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Bulgarian Chemical Communications, Volume 46, Special Issue A (pp. 94 – 99) 2014
Determination of Benzethonium Chloride in Grapefruit Seed Extracts - a GC/MS
alternative
D. V. Nedeltcheva-Antonova*, K. S. Tsandeva, R. D. Dimitrova
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev str.,
bl. 9, 1113 Sofia, Bulgaria
Received June 18, 2014; Revised July 03, 2014
Dedicated to Acad. Dimiter Ivanov on the occasion of his 120th birth anniversary
Commercial grapefruit seed extracts, distributed on the market as food supplements, were analysed by gas
chromatography-mass spectrometry (GC/MS). Presence of benzethonium chloride, a synthetic antimicrobial agent, in
the range of 0.14-22.2% was found in 4 of 5 analysed commercial samples (one of them - especially developed for
children). The presence of benzethonium chloride was additionally confirmed by high-performance liquid
chromatography with diode-array detection (HPLC/DAD) and direct infusion electrospray ionization mass spectrometry
(ESI-MS). This work demonstrates that GC/MS represents a simple, fast, selective and sensitive alternative approach
for identification and quantitation of benzethonium chloride in commercial grapefruit seed extracts.
Key words: benzethonium chloride, Grapefruit seed extract, GC/MS
INTRODUCTION
Nowadays, there is a growing interest toward
alternative medicine and consumption of herbal
drugs and additives. At the same time, many of the
herbal products available on the market have not
been assessed by regulatory authorities and their
chemical composition and pharmacological proper-
ties are not always well described. One of the
products with most controversial fame in the
natural products market is the “grapefruit seed
extract” (GSE), labelled as an extract of the seeds
and pulp of the common grapefruit (Citrus
paradisi, Rutaceae). GSE has been promoted over
the last three decades as a gentle non-toxic natural
product with healing power against a variety of
diseases, with very high antimicrobial efficacy and
has been used as an ingredient for cosmetic and
dermatological formulations, in dietary supple-
ments as well as food/cosmetic preservative.
However, in most of the cases, the compositions of
commercial GSEs are not defined, and the methods
of production being proprietary are not specified.
In 1991 the first analysis of commercial GSE
was published by Nishina et al. [1], where methyl
4-hydroxybenzoate (methyl paraben), a preserva-
tive, and 2,4,4-trichloro-2-hydroxydiphenyl ether
(triclosan), a microbicide and disinfectant were
found by means of preparative HPLC. Using HPLC
and ESI-MS, Sakamoto et al. [2] repeated the
analysis of GSE, and compared the commercial
GSE with ethanolic extracts of grapefruit seeds
prepared by themselves. The presence of methyl
paraben and triclosan in the commercial GSE
(1.66% and 1.97%, respectively) was confirmed
and no trace of these compounds was found in the
ethanolic extract of grapefruit seeds. The anti-
microbial activity as well as the content of presser-
vative agents (methyl paraben and triclosan) in 6
commercially available GSE were examined by von
Woedtke and co-workers [3], who additionally
found N-Benzyl-N,N-dimethyl-2-{2-[4-(2,4,4-tri-
methylpentan-2-yl)phenoxy]ethoxy}ethanaminium
chloride (benzethonium chloride), a synthetic
antimicrobial agent commonly used in cosmetics
and other topical applications, in these products.
