STABILITY INDICATING LIQUID …shodhganga.inflibnet.ac.in/bitstream/10603/82401/9/09_chapter 2.pdfStability indicating liquid chromatographic methods for ... reported a new HPLC approach

Stability indicating liquid chromatographic methods for the simultaneous determination of drug products.

Patil A.T. and Shaikh K.A. 26

CHAPTER – II

STABILITY INDICATING LIQUID

CHROMATOGRAPHIC METHODS FOR

THE SIMULTANEOUS

DETERMINATION OF DRUG

PRODUCTS.



2.1. INTRODUCTION:

Stability testing forms an important part of the drug product testing , it provide evidence

on how quality of a drug substance or drug product varies with time under the influence of a

variety of environmental factors such as temperature, humidity, light and enables

recommendation of storage conditions, retest period and shelf life to be established. The two

main aspects of drug products that play an important role in shelf life determination are assay of

active drug and degradants generated during the stability study. There are several stability-

indicating methods has been reported for assays of various drugs in drug products containing

only one active drug substance. Only few stability indicating methods are reported for the assay

of combination drug products containing two or more active drug substances.

The objective of the current study is to develop and validated stability indicating

reversed-phase HPLC method for the simultaneous determination of drug product. First HPLC

method is for simultaneous determination of Levocetirizine dihydrochloride and

Pseudoephedrine sulfate in Tablet dosage forms, second sensitive LC method for simultaneous

determination of Ciclesonide and Formoterol fumarate in dry powder inhaler and third Sensitive

LC method for the Simultaneous determination of Diacerein and Aceclofenac in tablet dosage

form.

Levocetirizine dihydrochloride is [2-[4-[(R)-(4-Chlorophenyl)phenylmethyl]-1-

piperazinyl]ethoxy]-acetic acid dihydrochloride is the pharmacologically active enantiomer of

cetirizine, and is a potent histamine H-1 receptor antagonist1 and Pseudoephedrine sulfate

chemically [(S-(R*,R*))-alpha-(1-(Methylamino)ethyl)benzenemethanol sulfate].

Pseudoephedrine sulfate is official in USP2. Levocetirizine dihydrochloride is official in Indian

Pharmacopoeia3. Levocetirizine Dihydrochloride is having antiallergic properties used Once

daily for the treatment of allergic rhinitis4.

A Fixed dose combination of 180 mg of Pseudoephedrine sulfate and 5 mg of

Levocetirizine dihydrochloride is available commercially as tablets and are widely used for the

symptomatic treatment of allergic rhinitis.

Formoterol fumarate is N-[2-Hydroxy-5-[[(1RS)-1-hydroxy-2-[[(1RS)-2-(4-

methoxyphenyl)-1-methylethyl] amino] ethyl] phenyl] formamide(E)-2-butenedioate is b2-



agonist with a long duration of action.5 Ciclesonide is (R)-11b,16a,17,21-Tetrahydroxypregna-

1,4-diene-3, 20-dione cyclic 16,17-acetal with cyclohexanecarboxaldehyde 21-isobutyrate is

effective and novel Inhaled corticosteroids, which has very low affinity for the glucocorticoid

receptor in its native form, but very high affinity when transformed to its active metabolite by

esterase in the lung. 6

Bronchodilator medications are central to the symptomatic management of

chronic obstructive pulmonary disease (COPD). Long-acting inhaled bronchodilators are more

convenient. Also, Systemic corticosteroids are beneficial in the management of acute

exacerbations of COPD. 7

As a result, the combination of b2-agonist and corticosteroids has been

a more useful tool in the management of asthma and COPD. Formoterol fumarate is reported in

British Pharmacopoeia8 whereas Ciclesonide is reported in Indian Pharmacopoeia

9. Fixed dose

combinations of 6 mg of Formoterol fumarate (FF) and 200 mg of Ciclesonide (CS) are available

commercially as dry powder inhaler and are widely used for the treatment of COPD.

Diacerein is chemically 1,8-diacetoxy-3-carboxyanthraquinone and is also known as

diacetylrhein. This drug is used in the treatment of osteoarthritis. After absorption, the drug is

metabolized to its active metabolite rhein10

. Diacerein and rhein are anthraquinone compounds

that ameliorate the course of osteoarthritis11-13

. Aceclofenac is chemically (2-[(2,6-

dichlorophenyl)amino] phenyl acetoxyacetic acid)14

. It has analgesic properties and a good

tolerability profile in a variety of painful conditions. A combined fixed dose of 50 mg Diacerein

and 100 mg Aceclofenac is available commercially as tablets and are widely used for the

treatment of osteoarthritis.

2.2 LITERATURE SURVEY:

In this section summarized some of the important analytical methods for the

determination of Levocetirizine dihydrochloride, Pseudoephedrine sulfate, Formoterol fumarate

Ciclesonide, Aceclofenac and Diacerein as a individual component or in combination with

another drug substance.

Feyyaz O., et. al., (2000)15

Feyyaz O et. al., reported Spectrophotometric method for the simultaneous

determination of Pseudoephedrine Sulfate, Dexbrompheniramine Maleate and Loratadine in



pharmaceutical preparations using derivative spectrophotometry and ratio spectra derivative

spectrophotometry. Two spectrophotometric methods are described for the simultaneous analysis

of pseudoephedrine sulfate-dexbrompheniramine maleate and pseudoephedrine sulfate-loratadine

combinations. The procedures do not require any separation step. Mean recoveries were found to

be >99% in the methods for these compounds in their synthetic mixtures.

Makhija S.N., et. al., (2001)16

Makhija S.N., reported HPTLC method for the simultaneous determination of

pseudoephedrine and cetirizine in pharmaceutical formulations. The solvent system consisted of

ethyl acetate-methanol-ammonia (7:1.5:1, v/v/v). This system was found to give compact spots

for both pseudoephedrine (Rf value of 0.69+/-0.01) and cetirizine (Rf value of 0.38+/-0.01).

Spectrodensitometric scanning-integration was performed at a wavelength of 240 nm.

Nian W., et. al., (2002)17

Nian W., et. al., reported HPLC method for the quantitative determination of

pseudoephedrine sulfate and its related compounds in pharmaceutical preparation. Isocratic

reversed-phase high performance liquid chromatograghic method for the separation of

pseudoephedrine and its related compounds in pharmaceutical formulations is described. The

separation is achieved on a C-18 column (4.6 mm x 25 cm length, 5µ particle size) using a

mobile phase consisting of a mixture of ammonium acetate and methanol. With run time 35

minutes.

Mabrouk M.M., et. al., (2003)18

Mabrouk M.M., et. al., reported Reversed phase liquid chromatographic and first

derivative spectrophotometric methods for determination of antihistaminic drug Loratadine and

nasal decongestant drug Pseudoephedrine sulfate. The HPLC method involves separation of

Loratadine and Pseudoephedrine sulfate on micro-BondaPak C18 column using mixture of

methanol:water:phosphoric acid:ammonium dihydrogen phosphate in the ratio of 220:300:2:3

(V/V/V/W), 60 and 40 acetonitrile as mobile phase flowing at 2 ml/min with ultraviolet detection



at 247 nm. The spectrophotometric method is based on recording the first derivative spectra for

Loratadine and Pseudoephedrine and at 307, 266 nm, respectively.

Tahraoui A., et. al., (2005)19

Tahraoui A., reported a new HPLC approach for the determination of hydrophilic and

hydrophobic components: the case of Pseudoephedrine sulfate and Loratadine in tablets.he

chromatographic behavior of Pseudoephedrine sulfate and Loratadine on RP C18 and C8

columns were studied in presence and absence of sodium lauryl sulfate (SLS). The effect of

combining two different types of stationary phases (cyano and C18 or C8) on the relative

retention of the two compounds was investigated. In conclusion, it was found that the

combination of a C18 column followed by a standard cyano column provides a stationary phase

that separates both compounds effectively and within a reasonable time.

Culzoni M.J., et. al., (2007)20

Culzoni M.J., reported determination of Loratadine and Pseudoephedrine sulfate in

pharmaceuticals based on non- linear second-order spectrophotometric data generated by a pH-

gradient flow injection technique and artificial neural networks, Loratadine

and Pseudoephedrine sulfate were determined in pharmaceutical samples by using non-linear

second-order data generated by a pH-gradient flow injection analysis system with diode-array

detection. Determination of both analytes was performed on the basis of differences between the

acid-base and spectral features of each drug species. Non-linearities were detected by using both

qualitative and quantitative tools.

Lakshmana P., et. al., (2008)21

Lakshmana P., et. al., reported UV spectrophotometric method for the estimation of

Ambroxol hydrochloride and Levocetirizine dihydrochloride. The method involved solving

simultaneous equations based on measurement of absorbance at two wavelengths 242 nm and

231 nm, the γ max of Ambroxol hydrochloride and Levocetirizine dihydrochloride, respectively.

Beer's law was obeyed in the concentration range 10–50 µg/ml and 8–24 µg/ml for Ambroxol



hydrochloride and Levocetirizine dihydrochloride respectively. Results of the method were

validated statistically and by recovery studies.

Hadad G. M., et. al., (2009)22

Hadad G. M., et. al., reported development and validation of a stability-indicating RP

HPLC method for the determination of Paracetamol with Dantrolene or/and Cetirizine

and Pseudoephedrine in two pharmaceutical dosage forms. A gradient mobile phase system

consisting of (A) 50 mmol L(-1) sodium dihydrogen phosphate, 5 mmol L(-1) heptane sulfonic

acid sodium salt, pH 4.2 and (B) acetonitrile was used with discovery reversed-phase HS C(18)

analytical column (250 mm x 4.6 mm i.d., 5 µ particle size). Quantitation was achieved with UV

detection at 214 nm, based on peak area.

Kalogria E., et. al., (2010)23

Kalogria E., et. al., reported a porous graphitized carbon column HPLC method for the

quantification of Paracetamol, Pseudoephedrine, and Chlorpheniramine in a pharmaceutical

formulation. Chromatographic separation was achieved isocratically on an RP porous graphitized

carbon analytical column (125 x 2.1 mm , 5µ) using 5.0 mM ammonium acetate-acetonitrile (35

: 65 v/v) mobile phase at a flow rate of 0.50 mL/min. UV spectrophotometric detection at 220

nm was used.

Reddy J. M., et. al., (2011)24

Reddy J. M., et. al., reported RP-HPLC for simultaneous estimation of

Diethylcarbamazine and Levocetirizine in tablet Formulation. UV detector was carried out at 224

nm, using Princeton Sphere-100 C18 (250×4.6 mm. 5 µ) column. The mobile phase used was

20mM potassium dihydrogen orthophosphate buffer (pH: 3.2):acetonitrile (50:50 v/v) with

isocratic flow rate 1 ml/min. The compounds Diethylcarbamazine, Levocetirizine and

Losartan otassium were eluted at 2.12, 4.27 and 5.96 min, respectively. The peaks were eluted

with better resolution.



Joshi S., et. al., (2012)25

Joshi S., et. al., reported quantization of Dextromethorphan and Levocetirizine in

combined dosage form using a novel validated RP-HPLC method the separation of these

compounds was achieved within 10 min on a Phenomenex (USA) C 18 analytical column,

250Χ4.0 mm i.d., using an isocratic mobile phase consisting of potassium dihydrogen phosphate

buffer (pH 2.5) - acetonitrile- tetrahydrofuran (70:25:5 v/v/v). The analysis was performed at a

flow rate of 1.2 ml/min and at a detection wavelength of 232 nm.

Srividya P., et. al., (2013)26

Srividya P., et. al., reported RP-HPLC method for the simultaneous analysis of

Levocetirizine dihydrochloride, Ambroxol hydrochloride, and Montelukast sodium. Analysis

was carried out on C18 reverse phase column (Phenomenox- RP Aqueous) of 250 × 4.6 mm

dimensions and 5 µm particle size with mobile phase containing 15 mm of Ammonium

acetate:Acetonitrile (40:60 v/v) by using isocratic mode and eluents were monitored at 215 nm.

The retention times for Levocetirizine dihydrochloride, Ambroxol hydrochloride, and

Montelukast sodium. are 2.21, 4.46, and 13.35 nm, respectively.

Other than above mentioned methods few more methods are also reported for the

quantification of Levocetirizine dihydrochloride in combination with another drug substances27,28

and Quantification of Levocetirizine dihydrochloride in human plasma29

. Similarly quantification

of Pseudoephedrine by LC–MS–MS 30

in human plasma and simultaneous quantification

Pseudoephedrine with another drug substances by HPLC31

are reported.

Butter J.J., et. al., (1996)32

Butter J.J., et. al., reported HPLC determination of Formoterol RR and SS

enantiomers in Urine. A method is described for the determination of the R,R- and S,S-

enantiomer of the long-acting ß2-adrenoceptor agonist formoterol, The sample clean-up from

urine takes place by liquid liquid extraction followed by solid phase extraction. An AGP-column

combined with electrochemical detection is used for the separation and detection of the

enantiomers.



