Chapter-3 Simultaneous Determination of Ambroxol ...shodhganga.inflibnet.ac.in/bitstream/10603/8513/10/10_chapter 3.pdf95 Chapter-3 Simultaneous Determination of Ambroxol Hydrochloride,

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Chapter-3

Simultaneous Determination of Ambroxol Hydrochloride,

Cetirizine Hydrochloride, Methylparaben and Propylparaben in

Liquid Pharmaceutical Formulation

Overview

The present chapter deals with the simultaneous determination of ambroxol hydrochloride,

cetirizine hydrochloride, methylparaben, and propylparaben in liquid pharmaceutical formulation

using the developed and validated, stability indicating, RP-UPLC method.

3.1 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 their (MP and PP) antimicrobial and antifungal

properties make them an integral part of the product formulation. This encourages the development

of new stability indicating method for simultaneous estimation of all compounds (AMB, CTZ, MP

and PP) to provide driving force in today’s pharmaceutical industry.

UPLC is a new category of separation technique based upon well-established principles of liquid

chromatography, which utilizes sub-2 µm particles for stationary phase. These particles operate at

Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation

96

elevated mobile phase linear velocities to affect dramatic increase in resolution, sensitivity and

speed of analysis. Owing to its speed and sensitivity, this technique is gaining considerable

attention in recent years for pharmaceuticals and biomedical analysis. In the present work, this

technology has been applied to the method development and validation study of assay

determination (AMB, CTZ, MP and PP) in liquid pharmaceutical formulation.

Several spectrophotometric methods have been reported for the qualitative and quantitative

determination of AMB from pharmaceuticals formulations [2-6]. Various HPLC [7-10], GLC [11,

12], sequential injection technique coupled with monolithic column [13] LC-MS [14], capillary

electrophoretic [15] and by capillary electrophoresis and fluorescence detection [16] are also

reported for its determination from biological fluids.

Literature survey revealed that several spectrophotometric [17-19] methods, HPLC methods [20-

23], HPLC coupled to tandem mass spectroscopy [24], capillary electrophoretic [25, 26] have been

also reported for determination of CTZ from Pharmaceutical formulations and biological fluids.

Detailed literature survey for MP and PP revealed that many existing analytical procedures are

available in literature for the determination of present preservatives studied, either alone or in

combination with other drugs by HPLC and other techniques [10, 27-35].

A detailed literature survey for AMB + CTZ revealed that few analytical methods are available

using spectrophotometric and HPLC where; Neela M. Bhatia et al. [36], describe RP-HPLC and

spectrophotometric estimation of AMB and CTZ in combined dosage form; Mukesh Maithani et al.

[37], simultaneous estimation of AMB and CTZ in tablet dosage form by RP-HPLC method;

Trivedi Aditya et al. [38], development of modified spectrophotometric and HPLC method for

simultaneous estimation of AMB and CTZ in tablet dosage forms; A. S. Birajdar et al. [39],

simultaneous analysis of AMB with CTZ and of AMB with levo-Cetirizine dihydrochloride in solid

dosage forms by RP-HPLC; NM Gowekar et al. [40], spectrophotometric estimation of AMB and

CTZ from tablet dosage form. HPTLC method is also reported by S.B. Bagade et al. [41].

Chapter-3

97

Ambroxol Impurity-A, B, C, D and E are official in British Pharmacopoeia [42]. Cetirizine

specified impurities A, B, C, D, E and F are also official in British Pharmacopoeia [43]. Cetirizine

CDH1 (impurity G as per British Pharmacopoeia) impurity is completely characterized in house

(Dr. Reddy’s Laboratory) by using IR, Mass and NMR.

3.2 THE SCOPE AND OBJECTIVES OF PRESENT STUDY

The combination of AMB and CTZ is not official in any pharmacopoeia. So far, no RP-UPLC

stability indicating method has been reported for the rapid simultaneous determination of AMB,

CTZ, MP and PP in liquid pharmaceutical formulation yet. Therefore, the research is undertaken to

develop a new rapid and stability-indicating method for simultaneous determination of four

compounds (AMB, CTZ, MP and PP) in liquid pharmaceutical formulation. The developed method

is able to separate AMB, CTZ, MP and PP with each other and from its all twelve (AMB impurities

A, B, C, D, E and CTZ impurities A, B, C, D, E, F, CDH1) known impurities/ degradation products

and one unknown degradation product within 3.5 min. Thereafter, this method is validated

according to the ICH guidelines [44] and successfully applied for separation and quantification of

all compounds of interest in the liquid and solid pharmaceutical formulation.

The objectives of the present work are as follow:

Development of rapid, stability indicating RP-UPLC method for simultaneous

determination of AMB, CTZ, MP, and PP in liquid pharmaceutical formulation.

Forced degradation study.

To separates AMB, CTZ, MP and PP with each other and from its all twelve (AMB

impurities A, B, C, D, E and CTZ impurities A, B, C, D, E, F, CDH1) known impurities/

degradation products and any unknown degradation product generated during forced

degradation study.

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

Application of developed and validated method on various pharmaceutical dosage forms

(various marketed products).


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3.3 AMBROXOL HYDROCHLORIDE

Ambroxol hydrochloride (AMB) is semi-synthetic derivative of vasicine obtained from Indian

shrub Adhatoda vasica. It is a metabolic product of bromhexine. AMB is a clinically proven

systemically active mucolytic agent. When administered orally onset of action occurs after about 30

minutes. The breakdown of acid mucopolysaccharide fibers makes the sputum thinner and less

viscous and therefore more easily removed by coughing. Although sputum volume eventually

decreases, its viscosity remains low for as long as treatment is maintained [45]. AMB is chemically

(1s,4s)-4-((2-amino-3,5-dibromocyclohexyl)methylamino)cyclohexanol hydrochloride [Figure 3.1].