The five extracts containing one to three of these
preservative agents showed high antimicrobial
activity. At the same time, the only GSE product
without synthetic preservatives along with the fresh
extracts prepared from grapefruit seeds with
glycerol, water and ethanol, did not exhibit any
antimicrobial activity. The authors concluded that
the antimicrobial activity attributed to GSE is due
to the synthetic preservative agents with benzetho-
nium chloride being responsible for the majority of
activity. Takeoka et al. subsequently published two
analyses of GSE formulations [4,5]. They have
found using HPLC, ESI-MS, nuclear magnetic
resonance (NMR) spectroscopy, and elemental
© 2014 Bulgarian Academy of Sciences, Union of Chemists in Bulgaria
* To whom all correspondence should be sent:
E-mail: dantonova@orgchm.bas.bg
D. V. Nedeltcheva-Antonova et al.: Determination of Benzethonium Chloride in Grapefruit Seed Extracts - a GC/MS alternative
95
analysis that benzethonium chloride is the main
constituent in the analysed commercial GSE in
form of a liquid concentrate and a concentrated
powder (as 8% of the mass of the liquid GSE,
higher amounts of benzethonium chloride were
found in powder). Sugimoto et al. [6] carried out
comprehensive research of the commercial GSE
products which are used in Japan as food additives
(13 products from 6 manufactures), dietary supple-
ments (5 products from 4 manufactures), cosmetic
materials (16 products from 10 manufactures) and
disinfectants (7 products from 7 manufactures). By
means of LC/MS and NMR analysis, synthetic
disinfectant agents such as benzethonium or
benzalkonium salts were detected in most of the
commercial GSE products. Simultaneous identifica-
tion and quantification of benzethonium chloride,
methyl paraben and triclosan in 9 commercial GSE
products, one pomegranate (Punica granatum,
Punicaceae) seed extract, and a freshly prepared
methanolic extract of grapefruit seeds, were perfor-
med by Avula et al. using HPLC/UV/MS [7].
Benzethonium chloride was found in 8 of 9 com-
mercial GSE preparations. Only the commercial
pomegranate seed extract (used as a control sample)
and the fresh grapefruit seed extract were free of
synthetic additives. An HPLC/UV/MS method was
developed and validated by Ganzera et al. for
simultaneous determination of 18 possible presser-
vatives, disinfectants, and microbicides in GSE and
tested on 9 commercial products used for eco-
farming [8]. A method for the quantitative analysis
of benzethonium chloride in GSE based on 1H-
NMR is also described in the literature [9].
Evidently, there is a serious problem with the
adulteration of the commercial GSE, which proves
the necessity of development and validation of new
methods for analysis of benzethonium chloride. As
seen, currently most of the methods are based on
high–performance liquid chromatography. In addi-
tion to the above described, benzethonium chloride
was determined by gas-liquid chromatоgraphy as
reduction product obtained by treatment of the
sample with sodium borohydrate and nickel (II)
chloride by Kawase et al. [10].
Benzethonium chloride being quaternary
ammonium salt (QAS) is non-volatile and at first
sight gas chromatography could not be the most
suitable method for its direct determination. How-
ever, the QAS are thermally unstable and decom-
pose at high temperatures by cleavage at the
quarternary nitrogen, forming tertiary amines and
other neutral molecules [11-13]. Gas chromato-
graphy seems to be an ideal analytical technique for
the determination of QAS because the heated inlet
system of the instrument allows in situ decom-
position of the salts. Consequently, thermal
decomposition to neutral molecules, vaporization,
and analysis of the characteristic products can be
performed in a single step. An injection port
pyrolysis method for the analysis of quaternary
ammonium compounds (QAC) is reported by
Lukazewsky et al. [14]. Direct injection GC/MS
was used for the analysis of benzyl diethyl (2,6-
xylylcarbamoylmethyl) ammonium benzoate, a
QAS, in various Canadian denatured alcohol
formulations [15].
Therefore, the aim of the current communication
is to describe a fast, simple and sensitive direct
GC/MS method for simultaneous identification and
quantification of benzethonium chloride, based on
the analysis of N,N-dimethyl-2-{2-[4-(2,4,4-tri-
methylpentan-2-yl)phenoxy]ethoxy}ethanamine as
main pyrolysis product.
EXPERIMENTAL
Materials
Samples of commersial GSE, four of them in
form of a liquid concentrate and one as a kid syrup,
were purchased from drug stores in Bulgaria,
Poland and UK. Benzethonium chloride reference
material (99%) was delivered from Sigma-Aldrich.
Solvents (methanol, chloroform, acetonitrile) of
HPLC gradient grade (Sigma-Aldrich) and de-
ionized water ASTM Type I were used. Solid-phase
extraction columns SOLA SCX, 10 mg/1ml were
purchased from Thermo Scientific.