Campestrini J., et. al., (1997) 33

Campestrini J., et. al., reported high-performance liquid chromatography and

electrochemical detection method for the determination of formoterol in human plasma. . The

compounds were eluted with pH 6 buffer solution-methanol (70:30, v/v) and the eluate was

further diluted with water. An aliquot of the extract solution was injected and analyzed by

HPLC. The extraction, dilution, injection and chromatographic analysis were combined and

automated using the automate (ASPEC) system. The chromatographic separations were achieved

on a 5 microm, Hypersil ODS analytical column (200 mm x 3 mm I.D.), using (pH 6 phosphate

buffer, 0.035 M + 20 mg/l EDTA)-MeOH-CH3CN (70:25:5, v/v/v) as the mobile phase at a

flow-rate of 0.4 ml/min. The analytes were detected with electrochemical detection at an

operating potential of +0.63 V.

Song J.Z., et. al., (1999)34

Song J.Z., et. al., reported capillary electrophoresis assay method for the determination

formoterol in dry syrup. The development of a capillary zone electrophoresis method with head-

column field-amplified sample stacking injection for the determination of formoterol in a low

dosage dry syrup form was described. To obtain the highest sensitivity, the sample solution was

prepared by high content of organic solvent with the presence of a small amount of H+ (60-100

µ) and the capillary inlet end was dipped in water before electroinjection.

Akapo S., et. al., (2003)35

Akapo S., et. al., reported RP-HPLC Assay of Formoterol and its Related Substances in

Formoterol Fumarate Dehydrate Drug Substance. A stability-indicating reversed-phase high

performance liquid chromatographic (HPLC) method has been developed and validated for

the assay offormoterol fumarate and the related substances, namely, formoterol

fumarate desformyl and formoterol fumarate acetamide analogs, in the active pharmaceutical

ingredient. The separation was achieved by isocratic elution using an Alltech Alltima C18 (150 x

4.6 mm) column, a mobile phase consisting of ammonium acetate (50 mM; pH 5.0)-ethanol

(65:35, v/v), a flow rate of 1.0 ml/min and UV detection at 242 nm.



Akapo S. O., et. al., (2004)36

Akapo S. O., et. al., reported gas chromatographic method for analysis of (RS,SR)-

astereoisomeric impurity in formoterol fumarate. The method involves silylation of formoterol

fumarate with N-(trimethylsilyl)imidazole in N,N-dimethylformamide at room temperature in an

autosampler vial to produce trimethylsilyl derivatives of the enantiomers prior to GC analysis.

The optimized silylation and separation conditions, respectively, produced good yield and

resolution of the analytes. The method appears to be convenient and fast, and permits accurate

determination of (RS,SR)-diastereoisomer in formoterol fumarate with adequate precision.

Nave R., et. al., (2005)37

Nave R., et. al., reported Formation of fatty acid conjugates of ciclesonide active

metabolite in the rat lung after 4-week inhalation of ciclesonide. Ciclesonide and des-CIC

concentrations were determined using solid-phase extraction and reverse-phase high-

performance liquid chromatography with tandem mass spectrometry (LC/MS/MS).

Concentrations of fatty acid ester conjugates were indirectly assessed using enzymatic de-

esterification before LC/MS/MS.

Prasad A.V.S.S., et. al., (2006)38

Prasad A.V.S.S., et. al., reported spectrophotometric determination of Formoterol

Fumarate and Budesonide in their combined dosage forms. Methanol is used as diluent for

carring out analysis. The γ max values for formoterol fumarte and budesonide are 217 nm and

252 nm respectively. The results of analysis by this method have been found to be precise and

accurate.

Kakubari I., et. al., (2007)39

Kakubari I., et. al., reported liquid chromatography-electrospray ionization mass

spectrometry in rat plasma using high performance liquid chromatographic separation with

tandem mass spectrometry. Samples were purified using liquid-liquid extraction and separated

on CAPCELL PAK C18 UG120 (2.0 x 150 mm) with a mobile phase consisting of a mixture of

methanol- 50 mM ammonium hydrogen carbonate (1:1 v/v). Detection was performed with a



TSQ 7000 mass spectrometer using positive ion electrospray ionisation, monitoring the shift

from precursor ions for formoterol at m/z 344.9 to product ions of m/z 121.0.

Mascher H.J., et. al., (2008)40

Mascher H.J., et. al., reported HPLC-MS/MS for the determination of Ciclesonide,

Ciclesonide-M1-Metabolite and Fluticasone Propionate in Human Serum. Serum was mixed

with the internal standards (IS) D11-CIC and D11-CIC-M1 and extracted with diisopropylether.

A gradient with acetonitrile (containing 10 mM of acetic acid and 10% of acetone) was used.

HPLC-MS/MS of the acetic acid adducts of the analytes was performed in negative mode. The

novel aspect of this method is that instead of the dopant being introduced directly into the source

by means of an external HPLC pump, it was added to the mobile phase. This provided

significantly better sensitivity than the usual method of in-source addition of the dopant, and

with no loss in HPLC performance.

Akapo S., et. al., (2009)41

Akapo S., et. al., reported Chiral HPLC analysis of Formoterol Stereoisomers. Analysis

was performed on a Chiral-AGP column (100 x 4-mm, 5-microm) using a variable mixture of

mobile phase A (50-mM sodium phosphate buffer, pH 7.0) and B (10% v/v IPA) at a flow rate of

1.3 ml min(-1), and UV detection at 242 nm. A chiral HPLC method was validated and

successfully applied for the determination of formoterol stereoisomers and their inversion

products in an aqueous matrix stored at 5-70 °C up to 3 weeks.

Dave N. H., et. al., (2010)42

Dave N.H., et. al., reported rapid Spectrophotometric methods for the determination of

two novel steroids Ciclesonide and Fluticasone propionate in bulk and pressurised metered -

dose preparations. The developed methods for determination of both the steroids are direct

spectrophotometric method and first derivative spectrophotometric method. The absorbance of

Ciclesonide was measured at 243 nm for direct spectrophotometric determination. Whereas

Fluticasone was determined by first derivative spectroscopy, the first derivative spectra were

plotted with delta lambda 8 nm and scaling factor 10.



Fei L., et. al., (2011)43

Fei L., et. al., reported Development of ciclesonide dry powder inhalers and the

anti-asthmatic efficacy in guinea pigs, The ciclesonide was measured with a high

performance liquid chromatography system with an ultraviolet detector.

The analysis was performed on ODS C18 column (Alltima, 250 mm×4.6 mm, 5µm) at 30 ºC

and the wavelength was set at 242nm.The mobile phase consisted of anhydrous alcohol

and water (61:39 v/v). The flow rate was 1.0 mL/min, and the injection volume was 20 µL.

Srinivasarao K., et. al., (2012)44

Srinivasarao K., et. al., reported HPLC method for determination of Formoterol

Fumarate and Mometasone Furoate in Metered Dose Inhaler. Separation was achieved on a

reversed-phase C18 column (150 mm×4.6 mm i.d., 5 µm) using a mobile phase consisting of

Sodium dihydrogen orthophosphate buffer/acetonitrile (50:50, v/v) at a flow rate of 1.0 mL/min

and UV detection at 220 nm.

Vaghela V., et. al., (2013)45

Vaghela V., et. al. reported optimized method for rapid estimation of ciclesonide in bulk

and its dosage form . The chromatographic method with isocratic elution by utilizing an inertsil

ODS-C18, 250 mm × 4.6 mm, 5 µm column. A mobile phase consisting of solvent [solution

containing Methanol:Water (95:05%, v/v)] endowed at a flow rate of 1.0 mL min−1

. The analyte

was detected and quantified at λmax 242 nm using UV detector.

Other than above mentioned methods few other methods are also reported for the

determination of formatoerol, such as capillary electrophoresis method46

, Spectophotometric

method47

and HPLC method48-49

for determination of formoterol in human serum and the assay of

Formoterol and Budesonide in Symbicort Turbuhaler.

Hinz B., et. al., (2003)50

Hinz B., et. al., reported HPLC method for simultaneous determination of Aceclofenac

and three of its metabolites in human plasma. A liquid–liquid extraction-based reversed-phase

HPLC method with UV detection was validated and applied for the analysis of Aceclofenac and



three of its metabolites (4′-hydroxy-Aceclofenac, Diclofenac, 4′-hydroxy-Diclofenac) in human

plasma. The analytes were separated using an Acetonitrile–Phosphate buffer gradient at a flow

rate of 1 mL/min, and UV detection at 282 nm. The retention times for Aceclofenac, Diclofenac,

4′-hydroxy-aceclofenac, 4′-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1,

60.9, 46.9, 28.4 and 21.2 min, respectively.

Chen H., et. al., (2004)51

Chen H., et. al., reported HPLC method for the determination of Aceclofenac in human

plasma by reversed-phase high performance liquid chromatography. Chromatography was

performed on an ODS column with methanol-0.1 mol/L ammonium acetate (pH 6.0) (7:3, v/v) as

the mobile phase. The flow rate was 1.0 mL/min. The UV-Vis detector was set at 275 nm.

Angelo Z., et. al., (2005)52

Angelo Z., et. al. reported separation of Aceclofenac and Diclofenac in human plasma

by free zone capillary electrophoresis using N-methyl-D-glucamine as an effective electrolyte

additive. The effect of increasing concentrations of N-methyl-D-glucamine organic base on

borate run buffer was investigated. A good separation was achieved using a 40 cm × 75 µm

uncoated silica capillary, 300 mmol/l sodium borate buffer, 200 mmol/l N-methyl-D-glucamine,

pH 8.9, in about 3 min. Momin M.Y., et. al., (2006)

53

Momin M.Y., et. al., reported HPLC method for determination of Aceclofenac and

Paracetamol in tablet dosage form. Chromatography was perform on reverse phase C-18 column

(Intersile 4.6 mm×25 cm, 10 µm) using Acetonitrile : 50 mM Sodium dihydrogen

orthophosphate in a ratio of 65:35 (pH adjusted to 3.0 with orthophosphoric acid) as a mobile

phase at a flow rate of 1.5 ml/min and detection at 276 nm. The retention time for Aceclofenac

and Paracetamol was found to be 1.58 and 4.01 min respectively. The method can be used for

estimation of combination of these drugs in tablets.



Han C.Y., et. al., (2007)54

Han C.Y., et. al., reported flow injection chemiluminescence method for sensitive

determination of Nanogram levels of Diacerein in a pharmaceutical formulation. It was based on

the greatly enhancive effect of diacerein on the CL reaction between luminal and hydrogen

peroxide in alkaline medium. The enhanced CL intensity was linear with the concentration of

diacerein over the range 1.0-500 ng/mL. The degradation of diacerein was also investigated

briefly.

Borgmann S. M., et. al., (2008)55

Borgmann S. M., et. al., reported spectrophotometric method for estimation of Diacerhein

in capsules dosage form. The dissolution established conditions were: 900 mL of sodium

phosphate buffer pH 7.0 with 0.75 % of sodium lauryl sulphate as dissolution medium, using a

basket apparatus at a stirring rate of 50 rpm. The drug release was evaluated by UV

spectrophotometric method at 258 nm. The method was validated to meet requirements for a

global regulatory filing.

Ojha A., et. al., (2009)56

Ojha A., et. al., reported HPLC method for determination of Rhein and Aeclofenac in

human plasma. Simple HPLC method with UV detection for simultaneous determination of

rhein and aceclofenac from human plasma samples. Sample preparation was accomplished

through liquid-liquid extraction with ethyl acetate and chromatographic separation was

performed on a reversed-phase ODS column. Mobile phase consisted of a mixture of acetate

buffer and acetonitrile run under gradient at flow rate of 1.0 ml/min. Wavelength was set at 258

nm.

Narade S., et. al., (2010)57

Narade S., reported UV spectrophotometric method for the determination of Diacerein in

capsules. DMF: Distilled water (1:4) used as a diluent and λmax was found to be 258.5 nm. The

solution was filtered through Whatman filter paper No. 41. The absorbance of standard and

sample solutions was measured at 258.5 nm using diluent as blank.



Maheshwari R. et. al., (2011)58

Maheshwari R. reported application of mixed-hydrotropy in titrimetric analysis of

Aceclofenac bulk drug sample, Investigation includes the enhancement of solubility of

Aceclofenac by more than 1155 fold in (20% N,N-dimethyl urea + 20% sodium citrate) solution

as compared to solubility in distilled water, utilizing the concept of mixed-hydrotropy. Mixed

hydrotropic solution was employed to solubilize a poorly water-soluble drug - aceclofenac, in

bulk to carry out titrimetric estimation precluding the use of organic solvents which are toxic,

eco-pollutant and costlier. Statistical data proved the accuracy, reproducibility and the precision

of the proposed method.