Its molecular weight is 414.6 g/mole with molecular formula C13H18Br2N2O.HCl. Its dissociation

constant (pKa) 8.2 is reported. Its melting point is 235°C to 240°C.

[Figure 3.1 Chemical structure of Ambroxol hydrochloride (AMB)]

3.3.1 Indications

All forms of tracheobronchitis, emphysema with bronchitis pneumoconiosis, chronic inflammatory

pulmonary conditions, bronchiectasis, bronchitis with bronchospasm asthma. During acute

exacerbations of bronchitis it should be given with the appropriate antibiotic [45].

3.3.2 Pharmacokinetics

Alteplase initiates local fibrinolysis and dissolution of clots by binding to fibrin in a thrombus and

the fibrin-bound plasminogen is converted to plasmin.

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99

3.3.3 Ambroxol dosage [45]

Adults: daily dose of 30 mg (one ambroxol tablets) to 120 mg (4 ambroxol tablets) taken in

2 to 3 divided dose.

Children up to 2 years: half a teaspoonful ambroxol syrup twice daily.

Children 2-5 years: half a teaspoonful ambroxol syrup 3 times daily.

Children over 5 years: one teaspoonful ambroxol syrup 2-3 times daily.

3.4 CETIRIZINE HYDROCHLORIDE (CETIRIZINE DIHYDROCHLORIDE)

Cetirizine hydrochloride (CTZ) is an orally active and selective H1-receptor antagonist. It is

piperazine derivative and metabolite of hydroxyzine. The drug substance, cetirizine

dihydrochloride, is commonly referred to as cetirizine hydrochloride or cetirizine HCl. The

chemical name is (±) - [2- [4- [ (4-chlorophenyl)phenylmethyl] -1- piperazinyl] ethoxy]acetic acid,

dihydrochloride. Cetirizine hydrochloride is a racemic compound with an empirical formula of

C21H25ClN2O3•2HCl. The molecular weight is 461.82 and the chemical structure is shown below

[Figure 3.2]. Cetirizine HCl has three ionizable moieties resulting in pKa values of 2.2, 2.9 and 8.0.

At physiological pH, it predominantly exists as a zwitterion or an anion. Cetirizine HCl is a white

or almost white powder that is freely soluble in water, practically insoluble in acetone and in

methylene chloride. Its melting point is 110°C to 115°C.

[Figure 3.2 Chemical structure of Cetirizine dihydrochloride (CTZ)]


100

3.4.1 Indications

For the relief of symptoms associated with seasonal allergic rhinitis, perennial allergic rhinitis and

the treatment of the uncomplicated skin manifestations of chronic idiopathic urticaria. [46].

3.4.2 Pharmacokinetics

Cetirizine undergoes rapid absorption where the maximum plasma concentration is reached at about

1 hour following oral administration of tablets, chewable tablets, and syrup. Comparable

bioavailability is found between the tablet and syrup dosage forms. No accumulation is observed

following multiple dosing of cetirizine HCI (10 mg tablets once daily for 10 days) in healthy

subjects. Cetirizine pharmacokinetics is linear for oral doses ranging from 5 to 60 mg. The mean

plasma protein binding of cetirizine hydrochloride is 93%, independent of concentration in the

range of 25 to 1000 ng/mL, which includes the therapeutic plasma concentrations. The mean

elimination half-life is 8.3 hours and the apparent total body clearance for cetirizine is 53 mL/min

following oral administration of cetirizine in healthy subjects. [47]

3.4.3 Ambroxol dosage

Cetirizine can be taken without regard to food consumption. Cetirizine is available as 5 mg and

10 mg tablets, 1 mg/mL syrup, and 5 mg and 10 mg chewable tablets which can be taken with or

without water [48].

Adults and children 12 years and older: The recommended initial dose of cetirizine is 5 mg

or 10 mg per day in adults and children 12 years and older, depending on symptom severity.

Children 6 to 11 years: The recommended initial dose of cetirizine in children aged 6 to

11 years is 5 mg or 10 mg once daily depending on symptom severity.

Children 2 to 5 years: The recommended initial dose of cetirizine in children aged 2 to 5

years is 2.5 mg (half teaspoon) syrup once daily. The dosage in this age group can be

increased to a maximum dose of 5 mg per day given as 1 teaspoon syrup once a day or one

½ teaspoon syrup given every 12 hours, or one 5 mg chewable tablet once a day.

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101

Children 6 months to <2 years: The recommended dose of cetirizine syrup in children 6

months to 23 months of age is 2.5 mg (half teaspoon) once daily. The dose in children 12 to

23 months of age can be increased to a maximum dose of 5 mg per day, given as half

teaspoon (2.5 mg) every 12 hours. Syrup is recommended for children under the age of 2

years.

3.5 PARABENS (METHYLPARABEN AND PROPYLPARABEN)

Parabens are a class of chemicals widely used as preservatives by cosmetic and pharmaceuticals

industries. Parabens are effective preservatives in many types of formulas. These compounds, and

their salts, are used primarily for their bactericidal and fungicidal properties. They can be found in

shampoos, commercial moisturizers, shaving gels, personal lubricants, topical/parenteral

pharmaceuticals, spray tanning solution, makeup, and toothpaste [49]. They are also used as food

additives.