Sample Preparation
Liquid-liquid extraction. Approximately 2 g of
each GSE sample was mixed with 10 ml of water
and extracted three times with 30 mL of
chloroform. The combined chloroform extracts
were evaporated either in a rotary vacuum evapo-
rator or under stream of nitrogen. The dry residues
were then dissolved in HPLC mobile phase and
filtered through a membrane filter (PTFE, 0.22 m)
before the chromatographic analysis.
Solid Phase Extraction. Approximately 0.1 g of
each sample was dissolved in 10 ml of methanol
and then an aliquot of 0.5 ml was used for solid
phase extraction. Methods
High-Performance Liquid Chromatography
(HPLC). The analyses were performed on a HPLC
system consisting of an HP1100 liquid chroma-
D. V. Nedeltcheva-Antonova et al.: Determination of Benzethonium Chloride in Grapefruit Seed Extracts - a GC/MS alternative
96
tograph equipped with a manual injector
(Rheodyne, model 7725), fitted with a 20 L
sample loop and a diode–array detector (G1365B
DAD), and controlled by ChemStation software
(Rev. B.04.03, Agilent Technologies). Analytical
column ChromSep SS, Inertsil 5 ODS-2 (250 x 4.6
mm i.d. 5 m) equipped with a ChromSep guard
column (Varian, Palo Alto, CA) was used. The
mobile phase was acetonitrile/water (80:20, v/v)
containing 0.1% formic acid (pH 3) at a flow rate of
1.0 ml/min. The detector signal was monitored at
215 and 275 nm.
Gas Chromatography-Mass Spectrometry (GC/
MS). The GC/MS analysis was performed on a HP
gas chromatograph 6890 Series Plus coupled with a
5973 mass - selective detector (Hewlett–Packard,
Palo Alto, CA). The ultra-inert fused silica capillary
column DB-5ms UI (J&W Scientific, Folsom, CA)
with 30 m column length, 0.25 mm i.d., 0.25 m
film thickness was used. The oven temperature was
programmed from 80 to 300oC at a rate of 5oC
/min, and a 10 min hold at 300oC was applied.
Helium (99.999%) was used as a carrier gas at a
constant flow rate of 0.8 ml/min. The split ratio was
1:10, the inlet temperature was set to 280oC and the
transfer line temperature was 300oC. Mass -
selective detector operated in electron impact
ionization (EI) mode at 70 eV electron energy, the
ion source temperature was set to 200oC, and the
quadrupole temperature was 150oC.
Electrospray Ionization-Mass Spectrometry
(ESI-MS). ESI-MS spectra were recorded on a DFS
High Resolution magnetic-sector mass spectro-
meter (Thermo Scientific, Bremen, Germany) under
the following operating conditions: positive ion
scan mode, capillary temperature of 260oC and
capillary voltage of 3.5 kV was applied. Direct
infusion mode was used for the introducing of
samples into the mass spectrometer via Harvard 11
Plus syringe pump (Harvard Apparatus, Holliston,
Massachusetts, USA). Acetonitrile/water (30:70%,
v.v) with 0.05% formic acid was used as a mobile
phase at a flow rate of 0.05 ml/min. The instrument
was controlled and the data was processed by
Xcalibur™ software (Rev. 2.0 SR 1, Thermo
Scientific).
Calibration Curves
The absolute calibration method (external
standard method) was used to establish the calib-
ration curve and quantify the analytes. 0.025 g of
benzethonium chloride standard was weight and
diluted in a 25 ml volumetric flask with methanol.
Five working standard solutions with concentra-
tions in the range 0.001-0.200 mg/ml were prepared
from the stock standard solution with concentration
1 mg/ml. Each working standard solution was then
analysed in triplicate by GC/MS, operating in full
scan mode and the results were presented graphi-
cally, plotting peak area (for the peak at tR =20.086
min) versus concentration.
RESULTS AND DISCUSSION
Benzethonium chloride, being quaternary
ammonium salt is non-volatile, but, as seen in Fig.
