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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.

Chapter-6

Determination of

Benzalkonium Chloride

in Pharmaceutical Formulation

184

Determination of Benzalkonium Chloride

in Pharmaceutical Formulation

6.1 LITERATURE REVIEW

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

driving force in today’s pharmaceutical industry.

The determination of low concentration preservative in pharmaceutical formulation constitutes a

challenging problem in current pharmaceutical analysis. Sparfloxacin eye drops contain

sparfloxacin, a synthetic broad-spectrum antimicrobial agent for ophthalmic solution

administration. Commercially available sparfloxacin ophthalmic solution is a clear, yellow

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

procedure for the quantification and estimation of BKC preservative in pharmaceutical

formulation of sparfloxacin. Moreover, BKC determination in sparfloxacin is not officially

represented in any pharmacopoeia to date.

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6.2 THE SCOPE AND OBJECTIVES OF PRESENT STUDY

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.

The objectives of the present work are as follow:

Development of rapid, stability indicating RP-HPLC method for determination of BKC in

liquid pharmaceutical formulation.

Forced degradation study.

To separates BKC from sparfloxacin and its placebo compounds.

Perform analytical method validation for the proposed method as per ICH guideline.

Application of developed method on marketed products.

6.3 BENZALKONIUM CHLORIDE

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.

Figure 6.1 Chemical structure of benzalkonium chloride (BKC)

Determination of benzalkonium chloride in pharmaceutical formulation

186

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:

Skin antiseptics Bactine to protect scrapes and cuts

Pharmaceuticals such as throat lozenges and various leave-on skin antiseptics

Hand sanitizers

Preservative in pharmaceuticals and personal care products such as eye, ear and nasal

drops, as a preservative

Hygienic towelettes and wet wipes

Cleaners for floor and hard surfaces as a disinfectant

Soak solutions for surgical/dental instruments prior to high-level sterilization

Spray disinfectants for hard surface sanitization

Over-the-counter single-application treatments for herpes, cold-sores, and fever blisters,

such as RELEEV and Viroxyn

Algaecide for clearing of algae, moss, lichens from paths, roof tiles, swimming pools,

masonry and in horticultural greenhouse disinfection

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,

which is not the case with ethanol-based antiseptics or hydrogen peroxide.

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Biological activity:

The greatest biocidal activity is associated with the C12 dodecyl and C14 myristyl alkyl

derivatives. The mechanism of bactericidal/microbicidal action is thought to be due to disruption

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

susceptible to deactivation. Critical intermolecular interactions and tertiary structures in such

highly specific biochemical systems can be readily disrupted by cationic surfactants.

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

be resistant. Solutions are bacteriostatic or bactericidal according to their concentration. Gram-

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

Metallobetalactamase 1} and other antibiotic resistant bacteria.

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

activity of quaternary ammonium-based disinfectants such as Virucide 100 to typical healthcare

infection hazards such as hepatitis and HIV. The use of appropriate excipients can also greatly

Determination of benzalkonium chloride in pharmaceutical formulation

188

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

organic and inorganic contamination.

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

disinfection performance by quaternary ammonium products.

6.4 EXPERIMENTAL

6.4.1 Materials and reagents

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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189

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

air oven (Cintex, Mumbai, India).

6.4.3 Preparation of mobile phase

Buffer preparation:

Buffer is prepared by dissolving 3.5 g of potassium dihydrogen orthophosphate and 3.0 g of 1-

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

filtered through a 0.45 µm PVDF membrane filter and degas.

6.4.4 Diluent preparation

Milli-Q water is used as a diluent.

6.4.5 Chromatographic conditions

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

conducted at room temperature. Milli-Q water is used as a diluent.

6.4.6 Standard solution preparation

Standard solution of BKC is prepared by using water as a diluent. Standard solution contains

0.1% of BKC (w/v).

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6.4.7 Sample solution preparation

As such solution is used (ophthalmic solution containing 0.1% BKC w/v) as sample.

6.4.8 Placebo solution preparation

The solution containing sparfloxacin and other excipients excluding BKC is used as placebo.

6.4.9 Sample solution preparation for market product

As such solution is used as sample solution.

6.5 METHOD VALIDATION

The method described herein has been validated for assay determination of BKC by RP-HPLC.

6.5.1 Specificity

Forced degradation studies are performed to demonstrate selectivity and stability-indicating

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.

6.5.2 System suitability

System suitability parameters are measured so as to verify the system performance. System

precision is determined on five replicate injections of standard preparation. All important

characteristics including % RSD of total area of homolog’s, tailing factor and theoretical plate

number of BKC-4 are measured.