Their efficacy as preservatives, in combination with their low cost, the long history of their use, and

the inefficacy of some natural alternatives like grapefruit seed extract (GSE) [50], probably

explains why parabens are so commonplace. They are becoming increasingly controversial,

however, because they have been found in extremely low concentrations in breast

cancer tumors (an average of 20 nanograms/g of tissue) [51]. Parabens have also displayed the

ability to slightly mimic estrogen (a hormone known to play a role in the development of breast

cancer) [51]. No effective direct links between parabens and cancer have been established, however

[52]. Another concern is that the estrogen-mimic aspect of parabens may be a factor in the

increasing prevalence of early puberty in girls [53].

3.5.1 Chemistry

Parabens are esters of para-hydroxybenzoic acid, from which the name is derived. Common

parabens include methylparaben (E number E218), ethylparaben (E214), propylparaben (E216) and

http://en.wikipedia.org/wiki/Bactericide

http://en.wikipedia.org/wiki/Fungicide

http://en.wikipedia.org/wiki/Moisturizer

http://en.wikipedia.org/wiki/Shaving_gel

http://en.wikipedia.org/wiki/Personal_lubricant



http://en.wikipedia.org/wiki/Grapefruit_seed_extract

http://en.wikipedia.org/wiki/Breast_cancer

http://en.wikipedia.org/wiki/Breast_cancer

http://en.wikipedia.org/wiki/Tumors

http://en.wikipedia.org/wiki/Estrogen

http://en.wikipedia.org/wiki/Ester

http://en.wikipedia.org/wiki/4-Hydroxybenzoic_acid

http://en.wikipedia.org/wiki/Methylparaben

http://en.wikipedia.org/wiki/E_number

http://en.wikipedia.org/wiki/Ethylparaben

http://en.wikipedia.org/wiki/Propylparaben


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butylparaben. Less common parabens include isobutylparaben, isopropylparaben, benzylparaben

and their sodium salts. The general chemical structure of a paraben is shown in Figure 3.3, where R

symbolizes an alkyl group such as methyl, ethyl, propyl or butyl.

[Figure 3.3 Chemical structure of Paraben]

3.5.2 Occurrence

Some parabens are found naturally in plant sources. For example, methylparaben is found

in blueberries [54-56], where it acts as an antimicrobial agent.

3.5.3 Synthesis

All commercially used parabens are synthetically produced, although some are identical to those

found in nature. They are produced by the esterification of para-hydroxybenzoic acid with the

appropriate alcohol. para-Hydroxybenzoic acid is in turn produced industrially from a modification

of the Kolbe-Schmitt reaction, using potassium phenoxide and carbon dioxide.

3.5.4 Toxicology

Studies on the acute, subchronic, and chronic effects in rodents indicate that parabens are

practically non-toxic [57, 58]. Parabens are rapidly absorbed, metabolized, and excreted [57]. The

major metabolites of parabens are p-hydroxybenzoic acid (pHBA), p-hydroxyhippuric acid

(M1), p-hydroxybenzoyl glucuronide (M3), and p-carboxyphenylsulfate (M4) [59].

3.5.5 Regulation

The European Scientific Committee on Consumer Products (SCCP) stated in 2006 that the

available data on parabens do not enable a decisive response to the question of whether propyl,

http://en.wikipedia.org/wiki/Butylparaben

http://en.wikipedia.org/wiki/Methyl

http://en.wikipedia.org/wiki/Ethyl_group

http://en.wikipedia.org/wiki/Propyl

http://en.wikipedia.org/wiki/Butyl

http://en.wikipedia.org/wiki/Blueberry

http://en.wikipedia.org/wiki/Esterification

http://en.wikipedia.org/wiki/4-hydroxybenzoic_acid

http://en.wikipedia.org/wiki/Alcohol

http://en.wikipedia.org/wiki/Kolbe-Schmitt_reaction

http://en.wikipedia.org/wiki/Phenol

http://en.wikipedia.org/wiki/Carbon_dioxide

http://en.wikipedia.org/wiki/Glucuronide

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butyl and isobutyl paraben can be safely used in cosmetic products at individual concentrations up

to 0.4%, which is the allowed limit in the EU [60].

3.5.6 Methylparaben

Methylparaben (MP) is effective preservative in many types of pharmaceutical formulations. It is

the Methyl ester of p-hydroxybenzoic acid. Methylparaben is chemically Methyl 4-

hydroxybenzoate and its chemical structure is shown in Figure 3.4.

[Figure 3.4 Chemical structure of Methylparaben (MP)]

Its molecular weight is 152.15 g/mole with molecular formula C8H8O3. MP is a white crystalline

powder that is easily soluble in diethyl ether, acetone and slightly soluble in cold water, hot water.

Its melting point is 126°C to 128°C.

3.5.7 Propylparaben

Propylparaben (PP) is effective preservative in many types of pharmaceutical formulations. It is the

Propyl ester of p-hydroxybenzoic acid. Propylparaben is chemically Propyl 4-hydroxybenzoate and

its chemical structure is shown in Figure 3.5.

[Figure 3.5 Chemical structure of Propylparaben (PP)]


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Its molecular weight is 180.2 g/mole with molecular formula C10H12O3. PP is a white crystalline

powder that is easily soluble in methanol, diethyl ether, acetone and slightly soluble in cold water,

hot water. Its melting point is 96°C to 99°C.

3.6 EXPERIMENTAL

3.6.1 Materials and reagents

Drug product, placebo solution, working standards and reference standards are provided by Dr.

Reddy’s laboratories Ltd., Hyderabad, India. HPLC grade acetonitrile and methanol are obtained

from J.T.Baker (NJ., USA). GR grade potassium dihydrogen phosphate, GR grade orhtophosphoric

acid and GR grade triethylamine are obtained from Merck Ltd. (Mumbai, India). 0.22 µm nylon

membrane filter and nylon syringe filters are purchased from Pall life science limited (India). 0.22

µm PVDF syringe filter is purchased from Millipore (India). High purity water is generated by

using Milli-Q Plus water purification system (Millipore®, Milford, MA, USA).

