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Determination of
Benzalkonium Chloride
in Pharmaceutical Formulation
Overview:
The present chapter deals with the determination of Benzalkonium
chloride in liquid pharmaceutical formulation using the developed and
validated, stability indicating, RP-HPLC method.
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Determination of Benzalkonium Chloride
in Pharmaceutical Formulation
Liquid preparations are particularly susceptible to microbial growth because of the nature of their
ingredients. Such preparations are protected by the addition of preservatives that prevent the
alteration and degradation of the product formulation [1]. The finished product release
specifications should include an identification test and a content determination test with
acceptance criteria and limits for each antimicrobial preservative present in the formulation [2].
The finished product self-life specification should also include an identification test and limits
for the antimicrobial preservatives present [2]. Hence BKC (benzalkonium chloride)
antimicrobial and antifungal properties make it an integral part of the product formulation. This
encourages the development of new stability indicating method for estimation of BKC to provide
colored solution of the drug in sterile water for injection; BKC is added as a preservative.
A comprehensive literature search revealed that review of available method for determination of
BKC by HPLC and other chromatographic methods [3-10] for the determination of sparfloxacin
and BKC are reported as a long run time and less resolution, as such there is lack of a suitable
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There is no stability-indicating RP-HPLC method reported in the literature that can adequately
separate BKC from sparfloxacin formulation and accurately quantify BKC in Sparfloxacin eye
drop, thus necessitating the development of a new stability-indicating method to assay BKC in
pharmaceutical formulation.
Perform analytical method validation for the proposed method as per ICH guideline.
BKC [Figure 6.1] is a mixture of alkyls, including all or some of the group beginning with n-
C8H17 and extending through higher homolog’s, n-C14H29 and n-C16H33 comprising the major
portion. BKC solutions are rapidly acting biocidal agents with a moderately long duration of
action. They are active against bacteria and some viruses, fungi and protozoa. Its use as a
preservative in cosmetic, eye and nasal drops attests to its general safety.
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Determination of benzalkonium chloride in pharmaceutical formulation
Benzalkonium chloride is readily soluble in ethanol and acetone. Although dissolution in water is
slow, aqueous solutions are easier to handle and are preferred. Solutions should be neutral to
slightly alkaline, with colour ranging from colourless to a pale yellow. Solutions foam profusely
when shaken, have a bitter taste and a faint almond-like odour which is only detectable in
concentrated solutions.
Applications:
The applications of BKC are extremely wide ranging, from disinfectant formulations, such as
being an active ingredient in Dettol and Lysol brand products, to microbial corrosion
inhibition in the oilfield sector, and a multi-surface mould, algae and moss remover.
It is used in:
Hand sanitizers
Preservative in pharmaceuticals and personal care products such as eye, ear and nasal
drops, as a preservative
Algaecide for clearing of algae, moss, lichens from paths, roof tiles, swimming pools,
Hand sanitizers based on BKC are more effective due to better residual activity and less irritant
than alcohol gels. As an antiseptic, it has the advantage of not burning when put on a wound,
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Biological activity:
The greatest biocidal activity is associated with the C12 dodecyl and C14 myristyl alkyl
of intermolecular interactions. This can cause dissociation of cellular membrane lipid bilayers,
which compromises cellular permeability controls and induces leakage of cellular contents. Other
biomolecular complexes within the bacterial cell can also undergo dissociation. Enzymes, which
finely control a wide range of respiratory and metabolic cellular activities, are particularly
BKC solutions are fast-acting biocidal agents with a moderately long duration of action. They are
active against bacteria and some viruses, fungi, and protozoa. Bacterial spores are considered to
positive bacteria are generally more susceptible than Gram-negative. Activity is not greatly
affected by pH, but increases substantially at higher temperatures and prolonged exposure times.
