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 necessit
Trang 1© Springer-Verlag Berlin Heidelberg 2005
Introduction
Surfactants can be classifi 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 Th 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 Th e quater-nary ammonium salt benzalkonium chlorides can be expressed as [C6H5CH2N(CH3)2R]Cl, where R is the mixture of C8H17-C18H37 In most cases, the major R’s are C12H25(C12), C14H29(C14) and C16H33(C16); especially according to the National Formulary XVI, C12, C14 and C12 + C14
count more than 40, 20 and 70 %, respectively [5] Th erefore, upon analysis of benzalkonium chlorides, it seems necessary to measure at least the C12 and C14 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 40%, C 14 20 %, and C 12 plus C 14 70 % [5].
⊡ Figure 2.1
Trang 2408 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 briefl y
GC/MS analysis
Reagents and their preparation
• A 1-mg aliquot each of benzyldimethyldodecylammonium (C12) bromide, benzyldimethyl-tetradecylammonium (C14) chloride and benzyldimethylhexadecylammonium (C16) chlo-ride (all from Sigma, St Louis, MO, USA) is dissolved in 100 mL methanol in a 100-mL volmetric fl 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 fl 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
fl ask
GC/MS conditions
GC column: SGE BPX35 a (30 m × 0.53 mm i d., fi 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: PTVb splitless high pressure injectionc, 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 fl 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
Trang 3i Liquid-liquid extraction
i A 50-µL volume each of C12, C14 and C16 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 solutiond for C12, C 14 and C16 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 Aft er voltex-mixing (or shaking) for 3 min, each tube is centrifuged at 3,000 rpm for 5 min
Th e upper organic phase is transferred to a vial
iv Th e phase is evaporated to dryness under a stream of nitrogen with warming at 50 °C
v Th e residue is dissolved in 100 µL ethyl acetate; a 10-µL aliquot is injected into GC/MS
vi Th e measurements are made in the scan mode (> Figure 2.3); quantitation is made by ion
chromatography using ions at m/z 134 for C12, C14 and C16 compounds of benzalkonium
chlorides and at m/z 58 for promazine (IS).
Mass spectra of benzalkonium chlorides.
⊡ Figure 2.3
Trang 4410 Benzalkonium chlorides
ii Extraction with Extrelut®
i Th e standard solutionsd for the C12, C14 and C16 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 left for
20 min Th e target compounds are eluted with 4 mL ethyl acetate
iv Th e eluate is evaporated to dryness under a stream of nitrogen with warming at 50 °C and reconstituted in 100 µL ethyl acetate
v Aft er centrifugatione at 3,500 rpm for 5 min, a 10-µL aliquot of the supernatant fraction is injected into GC/MS
vi Th e GC/MS detection method is the same as described at the step vi of the above liquid-liquid extraction
Assessment of the method
Th 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 diff erences, though the sensitivity becomes lower
to some extent Th 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
Trang 5HPLC analysis
Benzalkoniums in plasma can be extracted by solid-phase extraction with a C18 column, and analyzed by HPLC using a CN separation column, acetonitrile/propionate buff 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-NH2 column, chloroform/methanol (80:20) as a mobile phase and a post-column fl uorescence detector with 9,10-dimethoxy-2-anthracene sulfonate (DMAS)
as a fl 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 coff ee, by eating a quarter of a cigarette and then by slit-ting his left 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 fi 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 left wrist of about 4 cm length; X-ray photographs, no fi nding in the chest and intestinal gas observable in the abdomen
Aft er admission, gastrolavage and administration of activated charcoal and Nifl ec® (a mix-ture solution of NaCl, KCl, NaHCO3 and Na2SO4) 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 (C12: 52 ng/mL, C14: < 10 ng/mL, C16: < 10 ng/mL); nicotine h: < 10 ng/mL However, the con-centrations in gastric juice were: benzalkonium chlorides 54.4 µg/mL (C12: 50.7 µg/mL, C14: 3.6 µg/mL, C16: 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 poipoi-soning 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
Trang 6412 Benzalkonium chlorides
Th e survived patient might have ingested only a small amount of coff ee containing the com-pounds; and/or the dilution of the benzalkonium solution with coff ee might have resulted in
no protein-denaturing action
Notes
a) Any other column of slightly or intermediately polar types seems usable In this chapter, a wide-bore capillary column was used Th e column has a large capacity for loading of spec-imens, and the leading phenomenon does not appear, even when a trace amount of a drug
in serum is measured, followed by the measurement of a large amount of the compound present in stomach contents However, the wide-bore capillary column shows lower resolu-tion (separaresolu-tion) ability Since this type of column has a large capacity; an analyst some-times injects a large volume of a specimen extract, resulting in the carry-over of many re-sidual or impurity peaks during subsequent measurements