3.6.2 Equipments

Acquity UPLCTM

system (Waters, Milford, USA), consisting of a binary solvent manager, sample

manager and PDA (photo diode array) detector. System control, data collection and data processing

are accomplished using Waters EmpowerTM

-2 chromatography data software. Cintex digital water

bath is used for specificity study. Photo stability studies are carried out in a photo-stability chamber

(SUNTEST XLS+, ATLAS, Germany). Thermal stability studies are performed in a dry air oven

(Cintex, Mumbai, India).

3.6.3 Preparation of mobile phase and its gradient program

Mobile Phase-A (MP-A): Mixture of 0.01M phosphate buffer (KH2PO4) in 0.1% triethylamine.

Mobile phase-B (MP-B): Acetonitrile.

MP-A and MP-B is filtered through 0.22 µm nylon membrane filter and degassed under vacuum

prior to use.

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105

Table 3.1 Gradients program for elution

Time (min) Flow rate (mL/min) % MP-A % MP-B Curve

Initial 0.5 70 30 Isocratic

0.2 0.5 70 30 Isocratic

3.0 0.5 5 95 Linear

3.1 0.5 70 30 Isocratic

3.5 0.5 70 30 Equilibration

3.6.4 Diluent preparation

Mixture of water and methanol in the ratio of 50:50 (v/v) respectively.

3.6.5 Chromatographic conditions

The chromatographic condition is optimized using Agilent Eclipse Plus C18, RRHD 1.8 µm (50

mm x 2.1 mm) column. The finally selected and optimized conditions are as follows: injection

volume 4 µL, gradient elution [Table 3.1], at a flow rate of 0.5 mL/min at 50°C (column oven)

temperature, detection wavelength 237 nm. Under these conditions, the backpressure in the system

is about 6,000 psi. The stress degraded samples and the solution stability samples are analyzed

using a PDA detector covering the range of 200-400nm.

3.6.6 Standard solution preparation

Standard solution is prepared by dissolving standard substances in diluent to obtain solution

containing 120 µg/mL of Ambroxol hydrochloride, 20 µg/mL of Cetirizine hydrochloride, 40

µg/mL of Methylparaben and 4 µg/mL of Propylparaben.

3.6.7 Sample solution preparation

An accurately weighed 2 g of sample solution is taken into the 100 mL volumetric flask. About 70

mL of diluent is added to this volumetric flask and sonicated in an ultrasonic bath for 5 min. This


106

solution is then diluted up to the mark with diluent and mixed well. It is then filtered through 0.22

µm PVDF syringe filter and the filtrate is collected after discarding first few milliliters.

3.6.8 Placebo solution preparation

An accurately weighed 2 g of placebo solution is taken into the 100 mL volumetric flask. About 70

mL of diluent is added to this volumetric flask and sonicated in an ultrasonic bath for 5 min. This

solution is then diluted up to the mark with diluent and mixed well. It is then filtered through 0.22

µm PVDF syringe filter and the filtrate is collected after discarding first few milliliters.

3.6.9 Market product sample solution preparation (for oral solution)

An accurately weighed X g of sample solution is taken into 100 mL volumetric flask (where X= 4

gm for Xyzal® [UCB, India Pvt. Ltd.; B.No.-VO 10001], 2 gm of ZyrCold

® [UCB, India Pvt. Ltd.;

B.No.-LI10035] and 2 g of Relent® [Dr. Reddy’s Lab. Ltd. India; B.No.-L 0590]). About 70 mL of

diluent is added to this volumetric flask and sonicated in an ultrasonic bath for 3 min. This solution

is then diluted up to the mark with diluent and mixed well. It is then filtered through 0.22 µm

PVDF syringe filter and the filtrate is collected after discarding first few milliliters.

3.6.10 Market product sample solution preparation (for oral tablet)

Twenty tablets are crushed to fine powder. An accurately weighed portion of the powder equivalent

to 5 mg of CTZ is taken into 100 mL volumetric flask (Cetzine® Tablets [GSK Pharmaceuticals

Ltd.; B.No.-L473], Dio-1® Tablets [Unison pharmaceuticals; B.No.-2005] and ZyrCold

® Tablets

[UCB, India Pvt. Ltd.; B.No.-5829]). About 70 mL of diluent is added to this volumetric flask and

sonicated in an ultrasonic bath for 15 min. This solution is then diluted up to the mark with diluent

and mixed well. It is then filtered through 0.22 µm PVDF syringe filter and the filtrate is collected

after discarding first few milliliters.

3.6.11 Method Validation

The method described herein has been validated for simultaneous, assay determination by UPLC.

Chapter-3

107

3.6.11.1 Specificity

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

capability of the proposed method. The sample is exposed to acid hydrolysis [0.1N HCl (3 mL),

60°C, 1h], base hydrolysis [0.1N NaOH (3 mL), 60°C, 1h], oxidative [6% w/v H2O2 (3 mL), 60°C,

1h], thermal [60°C, 1h] and photolytic degradation [1.2 million Lux hours]. All exposed samples

are than analysed by the developed method.

3.6.11.2 System suitability

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

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

characteristics including % RSD, resolution (between CTZ and PP), tailing factor and theoretical

plate number are measured.

3.6.11.3 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 UPLC, and is

performed on different days.

3.6.11.4 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 AMB, CTZ, MP and PP at

each level and each replicate are determined. The mean of percentage recoveries (n = 9) and the

relative standard deviation are also calculated.

