Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 44) doc

5.25.2
© Springer-Verlag Berlin Heidelberg 2005
II.5.2 Benzalkonium chlorides
by Kazuhiro Koyama and Yoko Shimazu
Introduction
Surfactants can be classi ed into cationic, anionic and nonionic ones. Benzalkonium chlorides
are cationic surfectants and being widely used as a disinfectant and germicide using their
strong protein-denaturing action. Especially in hospitals, 10 % solution of benzalkonium chlo-
ride mixture is being usually used; it is diluted to 0.05–0.1 % solution to be used for various
types of disinfection.  e drugs are contained in gargles and preservative solutions for contact
lenses.
Fatal benzalkonium chloride poisoning cases, in which the 10 % solution had been ingested,
were reported [1, 2]; it can be regarded as one of the most important poisons in daily necessities.
As poisoning symptoms, a pungent sense of the oral mucous membranes, sore throat, cyanosis,
convulsion and coma can be mentioned [3, 4].
Benzalkonium chlorides are the mixture of multiple analogous compounds. To measure
the concentration, it is necessary to combine concentrations of each component.  e quater-
nary ammonium salt benzalkonium chlorides can be expressed as [C
6
H
5
CH
2
N(CH
3
)
2
R]Cl,
where R is the mixture of C
8
H

17
-C
18
H
37
. In most cases, the major R’s are C
12
H
25
(C
12
), C
14
H
29
(C
14
)
and C
16
H
33
(C
16
); especially according to the National Formulary XVI, C
12
, C
14
and C
12

+ C
14

count more than 40, 20 and 70 %, respectively [5].  erefore, upon analysis of benzalkonium
chlorides, it seems necessary to measure at least the C
12
and C
14
compounds (> Figure 2.1).
Structures of main components of benzalkonium chlorides. Benzalkonium chlorides are a mix ture
of quaternary ammonium salts showing the structures of [C
6
H
5
CH
2
N(CH
3
)
2
R]Cl, where R is a
mixture of C
8
H
17
–C
18
H
37
. However, main structures of R are C

12
H
25
(C
12
), C
14
H
29
(C
14
) and C
16
H
33
(C
16
);
C
12
м 40%, C
14
м 20 %, and C
12
plus C
14
м 70 % [5].
⊡ Figure 2.1
408 Benzalkonium chlorides
In this chapter, a method for wide-bore capillary GC/MS analysis of benzalkonium chlo-

rides is described and an HPLC method is also mentioned brie y.
GC/MS analysis
Reagents and their preparation
• A 1-mg aliquot each of benzyldimethyldodecylammonium (C
12
) bromide, benzyldimethyl-
tetradecylammonium (C
14
) chloride and benzyldimethylhexadecylammonium (C
16
) chlo-
ride (all from Sigma, St. Louis, MO, USA) is dissolved in 100 mL methanol in a 100-mL
volmetric  ask to prepare 10 µg/mL solution separately.

• A 4.3-g aliquot of NaOH is dissolved in distilled water to prepare 100 mL solution (1 M
NaOH) in a 100-mL volmetric  ask.

• A 1-mg aliquot of promazine hydrochloride (Sigma) is dissolved in distilled water to pre-
pare 100 mL solution (10 µg/mL promazine, internal standard IS) in a 100-mL volmetric
 ask.
GC/MS conditions
GC column: SGE BPX35
a
(30 m × 0.53 mm i. d.,  lm thickness 1.0 µm, Shimadzu GLC Center,
Tokyo, Japan).
GC/MS conditions; instrument: GCMS-QP 5050 Ancw (Shimadzu Corp., Kyoto, Japan);
column (oven) temperature: 100 °C (1.5 min) → 7 °C/min → 200 °C → 5 °C/min → 330 °C
(7 min); interface temperature: 280 °C; injection: PTV
b
splitless high pressure injection

c
, initial
injection temperature 50 °C (0.01 min) → 250 °C/min → 330 °C (10 min); initial pressure of
injection: 200 kPa, 1.5 min; injection volume: 10 µL; split ratio: 10 ; sampling time: 1.9 min;
column  ow rate: 15 mL/min; MS ionization: EI; scan range (
> Figure 2.2): m/z 50–509; scan
interval: 0.5 s [6].
Total ion chromatogram (TIC) for the authentic benzalkonium chlorides. C
12
: benzyldimethyl-
dodecylammonium; C
14
: benzyldimethyltetradecylammonium; C
16
: benzyldimethylhexadecyl-
ammonium.
⊡ Figure 2.2
409
Procedures
i. Liquid-liquid extraction
i. A 50-µL volume each of C
12
, C
14
and C
16
methanolic solutions is placed in a small test tube,
and evaporated to dryness under a stream of nitrogen with warming at 50 °C. To the com-
bined residue, 500 µL blank serum is added and mixed well to prepare 1 µg/mL standard
solution

d
for C
12
, C
14
and C
16
compounds of benzalkonium chlorides.
ii. A 500-µL volume each of specimens (serum, gastric juice or urine) and a 500-µL volume
of the above standard solution are placed in separate test tubes, followed by the addition of
2 mL ethyl acetate, 500 µL distilled water, 50 µL of 1 M NaOH solution and 50 µL IS solu-
tion, respectively.
iii. A er voltex-mixing (or shaking) for 3 min, each tube is centrifuged at 3,000 rpm for 5 min.
 e upper organic phase is transferred to a vial.
iv.  e phase is evaporated to dryness under a stream of nitrogen with warming at 50 °C.
v.  e residue is dissolved in 100 µL ethyl acetate; a 10-µL aliquot is injected into GC/MS.
vi.  e measurements are made in the scan mode (
> Figure 2.3); quantitation is made by ion
chromatography using ions at m/z 134 for C
12
, C
14
and C
16
compounds of benzalkonium
chlorides and at m/z 58 for promazine (IS).
Mass spectra of benzalkonium chlorides.
⊡ Figure 2.3
GC/MS analysis
410 Benzalkonium chlorides