1, its GC/MS chromatogram exhibits two main
peaks. The first one is benzyl chloride (1), at
retention time tR=5,54 min, and the second one has
been identified as N,N-dimethyl-2-{2-[4-(2,4,4-
trimethylpentan-2-yl)phenoxy]ethoxy}ethanamine
(2), at tR=20.08 min. These peaks are result of the
thermal dissociation of benzethonium chloride
(favored pathway is shown in Scheme 1) in the GC
inlet system, following the fact that QAS are
unstable at high temperatures. In general, the N-
benzyl cleavage is favored over the N-alkyl one in
this process. The main pyrolysis product 2 exhibits
GC/MS behavior, which makes it suitable for
identification and low level quantification of
benzethonium chloride.
Fig. 1. GC/MS total ion current (TIC) chromatogram of
the benzethonium chloride reference material.
Linear calibration curve with regression
coefficient of 0.9997 was established between the
EI-MS response of 2 as a function of the benzetho-
nium chloride standard solutions concentration. The
corresponding equation is given below:
74
Y 2.46 10 .X 4.74 10
,
where Y is the total ion current peak area of 2 and
X is the concentration of benzethonium chloride in
mg/ml.
The linearity was studied in concentration range
from 0.001 to 1 mg/ml with limit of quantification
D. V. Nedeltcheva-Antonova et al.: Determination of Benzethonium Chloride in Grapefruit Seed Extracts - a GC/MS alternative
97
(LOQ) of 0.005 mg/ml (S/N=10) and limit of
detection (LOD) of 0.001 mg/ml.
Scheme 1. Thermal dissociation of benzethonium
chloride.
Glycerin, used as carrier in the commercial GSE
formulations, is moderately volatile and need to be
removed from the sample before the GC/MS
analysis. Therefore two methods of extraction of
benzethonium chloride from the commercial
samples have been attempted, namely liquid-liquid
extraction with chloroform and solid phase extrac-
tion (SPE). In the first case it was impossible to
remove glycerin completely and always a white
viscous product, containing sufficient amount of
glycerin was obtained. The glycerin was fully
removed by using only SPE and, therefore, this
extraction method was finally selected for sample
preparation. The recovery of the SPE, determined
by standard addition method, was 0.98.
Using the above discussed procedure, five
commercial GSE formulations, available in phar-
macies as dietary supplements, with declared
ingredients extract of seeds and pulp of grapefruit,
plant glycerin and in some cases vitamin C, have
been analysed. It is worth to underline that one of
these products (sample 1) was introduced as stimu-
lating immune activity syrup, especially designed
for kids, with GSE content of 0.1%, containing in
addition rose hips extract. The GC/MS analysis has
shown availability of benzethonium chloride in four
of the samples in range from 0.14 to 22.2%.The
corresponding results are collected in Table 1.
These results are stunning, because according to
the opinion of the Scientific Committee on
Cosmetic Products and Non-Food Products, the
scientific advisory body to the European
Commission in matters of consumer protection
SCCNFP/0762/03, the use of benzethonium chlori-
de as a preservative in leave-on products is safe up
to a maximum concentration of 0.1% and abso-
lutely prohibited for internal use.
Table 1. Content of benzethonium chloride in the
analysed commercial GSE.
Sample
Benzethonium chloride [%]
1
0.14 ± 0.01
2
8.96 ± 0.39
3
10.6 ± 0.28
4
22.1 ± 0.02
5
< LOD
Benzethonium chloride was identified in the
commercial samples by using mass spectral
libraries (NIST08, Wiley 275 and MS Search
v.2.0), by the retention time of pyrolysis product 2
of the reference material and its mass spectrum
with characteristic fragment ions (Fig. 2). As an
additional piece of evidence the chromatograms of
the analysed samples are shown in Fig. 3.
Fig. 2. EI-MS (70 eV) spectrum of 2.
Fig. 3. GC/MS TIC chromatograms of commercial GSE
samples.