Shirwaikar A., et. al., (2012)59

Shirwaikar A., et. al., reported determination of Diacerein in Rabbit Plasma by LC -

Mass Spectroscopy. Chromatography was performed on C18 (4.6mm i.d.×50mm) analytical

column and operated at 40°C. The mobile phase was acetonitrile: 1mM ammonium formate

(70:30, v/v) at a flow-rate of 400µl/min. Detection was made at m/z 283.2/183 for Diacerein and

380/316.4 for internal standard (Celecoxib).

Somashekar P.L., et. al., (2013)60

Somashekar P.L. et .al. reported HPLC method for the quantification of Aceclofenac

Specified Impurity-B (Methyl[2-[(2, 6-dichlorophenyl)amino] phenyl]acetate) in Aceclofenac

Bulk Drug. Early chromatographic work was performed GraceSmart - RP-C18, 250 mm x 4.6

mm, 5 µm columns as stationary phase and various combinations of buffered (pH 4.5-5.0)

organic phases (Acetonitrile and /or Methanol). The flow rate of mobile phase was varied within

0.5-1.5 mL/min. All noted measurements were performed with an injection volume of 100 µL, 1

mL/min flow rate and UV detection at 275 nm using mobile phase- acetonitrile and phosphate

buffer pH-5 (60:40).

Other than above mentioned methods few other methods are also reported for the

determination of Aceclofenac, such as capillary Spectophotometric method61

and HPLC

method62-66

for determination of Aceclofenac in tablet dosage form or in combination with

another drug substance.



2.3. A STABILITY-INDICATING LC METHOD FOR THE SIMULTANEOUS

DETERMINATION OF LEVOCETIRIZINE DIHYDROCHLORIDE AND

PSEUDOEPHEDRINE SULFATE IN TABLET DOSAGE FORMS.

A comprehensive literature survey revealed the lack of suitable stability indicating assay

method for the determination of these two drugs in pharmaceutical dosage forms also Analytical

method for the determination of Levocetirizine dihydrochloride and Pseudoephedrine in tablet

dosage form not official in any pharmacopeia. Hence it is felt essential to develop HPLC

method for the determination of Levocetirizine dihydrochloride and Pseudoephedrine sulfate in

the tablet dosage form.

The mobile phase A consisted of Potassium dihydrogen phosphate Buffer 0.05M and 1-

Ocatne sulphonic acid sodium salt 0.25%, pH adjusted to 3.0 with orthophospheric acid. Mobile

Phase B: Acetonitrile, Gradient elution at flow rate of 1 mL/min and Column temperature at

40◦C. Detector wavelength of 242 nm using a photodiode array detector. The described method

shows excellent linearity over a range of 200–10 µg ml−1 for Levocetirizine dihydrochloride and

7200-360 µg ml−1 for Pseudoephedrine sulfate. The correlation coefficient for Levocetirizine

dihydrochloride and Pseudoephedrine sulfate are 0.9999.

The proposed stability indicating HPLC method for simultaneous determination of

Levocetirizine dihydrochloride and Pseudoephedrine sulfate in tablet dosage form is cost

effective, precise, accurate, linear and suitable for routine analysis and quality control stability

study of drug product.

2.3.1. DRUG PROFILE:

2.3.1.1. Levocetirizine dihydrochloride:

1. Chemical Name: 2-(2-{4-[(R)-(4-chlorophenyl)(phenyl)methyl]piperazin-1-

yl}ethoxy)acetic acid

2. Molecular Formula: C21H25ClN2O3•2HCl.

3. Molecular Weight: 461.82

4. Description: White crystalline powder



5. Chemical Structure:

6. Solubility: Soluble in water.

7. Melting Point: 215-220 ºC

8. Category: antihistaminic

2.3.1.2. Pseudoephedrine sulfate:

1. Chemical Name: (S-(R*,R*))-alpha-(1-(Methylamino)ethyl)benzenemethanol sulfate


3. Molecular Formula: (C10H15NO)2·H2SO4


5. Description: White crystalline powder

6. Solubility: Freely soluble in water

7. Melting Point: 174- 179 C

8. Category: sympathomimetic

2.3.2. EXPERIMENTAL:

2.3.2.1. Working standard:

The working standards were obtained from Dr. Reddys Laboratories Ltd Hyderabad,

India having following batch number and potency.



Working Standard Batch No. Potency (on as is basis)

Levocetirizine dihydrochloride LD1403 99.7 %

Pseudoephedrine sulfate P6672 100.0 %

2.3.2.2. Sample:

Levocetirizine dihydrochloride and Pseudoephedrine sulfate tablets were purchased from

Indian market.

Manufacturer Cipla Ltd

Product Name LEVORID-D (180+5) Tablets

Label Claim Levocetirizine dihydrochloride 5 mg and Pseudoephedrine sulfate 180 mg.

2.3.2.3. Instrument / Apparatus Used:

i) All the glassware used for the experiment were certified ‘A’ grade manufactured by

SCHOTT Glass India Pvt. Ltd. Mumbai, India

ii) A calibrated high performance liquid chromatography (HPLC), make Agilent-1100 series

was used for all the experiments.

iii) A calibrated digital pH meter, manufactured by Mettler-Toledo Inc, Columbus, OH.

Private Limited Mumbai, India.

iv) A calibrated analytical balance, manufactured by Sartorius, Germany.

v) A sonicator, manufactured by Amrut Enterprises, Pune, India.

2.3.2.4. Reagents and chemicals:

All reagents and chemicals were used from Merck chemicals. Orthophospheric acid ,

Potassium dihydrogen phosphate and Ocatane sulphonic acid sodium salt were used as GR

grade. Acetonitrile and Water were used as HPLC grade.

2.3.2.5. Levocetirizine dihydrochloride standard stock solutions:

50 mg Levocetirizine dihydrochloride working standard was accurately weighed and

transferred into a 100 mL volumetric flask, dissolved and diluted with Double distilled water.



2.3.2.6. Mixed standard solution:

A 90 mg Pseudoephedrine sulfate working standard was accurately weighed, transferred

into a 25 mL volumetric flask, transferred 5 ml Levocetrizine dihydrochloride Standard Stock

solution, dissolved and diluted with Double distilled water.

2.3.2.7. Preparation of sample solution:

Ten tablets were weighed and finely powdered. A quantity of powder equivalent to one

tablet containing 180 mg of Pseudoephedrine sulfate and 5 mg of Levocetirizine dihydrochloride was Transferred into a 50 mL volumetric flask. To this flask, 10 mL of methanol was added, and

the solution was sonicated for 20 min with intermittent shaking. The solution was cooled to

ambient Temperature, further added 20 mL of double distilled water and the solution was

sonicated for 20 min with intermittent shaking. The solution was cooled to ambient temperature.

Then the volume was made up with double distilled water and centrifuged at 4,000 rpm for 10

min. The Centrifuged solution filtered through a 0.45-um nylon filter.

2.3.2.8. Chromatographic conditions:

Mobile phase A: Potassium dihydrogen phosphate Buffer 0.05M and 1-Ocatne

Sulphonic acid sodium salt 0.25%, pH adjusted to 3.0 with

orthophospheric acid.

Mobile phase B: Acetonitrile

Column: Cosmosil C8, 250 x 4.6 mm, 5 µm

Column oven temperature: 40◦C

Flow: 1.0 mL/min

Wavelength: 242 nm

Injection volume: 20 µL

Runtime: 20 minutes.

Gradient program :



Time in minute Mobile phase A (%) Mobile phase B (%)

Initial 80 20

3 80 20

8 50 50

15 50 50

17 80 20

20 80 20

2.3.2.9. Procedure:

HPLC system was set up as described under chromatographic conditions. Standard and

sample solution was prepared as per above procedure and made single injection of diluent as a

blank, standard solution (five injections) and sample solution (duplicate injections) in to the

chromatographic system. Recorded the chromatograms at 242 nm and measured the peak area

counts for Pseudoephedrine and Levocetirizine peaks. Calculated Pseudoephedrine and

Levocetirizine against respective standard from standard solution.

2.3.2.10. Calculations:

A) For Levocetrizine dihydrochloride:

AT WS DT P AW

% Assay = -------- x ----- x --------x ------ x-------x 100

AS DS WT 100 LC

Where,

AT : Average area of Levocetrizine dihydrochloride peak in sample preparation.

AS : Average area of Levocetrizine dihydrochloride peak in standard preparation.

WS : Weight of Levocetrizine dihydrochloride working standard, in mg.

DS : Dilution of Standard preparation

DT : Dilution of Sample preparation

WT : Weight of sample taken, in mg.

P : potency of Levocetrizine dihydrochloride working standard,on as is basis.

L : Label claim of Levocetrizine dihydrochloride.

AW : Average weight of tablets



B) For Pseudoephedrine sulfate:

AT1 WS1 DT1 P1 AW1

% Assay = -------- x ----- x --------x ------ x-------x 100

AS1 DS1 WT1 100 LC1

Where,

AT1 : Average area counts of Pseudoephedrine sulfate peak in sample preparation.

AS1 : Average area counts of Pseudoephedrine sulfate peak in standard preparation.

WS1 : Weight Pseudoephedrine sulfate working standard, in mg.

DS1 : Dilution of Standard preparation

DT1 : Dilution of Sample preparation

WT1 : Weight of sample taken, in mg.

P1 : Percentage potency of Pseudoephedrine sulfate working standard, on as is basis.

LC1 : Label claim of Pseudoephedrine sulfate in mg per tablet.

AW1 : Average weight of tablets

2.3.3. RESULTS AND DISCUSSION:

2.3.3.1. Optimization of the chromatographic conditions:

The main criteria for development of a successful HPLC method for determination of

Levocetirizine dihydrochloride and Pseudoephedrine sulfate in tablet was the method should

be able to determine assay of both drugs in single run and should be accurate, reproducible,

robust, stability indicating, free of interference from degradation products, and straight forward

enough for routine use in the quality control laboratory67-75

. In order to optimize the LC

separation of Pseudoephedrine sulfate and Levocetirizine dihydrochloride initially, the retention

behavior of both the components was studied in the pH range of 2.5–6.8, using mobile phases of

buffer (pH 2.5–6.8) and acetonitrile, methanol as organic modifier. were found that

Pseudoephedrine sulfate eluted in void volume and more retention time Levocetirizine

dihydrochloride. Hence, it was decided to work by adding ion pairing reagent(1-Ocatne

sulphonic acid sodium salt) in the mobile phase.

To ensure that Pseudoephedrine sulfate gives better retention, resolution between

Levocetirizine dihydrochloride and Pseudoephedrine sulfate not less than 5 and the method was



as fast as possible, a gradient run was optimized using buffer pH 3.0 and acetonitrile, finally The

mobile phase A consisted of Potassium dihydrogen phosphate Buffer 0.05M and 1-Ocatne

sulphonic acid sodium salt 0.25%, pH adjusted to 3.0 with orthophospheric acid. Mobile Phase

B: Acetonitrile at Gradient elution at flow rate of 1 mL/min and Column temperature at 40◦C,

and detector wavelength of 242 nm using a photodiode array detector was selected as an

appropriate chromatographic conditions, which gave good resolution and acceptable peak

parameters for both Levocetirizine dihydrochloride and Pseudoephedrine sulfate peaks. Overline

spectra of Levocetirizine dihydrochloride and Pseudoephedrine sulfate is shown in figure 2.2,

Typical chromatogram of sample solution is as shown in figure 2.1.

Fig. 2.1: A typical chromatogram of a tablet sample solution.

Fig.2.2: Overlain UV spectra of 1) Levocetrizine Dihydrochloride, 2) Pseudophedrine Sulfate.



2.3.3.2. Procedure for forced degradation study of drug product:

Forced degradation studies were performed to demonstrate the selectivity and stability

indicating capability of the proposed method. The powdered samples of tablets were exposed to

acidic, alkaline, oxidizing and thermal degradation conditions. The stress conditions engaged for

degradation studies as per ICH recommendation

2.3.3.2.1. Acid degradation:

A quantity of powder equivalent to one tablet containing 180 mg of Pseudoephedrine

sulfate and 5 mg of Levocetirizine dihydrochloride was transferred into a 50 mL volumetric

flask. To this flask, 10 mL of methanol was added, and the solution was sonicated for 20min

with intermittent shaking. then 5 mL0 .1 N HCl added and the mixture kept at 60 ◦C for 45 min

in a water bath. The solution was allowed to attend ambient temperature, then it was neutralized

with 0.1 N NaOH and the volume made up to 50 mL with double distilled water. Typical

chromatogram of acid stressed sample solution is as shown in figure 2.3, where as results are

captured in table 2.1.