3.6.11.5 Linearity

Linearity is demonstrated from 1.5 to 150 % of standard concentration using a minimum of seven

calibration levels (25 %, 50 %, 75 %, 100 %, 125 %, 150 % and 200 %) for AMB, CTZ, MP and


108

PP. The method of linear regression is used for data evaluation. The peak areas of the standard

compounds are plotted against the respective AMB, CTZ, MP and PP concentrations. Linearity is

described by the linearity equation, correlation coefficient and Y-intercept bias is also determined.

3.6.11.6 Robustness

The robustness is a measure of the capacity of a method to remain unaffected by small but

deliberate changes in flow rate (± 0.05 mL/min) and change in column oven temperature (± 5 °C).

The theoretical plates, tailing factor and retention behaviour of all interested compounds (AMB,

CTZ, MP and PP) and the resolution (between CTZ and PP) are evaluated.

3.6.11.7 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 12 and 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 12 and 24h, and % RSD is calculated.

3.6.11.8 Filter compatibility

Filter compatibility is performed for nylon 0.22 μm syringe filter (Pall Life sciences) and PVDF

0.22 μm syringe filter (Millipore). To confirm the filter compatibility in proposed analytical

method, filtration recovery experiment is carried out by sample filtration technique. Sample is

filtered through both syringe filters and percentage assay is determined and compared against

centrifuged sample.

3.6.2 Application of the Method to Dosage Forms

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

available various dosage forms.

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109

3.7 RESULTS AND DISCUSSION

3.7.1 Method Development and Optimization

The main objective of the RP-UPLC method development is to rapid and simultaneous

determination of AMB, CTZ, MP and PP in liquid pharmaceutical formulation are: the method

should be able to determine assay of four compounds in single run and should be accurate,

reproducible, robust, stability indicating, filter compatible, linear, free of interference from blank /

placebo / impurities / degradation products and straightforward enough for routine use in quality

control laboratory.

The spiked solution of AMB (120 μg/mL), CTZ (20 μg/mL), MP (40 μg/mL) and PP (4 μg/mL) is

subjected to separation by RP-UPLC. Label claim of compounds and its working concentration is

presented in Table 3.2.

Table 3.2 Formulation label claim with its working concentration

Compound Formulation label claim per 5 mL Working concentration

mg/mL µg/mL

AMB Ambroxol hydrochloride 30 mg 0.12 120

CTZ Cetirizine hydrochloride 5 mg 0.02 20

MP Methylparaben 10 mg 0.04 40

PP Propylparaben 1 mg 0.004 4

Initially the separation of all compounds is studied using water as a MP-A and acetonitrile as a MP-

B on UPLC column (Eclipse Plus C18, RRHD, 50 x 2.1 mm; 1.8 μm) and Waters (UPLC) system

with the linear gradient program. The flow rate of 0.5 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 performed with above condition we observed broad peak of all the compounds.


110

Various types of MP-A and B are studied to optimize the method, which are summarized in Table

3.3 with the observation. Based on above solvent selection study optimized UPLC parameters are;

flow rate 0.5 mL/min; column oven temperature 50°C; gradient solvent program as per Table 3.1;

0.01M phosphate buffer in 0.1% triethylamine as a MP-A and acetonitrile as a MP-B.

Table 3.3 Summary of solvent used to optimize the method

MP-A MP-B Observation

Retention time (tR) USP tailing

Water Acetonitrile AMB=0.947; MP=1.373

CTZ=2.016; PP=2.748

AMB=2.8; MP=2.0

CTZ=2.5; PP=1.5

0.1M KH2PO4 Acetonitrile AMB=1.101; MP=1.451

CTZ=2.234; PP=2.851

AMB=1.8; MP=1.4

CTZ=1.3; PP=1.3

0.1M KH2PO4 buffer

(pH 3.0 with H3PO4)

Acetonitrile AMB=1.223; MP=1.477

CTZ=2.019; PP=2.835

AMB=1.7; MP=1.4

CTZ=1.3; PP=1.2

0.01M KH2PO4 + 0.1%

triethylamine

Acetonitrile AMB=2.185; MP=0.605

CTZ=1.217; PP=1.389

AMB=0.9; MP=1.3

CTZ=1.0; PP=1.0

USP = United state pharmacopoeia

In order to achieve symmetrical peak of all substances and more resolution between CTZ and PP

different stationary phases are explored. Peak merging (CTZ and PP) is observed with Acquity

BEH C8 (50 x 2.1 mm, 1.7µm) column. Poor resolution (RS=2.3 between CTZ and PP) is observed

with Acquity BEH C18 (50 x 2.1 mm, 1.7µm) column. Finally desired separation with symmetrical

peaks is obtained using Eclipse Plus C18, RRHD (50 x 2.1mm, 1.8µm) column. Column oven

temperature is also studied (at low temperature and 50°C) and found that 50°C is more appropriate

with respect to separation and peak shape. Based on compounds UV spectrums 237nm is found

more appropriate for the simultaneous determination. UV spectra and IUPAC name of AMB, CTZ,

MP and PP are presented in Figure 3.6.

Chapter-3

111

Ambroxol hydrochloride (AMB)

(1s,4s)-4-((2-amino-3,5-dibromocyclohexyl)methylamino)cyclohexanol hydrochloride

Cetirizine hydrochloride (CTZ); Cetirizine dihydrochloride

2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazine-1-yl)ethoxy)acetic acid

Methylparaben (MP): Methyl 4-hydroxybenzoate


112

Propylparaben (PP): Propyl 4-hydroxybenzoate

[Figure 3.6 UV spectra and IUPAC name of AMB, CTZ, MP and PP]

AMB, CTZ, MP and PP are well resolved with each other and also well resolved with all twelve

known impurities/ degradation products in reasonable time of 3.5 minutes which is presented in

Figure 3.7. There is no chromatography interference due to blank (diluent) and excipients (placebo)

at the retention time of AMB, CTZ, MP and PP which is presented in Figure 3.8.

