Journal of Chromatography, 463 (1989) 188-191
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
CHROM. 21 025
Note
Analysis of benzalkonium chlorides by gas chromatography
SUKEJI SUZUKI*, YOSHIAKI NAKAMURA, MASAMI KANEKO, KEN’ICHIRO MORI and
YOHYA WATANABE
Tokyo Metropolitan Research Laboratory of Public Health, 3-24-1, Hyakunincho, Shinjuku-ku, Tokyo 169
(Japan)
(First received June 4th, 1988; revised manuscript received October 4th, 1988)
Benzalkonium chlorides are widely used as an antimicrobial preservative in
pharmaceutical preparations and sanitary products, and are a mixture of pre-
dominantly C I 2, Cl4 and Cl6 alkylbenzyldimethylammonium chlorides.
Various analytical methods have been reported for the determination of
benzalkonium chlorides by high-performance liquid chromatography (HPLC)ip5 and
gas chromatography (GC) 6- I6 . HPLC analysis is available but difficult to use in
combination with mass spectrometry in our laboratory. We wished to establish a GC
method for the routine analysis of benzalkonium chlorides since their identification
can be facilitated using GC-mass spectrometry (MS). These compounds are non-
volatile and thus need to be converted into more volatile derivatives before GC”-’ 3.
The Hofmann degradation for ammonium compounds is one such conversion
method’“16. Tanakano et ~1.‘~ reported that a mixture of alkyldimethylamines,
alkylbenzylmethylamines and a-olefins is obtained from benzalkonium chlorides by
Hofmann degradation with sodium methoxide, but this approach requires too severe
reaction conditions to decompose them.
In previous papers17,‘*, we have reported that long-chain alkyltrimethyl- and
dialkyldimethylammonium compounds can be analyzed by GC as the corresponding
a-oletins and alkyldimethylamines which are formed by the Hofmann degradation
with potassium tert.-butoxide under mild conditions. In the present work, a GC
method for benzalkonium chlorides employing this degradation technique has been
developed.
EXPERIMENTAL
Dodecylbenzyldimethylammonium chloride (I) was obtained from Kao Soap
(Tokyo, Japan), tetradecylbenzyldimethylammonium chloride (II) and hexadecyl-
benzyldimethylammonium chloride (III) from Tokyo Kasei Kogio (Tokyo, Japan)
and octadecylbenzyldimethylammonium chloride (IV) from Aldrich (Milwaukee, WI,
U.S.A.). Standard solutions were prepared so as to contain 1 mg/ml of each compound
in benzene-dimethyl sulphoxide (DMSO) (8:2). All other chemicals were of analytical
grade.
0021-9673/89/$03.50 0 1989 Eisevier Science Publishers B.V.
NOTES 189
Degradation procedure
An aliquot of the standard solution containing 2-10 mg of compounds I-IV was
pipetted into a 25-ml Pyrex glass test-tube with a ground glass stopper. The total
volume was made up to 25 ml with benzene-DMSO (8:2). After the addition of 100 mg
of potassium tert.-butoxide, the solution was vigorously shaken and allowed to stand
for 10 min at room temperature. The reaction mixture was transferred to a IOO-ml
separating funnel. The test-tube was rinsed with two 5-ml portions of benzene. The
rinsing solutions were added to the reaction mixture in a separating funnel and then
shaken with 20 ml of 10% sodium chloride solution. The benzene layer was separated
and transferred to a lOO-ml round-bottom flask. After the solvent had been evaporated
to dryness under reduced pressure, the residue was dissolved in 5 ml of acetone and 5 ~1
of this solution were injected into the gas chromatograph.
Gas chromatography
GC analysis was carried out with a Shimadzu Model GC-15A gas chroma-
tograph equipped with an hydrogen flame ionization detector. Chromatography was
performed on a glass column (2 m x 3 mm I.D.) packed with 5% SE-30 on
Chromosorb W AW DMCS (So-100 mesh) under the following conditions: nitrogen
flow-rate 40 ml/min; injection port and detector temperature 250°C; column
temperature program from 140 to 230°C at S”C/min.