2.3.3.2.2. Base degradation:



flask. To this flask, 10 mL of methanol was added, and the solution was sonicated for 20min

with intermittent shaking.then 5 mL 1 N NaOH added and the mixture kept at 60 ◦C for 45 min

in a water bath. The solution was allowed to attend ambient temperature, then it was neutralized

with 1 N HCL and the volume made up to 50 mL with double distilled water. Typical



2.3.3.2.3. Oxidative degradation:



flask. To this flask, 10 mL of methanol were added, and the solution was sonicated for 20min



with intermittent shaking. Then 5 mL 5% H202 added and the mixture kept at Ambient

Temperature for 45 min and the volume made up to 50 mL with double distilled water. Typical



2.3.3.2.4. Thermal degradation:

About 1000 mg of Tablet powder was kept at 105oC for 24 h. Then A quantity of powder

equivalent to one tablet containing 180 mg of Pseudoephedrine sulfate and 5 mg of

Levocetirizine dihydrochloride was transferred into a 50 mL volumetric flask. To this flask, 10

mL of methanol was added, and the solution was sonicated for 20min with intermittent shaking.

The solution was cooled to ambient temperature. Then added 20 mL of double distilled water,

and the solution was sonicated for 20min with intermittent shaking. The solution was cooled to

ambient temperature.Then the volume was made up with double distilled water. Typical

chromatogram of acid stressed sample solution is as shown in figure 2.6 where as results are


Fig. 2.3: chromatogram of the acid stressed sample solution



Fig. 2.4: Chromatogram of the Base stressed sample solution

Fig.2.5. Chromatogram of the Oxidative stressed sample solution

Fig. 2.6. Chromatogram of the Thermal stressed sample solution.



Table 2.1: Summary of forced degradation results

Stress condition/media/duration

Levocetirizine dihydrochloride

Degradation (%)

Pseudoephedrine

Sulfate

Degradation (%)

Acidic/0.1 N HCl/600C/45 min 6.0 0.9

Alkaline/1 NaOH/600C/45 min 1.2 0.9

Oxidative/5%H2O2/ambient/45 min 3.3 0.1

Thermal/1050C/24hrs 3.0 6.2

2.3.3.3. Validation of method:

As per the ICH guidelines76-78

. the method validation parameters were checked for

linearity, precision, accuracy, limit of detection, limit of quantitation, and robustness.

2.3.3.3.1. Specificity:

The specificity of the HPLC method is illustrated in Fig. 2.2 where complete separation

of Levocetrizine and Pseudoephedrine was noticed in presence of tablet excipients. In addition

there was no any interference at the retention time of Levocetrizine and Pseudoephedrine in the

chromatogram of placebo solution. In peak purity analysis with photo diode detector, purity

angle was less than purity threshold for both the analytes. This shows that the peak of analytes

was pure and excipients in the formulation did not interfere the analytes. System suitability

parameters are given in Table 2.2.

Table 2.2: System suitability parameters

S. No. Compound Theoretical

plates

(NLT 2000)

Resolution

(NMT 2)

Asymmetry

factor

(NMT 2)

% RSD

of Peak area

(NMT 2)

1. Levocetirizine

dihydrochloride 99220 13.93 1.10 0.10

2. Pseudoephedrine

sulfate 53335 - 0.90 0.10

2.3.3.3.2. Linearity:

Linearity of the method was tested from 10 to 200% of the targeted level of the assay

concentration ( Levocetirizine dihydrochloride 100 ug mL-1

Pseudoephedrine sulfate 3600 ug



mL-1

) for both analytes. Mixed standard solutions contained 10-200 ug mL-1

of Levocetirizine

dihydrochloride and 360-7200 ug mL-1

of Pseudoephedrine sulfate. Linearity solutions were

injected. The calibration graphs were obtained by plotting peak area ratio against the

concentration of the drugs. The equations of the calibration curves for Levocetirizine

dihydrochloride and Pseudoephedrine sulfate obtained were y = 14184x +15373 and y = 332.2x

+ 3534 respectively. In the simultaneous determination, the calibration graphs were found to be

linear in the aforementioned concentrations with correlation coefficient for Levocetirizine

dihydrochloride and Pseudoephedrine sulfate are 0.9999.Linearity graphs are given in figure 2.7.

Fig. 2.7. Linearity graph of Levocetrizine dihydrochloride and Pseudoephedrine sulfate.



2.3.3.3.3. Precision:

The repeatability of the analytical method was evaluated by assaying six samples

solutions of Levocetirizine dihydrochloride 100 ug mL-1

Pseudoephedrine sulfate 3600 ug mL-1

,

during the same day, under the same experimental conditions. Intermediate precision was

evaluated by assaying solutions on different days. Peak areas were determined and compared.

Precision was expressed as percentage relative standard deviation (R.S.D%<2). From the data

obtained, the developed RP-HPLC methods was found to be precise and are reported in Table

2.3.

Table 2.3. Results from validation studies

Precision (%RSD) Accuracy

Compound Intra

day

Inter

day

LOD

µg/ml

LOQ

µg/ml

50% 100% 150%

Co-relation

coefficient

(r)

Levocetirizine

dihydrochloride 0.8 0.6 0.012 0.036 100.4 99.9 99.1 0.9999

Pseudoephedrine

sulfate 1.1 1.0 0.462 1.4 100.5 100.8 101.3 0.9999

2.3.3.3.4. Accuracy,(Recovery,test):

Accuracy of the method was studied by recovery experiments. The recovery experiments

were performed by spiking solution of known amounts of the drugs in the placebo.

The recovery was performed at three levels, 50, 100 and 150% of the label claim of the tablet

(180 mg of Pseudoephedrine sulfate and 5 mg of Levocetirizine dihydrochloride). Placebo

equivalent to one tablet was transferred into a 50 mL volumetric flask, and the 50, 100 and 150%

of the label claim amounts of Pseudoephedrine sulfate and Levocetirizine dihydrochloride tablet

were spiked . six samples were prepared for each recovery level. The solutions were then

analyzed, and the Percentage recoveries were calculated. The recovery values for Levocetirizine

dihydrochloride and Pseudoephedrine sulfate are as shown in Table 2.3.



2.3.3.3.5. LOD and LOQ:

The LOD and LOQ for Levocetirizine dihydrochloride and pseudoephedrine sulfate were

determined at a signal to-noise ratio of 3:1 and 10:1, respectively by injecting a series of dilute

solutions with known concentrations. The LOD and LOQ are as shown in Table 2.3.

2.3.3.3.6. Robustness:

The robustness of a method is the ability to remain unaffected by small changes in

parameters. The evaluation of robustness should be considered during the development phase

and depends on the type of procedure under study. It should show the reliability of an analysis

with respect to deliberate variations in method parameters. If measurements are susceptible to

variations in analytical conditions, the analytical conditions should be suitably controlled or a

precautionary statement should be included in the procedure. One consequence of the evaluation

of robustness should be that a series of system suitability parameters is established to ensure that

the validity of the analytical procedure is maintained whenever used. In the case of liquid

chromatography, typical variations are influence of variations of pH in a mobile phase; influence

of variations in mobile phase composition; Column temperature; mobile phase flow rate.

System suitability testing is an integral part of many analytical procedures hence to

determine robustness of the method, experimental conditions were purposely altered and

chromatographic resolution between Levocetirizine dihydrochloride and Pseudoephedrine sulfate

were evaluated. The flow rate of the mobile phase was 1.0 mLmin-1

. To study the effect of flow

rate on the resolution of Levocetirizine dihydrochloride and Pseudoephedrine sulfate, it was

changed to 0.2 units from 1.0 mL min-1

to1.2 mL min-1

and 0.8 mL min-1

. The effect of column

temperature on the resolution was studied at 45 and 35 0C instead of 40

0C and The effect of pH

Variation of Mobile phase A( Buffer) on the resolution was studied at pH 3.2 and pH 2.8 instead

of pH 3.0. Robustness results are as shown in Table 2.4.

54

Table

2.4

. S

yste

m s

uit

abil

ity

para

met

ers

an

d r

obu

stn

ess

Vari

ati

on

in

Ch

rom

ato

gra

ph

ic p

ara

met

ers

to s

tud

y i

mp

act

on

sy

stem

su

itab

ilit

y

para

met

ers

Flo

w r

ate

in

ml

pH

of

bu

ffer

T

emp

era

ture

in

°C

C

om

pou

nd

S

yst

em

su

itab

ilit

y

Para

met

er

0.8

1.0

1.2

2.8

3.0

3.2

35

40

45

Res

olu

tion

(NL

T 1

0)

13.9

15.6

17.6

16.2

15.6

15.2

15.2

15.6

16.1

Asy

mm

etry

fac

tor

(NM

T 2

) 1.1

1.1

1.1

1.1

1.1

1.1

1.1

1.1

1.1

Lev

oce

tiri

zine

dih

ydro

chlo

ride

Theo

reti

cal

pla

te

(NL

T 2

000)

123940

137882

143924

142610

137882

143924

137282

137882

144411

Vari

ati

on

in

Ch

rom

ato

gra

ph

ic p

ara

met

ers

to s

tud

y i

mp

act

on

sy

stem

su

itab

ilit

y

para

met

ers

Flo

w r

ate

pH

of

bu

ffer

T

emp

era

ture

C

om

pou

nd

S

yst

em

su

itab

ilit

y

Para

met

er

0.8

ml

1.0

ml

1.2

ml

2.8

3.0

3.2

35

° C

40

° C

45

° C

Asy

mm

etry

fac

tor

(NM

T 2

) 0.9

0.9

0.9

0.9

0.9

0.9

0.9

0.9

0.9

P

seudoep

hed

rine

sulf

ate

Theo

reti

cal

pla

te

(NL

T 2

000)

62042

65295

65275

66080

65295

65275

63594

65295

66076



2.3.4. CONCLUSION:

The developed simple LC method for assay determination of Levocetirizine

dihydrochloride and Pseudoephedrine sulfate is linear, precise, accurate and specific. The

method was validated to the requirements of ICH and the results were satisfactory. The

developed stability-indicating analytical method can be used for the routine analysis of

production samples, where sample load is higher and high throughput is essential for faster

delivery of results. Overall, the method provides a high throughput solution for determination of

Levocetirizine dihydrochloride and Pseudoephedrine sulfate in tablet dosage form with excellent

selectivity, precision, and accuracy.

2.4. SENSITIVE LC METHOD FOR SIMULTANEOUS DETERMINATION OF

CICLESONIDE AND FORMOTEROL FUMARATE IN DRY POWDER INHALER.

To the best of our knowledge, a stability indicating RP-HPLC method for the

simultaneous determination of Formoterol fumarate and Ciclesonide in dry powder inhaler is not

available in any pharmacopoeia. Hence, it was felt essential to develop and validate a sensitive,

accurate, and stability indicating RP-HPLC method for the simultaneous determination of

Formoterol fumarate and Ciclesonide in dry powder inhaler.

The chromatographic separation was achieved on Hypersil BDS C8 250x4.6 mm, 5 um

column using a mobile phase consisting of 0.1% orthophospheric acid and acetonitrile in the

ratio of 35:65 (v/v), at a flow rate of 2.0 mLmin-1

. The column compartment temperature was set

at 40°C. The typical HPLC chromatograms was extracted at 214 nm using photodiode array

detector (PDA). The described method shows excellent linearity over a range of 3.6 µg mL-1

to

1.1 ng mL-1

for formoterol fumarate and 120 µg mL-1

to 34 ng mL-1

for ciclesonide. The

correlation coefficient for formoterol fumarate and ciclesonide was 0.9998. The limit of

detection and limit of quantification for formoterol fumarate was 0.33 ng mL-1

, 1.1 ng mL-1

and

for ciclesonide 10.2 ng mL-1

, 34 ng mL-1

, respectively. The proposed method was found to be

very sensitive and accurate for the determination of Formoterol Fumarate and ciclesonide in dry

powder inhaler.




2.4.1.1. Formetrol fumarate:

1. Chemical Name: N-[2-Hydroxy-5-[[(1RS)-1-hydroxy-2-[[(1RS)-2-(4-methoxyphenyl)-1-

methylethyl] amino] ethyl] phenyl] formamide(E)-2-butenedioate


3. Molecular Formula: (C19H24N2O4)2•C4H4O4•2H2O.

4. Molecular weight: 840.92

5. Description: white to yellowish crystalline powder

6.Solubility: Freely soluble in glacial acetic acid, soluble in methanol, sparingly soluble in

ethanol and isopropanol, slightly soluble in water, and practically insoluble in acetone, ethyl

acetate, and diethyl ether.

7. Melting Point: 138-140°C

8. Category: beta2-adrenergic bronchodilator.

2.4.1.2. Ciclesonide:

1. Chemical Name: (R)-11b,16a,17,21-Tetrahydroxypregna-1,4-diene-3, 20-dione cyclic 16,17-

acetal with cyclohexanecarboxaldehyde 21-isobutyrate


3. Molecular Formula: C32H44O7



4. Molecular weight: 540.688

5. Description: white to yellow-white powder

6. Solubility: practically insoluble in water and freely soluble in ethanol and Acetone.

7. Melting Point: 207 ºC

8. Category: non-halogenated glucocorticoid



The working standards were obtained from Indian market with following batch number

and potency.