[Figure 3.7 Overlaid chromatograms of placebo, blank and spiked impurities

along with analytes]

Chapter-3

113

[Figure 3.8 Overlaid specimen chromatograms of blank, placebo and standard

preparation]

3.7.2 Analytical Parameters and Validation

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

guidelines [44]. The method is validated to demonstrate that it is suitable for its intended purpose

by the standard procedure to evaluate adequate validation characteristics (system suitability,

accuracy, precision, linearity, robustness, solution stability, filter compatibility and stability

indicating capability).

3.7.2.1 Specificity

Specificity is the ability of the method to measure the analyte response in the presence of its

potential impurities [44]. There is no any interferences at the RT (retention time) of AMB, CTZ,

MP and PP due to blank, placebo, impurities and degradation products [Figure 3.7 and 3.8]. Peak

purity plot of AMB, CTZ, MP and PP are presented in Figure 3.9.

Ambroxol HCl (AMB) Cetirizine HCl (CTZ)


114

Methylparaben (MP) Propylparaben (PP)

[Figure 3.9 Spectral purity plot of AMB, CTZ, MP and PP]

Chemical name of all impurities with its purity plot are presented in Figure 3.10.

AMB IMP-A

(2-amino-3,5-dibromophenyl)methanol

AMB IMP-B

trans-4-(6,8-dibromo-1,4-dihydroquinazolin-

3(2H)-yl)cyclohexanol

AMB IMP-C

trans-4-[[(E)-2-amino-3,5-

dibromobenzyliden] amino]cyclohexanol

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115

AMB IMP-D

cis-4-[(2-amino-3,5-dibromobenzyl)amino]

cyclohexanol

AMB IMP-E

2-amino-3,5-dibromobenzaldehyde

CTZ IMP-A

R1 = R2 = H, R3 = Cl:

(RS)-1-[(4-chlorophenyl)phenylmethyl]

piperazine

CTZ IMP-B

R1 = CH2-CO2H, R2 = H, R3 = Cl:

(RS)-2-[4-[(4-chlorophenyl)phenylmethyl]

piperazin-1-yl]acetic acid


116

CTZ IMP-C

R1=CH2-CH2-O-CH2-CO2H, R2=Cl, R3=H:

(RS)-2-[2-[4-[(2-chlorophenyl)phenylmethyl]

piperazin-1-yl]ethoxy]acetic acid

CTZ IMP-D

1,4-bis[(4-chlorophenyl)phenylmethyl]

piperazine

CTZ IMP-E

R1=CH2-[CH2-O-CH2]2-CO2H, R2=H, R3=Cl:

(RS)-2-[2-[2-[4-[(4-chlorophenyl)

phenylmethyl]piperazin-1-yl]ethoxy]ethoxy]

aceticacid (ethoxycetirizine)

CTZ IMP-F

R1 = CH2-CH2-O-CH2-CO2H, R2 = R3 = H:

[2-[4-(diphenylmethyl)piperazin-1-

yl]ethoxy]acetic acid

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117

CTZ IMP-G

R1 = CH2-CH2-OH, R2 = H, R3 = Cl:

2-[4-[(RS)-(4-chlorophenyl)phenylmethyl]

piperazin-1-yl]ethanol

[Figure 3.10 Chemical structure, IUPAC name and purity plot of all impurities]

Degradation are observed when the drug product is subjected to acid hydrolysis (0.1N HCl, 60°C,

1h, Figure 3.11), base hydrolysis (0.1N NaOH, 60°C, 1h, Figure 3.12), oxidative (6% H2O2, 60°C,

1h, Figure 3.13), thermal (60°C, 1h, Figure 3.14) and photolytic degradation (1.2 million Lux

hours, Figure 3.15). Significant degradation is observed when the drug product is subjected to base

hydrolysis leading to the formation of unknown impurity after the peak of MP, CTZ impurity-D, E

and AMB impurity-B (Figure 3.12). Peaks due to AMB, CTZ, MP and PP are investigated for

spectral purity in the chromatogram of all exposed samples and found spectrally pure [Table 3.4].

[Figure 3.11 Overlaid chromatograms of acid hydrolysis study]


118

[Figure 3.12 Overlaid chromatograms of base hydrolysis study]

[Figure 3.13 Overlaid chromatograms of peroxide degradation study]

[Figure 3.14 Overlaid chromatograms of heat degradation study]

Chapter-3

119

[Figure 3.15 Overlaid chromatograms of photolytic degradation study]

Table 3.4 Peak purity data obtained from forced degradation study

Stress conditions Purity flag

$ (Peak purity)

AMB CTZ MP PP

Unstressed sample No No No No

0.1N HCl at 60°C for 1h No No No No

0.1N NaOH at 60°C for 1h No No No No

6 % H2O2 at 60°C for 1h No No No No

Heat at 60°C for 1h No No No No

Photolytic (1.2 million Lux hours) No No No No

$=For waters UPLC system,

Purity flag: No, which indicates that purity angle is less than purity threshold and

Purity flag: Yes, which indicates that purity angle is more than purity threshold

3.7.2.2 System suitability

System suitability results from precision and intermediate precision study are summarized in Table

3.5 with its proposed acceptance criteria. The percentage RSD of area counts of six 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. Overlaid chromatograms of six

replicate standards are presented in Figure 3.16.