In a 1998 study utilizing the FDA protocol, a non-alcohol sanitizer utilizing the active ingredient
BKC met the FDA performance standards, while Purell, a popular alcohol-based sanitizer, did
not. The study found that a BKC-based sanitizer was the most favorable non-alcohol-based hand
sanitizer. Advancements in the quality and efficacy of BKC in current non-alcohol hand
sanitizers has addressed the CDC concerns regarding gram negative bacteria, with the leading
products being equal if not more effective against gram negative, particularly NDM1{New Delhi
Newer formulations using BKC blended with various quaternary ammonium derivatives can be
used to extend the biocidal spectrum and enhance the efficacy of BKC based disinfection
products. Formulation techniques have been used to great effect in enhancing the virucidal
infection hazards such as hepatitis and HIV. The use of appropriate excipients can also greatly
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Determination of benzalkonium chloride in pharmaceutical formulation
enhance the spectrum, performance and detergency, and prevent deactivation under use
conditions. Formulation can also help minimise deactivation of BKC solutions in the presence of
Effectively formulated Quaternary ammonium disinfectants are effective at very low ppm levels,
and are now the disinfectants of choice for hospitals. This is on account of user and patient safety
on contact with treated surfaces and the absence of harmful fumes. BKC solutions for hospital
use tend to be neutral to alkaline, non-corrosive on metal surfaces, non-staining, and safe to use
on all washable surfaces. Solutions are incompatible with soaps, and must not be mixed with
anionic surfactants. Hard water salts can also reduce biocidal activity. As with any disinfectant, it
is recommended that surfaces are free from visible dirt and interfering materials for maximal
6.4 EXPERIMENTAL
Sparfloxacin formulation and placebo are provided by Cadila Pharmaceutical Ltd., Ahmedabad,
India. The working standard of BKC (Batch No- B6295) is purchased from Sigma Aldrich
(Milan, Italy). HPLC grade acetonitrile is obtained from J.T. Baker (NJ., USA). HPLC grade
potassium dihydrogen orthophosphate, 1- octane sulphonate and sodium hydroxide are obtained
from Merck Ltd. (Mumbai, India). 0.45 µm PVDF membrane filter and PVDF syringe filters are
purchased from Milipore, India. High purity water is generated using Milli-Q Plus water
purification system (Millipore®, Milford, MA, USA). All other chemicals used are of analytical
grade.
6.4.2 Equipments
Acquity HPLCTM system (Waters, USA), consisting of a binary solvent manager, sample
manager and PDA (photo diode array) detector. System control, data collection and data
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processing are accomplished using Waters Empower chromatography data software. Cintex
digital water bath is used for specificity study. Thermal stability studies are performed in a dry
Buffer preparation:
octane sulphonate in 500 mL milli-Q water. The pH 6.3 is adjusted with diluted sodium
hydroxide solution.
Mobile phase:
The mixture of acetonitrile and buffer in 65:35 ratio used as a mobile phase. The mobile phase is
The chromatographic condition is optimized using a column Purospher Star RP-18e (75 x 4.0
mm, 3.0µ). The mobile phase consisted of acetonitrile: buffer (65:35, v/v). The mobile phase is
filtered through a 0.45 µm PVDF filter and degassed under vacuum prior to use. The flow rate is
1.8 mL/min. The monitoring wavelength is 215 nm and the injection volume is 50 µL with
maintaining column oven temperature with 25 ºC. Peak area is measured and HPLC analysis is
Standard solution of BKC is prepared by using water as a diluent. Standard solution contains
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Determination of benzalkonium chloride in pharmaceutical formulation
6.4.7 Sample solution preparation
As such solution is used (ophthalmic solution containing 0.1% BKC w/v) as sample.
The solution containing sparfloxacin and other excipients excluding BKC is used as placebo.
The method described herein has been validated for assay determination of BKC by RP-HPLC.
6.5.1 Specificity
capability of the proposed method. The sample is exposed to acid hydrolysis, base hydrolysis,
oxidative and thermal. All exposed samples are than analysed by the developed method.
System suitability parameters are measured so as to verify the system performance. System
characteristics including % RSD of total area of homolog’s, tailing factor and theoretical plate
6.5.3 Precision
The precision of the system is determined using the sample preparation procedure described
above for six real samples of liquid formulation and analysis using the same proposed method.
Intermediate precision is studied by other scientist, using different columns, different HPLC, and
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6.5.4 Accuracy
To confirm the accuracy of the proposed method, recovery experiments are carried out by the
standard addition technique. Three levels (50 %, 100 % and 150 %) of standards are added to
pre-analyzed placebo samples in triplicate. The percentage recoveries of BKC at each level and
each replicate are determined. The mean of percentage recoveries (n=9) and the relative standard
6.5.5 Linearity
Linearity is demonstrated from 50% to 150 % of standard concentration using a minimum of five
calibration levels (50 %, 75 %, 100 %, 125 % and 150 %) for BKC. The method of linear
regression is used for data evaluation. The peak area of the standard compound is plotted against
the BKC concentrations. Linearity is described by the linearity equation, correlation coefficient
6.5.6 Robustness
The robustness is a measure of the capacity of a method to remain unaffected by small but
deliberate changes in column oven temperature (+ 5°C), change in flow rate (± 0.2 mL/min),
change in wavelength (± 2 nm) and change in buffer pH (± 0.2 units). The theoretical plates,
tailing factor and retention behaviour of BKC are evaluated. All important characteristics
including % RSD of total area of homolog’s, tailing factor and theoretical plate number of BKC-
4 are measured.