Working Standard Batch No. Potency (on as is basis)

Formetrol fumarate FF3121 94.5 % w/w

Ciclesonide CN4517 98.9 % w/w

2.4.2.2. Sample:

Formetrol fumarate and Ciclesonide capsules were purchased from Indian market.

Manufacturer Cipla Ltd

Product Name Simply One Rotacaps

Label Claim Formoterol fumarate 6mcg, Ciclesonide 200 mcg capsules




ii) A calibrated high performance liquid chromatography (HPLC), make Agilent-1100 series

was used for all the experiments.







2.4.2.4.. Reagent, chemicals and diluent:

All reagents and chemicals were used from Merck chemicals. Orthophospheric acid and

Potassium dihydrogen phosphate were used as GR grade. Acetonitrile and Water were used as

HPLC grade. Purified water and Acetonitrile in the ratio of 50:50 v/v were used as diluent for

the experiment.

2.4.2.5. Preparation of standard stock solution A :

Accurately weighed 24mg working standard of Formoterol fumarate is transfer to a

100ml volumetric flask, 70 ml diluent is added and sonicate to dissolve it completely. Make up

the volume to 100 ml with diluent and mix.

2.4.2.6. Preparation of standard stock solution B :

Accurately weighed 80.0 mg working standard of Ciclesonide is transfer to a 100 mL

volumetric flask, 70 mL diluent is added and sonicate to dissolve it completely. Make up the

volume to 100 mL with diluent and mix.

2.4.2.7. Preparation of standard solution:

Transferred 2.0 ml of the standard stock solution A and 10.0 ml of the standard stock

solution B into a 100ml volumetric flask and make up the volume with diluent and mix.

2.4.2.8. Preparation of Sample solution:

The average fill weight of 20 capsules was determined. Accurately weighed sample

powder was transferred in the amount of 120 mg of FF and 4000 mg of CS in to a 50mL

volumetric flask, to which was added 20mL of diluent and sonicated for about 20 min to

dissolve; then, diluent was added to obtain a volume of 50 mL.


Buffer solution : 0.1% orthophospheric acid

Mobile Phase : Mixture of buffer and acetonitrile in a ratio of (35:65)v/v.

Column: Hypersil BDS C8, 250x4.6 mm, 5 um




Flow: 2.0 mL/min

Wavelength: 214 nm


Runtime: 10 minutes

2.4.2.10. Procedure:




chromatographic system.Recorded the chromatograms at 214 nm for Formoterol fumarate and

Ciclesonide peaks. Calculated % assay of Formoterol fumarate and Ciclesonide against

respective standard from standard solution.

2.4.2.11. Calculations:

A) For Formoterol fumarate (% Assay ) :

AT WS DT P AW

% Assay = -------- x ----- x -----x ------ x-------x 100

AS DS WT 100 LC

Where,

AT :Average area of Formoterol fumarate peak in sample preparation.

AS :Average area of Formoterol fumarate peak in standard preparation.

WS :Weight of Formoterol fumarate working standard, in mg.

DS :Dilution of Standard preparation


WT :Weight of sample taken, in mg.

P :Percentage potency of Formoterol fumarate working standard,

on as is basis.

L : Label claim of Formoterol fumarate.




B) For Ciclesonide:

AT1 WS1 DT1 P1 AW1

% Assay = -------- x ----- x --------x ------ x-------x 100

AS1 DS1 WT1 100 LC1

Where,

AT1 : Average area counts of Ciclesonide peak in sample preparation

AS1 : Average area counts of Ciclesonide peak in standard preparation

WS1 : Weight Ciclesonide working standard, in mg



WT1 : Weight of sample taken, in mg

P1 : Percentage potency of Ciclesonide working standard, on as is basis

LC1 : Label claim of Ciclesonide in mg per tablet




The main objective of development of a HPLC method for determination of FF and CS in

dry powder inhaler was that the method should be able to determine assays of both drugs in

single run with run time <10 min. As dose strength is at mcg level, method should be more

sensitive, stability indicating, free of interference from degradation products, and straight

forward enough for routine use in the quality control laboratory25-33

.

During optimization of chromatographic conditions, different mobile phase compositions,

different HPLC columns, organic modifiers such as acetonitrile and methanol, and flow rate

were tried to achieve acceptable system suitability parameters, as well as good separation

between FF and CS. Optimum wavelength selected was 214nm because of higher sensitivity of

FF at this wavelength and also good absorption shown by CS at this wavelength (Figure 2.8).



2.4.3.2. Effect of the organic modifier:

0.1% orthophosphoric acid was used as the buffer for the mobile phase preparation.

Different combinations of buffer and acetonitrile within the range of 10:90–90:10, as well as

buffer and methanol within the range of 10:90–90:10 were tested. It was observed that methanol

in combination with a buffer leads to more retention of CS peak and asymmetric peak shape. An

increase in the organic modifier volume in the mobile phase produces a reduced retention time of

CS. The mobile phase combination of buffer: Acetonitirle (35:65) gives a symmetric peak shape,

an acceptable tailing factor, and a shorter run time up to 10 min.

2.4.3.3. Effect of stationary phase and pH of mobile phase:

Reversed-phase columns are silica based bonded phases, and C18-type bonded phase is

most frequently used. On an Inertsil ODS 3 V 250mmx 4.6 mm, 5 mm column FF was eluted

with a solvent front peak, as the basic polar compound was protonated in acidic pH and eluted

more quickly. Polar compounds were eluted slowly on C8 column as it is less hydrophobic than

C18 and by use of base deactivated silica column (Hypersil BDS C8 250x4.6 mm, 5 um). Also,

by keeping the pH of the mobile phase on the acidic side, the silanol interaction with basic

analyte was minimized and peak symmetry and sensitivity were improved. The flow rate of the

mobile phase was changed to obtain the expected retention time of the analyte. An increase in

the flow rate led to reduced retention time of the analyte. Typical chromatogram of formoterol

fumarate and Ciclesonide tablet is as show in figure 2.9, where as system suitability results are

reported in table 2.5.



Fig. 2.8. Overlain UV spectra of diacerein and aceclofenac.

Fig. 2.9. A typical chromatogram of the tablet: Formoterol fumarate and Ciclesonide.

Table 2.5 : Results from system suitability studies


plates

(NLT 2000)

Asymmetry

factor

(NMT 2)

% RSD

of Peak area

(NMT 2)

1. Formoterol fumarate 7770 1.10 0.15

2. Ciclesonide 8791 1.00 0.10



2.4.3.4. Aerodynamic particle size distribution:

Anderson cascade impactor has been used for determination of Aerodynamic particle size

distribution from formoterol fumarate and ciclesonide dry powder inhaler. Fine particle dose

obtained for formoterol fumarate and ciclesonide was 1.284mcg and 40.961mcg respectively.

Mass median aerodynamic Diameter and Geometrical standard deviation for formoterol fumarate

was 2.47 and 2.58 respectively, and for ciclesonide 3.20 and 2.01respectively.Results are

reported in table no table 2.6.

Table 2.6: Results of aerodynamic particle size distribution:

Formoterol

fumarate Area

Recovery

In mcg

Recovery

in % Ciclesonide Area

Recovery

In mcg

Recovery in

In %

Standard area 1012325 Standard area 4553891

Devise 52420 0.592 9.9 Devise 196879 17.295 8.6

Mouthpiece 10420 0.118 2.0 Mouthpiece 16586 1.457 0.7

Throat 25300 0.286 4.8 Throat 97895 8.600 4.3

Preseparator 248500 2.808 46.8 Preseparator 1335238 117.296 58.6

Stage-0 12353 0.140 2.3 Stage-0 25300 2.223 1.1

Stage-1 13380 0.151 2.5 Stage-1 28598 2.512 1.3

Stage-2 16500 0.186 3.1 Stage-2 110567 9.713 4.9

Stage-3 10785 0.122 2.0 Stage-3 86598 7.607 3.8

Stage-4 28982 0.327 5.5 Stage-4 138650 12.180 6.1

Stage-5 18950 0.214 3.6 Stage-5 69852 6.136 3.1

Stage-6 17015 0.192 3.2 Stage-6 29860 2.623 1.3

Stage-7 11313 0.128 2.1 Stage-7 19856 1.744 0.9

Filter 10089 0.114 1.9 Filter 10894 0.957 0.5

Total 5.379 89.6 Total 190.344 95.2


Forced degradation study of drug product is helpful to investigate the likely degradation

products, which in turn helps to establish the degradation pathways and the intrinsic stability of

the drug molecule. Which is helpful for developing a suitable stability indicating method. The

stress studies were performed as per International Conference on Harmonisation (ICH)



recommendation. Following stressed conditions has been carried out in order to establish

degradation pathway of drug product.

2.4.3.5.1. Acid stressed condition:

Transferred sample powder equivalent to 120 mg of FF and 4000 mg of CS was put into

a 50mL volumetric flask, 20mL of water and Acetonitrile in the ratio of 1:1 (v/v) was added and

sonicated for about 20 min to dissolve and treated with 5 mL of 0.1 N HCl and kept on water

bath at 600C for 15 minutes cool to room temperature and neutralized with 5 mL of 0.1 N

NaOH and makeup the volume with water and Acetonitrile in the ratio of 1:1 (v/v) and mix.

2.4.3.5.2. Base stressed condition:



sonicated for about 20 min to dissolve and treated with 5 mL of 0.1 N NaOH and kept on water

bath at 600C for 15 minutes cool to room temperature and neutralized with 5 mL of 0.1 N HCl

and makeup the volume with water and Acetonitrile in the ratio of 1:1 (v/v) and mix.

2.4.3.5.3. Peroxide stressed condition:



sonicated for about 20 min to dissolve and treated with 5 mL of 30% H2O2 and kept ambient

condition for 45 minutes cool to room temperature and makeup the volume with water and

Acetonitrile in the ratio of 1:1 (v/v) and mix.

Table 2.7 : Summary of forced degradation results

Stress condition/media/duration

Formoterol Fumarate

Degradation (%)

Ciclesonide

Degradation (%)

Acidic/0.1 N HCl/600C/15 min 20.20 1.39

Alkaline/0.1 NaOH /600C/15 min 21.64 39.64

Oxidative/30%H2O2/ambient/45 min No No



Fig. 2.10: Typical HPLC chromatogram of stressed samples(A) sample treated with acid,

(B)sample treated with base and (C) sample treated with peroxide.


The developed method was validated according to ICH guidelines22-24

with respect to

specificity, linearity, accuracy, precision, limit of detection, limit of quantitation, solution

stability, and robustness.


The specificity of the method was checked by injecting solution containing excipient

without drug substances, and no chromatographic interference was observed from excipients at

the retention time of the analyte peaks. Peak purity was verified by confirming homogeneous



spectral data for FF and CS. The specificity of the method was also checked by performing the

stressed study and no interference from degradation products at the retention time of FF and CS

was found.

2.4.3.6.2. Precision:

The repeatability of the analytical method was evaluated by analyzing six test samples

solutions of Formoterol FumarateþCiclesonide dry powder for Inhaler, during the same day,

under the same experimental conditions. Intermediate precision was evaluated by analyzing six

test solutions on different days. The percentage assay for each component were determined and

compared. Precision was expressed as percentage relative standard deviation of percentage

assay, which was found to be well within the limit (<2). Hence, the method was found to be

precise.

2.4.3.6.3. Accuracy:

Accuracy was evaluated by the simultaneous determination of analytes in solution

prepared by standard addition method. The experiment was carried out by adding known amount

of each component corresponding to three concentration levels of 50%, 100%, and 150% of

target analyte concentration in placebo solution. The samples were prepared in triplicate at each

level. The solutions were then analyzed as per the proposed method and the quantification of

added analyte (% weight/weight) was carried out by using an external standard of corresponding

main drug prepared at the analytical concentration. The recovery values for Formoterol Fumarate

and Ciclesonide are as shown in Table 2.8.

Table 2.8: Recovery data

Formoterol Fumarate Ciclesoinde Recovery levels

Recovery (%) RSD (%) Recovery (%) RSD (%)

50 99.4 0.43 99.5 0.31

100 99.1 0.37 99.7 0.24

150 99.8 0.54 100.3 0.48




Linearity of the method was tested from LOQ to 150% of the targeted level of the assay

concentration (FF 2.4 µg mL-1

, CS 80 µg mL-1

) for both analytes. The calibration graphs were

obtained by plotting peak area ratios against the concentration of the drugs and the results are

presented in Table 2.9 where as Linearity graphs are as shown in figure 2.11.