b) In the usual split/splitless injection mode, a sample solution is injected into an insert at a high temperature to vaporize it immediately In contrast, the programed temperature vapo-rizing ( PTV) device enables the injection of a liquid sample without immediate evapora-tion; gradual elevation of its temperature for mild evaporation Th is is useful for more sensitive analysis of thermolabile compounds without heat decomposition to some extent [9] In this respect, the PTV device seems very useful in clinical analysis cases, in which many thermolabile compounds are dealt with Th e glass insert and silica wool to be used should be silanized to prevent drugs from their decomposition [10]
c) Th e high pressure injection is used for suppressing the broadening of a peak due to the large volume injection
d) In this method, semiquantitative measurements with one point standard are being used, because of too limited times available for analysis at a critical care medical center For accurate quantitation, a calibration curve made of not less than 3 plots at diff erent concen-trations becomes necessary
e) When the Extrelut NT1® is used for extraction, clogging of a pre-fi lter for HPLC usually takes place in a relatively early stage; the movement of a plunger of a syringe for autoinjec-tor of GC also becomes not smooth To prevent these phenomena, insoluble components should be removed by centrifugation
f) In this method, large volume injection is being used to enhance the sensitivity for enabling identifi cation of a trace amount of a drug being contained in a biomedical specimen Our system can also quantitate a drug at the levels of > 10 ng/mL Th e large volume injection together with PTV seems to be a powerful tool for analysis of samples obtained from drug-poisoned patients at a very busy critical care medical center, while it requires a time-con-suming labor for its maintenance; when a large number of samples are analyzed, the wash-ing of the ion source is necessary several times a week
g) Benzalkonium chlorides can be derivatized with 100 mg of potassium tert-butoxide in the
presence of benzene/dimethyl sulfoxide (DMSO) by its reaction at room temperature for
10 min [11] Th ey cannot be converted into t- BDMS or TMS derivatives in their unchanged
forms
h) Nicotine can be analyzed by HPLC as follows A 500-µL volume of a specimen solution (serum, gastric juice or urine), 2 mL diethyl ether, 500 µL of 5 M NaOH solution, 50 µL of
Trang 7chlordiazepoxide solution (10 µg/mL, IS) are shaken for 3 min and centrifuged Th e upper organic (diethyl ether) phase is mixed with 50 µL of 5 M HCl solution, shaken for back-extraction and centrifuged Th e lower aqueous phase is evaporated to dryness under a stream of nitrogen with warming at 50 °C Th e residue is dissolved in 100 µL of a mobile phase; a 20-µL aliquot is injected into HPLC [12] HPLC conditions are: column: Shim-pack CLC-ODS (M) (25 cm × 4.6 mm i.d., Shimadzu Corp.); mobile phase: 10 mM NaH2PO4/10 mM Na2HPO4/methanol (20:20:60); fl ow rate: 1 mL/min; column (oven) temperature: 40 °C; detection: UV
References
1) Akutsu I, Motojima S, Ogata H et al (1989) A case of benzalkonium chloride poisoning J Jpn Soc Intern Med 78: 1613–1614 (in Japanese)
2) Hitosugi M, Maruyama K, Takatsu A (1998) A case of fatal benzalkonium chloride poisoning Int J Legal Med 111: 265–266
3) Kotaki M, Fujimura N, Omote T (1996) A case of asphyxia by severe oropharyngeal edema and erosion with
10 % benzalkonium chloride (Osvan®) ingestion Jpn J Toxicol 9:427–429 (in Japanese with an English abs-tract)
4) Berkel MV, Wolff AD (1988) Survival after acute benzalkonium chloride poisoning Hum Toxicol 7:191–193
5) Suzuki I, Inoue T, Fukuda H (1991) Benzalkonium chloride In: Manual of the 12th Edition of Japanese Pharma-copoeia Hirokawa Publishing Co., Tokyo, pp c366–c369 (in Japanese)
6) Cybulski ZR (1984) Determination of benzalkonium chloride by gas chromatography J Pharm Sci 73: 1700– 1702
7) Bleau G, Desaulniers M (1989) High-performance liquid chromatographic assay of benzalkonium in plasma
J Chromatogr 487:221–227
8) Kummerer K, Eitel A, Braun U et al (1997) Analysis of benzalkonium chloride in the effluent from European hospitals by solid-phase extraction and high-performance liquid chromatography with post-column pairing and fluorescence detection J Chromatogr A 774: 281–286
9) Study group of Gas chromatography of Japanese Society of Analytical Chemistry (translation) (1999) PTV split-less injection In: Guidebook of Sample Introduction Techniques in CGC Maruzen, Tokyo, pp 256–266 (in Japa-nese)
10) Study Group of Gas chromatography of Japanese Society of Analytical Chemistry (translation) (1989) Inactiva-tion of the insert and packing materials In: Guidebook of Sample IntroducInactiva-tion Techniques in CGC Maruzen, Tokyo, pp 49–54 (in Japanese)
11) Suzuki S, Nakamura Y, Kaneko M et al (1989) Analysis of benzalkonium chlorides by gas chromatography
J Chromatogr 463:188–191
12) Saito M, Tamura Z (ed and translation) (1982) Nicotine In: Drug Level Monitoring Hirokawa Publishing Co., Tokyo, pp 254–258 (in Japanese)