The stability of the sample solution is established by storage of the sample solution at ambient
temperature for 24h. The sample solution is re-analyzed after 24h, and the results of the analysis
are compared with the results of the fresh sample. The stability of standard solution is established
by the storage of the standard solution at ambient temperature for 24h. The standard solution is
re-injected after 24h, and % RSD of total area of homologs are calculated.
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Determination of benzalkonium chloride in pharmaceutical formulation
6.5.8 Application of the method to dosage forms
The present method is applied for the estimation of preservatives in the commercially available
The main objective of the RP-HPLC method development is to rapid determination of BKC, in
liquid pharmaceutical formulation. The developed method should be able to determine assay of
BKC compound in single run and should be accurate, reproducible, robust, stability indicating,
linear, specific and enough for routine use in quality control laboratory.
The spiked solution of BKC (100 μg/mL) in placebo solution is subjected to separation by RP-
HPLC. Label claim of compounds and its working concentration is presented in Table 6.1.
Working concentration
Compound Formulation label claim per 5 mL
mg/mL µg/mL
Initially the separation of all homolog’s and placebo peaks are studied using water as a MP-A
and acetonitrile as a MP-B on HPLC column (Hypersil BDS C18, 150 x 4.6 mm; 5μm) and
Waters (HPLC) system with the linear gradient program. The flow rate of 1.0 mL/min is selected
with regards to the backpressure and analysis time as well. During this study column oven
temperature is capped at 50°C. When study is performed with above condition we observed that
all homolog’s peaks are merged with each other and with placebo. Various types of mobile phase
are used with various gradient and isocratic program, which are summarized in Table 6.2 with
the observation. Based on above solvent selection study optimized HPLC parameters are; flow
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Chapter-6
rate 1.8 mL/min; column oven temperature 25°C; isocratic solvent program; buffer (0.05M
phosphate buffer, 0.013M 1-octane sulfonic acid sodium salts (adjusted pH 6.3 with diluted
0.05M KH2PO4, 0.013M 1-octane Acetonitrile Homolog’s peaks are well separated
sulfonic acid sodium salts, from each other and placebo peak
adjusted pH 6.3 with NaOH
In order to achieve symmetrical peak of all homologs and more resolution between homologs
and placebo peaks in different stationary phases are explored. Finally desired separation with
symmetrical peaks is obtained using purospher star RP-18e, (75 x 4.0mm, 3.0µ) column. Column
oven temperature is also studied and found that 25°C is more appropriate with respect to
separation. Based on compounds UV spectrums 215 nm is found more appropriate for the BKC
determination.
ICH guidelines [11]. The method is validated to demonstrate that it is suitable for its intended
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Determination of benzalkonium chloride in pharmaceutical formulation
6.6.2.1 Specificity
From the BKC standard chromatogram, it is observed that the homologs eluted at a retention
time of 1.6, 1.9, 3.1 and 3.8 min for BKC-1, BKC-2(C12), BKC-3 and BKC-4(C14) respectively
[Figure 6.2]. The peak purity is performed for two major responses (at 1.9 min and 3.8 min). The
peak purity angle should be less than peak purity threshold for waters HPLC system. It’s
indicating that all peaks are pure. Peak purity data obtained from force degradation study are
presented in Table 6.3. According to the areas obtained, it can be concluded that all are stable in
these conditions. The purity factor for the drug assures that there is no co elution of other peaks.
Therefore, the method is specific and suitable for routine work. The specimen chromatogram of
sample, blank, placebo and standard are presented into Figure 6.2 to 6.5.
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Table 6.3 Peak purity data obtained from forced degradation study
Oxidation Passed
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Determination of benzalkonium chloride in pharmaceutical formulation
6.6.2.2 System suitability
System suitability results from precision, intermediate precision and robustness study are
summarized in Table 6.4 with its proposed acceptance criteria. The percentage RSD of total area
of homologs of five replicate injections is below 1.0 %, which indicates that the system is
precise. The parameters all complied with the acceptance criteria and system suitability is
established.
Table 6.4 System suitability results (precision, intermediate precision and robustness study)
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NLT= Not less than; NMT= Not more than; USP=United State Pharmacopeia
6.6.2.3 Precision
The precision of the assay method is evaluated by carrying out six independent determination of
BKC (100 µg/mL) test samples against qualified working standard. The method precision study
shows the repeatability of the results obtained by the testing method. The % RSD (n=6) is 1.2 %
for BKC, which are well within the acceptable limit of 2.0%. It is confirmed from results that the
The purpose of intermediate study is to demonstrate the reliability of the test results with
variations. The reproducibility is checked by analyzing the samples by different analyst using
different chromatographic system and column on different day. The analysis is conducted in the
same manner as the method precision and the % RSD of all six sets of sample preparations is
determined [Table 6.5]. The % RSD is 1.2 % for BKC, which are well within the acceptance
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Determination of benzalkonium chloride in pharmaceutical formulation
criteria of 2.0%. The difference in % is 0.3 between method precision and intermediate precision
result, which proves that the method to be rugged enough for day to day use.