Figure 2.11.: Linearity graph of Formoterol fumarate and Ciclesonide.

Table 2.9: Results from validation studies

Compound LOD

ng/ml

LOQ

ng/ml

Linearity

Range

(ng mL-1

– µg mL-1)

Co-relation

coefficient (r) Regression equation

Formoterol

Fumarate 0.33 1.1 1.1 - 3.6 0.9998 y=42338x +1898

Ciclesonide 10.2 34 34 - 120 0.9998 y=57664x +11643



2.4.3.6.5. Limit of Quantification (LOQ) and Detection (LOD:

The limit of detection (LOD) was estimated as three times the signal to noise ratio and

the limit of quantification (LOQ) was estimated as ten times the signal to noise ratio. LOD and

LOQ were achieved by injecting a series of dilute solutions of FF and CS. The precision of the

developed method for FF and CS was checked by analyzing six test solutions prepared at LOQ

and LOD level and determined the percentage relative standard deviation of peak area. The LOD

and LOQ values are as shown in Table 2.9.

2.4.3.6.6. Stability in Analytical Solution:

Standard solution and sample solution were found to be stable for about five days at room

temperature. Similarity factor between freshly prepared standard and the fifth day standard

solution was found to be 1. Percentage assay was checked for initial sample preparation, and on

the fifth day at room temperature against the freshly prepared standard, the percentage difference

between the fifth day sample and initial sample assay was less than 1.



parameters. To determine robustness of the method, experimental conditions were deliberately

altered and percentage assay was checked for FF and CS. The altered parameters were change in

flow rate (+0.2mLmin_1), column compartment temperature (+5°C), and absolute organic

composition (+10%). The percentage assay for both the components was found to be well within

the limits.

2.4.4. CONCLUSION:

The proposed HPLC method is specific, accurate, and precise for simultaneous

determination of FF and CS from its pharmaceutical dosages form. The method was validated as

per ICH guidelines and the results showed an the stability-indicating property of the method and,

hence, the method is suitable for routine analysis and quality control of pharmaceutical

preparation containing FF and CS as active pharmaceutical ingredients.



2.5. SENSITIVE LC METHOD FOR THE SIMULTANEOUS DETERMINATION OF

DIACEREIN AND ACECLOFENAC IN TABLET DOSAGE FORM.

A comprehensive literature survey revealed the lack of suitable stability indicating assay

method for the determination of Aceclofenac and Diacerein in tablet dosage form also this

method is not official in any pharmacopeia. Hence, it was felt essential to develop and validate a

sensitive, accurate and stability indicating RP-HPLC method for the simultaneous determination

of Aceclofenac and Diacerein in tablet dosage form. the chromatographic separation was

achieved on Kromasil C-18, 150 × 4.6 mm, 3.5-µm analytical column using mobile phase double

distilled water (pH 2.7 with glacial acetic acid)-acetonitrile (45:55 v/v). Detector was set at 256

nm. The described method shows excellent linearity over a range of 75–2.5 µg/mL for diacerein

and 150–5 µg/mL for aceclofenac. The correlation coefficient for diacerein is 0.9999 and for

aceclofenac is 0.9997. The proposed method was found to be suitable for simultaneous

determination and stability study of diacerein and aceclofenac in tablet dosage form.


2.5.1.1. Diacerein:

1. Chemical Name: 1,8-diacetoxy-3-carboxyanthraquinone


3. Molecular Formula: C19H12O8


5. Description: Orange-yellow needle crystalline powder

6. Solubility: Soluble in dimethyl sulfoxide, dimethyl acetamide; difficult to dissolve in water,

methanol, chloroform.

7. Melting Point: 217-218°C

{8. Category: anti-inflammatory agent prescribed for osteoarthritis



2.5.1.2. Aceclofenac:

1. Chemical Name: 5-chloro-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl) propyl]--4-yl]-

1,3-dihydro-2H-benzimidazol-2-one


3. Molecular Formula: C16 H13Cl2NO4


5. Description: white or off-white crystalline powder

6. Solubility: Practically insoluble in water, freely soluble in acetone, soluble in alcohol.

7. Melting Point:149-153 ºC

8. Category: Antipyretic and Analgesics



The working standards were obtained from Glenmark pharmaceuticals Ltd., India having

following batch number and potency.

Working Standard Batch Number Potency (on as is basis)

Diacerein DAC2145 99.00 % w/w

Aceclofenac ACE3412 99.12 % w/w

2.5.2.2. Sample:

Tablets were purchased from Indian market.

Manufacturer Glenmark pharmaceuticals

Product Name Dycerin-A

Label Claim Diacerein 50 mg and Aceclofenac 100 mg per tablet






ii) A calibrated high performance liquid chromatography (HPLC), make Agilent-1100 was

used for all the experiments.





2.5.2.4. Reagents and chemicals:

All used reagents and chemicals from Merck fine chemicals. Glacial acetic acid GR

grade, Acetonitrile HPLC grade, Dimethyl acetamide GR grade, Water HPLC grade from Milli-

Q water purification system, was used through the experiment.

2.5.2.5. Preparation of standard stock solution :

Accurately weighed 25mg working standard of Diacerein and 50mg working standard of

Aceclofeanc and transferred it into a 100ml volumetric flask, 10 mL Dimethyl acetamide added

and sonicate to dissolve it completely. 70 ml of mobile phase added and sonicate for 5 minutes.

Made up the volume with mobile phase and mix.

2.5.2.6. Preparation of standard solution:

Transferred 5.0 ml of the Standard stock Solution into a 50ml volumetric flask and made

up the volume with mobile phase.

2.5.2.7. Preparation of Sample solution:

Ten tablets were weighed and finely powdered. A quantity of powder equivalent to one

tablet containing 100 mg of Aceclofenac and 50 mg of Diacerein was Transferred into a 200 mL

volumetric flask. To this flask 20 mL of Dimethyl acetamide were added and the solution was

sonicated for 20min with intermittent shaking. The solution was cooled to ambient temperature.



Then added 70 mL of mobile phase and sonicate for 5 minutes and volume was made up with

mobile phase. The Centrifuged solution filter through 0.45µ nylon filter further dilute 5ml

solution was transferred into a 50 mL volumetric flask and diluted to volume mobile phase.


Buffer solution: Double distilled water pH adjusted 2.7 with glacial acetic acid.

Mobile Phase : Prepared a mixture of buffer and acetonitrile in a ratio of (45:55) v/v.

Column: Kromasil C18, 150 x 4.6 mm, 3.5 µm


Flow: 1.0 mL/min

Wavelength: 256 nm


Runtime: 10 minutes.

2.5.2.9. Procedure:




chromatographic system. Recorded the chromatograms at 256 nm for Diacerein and Aceclofenac

peaks. Calculated % assay of Diacerein and Aceclofenac against respective standard from

standard solution.

2.5.2.10. Calculation:

A) For Diacerein:

AT WS DT P AW

% Assay = -------- x ----- x -----x ------ x-------x 100

AS DS WT 100 LC

Where,

AT : Average area of Diacerein peak in sample preparation

AS : Average area of Diacerein peak in standard preparation

WS : Weight of Diacerein working standard, in mg



DS : Dilution of Standard preparation


WT : Weight of sample taken, in mg

P : Percentage potency of Diacerein working standard, on as is basis.

L : Label claim of Diacerein


B) For Aceclofenac:

AT1 WS1 DT1 P1 AW1

% Assay= -------- x ----- x --------x ------ x-------x 100

AS1 DS1 WT1 100 LC1

Where

AT1 : Average area counts of Aceclofenac peak in sample preparation

AS1 : Average area counts of Aceclofenac peak in standard preparation

WS1 : Weight Aceclofenac working standard, in mg



WT1 : Weight of sample taken, in mg

P1 : Percentage potency of Aceclofenac working standard, on as is basis

LC1 : Label claim of Aceclofenac in mg per tablet




The main criteria for development of a successful HPLC method for determination of

Diacerein and Aceclofenac in Tablet were the method should be able to determine assay of

both drugs in single run and should be accurate, reproducible, robust, stability indicating, free of

interference from degradation products, and straight forward enough for routine use in the

quality control laboratory67-75

.



Our objective of the chromatographic method development was to achieve a peak tailing

factor <2, Run time up to 10 min, along with a resolution between Diacerein and Aceclofenac

>5. In order to optimize the LC separation of Aceclofenac and Diacerein initially the retention

behavior of both the components was studied in the pH range of 2.5–6.8, using mobile phases of

buffer (pH 2.5–6.8) and acetonitrile, methanol as organic modifier.

To ensure resolution between Diacerein and Aceclofenac not less than 5, the method

was as fast as possible, a Isocratic run was optimized by using the mobile phase consisted of

buffer–acetonitrile (45:55 v/v). The buffer used in mobile phase contains double distilled water

pH adjusted 2.7 with glacial acetic acid. Isocratic elution at flow rate of 1 mL/min and Column

temperature at 25◦C. Detector wavelength Kept 256 nm using a photodiode array detector. This

was selected as an appropriate chromatographic conditions, which gave good resolution,

acceptable peak parameters for both Diacerein and Aceclofenac. The analytes of this

combination had adequate retentions, peak shape, less tailing, more resolution and the

chromatographic analysis time was 10 min. In optimized conditions Diacerein, Aceclofenac and

their degradants were well separated. Overlain UV spectra of diacerein and aceclofenac is shown

in figure 2.12, typical chromatogram of tablet is shown in figure 2.13, system suitability results

are shown in table 2.10.

Table 2.10: Results of system suitability studies.


plates

(NLT 2000)

Resolution

(NMT 2)

Asymmetry

factor

(NMT 2)

% RSD

of Peak area

(NMT 2)

1. Diacerein 9687 25.53 1.2 0.10

2. Aceclofenac 17795 - 1.2 0.10



Fig. 2.12. Overlain UV spectra of diacerein and aceclofenac.

Fig. 2.13. A typical chromatogram of the tablet: diacerein and aceclofenac.


Forced degradation studies were performed to demonstrate the selectivity and stability

indicating capability of the proposed method. The powdered samples of Tablets were exposed to

acidic, alkaline, and oxidizing degradation conditions. The stress conditions engaged for

degradation studies as per ICH recommendation, summary of forced degradation results is




2.5.3.2.1. Acid degradation:

A quantity of Tablet powder equivalent 50 mg of Diacerein was weighed and dissolved

in 20 mL of Dimethyl acetamide. Then 2 mL 1 N HCl added and the mixture kept at 60◦C for 20

min in a water bath. Then solution was neutralized with 1 N NaOH. Then added 70 mL of

mobile phase and sonicate for 5 minutes. Then the volume was made up to 200ml with mobile

phase, 5 mL were transferred into a 50 mL volumetric flask and diluted to volume mobile phase.

Peak purity of the principal peak in the chromatogram of stressed samples was checked using

photo diode array detection. The chromatogram of the Acid degraded sample solution is as

shown in Fig. 2.14.

Fig. 2.14: A Typical chromatogram of the Acid degraded sample solution.

2.5.3.2.2. Base degradation:


in 20 mL of Dimethyl acetamide. Then 1 mL 0.1 N NaOH added and the mixture kept at 60◦C

for 5 min in a water bath. Then solution was neutralized with 0.1 N HCl. Then added 70 mL of

mobile phase and sonicate for 5 minutes. Then the volume was made up to 200ml with mobile

phase, 5 mL were transferred into a 50 mL volumetric flask and diluted to volume mobile phase.

Peak purity of the principal peak in the chromatogram of stressed samples was checked using

photo diode array detection.The chromatogram of the Acid degraded sample solution is as shown

in Figure 2.15.



Fig. 2.15: A Typical chromatogram of the Base degraded sample solution.

2.5.3.2.3. Oxidative,degradation:


in 20 mL of Dimethyl acetamide. 5 mL 3%H2O2 added and the mixture kept at Ambient Temp.

for 45 min on bench top. Then added 70 mL of mobile phase and sonicate for 5 minutes. Then

the volume was made up to 200ml with mobile phase, 5 mL were transferred into a 50 mL

volumetric flask and diluted to volume mobile phase. Peak purity of the principal peak in the

chromatogram of stressed samples was checked using photo diode array detection. The

chromatogram of the Peroxide stressed sample solution is as shown in Figure 2.16.

Table 2.11 : Summary of forced degradation results

Stressed condition/ Media/duration Diacerein

degradation (%)

Aceclofenac

degradation (%)

Acidic/1N HCl/60°C/20 min 30.1 28.6

Alkaline/1N HCl/60°C/20 min 14.7 13.5

Oxidative/ 3% H2O2/ambient/ 45 min 0.5 0.2



Fig. 2.16: A Typical chromatogram of the Peroxide stressed sample solution.


As per the ICH guidelines76-78

the method validation parameters were checked for

linearity, precision, accuracy, limit of detection, limit of quantitation, and robustness.