ii. Extraction with Extrelut
®
i.  e standard solutions
d
for the C
12
, C
14
and C
16
compounds of benzalkonium chlorides are
prepared according to the above i) step of the liquid-liquid extraction.
ii. A 500-µL volume each of specimens (serum, gastric juice or urine) and a 500-µL volume
of the above standard solution are placed in separate test tubes, followed by the addition of
500 µL distilled water, 50 µL of 1 M NaOH solution and 50 µL IS solution, respectively.
iii. Each mixture is poured into Extrelut NTI
®
(Merck, Darmstadt, Germany) and le for
20 min.  e target compounds are eluted with 4 mL ethyl acetate.
iv.  e eluate is evaporated to dryness under a stream of nitrogen with warming at 50 °C and
reconstituted in 100 µL ethyl acetate.
v. A er centrifugation
e
at 3,500 rpm for 5 min, a 10-µL aliquot of the supernatant fraction is
injected into GC/MS.
vi.  e GC/MS detection method is the same as described at the step vi. of the above liquid-
liquid extraction.
Assessment of the method
 e detection limits by this method required for library research of a compound in the scan
mode were 1–2 µg/mL; those for quantitation by mass chromatography were about 10 ng/mL

(
> Figure 2.4). If higher sensitivity is required, the measurements in the SIM mode become
necessary. In the present analytical system, the sensitivity is being increased by large-volume
injection
f
using a wide-bore capillary column and the PTV sample injection device; but usual
capillary GC/MS can be used without great di erences, though the sensitivity becomes lower
to some extent.  e lower sensitivity can be overcome by using the SIM mode and/or de-
rivatization
g
of the analytes.
In this section, both liquid-liquid and Extrelut extractions are described. According to the
experience of the authors, the Extrelut extraction is superior to the liquid-liquid extraction in
view of stability and reproducibility.
TIC and mass chromatograms for an extract of plasma, into which benzalkonium chlorides had
been spiked. C
12
: benzyldimethyldodecyl-ammonium; C
14
: benzyldimethyltetradecylammonium;
C
16
: benzyldimethyl-hexadecylammonium. The concentration of each compound spiked was
1 µg/mL; GC/MS analysis was made after liquid-liquid extraction.
⊡ Figure 2.4
411
HPLC analysis
Benzalkoniums in plasma can be extracted by solid-phase extraction with a C
18
column, and

analyzed by HPLC using a CN separation column, acetonitrile/propionate bu er solution
(0.161 M, pH 5.4) (90:10) as a mobile phase and a UV detector [7]. In another method [8],
dimethyldodecylammonium chloride (DDMAC) was used as IS; benzalkoniums and IS were
analyzed by HPLC using a CN-NH
2
column, chloroform/methanol (80:20) as a mobile phase
and a post-column  uorescence detector with 9,10-dimethoxy-2-anthracene sulfonate (DMAS)
as a  uorogenic reagent with excitation at 383 nm and emission at 459 nm.
Poisoning case, and toxic and fatal concentrations
Poisoning case
A 22-year-old male attempted suicide by taking 20–100 mL of 10 % benzalkonium chloride
solution, which had been mixed with co ee, by eating a quarter of a cigarette and then by slit-
ting his le wrist (about 4 cm long injury). His consciousness was clear and he walked to an
emergency room of a hospital by himself.
Upon admission to the hospital, his physical  ndings were: blood pressure 120/60 mmHg;
heart beat rate 94/min; body temperature 36.4 °C; respiration rate 16/min; consciousness, clear
(O/JCS, E4V5M6/GCS); light reaction, positive; pupils, equally 3.5 mm; wound, cut injury in
the le wrist of about 4 cm length; X-ray photographs, no  nding in the chest and intestinal gas
observable in the abdomen.
A er admission, gastrolavage and administration of activated charcoal and Ni ec
®
(a mix-
ture solution of NaCl, KCl, NaHCO
3
and Na
2
SO
4
) were made. During his admission, no poi-
soning symptoms due to benzalkonium chlorides could be found; he was discharged on the

next day.
Serum concentrations upon admission were: benzalkonium chlorides, 52 ng/mL in total
(C
12
: 52 ng/mL, C
14
: < 10 ng/mL, C
16
: < 10 ng/mL); nicotine
h
: < 10 ng/mL. However, the con-
centrations in gastric juice were: benzalkonium chlorides 54.4 µg/mL (C
12
: 50.7 µg/mL, C
14
:
3.6 µg/mL, C
16
: 0.06 µg/mL); nicotine
h
5.75 µg/mL.
Toxic and fatal concentrations
In fatal benzalkonium chloride poisoning cases, solution products, containing not less than
10 % benzalkonium chlorides, were ingested. Human oral fatal doses are 100–400 mg/kg. In
cases of non-oral administrations (i.v. and i.m. injections or intrauterine injection) its toxicity
is much enhanced; the fatal doses were reported to be 5–15 mg/kg [1, 2].
However, the number of reports describing blood concentrations of benzalkoniums in poi-
soning cases are very limited. Hitosugi et al. [2] reported a fatal poisoning case, in which a
84-year-old female had taken 50 mL (96.2 mg/kg) of 10 % benzalkonium chloride solution and
died 3 h later; her serum benzalkonium chloride concentration was reported to be 1.15 mg/

mL. In the above survived case described in the (1) section, the serum benzalkonium chloride
concentration upon admission was 52 ng/mL, which is much lower than that in the fatal case.
Poisoning case, and toxic and fatal concentrations