chapter-3 simultaneous determination of ambroxol...
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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
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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].
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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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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)]
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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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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
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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,
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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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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
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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.
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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
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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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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.
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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.
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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
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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]
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[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)
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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
Chapter-3
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
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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
Chapter-3
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]
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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.
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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 resolution -- -- 4.35 -- NLT 3.5
USP tailing 1.3 1.0 1.0 0.9 NMT 1.5
USP plate count 4656 23951 18444 33537 NLT 3000
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]
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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.
Chapter-3
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.
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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 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
Retention time in min 0.603 1.224 1.385 2.183 --
At flow rate
0.45 mL/min
USP resolution -- -- 4.79 -- NLT 3.5
USP tailing 1.3 1.0 1.0 0.9 NMT 1.5
USP plate count 4657 24838 18993 34634 NLT 3000
Retention time in min 0.667 1.309 1.498 2.319 --
At flow rate
0.55 mL/min
USP resolution -- -- 3.88 -- NLT 3.5
USP tailing 1.3 1.0 1.0 0.9 NMT 1.5
USP plate count 4790 22386 17442 32067 NLT 3000
Retention time in min 0.549 1.160 1.300 2.081 --
At 45°C
column oven
temp.
USP resolution -- -- 5.3 -- NLT 3.5
USP tailing 1.3 1.0 1.0 0.9 NMT 1.5
USP plate count 5310 23455 19314 32483 NLT 3000
Retention time in min 0.628 1.224 1.434 2.186 --
At 55°C
column oven
temp.
USP resolution -- -- 3.6 -- NLT 3.5
USP tailing 1.3 1.0 1.0 0.9 NMT 1.5
USP plate count 4481 22939 16790 32440 NLT 3000
Retention time in min 0.580 1.210 1.337 2.172 --
Chapter-3
127
[Figure 3.25 Chromatograms of column oven temperature study]
[Figure 3.26 Chromatograms of flow rate study]
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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]
Chapter-3
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.
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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.
Chapter-3
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]
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
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.
Simultaneous Determination of AMB, CTZ, MP and PP in Liquid Pharmaceutical Formulation
134
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