Photodiode array detection was used as an evidence of the specificity of the method to

evaluate Where complete separation of Diacerein and Aceclofenac was noticed in presence of

tablet excipients. In addition there was no any interference at the retention time of Diacerein and

Aceclofenac in the chromatogram of placebo solution. In peak purity analysis with photo diode

detector, purity angle was less than purity threshold for both the analytes. This shows that the

peak of analytes was pure and excipients in the formulation does not interfere the analytes.

2.5.3.3.2..Precision:

The repeatability of the analytical method was evaluated by assaying six samples

solutions of Diacerein 25 ug mL-1

and Aceclofenac 50 ug mL-1

, during the same day, under

the same experimental conditions. Intermediate precision was evaluated by assaying solutions

on different days. Peak areas were determined and compared. Precision was expressed as

percentage relative standard deviation (R.S.D%<2). From the data obtained, the developed RP-

HPLC method was found to be precise and results are reported in Table 2.16. The results



obtained by the proposed method for repeatability were compared with those of the literature

HPLC method [20] and found that %difference between mean %assay (n=6) is 0.2% and

reported in table 2.12.

Table 2.12: Determination of Aceclofenac in tablets and comparison with the reference.

2.5.3.3.3. Accuracy:

Accuracy of the method was studied by recovery experiments. The recovery experiments

were performed by spiking solution of known amounts of the drugs in the placebo. The

recovery was performed at three levels, 50, 100 and 150% of the label claim of the tablet (100

mg of Aceclofenac and 50 mg of Diacerein ). Placebo equivalent to one tablet was transferred

into a 200 mL volumetric flask, and the amounts of Aceclofenac and Diacerein at 50, 100 and

150% of the label claim of the tablet were added. Three samples were prepared for each

recovery level. The solutions were then analyzed, and the percentage recoveries were calculated.

The recovery values for Diacerein and Aceclofenac are as shown in Table:2.13.


Linearity of the method was tested from 10 to 300% of the targeted level of the assay

concentration (Diacerein 25 ug mL-1

, Aceclofenac 50 ug mL-1

) for both analytes. Mixed standard

solutions contaning 2.5-75 ug mL-1

of Diacerein and 5-150 ug mL-1

of Aceclofenac. Linearity

solutions were injected. The calibration graphs were obtained by plotting peak area ratio against

the concentration of the drugs. The equations of the calibration curves for Diacerein and

S. No. Reference method % Assay [20] proposed method % Assay

%difference

1. 100.3 100.4

2. 100.2 100.3

3. 99.9 99.9

4. 100.5 99.8

5. 100.3 99.9

6. 100.3 100.3

-

Mean 100.3 100.1 0.2

%RSD 0.2 0.3 -



Aceclofenac obtained were y = 11194x + 2477and y = 11926x - 2248 respectively. In the

simultaneous determination, the calibration graphs were found to be linear in the

aforementioned concentrations with correlation for Diacerein is 0.9999 and Aceclofenac is

0.9997. Linearity graph of Diacerine and Aceclofenac are as shown in figure 2.17.

Fig. 2.17. Linearity graph of Diacerein and Aceclofenac

2.5.3.3.5. LOD and LOQ:

The LOD and LOQ for Diacerein and Aceclofenac were determined at a signal to-noise

ratio of 3:1 and 10:1, respectively by injecting a series of dilute solutions with known

concentrations . The LOD and LOQ are as shown in Table 2.13.



Table 2.13. Results from validation studies

Precision (%RSD) Accuracy

Compound Intra

day

Inter

day

LOD

µg/ml

LOQ

µg/ml

50% 100% 150%

Co-relation

coefficient

(r)

Diacerein 0.26 0.52 0.0033 0.01 99.5 99.8 100.3 0.9999

Aceclofenac 0.35 0.64 0.007 0.021 100.2 100.5 100.0 0.9997



parameters. The evaluation of robustness should be considered during the development phase

and depends on the type of procedure under study. One consequence of the evaluation of

robustness should be that a series of system suitability parameters is established to ensure that

the validity of the analytical procedure is maintained whenever used. In the case of liquid

chromatography, typical variations are influence of variations of pH in a mobile phase; influence

of variations in mobile phase composition; Column temperature; mobile phase flow rate.

To determine robustness of the method, experimental conditions were purposely altered

and Tailing Factor, %RSD of Five replicate injection of Diacerein and Aceclofenac standard

solution were evaluated. The flow rate of the mobile phase was 1.0 mL min-1

. To study the

effect of flow rate on the Tailing Factor, %RSD of Five replicate injection of Diacerein and

Aceclofenac standard solution, it was changed to 0.2 units from 1.0 to1.2 mL min-1

and 0.8 mL

min-1

. The effect of column temperature on the Tailing Factor, %RSD of Five replicate injection

of Diacerein and Aceclofenac standard solution was studied at 20 and 30 0C instead of 25

0C.

The effect of pH Variation of Buffer on the Tailing Factor, %RSD of Five replicate injection of

Diacerein and Aceclofenac standard solution was studied at pH 2.5 and pH 2.9 instead of pH

2.7. The effect of Organic Variation (Acetonitrile) in mobile phase on the Tailing Factor, %RSD

of Five replicate injection of Diacerein and Aceclofenac standard solution was studied at 90%

Organic (Acetonitrile) in mobile phase and 110% Organic (Acetonitrile) in mobile phase instead

of 100% Organic (Acetonitrile) in mobile phase. Results are reported in table 2.14.

82

Table

2.1

4.

Sys

tem

su

itabil

ity

para

met

ers

an

d r

obu

stn

ess

Vari

ati

on

in

Ch

rom

ato

gra

ph

ic p

ara

met

ers

to s

tud

y i

mp

act

on

sy

stem

su

itab

ilit

y

para

met

ers

Flo

w r

ate

in

ml

pH

of

bu

ffer

T

emp

era

ture

in

°C

Org

an

ic v

ari

ati

on

in

%

Co

mp

ou

nd

S

yst

em

su

itab

ilit

y

Para

met

er

0.8

1.0

1.2

2.5

2.7

2.9

20

25

30

90

100

150

Asy

mm

etry

fac

tor

(NM

T 2

) 1.2

1.1

1.1

1.1

1.1

1.2

1.1

1.1

1.2

1.1

1.2

1.1

Dia

cere

in

%

RS

D o

f pea

k

area

(NM

T 2

)

0.3

1

0.2

1

0.3

5

0.2

4

0.2

6

0.1

5

0.1

4

0.3

6

0.3

0

0.1

8

0.3

4

0.3

1

Vari

ati

on

in

Ch

rom

ato

gra

ph

ic p

ara

met

ers

to s

tud

y i

mp

act

on

sy

stem

su

itab

ilit

y

para

met

ers

Flo

w r

ate

in

ml

pH

of

bu

ffer

T

emp

era

ture

in

°C

Org

an

ic v

ari

ati

on

in

%

Co

mp

ou

nd

S

yst

em

su

itab

ilit

y

Para

met

er

0.8

1.0

1.2

2.5

2.7

2.9

20

25

30

90

100

150

Asy

mm

etry

fac

tor

(NM

T 2

) 1.2

1.1

1.1

1.2

1.1

1.2

1.1

1.2

1.2

1.2

1.1

1.2

Ace

clofe

nac

%

RS

D o

f pea

k

area

(NM

T 2

)

0.2

4

0.1

9

0.1

5

0.3

1

0.2

2

0.2

7

0.2

1

0.3

2

0.1

7

0.2

2

0.3

6

0.2

9



2.5.4. CONCLUSION:

The developed simple LC method for assay determination of Diacerein and Aceclofenac

is linear, precise, accurate and specific. The method was validated to the requirements of ICH

and the results were satisfactory. The developed stability-indicating analytical method can be

used for the routine analysis of production samples, where sample load is higher and high

throughput is essential for faster delivery of results. Overall, the method provides a high

throughput solution for determination of Diacerein and Aceclofenac.

2.6. REFERENCES:

[1] Levocetirizine: a new selective H-1 receptor antagonist for use in allergic disorders,

Day J.H., Ellis A.K., Rafeiro E., Drugs Today (2004)40, 415–421.

[2] U.S. Pharmacopoeia USP31 NF26, VOL 3, 129.

[3] Indian Pharmacopoeia (2007)2, 291.

[4] Once daily Levocetirizine for the treatment of allergic rhinitis and chronic idiopathic,

Urticaria N.E., Calogiuri G.F., Di L.E., Cardinale F., Macchia L., Ferrannini A.,

Vacca1 A., J. Asth. Allergy (2009)2, 17–23.

[5] Formoterol Used as Needed-Clinical Effectiveness, Selroos O., Respir. Med. (2001)95,

17–20.

[6] Ciclesonide has Minimal Oropharyngeal Side Effects in the Treatment of Patients with

Moderate-to-Severe Asthma, Bernstein J.A., Noonan M.J., Rim C., Fish J., Kundu S.,

Williams J., Banerji D.D., Hamedani P., J. Allergy Clin. Immunol. (2004)113-2, 113.

[7] Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive

Pulmonary Disease, Pauwels R.A., Buist A.S., Calverley P.A., Jenkins C.R., Hurd S.

S., Am. J. Respir. Crit. Care. Med. (2001)163, 1256–1276.

[8] British Pharmacopoeia (2009)1, 913.

[9] Indian Pharmacopoeia (2007)2, 931.

[10] An active metabolite of Diacerein, suppresses the interleukin-1alpha-induced

proteoglycan degradation in cultured rabbit articular chondrocytes, Tamura T., Ohmori

K.R., Jpn. J. Pharmacol. (2001)85, 101–104.

[11] New Symptomatic Slow Acting Drug for Osteoarthritis : Diacerein, Mahajan A., Singh



K., Tandon V.R., Kumar S., Kumar H., A JK Science (2006)8, 173-175.

[12] Diacerhein and Rhein Prevent Interleukin-1β-Induced Nuclear Factor-κB Activation by

Inhibiting the Degradation of Inhibitor κB-α, Alexandrina F.M., Caramona M.M.,

Carvalho A.P., Lopes M.C., Pharma. & Toxi. (2002)91, 22–28.

[13] Bioequivalence pharmacokinetic evaluation of two branded formulations of aceclofenac

100 mg: a single-dose, randomized, open-label, two-period crossover comparison in

healthy Korean adult volunteers, Rhim S.Y., Park J.H., Park Y.S., Lee M.H., Shaw L.

M., and Kang J.S., Clil Therap. (2008)30, 633-640.

[14] Simultaneous HPLC–UV determination of Rhein and Aeclofenac in human plasma,

Ojha A., Rathod R., Padh H., J. Chrom. B (2009)877, 1145- 1148.

[15] Simultaneous determination of Pseudoephedrine Sulfate, Dexbrompheniramine Maleate

and Loratadine in pharmaceutical preparations using derivative spectrophotometry and

ratio spectra derivative spectrophotometry, Feyyaz O., Yücesoy C., Dermi S., Kartal M.,

Kokdil G.,Talanta (2000)51, 269-279.

[16] Stability indicating HPTLC method for the simultaneous determination of

pseudoephedrine and cetirizine in pharmaceutical formulations. Makhija S.N., Vavia

P.R., J Pharm Biomed Anal. (2001) 25, 663-667.

[17] Quantitative and structural determination of pseudoephedrine sulfate and its related

compounds in pharmaceutical preparations using high-performance liquid

chromatography, Nian W., Wenqing F., Elizabeth L., Guodong C., Jayashree P.,

Tze-Ming C., and Birendra P., J. Pharm. Biomed. Anal. (2002)30, 1143-1155.

[18] Simultaneous determination of Loratadine and Pseudoephedrine sulfate in pharmaceutical

Formulation by RP-LC and derivative spectrophotometry, Mabrouk M.M., El-Fatatry H.

M., Hammad S., Abdel A., Wahbi M., J. Pharm. Biomed. Anal. (2003)33, 597-604.

[19] A new HPLC approach for the determination of hydrophilic and hydrophobic

components: the case of pseudoephedrine sulfate and loratadine in tablets. Abu L.

A., Hamdan I.I., Tahraoui A., Drug Dev Ind Pharm (2005)31, 577-588.



[20] Determination of loratadine and pseudoephedrine sulfate in pharmaceuticals based on

non- linear second-order spectrophotometric data generated by a pH-gradient flow

injection technique and artificial neural networks, Culzoni M.J., Goicoechea H.C., Anal

Bioanal Chem., (2007)389, 2217-2225.

[21] Simultaneous UV spectrophotometric estimation of ambroxol hydrochloride and

Levocetirizine dihydrochloride, Lakshmana P., Shirwaikar A.A., Annie S., Kumar C.D.,

Aravind K., Indian J. pharm. Sci. (2008)70-2, 236-238.