6.6.2.4 Accuracy
The accuracy of an analytical method is the closeness of test results obtained by that method
compared with the true values. To confirm the accuracy of the proposed method, recovery
experiments are carried out by standard addition technique. The accuracy of the method is
carried out by adding known amounts of BKC to three concentration levels; 50, 100, and 150%
of the label claim [Table 6.1] along with the excipients in triplicate. The accuracy samples are
given the same treatment as described in sample preparation. The percentage recoveries of BKC
at each level and each replicate are determined. The mean of percentage recoveries (n=3) and the
relative standard deviation is calculated. The amount recovered is within ± 2.0 % of amount
added, which indicates that there is no interference due to excipients present in liquid
formulation. It is confirmed from results that the method is highly accurate [Table 6.6].
6.6.2.5 Linearity
The linearity of an analytical method is its ability to elicit test results that are directly, or by a
within a given range. The nominal concentrations of standard and test solutions for BKC are
100μg/mL. The response function is determined by preparing standard solutions at five different
concentration levels ranging from 50 to 150 μg/mL for BKC. The response is found linear from
50% to 150% of standard concentration. For all compounds the correlation coefficient is greater
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Chapter-6
than 0.999. The regression statistics are shown in Table 6.7 and linearity curve shown in Figure
6.6.
6.6.2.6 Robustness
small, but deliberate variations in method parameters and provides an indication of its reliability
during normal usage. Robustness parameters are selected based on critical method attribute. The
effect of change in column oven temperature (+ 5°C), change in flow rate (± 0.2 mL/min),
change in wavelength (± 0.2 nm) and change in pH of buffer (± 0.2 units) all important
characteristics including % RSD of total area of homolog’s, tailing factor and theoretical plate
number of BKC-4 are measured. During study other chromatographic conditions are kept same
as per the experimental section. It is conformed from results that the method is robust with
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Determination of benzalkonium chloride in pharmaceutical formulation
6.6.2.7 Solution stability
properties of the drug, preservative and its impurities. Condition used in stability experiments
should reflect situations likely to be encountered during actual sample handling and analysis.
Stability data is required to show that the concentration of analyte in the sample at the time of
analysis corresponds to the concentration of analyte at the time of sampling [12-17]. Stability of
sample solution is established by storage of sample solution at ambient temperature (25°C) for
24h. Sample solution is re-analyzed after 24h time intervals and assay is determined for BKC and
compared against fresh sample. Sample solution does not show any appreciable change in assay
value when stored at ambient temperature up to 24h, which are presented in Table 6.8. The
results from solution stability experiments confirmed that sample solution is stable for up to 24h
during assay determination. Standard solution is re-injected after 24h time intervals and % RSD
of all injected standard injections are calculated. Standard solution does not show any
appreciable change in % RSD (less than 1.0 %) value when stored at ambient temperature up to
24h.
The present method is applied for the estimation of preservative in the commercially available
various dosage forms. The results obtained are as shown in Table 6.9. Developed method also
proves the suitability for preservative determination in various liquid dosage forms.
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Table 6.9 Results of market products
Product Name and Labeled claim of BKC in eye drop (ED) BKC
(in mg)
Latanoprost eye drops (0.2 mg/mL) 98.3%
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Determination of benzalkonium chloride in pharmaceutical formulation
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Determination of benzalkonium chloride in pharmaceutical formulation
Calculated the quantity, in mg, of BKC (total area of homolog’s) in the portion of liquid
Where,
Rs = Compound peak response (sum of homolog’s) obtained from the sample preparation
Rstd = Compound peak response (sum of homolog’s, mean peak area) obtained from the
standard preparation
It is expressed by the following formula and calculated using Microsoft excel program in a
computer.
Where,
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6.8 CONCLUSION
A reversed phase liquid chromatography method is developed and validated for the
This method has significant advantages, in terms of shorter analysis time with excellent
resolution, selectivity and accuracy than previously reported analytical methods. This stability-
indicating method can be applied for the routine analysis in quality control laboratory. Moreover,
it can be applied for determination of assay, filter compatibility and preservative efficacy study,
where sample load is higher and high throughput is essential for faster delivery of results.
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Determination of benzalkonium chloride in pharmaceutical formulation
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