.9
H.E.T.P. (mm): 0.111 0.123
Tailing factor (%): 70 70
•Chromatographic conditions: •Bondapak alkyl phenyl column, 1:1 (V/V)Acetonitrile:water, mobile phase;
flow rate 2 ml/min., detection wavelength 280 nm. For calculation of tailing factor see Ref. (6).
Both triclosan and TCC were tested for their linear detection range. The data were
obtained using three calibration standards and each standard was injected in duplicate.
The results are summarized in Table II.
Table II
Linearity Data of Triclosan and TCC •
Amount Injected Slope Correlation
Compound range 0tg) (counts, ng) Intercept Coefficient
Triclosan 0.63- 1.89 467 +6600 0.99999
TCC 0.15 - 0.62 1,354 - 7800 0.99994
•Chromatographic conditions: As in Table I. For regression analysis, area under the curve was obtained in
electronic counts.
Chromatograms of a reference triclosan standard and that of a deodorant stick are
shown in Figure 1. The chromatogram clearly demonstrates the separation of triclosan
from perfumes and other ingredients. None of the deodorant sticks contained any
TCC. Similarly, chromatograms showing the separation of TCC are shown in Figure 2.
All of the commercial deodorant soaps except one analyzed in this study contained
only TCC as shown in the product label; however, both TCC and triclosan can be
separated using a modified solvent system as shown in Figure 3.

The precision and accuracy of the procedure were determined. The within-run and
between-run precisions of the chromatographic responses were determined by repli-
HPLC ANALYSIS OF BACTERIOSTATS 167
Inject
A
Triclosan
B
Triclosan
Figure 1. Chromatograms of (a) reference triclosan standard, and (b) that of a deodorant stick.
cate injections of reference standard solutions of triclosan and TCC in a single day,
and over a period of three to four days. Chromatographic response precision data are
shown in Table III. Data indicate that precision of the procedure is very good.
Accuracy of the procedure was tested by addition of known amounts of triclosan and
TCC to deodorant sticks and deodorant soaps, respectively. For this study triclosan
was added to a deodorant stick placebo sample, whereas TCC was added to a
deodorant soap sample which was previously assayed. The data are presented in Table
IV.
168 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Inject
A
•dTCC
Iniect
B
Figure 2. Chromatograms of (a) reference TCC standard, and (b) that of a deodorant soap.
The recovery data indicate that the accuracy of the •rocedure is excellent. The
precision of the assay procedure was further determined by analyzing a commercial
deodorant stick for triclosan and a deodorant soap for TCC. For five replicate assays
the coefficient of variation for triclosan was + 3% and that for TCC was 0.38%.
SPECIFICITY OF THE ASSAY PROCEDURE
Although it was established that the procedure is capable of discriminating the

bacteriostats from the inactive matrix, perfumes, etc., it was necessary to show that the
procedure has the capability of discriminating the bacteriostats from the degradation
products of the bacteriostats and from those of the perfumes or any other ingredient
present in the deodorants.
Table III
Reproducibility Data of Chromatographic Responses •
Within-Run (n = 6)
Between-Run (n = 4)
Retention time Area Retention Time Area
Triclosan 483 _+ 8.1 572,870 _+ 10,983 496 _+ 18 548,166 _+ 24,654
% CV 0.84 0.96 1.8 4.5
TCC 468 _+ 2.4 at 340,877 _+ 1,811 473 _+ 9.8 365,393 + 29,748
% CV 0.26 0.27 1.04 8.1
•Retention time and area are expressed in seconds and electronic counts, respectively. The amounts of triclosan
and TCC injected were 1.26 and 0.258/zg respectively.
HPLC ANALYSIS OF BACTERIOSTATS 169
Inject
TCC
Figure 3. Chromatogram of (a) deodorant soap containing TCC and triclosan. Chromatographic
conditions: mobile phase, 60:30:10 (V/V), methanol:water:ethylacetate; flow rate, 1 ml/min., other
conditions as in Table I.
For this purpose the bacteriostats and the deodorant samples containing bacteriostats
were artificially degraded as described in the experimental section, and the degraded
solutions were analyzed. In each case the identity and the purity of the bacteriostats
were confirmed by the absorbance ratio technique. Based on the UV spectra of the
170 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
HPLC ANALYSIS OF BACTERIOSTATS 171
Table IV
Recoveries of Triclosan and TCC from Spiked Samples •
Triclosan 2 TCC 3

Amount Added Amount Added
(/•g/injection) % Recovered (/•g/injection) % Recovered
1.007 100.6 0.155 100.2
1.26 100.3 0.206 99.9
1.51 100.4 0.258 99.9
•AII recovery data represent an average of three determinations. Range refers to recoveries from all
determinations.
2Range: 99.8-101.1.
3Range: 99.4-100.6.
bacteriostats in the HPLC mobile phase (Figure 4), the wavelengths selected for
absorbance ratio determination were 266, 288, 300 nm for triclosan and 240, 270, 280
nm for TCC, respectively. The results of the chemical degradation of TCC and
triclosan are described below:
TCC
No significant degradation was found upon heating of TCC. Some degradation
products were observed upon hydrolysis (acid and base) probably from p-chloroanil-
ine, 3, 4-dichloroaniline.
Oxidation by potassium monopersulfate showed some degradation products.
However, the identity of the degradation products has not been established here.
Photolysis produced several degradation products. The degradation products were not
identified in this study but previous studies on the photolysis of TCC in methanol have
indicated that a loss of chlorine from TCC occurs, confirmed by the identification of
chloride ion (7). This will probably result in yielding several dechlorinated carbanilide
products.
In all the degradation studies carried out here no interference was rendered to the
analytical peak by any degradation products as determined by the absorbance ratios
summarized in Table V.
Data shown in Table V clearly demonstrate that the present procedure is stability-
indicating for TCC. The analytical peak identified as TCC is pure and free from
interferences in samples that have been subjected to vigorous degradation conditions