120

Table 3.5 System suitability results (precision and intermediate precision study)

Test Parameters MP CTZ PP AMB Proposed

criteria

Precision

(n=6)

USP resolution -- -- 4.34 -- NLT 3.5

USP tailing 1.3 1.0 1.0 0.9 NMT 1.5

USP plate count 4549 24009 18630 33643 NLT 3000

Area % RSD 0.2 0.2 0.4 0.1 NMT 2.0%

Intermediate

precision

(n=6)


USP tailing 1.3 1.0 1.0 0.9 NMT 1.5


Area % RSD 0.3 0.2 0.5 0.2 NMT 2.0%

NLT= Not less than; NMT= Not more than; USP=United State Pharmacopeia

[Figure 3.16 Overlaid chromatograms of six replicate standard injections]

3.7.2.3 Precision

The precision of the assay method is evaluated by carrying out six independent determination of

AMB, CTZ, MP and PP (120 µg/mL of AMB, 20 µg/mL of CTZ, 40 µg/mL of MP and 4 µg/mL of

PP) 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 0.3 % for AMB, 0.5

Chapter-3

121

% for CTZ, 0.4 % for MP and 0.7 % for PP, 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 3.6].

The purpose of this 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 3.6]. The % RSD is 0.4 % for AMB, 0.6 % for CTZ, 0.7 % for MP and 0.9 % for PP, which

are well within the acceptance criteria of 2.0%, so this study proves that the method to be rugged

enough for day to day use. Overlaid chromatograms of precision and intermediate precision study

are presented in Figure 3.17 and Figure 3.18 respectively.

Table 3.6 Precision (n=6) and Intermediate precision (n=6) results

Substance Precision at 100% Intermediate precision

Mean % assay % RSD Mean % assay % RSD

AMB 101.1 0.3 101.0 0.4

CTZ 99.3 0.5 99.5 0.6

MP 98.1 0.4 98.3 0.7

PP 97.5 0.7 97.2 0.9

[Figure 3.17 Overlaid chromatograms of precision study]


122

[Figure 3.18 Overlaid chromatograms of intermediate precision study]

3.7.2.4 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 each drug corresponding to three concentration levels; 50, 100,

and 150% of the label claim [Table 3.2] along with the excipients in triplicate. The samples are

given the same treatment as described in sample preparation. The percentage recoveries of AMB,

CTZ, MP and PP 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 ±

1.0 % of amount added, which indicates that there is no interference due to excipients present in

liquid oral formulation. It is confirmed from results that the method is highly accurate [Table 3.7].

Accuracy study chromatograms are presented in Figure 3.19.

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123

Table 3.7 Accuracy results

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

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

AMB 100.1 0.3 99.8 0.2 99.8 0.2

CTZ 99.7 0.4 99.9 0.3 100.2 0.2

MP 100.2 0.3 99.8 0.2 99.7 0.2

PP 100.6 0.5 100.4 0.4 99.6 0.6

[Figure 3.19 Overlaid chromatograms of accuracy study]

3.7.2.5 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 AMB, CTZ, MP and

PP are 120, 20, 40 and 4 μg/mL, respectively. The response function is determined by preparing

standard solutions at seven different concentration levels ranging from 30.04-240.32 μg/mL for

AMB, 5.01-40.08 μg/mL for CTZ, 9.97-79.76 μg/mL for MP and 1.005-8.04 μg/mL for PP (25 to

200% of analyte concentration). The response is found linear from 25% to 200% of standard

concentration. For all compounds the correlation coefficient is greater than 0.999. The regression

statistics are shown in Table 3.8. Overlaid specimen chromatograms of linearity study are presented

in Figure 3.20. Linearity plot are also presented in Figure 3.21-3.24.


124

Table 3.8 Regression statistics

Compound Linearity range

(µg/mL)

Correlation

coefficient (r2)

Linearity (Equation) Y- intercept

bias in %

AMB 30.04 to 240.32 0.9996 y =9020.7497(x) + 6130.5328 0.557

CTZ 5.01 to 40.08 0.9998 y =11111.1220(x) – 263.1311 -0.117

MP 9.97 to 79.76 0.9997 y =18542.8499(x) + 5084.6885 0.676

PP 1.005 to 8.04 0.9997 y =16185.3682(x) – 118.5082 -0.181

[Figure 3.20 Overlaid chromatograms of linearity study]

[Figure 3.21 Linearity of Ambroxol hydrochloride]

y = 9020.7497x + 6130.5328

R² = 0.9996

0

500000

1000000

1500000

2000000

2500000

0 50 100 150 200 250 300

Are

a

Conc. in ppm

Chapter-3

125

[Figure 3.22 Linearity of Cetirizine hydrochloride]

[Figure 3.23 Linearity of Methylparaben]

[Figure 3.24 Linearity of Propylparaben]

y = 11111.1220x - 263.1311

R² = 0.9998

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

0 5 10 15 20 25 30 35 40 45

Are

a

Conc. in ppm

y = 18,542.8499x + 5,084.6885

R² = 0.9997

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

0 10 20 30 40 50 60 70 80 90

Are

a

Conc. in ppm

y = 16185.3682x - 118.5082

R² = 0.9997

0

20000

40000

60000

80000

100000

120000

140000

0 1 2 3 4 5 6 7 8 9

Are

a

Conc. in ppm


126

3.7.2.6 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 flow rate (± 0.05 mL/min) and column oven temperature (± 5°C) on the

retention time, resolution (between CTZ and PP), theoretical plates and tailing factor are studied.

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 3.9]. Robustness study chromatograms are presented in Figure 3.25 and 3.26.