The identification of benzethonium chloride was
additionally confirmed by means of HPLC/DAD
and ESI-MS. It is worth to underline that in both
cases the samples were analysed at room
temperature, which excludes thermal dissociation.
D. V. Nedeltcheva-Antonova et al.: Determination of Benzethonium Chloride in Grapefruit Seed Extracts - a GC/MS alternative
98
When HPLC/DAD (215 and 275 nm) was used
a single peak at tR = 8.35 min has been detected in
the samples 1-4, which corresponds to the retention
time of the benzethonium chloride standard. The
commercial samples and the standard solution of
benzethonium chloride in suitable concentrations
have been analysed by direct infusion ESI-MS in
full scan positive mode. The ESI-MS spectra of the
reference benzethonium chloride and the samples
1-4, shown in Fig. 4, are identical, which once
again confirms the presence of benzethonium
chloride in the samples.
Fig. 4. Positive mode ESI-MS spectrum of benzethoni-
um chloride as reference material and in the samples 1-4.
CONCLUSION
This work demonstrates that direct GC/MS
represents a simple, fast, selective and sensitive
alternative approach for identification and
quantification of benzethonium chloride in
commercial GSE. The proposed method seems to
be suitable, with small modifications of the sample
preparation procedure, for determination of
benzethonium chloride and/or other synthetic
microbicides based on quaternary ammonium
compounds in various matrices (food additives,
cosmetics, etc.).
Acknowledgements: The technical support from
Ms E. Dimitrova is gratefully acknowledged.
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6. N. Sugimoto, A. Tada, M. Kuroyanagi, Y. Yoneda,
S. Y. Yun, A. Kunugi, K. Sato, T. Yamazaki, K.
Tanamoto, Shokuhin Eiseigaku Zasshi, 49, 56 (2008).
7. B. Avula, S. Dentali, A. I. Khan, Pharmazie, 62, 593
(2007).
8. M. Ganzera, A. Aberham, H. Stuppner, J. Agr. Food
Chem., 54, 3768 (2006).
9. S. Bekiroglu, O. Myrberg, K. Ostman, M. Ek, T.
Arvidsson, T. Rundlöf, B. Hakkarainen, J. Pharm.
Biomed. Anal., 47, 958 (2008).
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273 (1982).
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D. V. Nedeltcheva-Antonova et al.: Determination of Benzethonium Chloride in Grapefruit Seed Extracts - a GC/MS alternative
99
ОПРЕДЕЛЯНЕ НА БЕНЗЕТОНИЕВ ХЛОРИД В ЕКСТРАКТИ ОТ СЕМЕНА НА
ГРЕЙПФРУТ – АЛТЕРНАТИВЕН ПОДХОД ЧРЕЗ ИЗПОЛЗВАНЕ НА ГАЗОВА
ХРОМАТОГРАФИЯ С МАССПЕКТРАЛНА ДЕТЕКЦИЯ
Д. В. Неделчева-Антонова*, К. С. Цандева, Р. Д. Димитрова
Институт по Органична химия с Център по Фитохимия, Българска Академия на Науките, ул. Акад. Г. Бончев,
бл. 9, 1113 София, България
Постъпила на 18 юни 2014 г.; Коригирана на 03 юли 2014 г.
(Резюме)
Търговски екстракти от семена на грейпфрут, разпространени в търговската мрежа като хранителни
добавки, бяха анализирани чрез газова хроматография – масспектрометрия (ГХ/МС). В четири от пет
анализирани търговски продукта (един от които специално разработен за деца) беше намерено присъствие на
бензетониев хлорид, синтетичен антимикробен агент, със съдържание от 0.14-22.2%. Наличието на бензетониев
хлорид беше допълнително потвърдено чрез високо ефективна течна хроматография с фотометричен детектор с
диодна матрица и масспектрометрия с електроспрей йонизация. Публикацията демонстрира възможностите на
ГХ/МС като лесен, бърз, селективен и чувствителен алтернативен подход за идентификация и количествено
определяне на бензетониев хлорид в търговски екстракти от семена на грейпфрут.