of heat, acid and base hydrolysis, oxidation and photolysis. TCC degradation was
observed in the latter cases.
Triclosan
Similar degradation studies were carried out for triclosan and the deodorant sticks
containing triclosan. The absorbance ratios of the analytical peak were determined in
all cases. The absorbance ratios of a reference triclosan standard between 288/266 nm
and 288/300 nm were 3.17 and 2.78, respectively. No interference was observed in any
of these studies and the triclosan peak was found to be pure in all cases. The
absorbance ratios of the bacteriostat peak did not differ more than + 10% from those
of the reference standard in any of the degraded samples.
172 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Table V
Absorbance Ratios of Reference TCC and Those of the Analytical Peak in the
Degraded Solutions of TCC and Deodorant Soaps
Absorbance Ratio
Sample 270/240 (nm) 270/280 (nm)
TCC Ref. standard 6.28 1.55
Degraded samples
TCC (heat) 6.21 1.60
Soap 5.92 1.56
TCC (acid hydrolysis) 6.15 1.59
Soap 6.44 1.51
TCC (base hydrolysis) 6.13 1.58
Soap 6.18 1.56
TCC (oxidation) 6.03 1.55
Soap 6.31 1.55
TCC (photolysis) • 6.22 1.58
Soap 6.21 1.55
•Data from methanolic solution. The UV exposure to TCC on plates resulted in complete degradation of TCC
and hence no absorbance ratios of the analytical peak could be provided.

Three deodorant stick samples prepared in the laboratory (A, B & C) to contain 0.25%
triclosan and two samples obtained commercially (D & E) were analyzed. The results
are shown in Table VI.
Table VI
Assay of Triclosan in Deodorant Sticks (% W/W)
Product Triclosan
Deodrant Stick A 0.258
B 0.256
C 0.254
D 0.265
E 0.261
A number of deodorant soaps were analyzed for TCC. The results are summarized in
Table VII.
SUMMARY AND CONCLUSION
The procedure described above is precise, accurate and specific for both qualitative
and quantitative assay of triclosan and TCC. No interferences or any special difficulty
have been encountered for the assay of the above bacteriostats in the commercial
products we analyzed. However, it must be borne in mind that the deodorant samples
(stick or soaps) contain many lipophillic components, and over a period of time these
components tend to be deposited in the column. This could change both the nature
and the efficiency of the chromatographic column. Thus, one should monitor the
efficiency of the column periodically, and some form of clean up procedure should be
adopted to regenerate the column. Also, wide varieties of perfumes are used in these
products and these perfumes are composed of several components of different
HPLC ANALYSIS OF BACTERIOSTATS 173
Table VII
Assay of TCC in Deodorant Soaps (% W/W) 1
Name TCC
A 1.6
B 0.6

C 0.5
D 1.4
E 0.5
F 1.03
G 0.47
H 1.04
I 0.39
J 0.16
tall soaps were common brand names obtained from local supermarkets. Soap "C" also contained 0.14% W/W
of triclosan. Sample "F" was a laboratory prepared sample to contain 1.0% TCC.
hydrophobicity which tend to interfere with the analytical components. It is strongly
recommended that when a product is analyzed and the composition of such a product
is not fully known, absorbance ratio determination of the analytical peak should be
done to confirm both identity and the purity of the analytical peak; alternately, the
analytical peak should be collected and the 1st or 2nd derivative spectrum taken. In
many instances this procedure may help in finding interfering components in the
analytical peak. These spectra are shown in Figure 4. If any interference is noticed a
slight modification of the solvent system may alleviate the problem in most cases. In
certain circumstances a shoulder may be observed either in the leading or tailing edge
of the analytical peak. Quantitation by peak height method will prove more accurate in
these cases. In this report we have provided data on linearity, precision, etc., by peak
area only; however, we have tested the above properties by peak height method as well.
The peak height method was found to be as accurate and precise as the peak area
method.
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(3) F. X. Demers and R. L. Yates, Antimicrobials: Identification of 3,4,4'-trichlorocarbanilide and

4,4'-dichloro-3-(trifluoromethyl) carbanilide in deodorant bars,J. Soc. Cosmet. Chem., 28, 659 (1977).
(4) T. Wolf and D. Semionow, Rapid liquid chromatography of bacteriostats, J. Soc. Cosmet. Chem., 24,
363 (1973).
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reverse phase high pressure liquid chromatography,J. Amer. Oil Chem. $oc., 57, 131 (1980).
(6) T. R. Koziol, J. T. Jacob and R. G. Achari, Ion-pair liquid chromatographic assay of decongestants
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(7) Personal communication, Monsanto Chemical Co., St. Louis, Mo.