Table 3.9 Robustness study results

Condition Parameters MP CTZ PP AMB Proposed

criteria

Normal

methodology


USP tailing 1.3 1.0 1.0 0.9 NMT 1.5


Retention time in min 0.603 1.224 1.385 2.183 --

At flow rate

0.45 mL/min


USP tailing 1.3 1.0 1.0 0.9 NMT 1.5



At flow rate

0.55 mL/min


USP tailing 1.3 1.0 1.0 0.9 NMT 1.5



At 45°C

column oven

temp.


USP tailing 1.3 1.0 1.0 0.9 NMT 1.5



At 55°C

column oven

temp.


USP tailing 1.3 1.0 1.0 0.9 NMT 1.5



Chapter-3

127

[Figure 3.25 Chromatograms of column oven temperature study]

[Figure 3.26 Chromatograms of flow rate study]


128

3.7.2.7 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 and purity of analyte in the sample at the

time of analysis corresponds to the concentration and purity of analyte at the time of sampling.

Stability of sample solution is established by storage of sample solution at ambient temperature

(25°C) for 24h. Sample solution is re-analyzed after 12 and 24h time intervals and assay are

determined for the compounds (AMB, CTZ, MP and PP) 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 3.10. 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 12 and 24h time intervals and % RSD of all injected standard

injections are calculated. Standard solution does not show any appreciable change in % RSD (RSD

for AMB, CTZ, MP and PP are less than 1.0%) value when stored at ambient temperature up to

24h. Overlaid specimen chromatograms for standard and sample solution stability are presented in

Figure 3.27 and 3.28 respectively.

[Figure 3.27 Overlaid specimen chromatograms of standard solution stability]

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129

[Figure 3.28 Overlaid specimen chromatograms of sample solution stability]

Table 3.10 Solution stability results

Time intervals AMB CTZ MP PP

% Assay Initial 100.7 99.5 98.0 97.6

% Assay after 12h 100.3 99.6 98.2 97.7

% Assay after 24h 100.4 99.3 98.1 97.4

3.7.2.8 Filter compatibility

Filter compatibility is performed for nylon 0.22 μm syringe filter (Pall Life sciences) and PVDF

0.22 μm syringe filter (Millipore). To confirm the filter compatibility in proposed analytical

method, filtration recovery experiment is carried out by sample filtration technique. Sample is

filtered through both syringe filters and percentage assay is determined and compared against

centrifuged sample. Sample solution does not show any significant changes in assay percentage

with respect to centrifuged sample. Percentage assay results are presented in Table 3.11. In

displayed result difference in % assay is not observed more than ±0.5, which indicates that both

syringe filters having a good compatibility with sample solution. Overlaid chromatograms of filter

compatibility study are presented in Figure 3.29.


130

Table 3.11 Filter compatibility results (Assay % w/w)

Compound Centrifuged PVDF filter 0.22µm

(Millipore)

Nylon filter 0.22µm

(Pall Life Sciences)

AMB 100.5 100.3 100.3

CTZ 99.7 99.5 99.4

MP 98.3 98.3 98.5

PP 97.5 97.6 97.3

[Figure 3.29 Overlaid specimen chromatograms of filter compatibility study]

3.7.3 Application of the method to dosage forms

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

available various dosage forms. The results obtained are as shown in Table 3.12. Based on obtained

results developed method is suitable for the various marketed dosage forms. Developed method

also proves the suitability for preservatives determination in various liquid dosage forms.

Representative chromatograms of analysed marketed products are presented in Figure 3.30.

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131

Table 3.12 Results of market products (mg/ 5 mL for syrup and mg/ tablets for

tablets)

Product Name and

Labeled claim (in mg) AMB CTZ MP PP LCTZ

ZyrCold Syrup [AMB(30); CTZ(2.5)] 29.8 2.47 9.53 0.94 N.A.

Relent Syrup [AMB(30); CTZ(2.5)] 30.1 2.48 10.10 1.01 N.A.

ZyrCold Tablets [AMB(30); CTZ(2.5)] 29.7 2.46 N.A. N.A. N.A.

Cetzine Tablets [CTZ(10)] N.A. 9.93 N.A. N.A. N.A.

DOI-1 Tablets [CTZ(10)] N.A. 9.72 N.A. N.A. N.A.

Xyzal Syrup [LCTZ (2.5)] N.A. N.A. 10.5 1.07 2.51

N.A.; not applicable

[Figure 3.30 Specimen chromatograms of analysed marketed products]


132

3.8 CALCULATION FORMULA

3.8.1 Assay (% w/w)

Calculated the quantity, in mg, of compound (AMB, CTZ, MP and PP) 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 obtained from the sample preparation

Rstd = Compound peak response (mean peak area) obtained from the standard preparation

3.8.2 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

3.8.3 Accuracy (% Recovery)

It is calculated using the following equation:

Chapter-3

133

3.9 CONCLUSION

A gradient RP-UPLC method is successfully developed for the simultaneous estimation of AMB,

CTZ, MP and PP in liquid pharmaceutical formulation. The developed method is selective, precise,

accurate, linear, filter compatible and robust. Forced degradation data proves that the method is

specific for the analytes and free from the interference of placebo / known impurities / degradation

products and unknown degradation products. The run time (3.5 min) enables for rapid

determination of drugs and preservatives. Moreover, it may be applied for individual and

simultaneous determination of AMB, CTZ, LCTZ, MP and PP compound in pharmaceutical drug

product and substance. Also it can be utilized for determination of assay, blend uniformity and

content uniformity of pharmaceutical products (CTZ tablets and AMB+CTZ tablets), where sample

load is higher and high throughput is essential for faster delivery of results.

Note: Intellectual Property Management (IPM) clearance number for the present research

work is: PUB-00099-11.


134

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