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

is performed on different days.

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191

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

deviation are calculated.

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

and Y-intercept bias is also determined.

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.

6.5.7 Solution stability

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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6.5.8 Application of the method to dosage forms

The present method is applied for the estimation of preservatives in the commercially available

various dosage forms.

6.6 RESULTS AND DISCUSSION

6.6.1 Method Development and Optimization

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.

Table 6.1 Formulation label claim with its working concentration

Compound Formulation label claim per 5 mL Working concentration

mg/mL µg/mL

BKC 0.5 mg 0.1 100

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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193

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

NaOH) and acetonitrile in ratio of 35:65 is used as a mobile phase.

Table 6.2 Summary of solvent used to optimize the method

MP-A MP-B Observation

Water Acetonitrile Homologs peak shape is not proper

0.05M KH2PO4 Acetonitrile Retention time decrease but

separations between homologs are

not increased

0.05M KH2PO4, 0.013M 1-octane

sulfonic acid sodium salts,

adjusted pH 6.3 with NaOH

Acetonitrile Homolog’s peaks are well separated

from each other and placebo peak

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.

6.6.2 Analytical Parameters and Validation

After satisfactory development of RP-HPLC method it is subjected to method validation as per

ICH guidelines [11]. The method is validated to demonstrate that it is suitable for its intended

purpose by the standard procedure to evaluate adequate validation characteristics (specificity,

system suitability, precision, accuracy, linearity, robustness and solution stability).

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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.

Figure 6.2 Specimen chromatogram of standard solution

Figure 6.3 Specimen chromatogram of blank

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195

Figure 6.4 Specimen chromatogram of placebo preparation

Figure 6.5 Specimen chromatogram of sample solution

Table 6.3 Peak purity data obtained from forced degradation study

Stress conditions Peak purity

Unstressed sample Passed

Acid Hydrolysis Passed

Base Hydrolysis Passed

Oxidation Passed

Thermal degradation Passed

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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)

Test Parameters BKC Proposed

criteria

Precision USP tailing of BKC-4 2.2 NMT 2.5

USP plate count of BKC-4 2124 NLT 1000

% RSD of Total Area of Homologs 0.1% NMT 2.0%

Intermediate

precision

USP tailing of BKC-4 1.9 NMT 2.5

USP plate count of BKC-4 2065 NLT 1000

% RSD of Total Area of Homologs 0.5% NMT 2.0%

Column

temperature

30°C

USP tailing of BKC-4 1.6 NMT 2.5

USP plate count of BKC-4 1676 NLT 1000

% RSD of Total Area of Homologs 0.2% NMT 2.0%

Flow rate

1.6 mL/min

USP tailing of BKC-4 1.6 NMT 2.5

USP plate count of BKC-4 1584 NLT 1000

% RSD of Total Area of Homologs 0.1% NMT 2.0%

Flow rate

2.0 mL/min

USP tailing of BKC-4 1.6 NMT 2.5

USP plate count of BKC-4 1575 NLT 1000

% RSD of Total Area of Homologs 0.2% NMT 2.0%

Wavelength

213 nm

USP tailing of BKC-4 1.6 NMT 2.5

USP plate count of BKC-4 1636 NLT 1000

% RSD of Total Area of Homologs 0.8% NMT 2.0%

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197

Wavelength

217 nm

USP tailing of BKC-4 1.6 NMT 2.5

USP plate count of BKC-4 1636 NLT 1000

% RSD of Total Area of Homologs 0.8% NMT 2.0%

pH of buffer

6.1

USP tailing of BKC-4 1.7 NMT 2.5

USP plate count of BKC-4 1946 NLT 1000

% RSD of Total Area of Homologs 0.2% NMT 2.0%

pH of buffer

6.5

USP tailing of BKC-4 1.7 NMT 2.5

USP plate count of BKC-4 1969 NLT 1000

% RSD of Total Area of Homologs 1.0% NMT 2.0%

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

method is precise for the intended purpose [Table 6.5].

Table 6.5 Precision (n=6) and intermediate precision (n=6) results

Substance Precision at 100% Intermediate precision

Mean % assay % RSD Mean % assay % RSD

BKC 97.6 1.2 97.9 1.2

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

Determination of benzalkonium chloride in pharmaceutical formulation

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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].

Table 6.6 Accuracy results

Substance

At 50% (n=3) At 100% (n=3) At 150% (n=3)

%Recovery %RSD %Recovery %RSD %Recovery %RSD

BKC 100.6 1.1 99.1 0.3 99.2 0.7

6.6.2.5 Linearity

The linearity of an analytical method is its ability to elicit test results that are directly, or by a

well-defined mathematical transformation, proportional to the concentration of analyte in sample

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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199

than 0.999. The regression statistics are shown in Table 6.7 and linearity curve shown in Figure

6.6.