RESULTS AND DISCUSSION
Benzalkonium chlorides shown in Fig. 1 were degraded with potassium
tert.-butoxide by essentially the same manner as described in previous papers7T8.
However, the formation of cl-olefins and alkyldimethylamines as degradation products
was found to vary with the reaction temperature. In searching for an optimum
condition for alkyldimethylamine formation the effect of the reaction temperature for
compounds I-IV was examined. As shown in Fig. 2, alkyldimethylamine formation
was found to be accompanied by a decrease in that of a-olefin at temperatures lower
than 40°C. These results indicate that the debenzylation of benzalkonium chlorides
with potassium tert.-butoxide proceeds selectively at lower temperature to give
akyldimethylamine, and on the other hand the p-elimination reaction at the
nitrogen-bonded alkyl chain predominates at temperatures higher than 80°C to give
> Cy(Cy) ” _3CH=CH
2
I : n=12
II : n=14 o(-olefin
III : n=I6
IV : n=18
Fig. 1. Degradation scheme of benzalkonium chlorides with potassium tert.-butoxide in benzene-DMSO
(8:2).
190 NOTES
8 B-
(B)
rz 6 6-
s
E 4 4-
%
B
2 2-
M:: l.f I I I
0 20 40 60 80 100 0 20 40 60 80 Id0
ReactiontemPerawe Reactlontemperature(Y)
Fig. 2. Effects of reaction temperature on the formation of alkyldimethylamines (A) and a-oletins (B) from
the four benzalkonium chlorides, compounds I (0). II (x ), III (A) and IV (W).
a-olelin (Fig. I). Thus, room temperature was selected for the GC analysis of
benzalkonium chlorides as their alkyldimethylamines. The reaction was rapid under
mild conditions in comparison with debenzylation in the previously reported
conversion methods’i-i6 and was complete within 10 min at room temperature.
A typical gas chromatogram of compounds I-IV as their alkyldimethylamines
on a 5% SE-30 column is shown in Fig. 3. Calibration graphs were constructed by
plotting the peak heights of the alkyldimethylamines veTSuSthe concentrations of
compounds I-IV. They showed good linearity over the concentration range 0.4-2
mg/ml. The minimum detectable amounts for compounds I-IV were found to be about
0.1 mg/ml.
For practical application e.g., to a wet wiper containing benzalkonium chlorides,
recovery tests were performed by adding 4 and 8 mg of compounds I-IV to an
absorbent cotton (Table I). The cotton was extracted with ethanol under reflux for 60
min. The ethanol extract was filtered and evaporated to dryness. The analysis of
IV
i
I I I I I
0 5 10 15 20
Time(mln)
Fig. 3. Temperature-programmed gas chromatogram of the four benzalkonium chlorides as their
alkyldimethylamines after degradation with potassium tert.-butoxide at room temperature, compounds
I-IV: 6 mg.
NOTES 191
TABLE I
RECOVERIES OF BENZALKONIUM CHLORIDES FROM ABSORBENT COTTON
Compound* Amount added Average recovery (n = 3) c. v.
lmgl (%) l”/o)
I 4 98.3 3.9
8 103.4 1.9
II 4 104.2 1.4
8 102.1 1.4
111 4 96.7 1.5
8 92.4 3.3
IV 4 98.3 2.9
8 96.3 4.5
l Benzalkonium chlorides (I-IV) were dissolved in 10 ml of water, added to 1.7g of absorbent cotton
(The Pharmacopoeia of Japan, 1Ith ed.) and autoclaved at 120°C for 30 min.
compounds I-IV was performed by the method described. As shown in Table I, the
average recoveries in three experiments were 92.4104.2% and a good reproducibility
was obtained with coefficients of variation (C.V.) of l&4.5%.
We have applied this method to the determination of benzalkonium chlorides in
36 samples of commercial wet wipers. These were found to contain 0.06-0.28% in 10
samples as a mixture of Ci2, Cl4 and Cl6 alkylbenzyldimethylammonium chlorides.
These experiments have demonstrated that the method proposed can be applied
to routine analysis of benzalkonium chlorides.
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