Table 6.7 Regression statistics

Compound Linearity range

(µg/mL)

Correlation

coefficient (r2)

Linearity (Equation) Y- intercept

bias in %

BKC 50 to 150 0.999 y =28.96(x) + 18.27 0.6%

Figure 6.6 Linearity of BKC

6.6.2.6 Robustness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by

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

respect to variability in above conditions [Table 6.4].

Determination of benzalkonium chloride in pharmaceutical formulation

200

6.6.2.7 Solution stability

Drug stability in pharmaceutical formulations is a function of storage conditions and chemical

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.

Table 6.8 Solution stability results

Time intervals BKC

% Assay Initial 98.3

% Assay after 24h 98.5

6.6.3 APPLICATION OF THE METHOD TO DOSAGE FORMS

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.

Chromatogram of analysed sample presented in Figure 6.7- 6.14.

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201

Table 6.9 Results of market products

Product Name and Labeled claim of BKC in eye drop (ED)

(in mg)

BKC

Latanoprost eye drops (0.2 mg/mL) 98.3%

Dexamethasone eye drops (0.1 mg/mL) 92.4%

Gatifloxacin eye drops (0.1 mg/mL) 98.6%

Moxifloxacin and Dexamethasone eye drops (0.05 mg/mL) 109.3%

Timolol eye drops (0.1 mg/mL) 99.0%

Nephazoline, Zinc sulphate, Chlorpheniramine (0.025 mg/mL) 90.6%

Tobramycin and Dexamethasone eye drops (0.1 mg/mL) 108.6%

Phenylephrine, Nephazoline, Menthol, Camphor (0.1 mg/mL) 109.5%

Figure 6.7 Specimen chromatogram of Latanoprost eye drop

Figure 6.8 Specimen chromatogram of Dexamethasone eye drop

Determination of benzalkonium chloride in pharmaceutical formulation

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Figure 6.9 Specimen chromatogram of Gatifloxacin eye drops

Figure 6.10 Specimen chromatogram of Moxifloxacin and Dexamethasone eye drops

Figure 6.11 Specimen chromatogram of Timolol eye drops

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203

Figure 6.12 Specimen chromatogram of Nephazoline, Zinc sulphate, Chlorpheniramine ED

Figure 6.13 Specimen chromatogram of Tobramycin and Dexamethasone eye drops

Figure 6.14 Specimen chromatogram of Phenylephrine, Nephazoline, Menthol, Camphor ED

Determination of benzalkonium chloride in pharmaceutical formulation

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6.7 CALCULATION FORMULA

6.7.1 Assay (% w/w)

Calculated the quantity, in mg, of BKC (total area of homolog’s) in the portion of liquid

pharmaceutical formulation using the following formula:

Where,

Cstd = Concentration of standard solution in mg/mL

Cs = Concentration of sample solution in mg/mL

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

6.7.2 Relative standard deviation (% RSD)

It is expressed by the following formula and calculated using Microsoft excel program in a

computer.

Where,

SD= Standard deviation of measurements

= Mean value of measurements

6.7.3 Accuracy (% Recovery)

It is calculated using the following equation:

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205

6.8 CONCLUSION

A reversed phase liquid chromatography method is developed and validated for the

determination of benzalkonium chloride (BKC)/ preservative in pharmaceutical formulation.

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.

Determination of benzalkonium chloride in pharmaceutical formulation

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6.9 REFERENCES

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of Pharmacy” Lippincott Williams & Wilkins, Philadelphia, 21st ed., 2006.

[2] European Medicines Agency, “Guideline on Excipients in the Dossier for Application

for Marketing Authorisation of a Medicinal Product” Doc. Ref. EMEA/CHMP/

QWP/396951 /2006, London, 6 November 2006.

[3] Shen Y, Xu SJ, Wang SC, Tu JS, “Determination of benzalkonium chloride in viscous

ophthalmic drops of azithromycin by high performance liquid chromatography”

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[4] Rojsitthisak P, Wichitnithad W, Pipitharome O, Sanphanya K, Thanawattanawanich P,

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332- 337, PMID: 16316068.

[5] Jadwiga DW, Jadwiga T, Iza R, “Application of the HPLC method for benzalkonium

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[6] Shelly JP, Hei-Jen M, Loyd VA, Phil M, “Analysis of benzalkonium chloride and its

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