[22] Development and validation of a stability-indicating RP-HPLC method for the

determination of paracetamol with dantrolene or/and cetirizine and pseudoephedrine in

two pharmaceutical dosage forms. Hadad G. M., Emara S., Mahmoud W.M.,

Talanta (2009)79, 1360-1367.

[23] A porous graphitized carbon column HPLC method for the quantification of

paracetamol, pseudoephedrine, and chlorpheniramine in a pharmaceutical formulation.

Kalogria E., Koupparis M., Panderi I., J AOAC Int. (2010) 93, 1093-1101.

[24] A Sensitive RP-HPLC Method for Simultaneous Estimation of Diethylcarbamazine and

Levocetirizine in Tablet Formulation, Reddy J. M., Jeyaprakash M. R., Madhuri

K., Meyyanathan S. N., Elango K., Indian J Pharm Sci. (2011) 73, 320–323.

[25] Quantization of Dextromethorphan and Levocetirizine in combined dosage form using a

novel validated RP-HPLC method, Joshi S., Bhatia C., Bal C.S., Indian J Pharma. sci.,

(2012)74, 83-86.

[26] Simultaneous analysis of Levocetirizine dihydrochloride, Ambroxol hydrochloride, and

Montelukast sodium by RP-HPLC - PDA method, Srividya P., Buchi N. N., Tejaswini

M., Sravanthi D., J. Liq. Chrom. Rel. Techno. (2013) 36, 2871-2881.

[27] Simultaneous Quantification of Cefpirome and Cetirizine or Levocetirizine in

Pharmaceutical Formulations and Human Plasma by RP- HPLC, Arayne M.S., Sultana

N., and Nawaz M., J of Anal. Chem. (2008)63-9, 881–887.

[28] Simultaneous Analysis of Ambroxol HCl with Cetirizine HCl and of Ambroxol HCl with

levo-Cetirizine Dihydrochloride in Solid Dosage Forms by RP-HPLC, Birajdar A.S.,



Meyyanathan S.N., Raja R.B., Krishanaveni N., and Suresh B., Acta Chromatog. (2008)

3, 411–421.

[29] Determination of levocetirizine in human plasma by liquid chromatography–electrospray

tandem mass spectrometry: Application to a bioequivalence study, Morita M. R., Berton

D., Boldin R., Barros A.P., Meurer E. C., Amarante A.R., Campos D.R., Calafatti S.A.,

Pereira R., Abib J.E., and Pedrazolli J., J. Chrom. B (2008)862, 132-139.

[30] LC–MS–MS Assay for Simultaneous Quantification of Fexofenadine and

Pseudoephedrine in Human Plasma, Vijaya D., Bharathi K.R., Banda J., Gajjela R., Indu

B., Naidu A., Mullangi R., Chromatographia (2008)67, 461-466.

[31] Development and validation of a HPLC method for the simultaneous determination of

Bromhexine, Chlorpheniramine, Paracetamol, and Pseudoephedrine in their combined

cold medicine formulations, Silvana E. V., Teodoro S. K., J. Liq. Chrom. Rel. Techno.

(2013) 36, 2829-2843.

[32] Determination by HPLC with Electrochemical Detection of Formoterol RR and SS

Enantiomers in Urine, Butter J.J., Van den Berg B.T.J., Portier E.J.G., Kaiser G., Boxtel

C.J.V., J. Liq. Chromatogr. R. T. (1996)19, 993–1005.

[33] Automated and sensitive method for the determination of formoterol in human plasma by

high-performance liquid chromatography and electrochemical detection. Campestrini J.,

Lecaillon J.B., Godbillon J., J Chromatogr B Biomed Sci Appl. (1997) 704, 221-229.

[34] Assay for the determination of low dosage form of formoterol dry syrup by capillary

electrophoresis with head-column field-amplified sample stacking. Song J.Z., Chen

J., Tian S.J., Sun Z.P., J Pharm Biomed Anal. (1999) 21, 569-576.

[35] Validation of a RP-HPLC Method for the Assay of Formoterol and Its Related Substances

in Formoterol Fumarate Dehydrate Drug Substance, Akapo S., Asif M., J. Pharm.

Biomed. Anal. (2003)33, 935–945.

[36] Optimization and validation of a gas chromatographic method for analysis of (RS,SR)-

diastereoisomeric impurity in formoterol fumarate. Akapo S.O., Wegner M., Mamangun

A., McCrea C., Asif M., Dussex J.C., J Chromatogr A. (2004) 1045, 211-216.



[37] Formation of fatty acid conjugates of ciclesonide active metabolite in the rat lung after 4-

week inhalation of ciclesonide. Nave R., Meyer W., Fuhst R., Zech K.. Pulm Pharmacol

Ther. (2005)18 390-396.

[38] Simutaneous Spectrometric Determination of Formoterol Fumarate and Budesonide in

Their Combined Dosage Forms, Prasad A.V.S.S., Indian J. Chem. Technol. (2006)13,

81–83.

[39] Determination of formoterol in rat plasma by liquid chromatography-electrospray

ionisation mass spectrometry, Kakubari I., Dejima H., Miura K., Koga Y., Mizu

H., Takayasu T., Yamauchi H., Takayama S., Takayama K., Pharmazie (2007) 62

62,94-95.

[40] Sensitive Simultaneous Determination of Ciclesonide, Ciclesonide-M1-Metabolite and

Fluticasone Propionate in Human Serum by HPLC-MS/MS with, Mascher H.J., Zech K.,

Mascher D. J., APPI. J. Chrom. B (2008)869, 84–92.

[41] Chiral HPLC Analysis of Formoterol Stereoisomers and Thermodynamic Study of Their

Interconversion in Aqueous Pharmaceutical Formulations, Akapo S., McCrea C., Gupta

J., Roach M., Skinner W., J. Pharm. Biomed. Anal. (2009)49, 632–637.

[42] Rapid Spectrophotometric methods for the determination of two novel steroids in bulk

and pressurised metered - dose preparations, Dave N. H., Makwana A. G., Int.J.Pharm.&

Health Sci.(2010)1, 68-76.

[43] Development of ciclesonide dry powder inhalers and the anti-asthmatic efficacy in

guinea pigs, Fei L., Wang G.L., Zhang Y., et. al., J Chin Pharma Sci. (2011)20, 473-

482.

[44] Validated Method Development for Estimation of Formoterol Fumarate and

Mometasone Furoate in Metered Dose Inhalation Form by High Performance Liquid

Chromatography, Srinivasarao K., Gorule V.,et. al. J Anal Bioanal Techniques (2012)7,

1-4.

[45] Optimized method for rapid estimation of ciclesonide in bulk and its dosage form

(rotacap) by RP-HPLC, Vaghela V., J Liq Chrom Rel Tech. (2013)36, 1678-1685.



[46] Assay for the Determination of Low Dosage Form of Formoterol Dry Syrup by

Capillary Electrophoresis with Head-Column Field-Amplified Sample, Song J.Z., Chen

J., Tian S.J., Sun Z.P., J. Pharm. Biomed. Anal. (1999)21, 569–576.

[47] Spectrophotometric Determination of Formoterol Fumarate in Rotacap Formulation, Pai

P.N.S., Sandeep S.K., Sekhar A.B., IJPS, (2003)65, 649–650.

[48] Ultra-Sensitive Determination of Fomoterol in Human Serum by HPLC and

Electrospray Tandem Mass Spectrometry, Mascher D.G., Zech K., Nave R.,

Kubesch K.M.H.J., J. Chrom. B (2006 )830, 25–34.

[49] High Performance Liquid Chromatography Assay Method for Simultaneous Quantitation

of Formoterol and Budesonide in Symbicort Turbuhaler, Assi K.H., Tarsin W., Chrystyn

H., J. Pharm. Biomed. Anal. (2006)41, 325–328.

[50] Simultaneous determination of Aceclofenac and three of its metabolites in human plasma

by high- performance liquid chromatography, Hinz B., Auge D., Rau T., Rietbrock S.,

Brune K., Werner U., Bio. Chromate. (2003)17, 268-275.

[51] Determination of aceclofenac in human plasma by reversed-phase high performance liquid

chromatography. Jin Y., Chen H., Gu S., Zeng F., Se Pu. (2004)22, 252-254.

[52] Separation of aceclofenac and diclofenac in human plasma by free zone capillary

electrophoresis using N-methyl-D-glucamine as an effective electrolyte additive, Angelo

Z., Ciriaco C., Salvatore S., Emanuela P., Paolo E., Luca D., Euro j Pharma Sci. (2005)

24,375-380.

[53] Reverse phase HPLC method for determination of aceclofenac and paracetamol in tablet

dosage form, Momin M.Y., Yeole P.G., Puranik M.P., Wadher S.J., Ind. J. Pharma.

Sci. (2006)68-3, 387-389.

[54] Sensitive Determination of Nanogram Levels of Diacerein in a Pharmaceutical

Formulation by Flow Injection Chemiluminescence Analysis, Han-C. Y., Xiao F. Y., Hua

L., J Chin Chemi Soc (2007) 54, 949-956.

[55] Development and Validation of a Dissolution Method with Spectrophotometric Analysis



for Diacerhein Capsules, Borgmann S. M., parcianello L., Marcela Z., Bajerski L.,

Simone G., Sci. Pharma. (2008)76, 541–554.

[56] Simultaneous HPLC–UV determination of Rhein and Aeclofenac in human plasma,

Ojha A., Rathod R., Padh H., J. Chrom. B (2009)877, 1145- 1148.

[57] Development and validation of UV Spectrophotometric method for the determination of

Diacerein in capsules, Narade S., Patil S., Surve S., Shete D., Pore Y., Dig J Nano

Biostru. (2010)5, 113-118.

[58] Application of mixed-hydrotropy in titrimetric analysis of Aceclofenac bulk drug sample,

Maheshwari R. K., Jawalker S., Jahan F., Patel S., Mehtani D., Bulletin of Pharma Res.

(2011)1, 44-46.

[59] Determination of Diacerein in Rabbit Plasma by Liquid Chromatography - Mass

Spectroscopy and Its Application to Bioavailability Study, Shirwaikar A., Sarala A. D.,

Premalatha K., Sreejith K. R., Int J Res Pharma Biom Sci. (2012) 3, 1738-1744.

[60] A Quantitative and Qualitative High Performance Liquid Chromatographic etermination

of Aceclofenac Specified Impurity-B (Methyl[2-[(2, 6-dichlorophenyl)amino]

phenyl]acetate) in Aceclofenac Bulk Drug, Somashekar P.L., Sanjay Pai., Gopalkrishna

R., Chem Sci Trans., (2013)1, 1-9.

[61] Simple spectrophotometric methods for estimation of aceclofenac from bulk and

formulations, Bose A., Dash P.P., Sahoo M.K., Pharma. Methd. (2010) 1, 57-60.

[62] A simple and sensitive stability-indicating RP-HPLC assay method for the determination

of Aceclofenac, Bhinge J.R., Kumar R.V., Sinha V.R., J chroma sci (2008)46, 440-444.

[63] Single RP-HPLC method for the quantification of Aceclofenac, Paracetamol and

Chlorozoxazone in formulation, Reddy H.K.G., Reddy H.B., Maram R.K., Reddy UM.,

Int. J Sci. Inno. and Disco. (2012) 2, 471-490.

[64] Validated LC method for simultaneous analysis of tramadol hydrochloride and

aceclofenac in a commercial tablet, Kachhadia P.K., Doshi A.S., Ram V.R., Joshi H.S.,

Chromatographia (2008)68, 997–1001.



[65] Reverse Phase HPLC Method for Determination of Aceclofenac and Paracetamol in

Tablet Dosage Form, Godse V.P., Deodhar M.N., Bhosale A.V., Sonawane R.A., Sakpal

P.S., Borkar D.D., and Bafana Y.S., A. J. Res. Chem (2009)2-1, 37-40.

[66] Simultaneous Estimation of Paracetamol, Chlorzoxazone and Aceclofenac in

pharmaceutical formulation by HPLC Method, Karthikeyan V., Yuvaraj V., Nema R.

K., Int. J. ChemTech Res. (2009)1, 457-460.

[67] A Review – Importance of RP-HPLC in Analytical Method Development, Prathap B.,

Akalanka D., Srinivasa G.H., Johnson P. & Arthanariswaran P., int. j. no. tre. in Pharma.

Sci. (2013)3-1,15-23.

[68] On the Reproducibility of Column Performance in Liquid Chromatography and the Role

of the Packing Density, Stanley B.J., Foster C.R., Guiochon G., J. Chrom. A. (1997)

761, 41-51.

[69] Liquid phase separation methods: HPLC, FFF, electrophoresis, Kirkland J.J.,

McCormick R.M., Chromatographia (1987)24-1, 58-76.

[70] On the Reproducibility of Column Performance in Liquid Chromatography and the Role

of the Packing Density, Stanley B.J., Foster C.R., Guiochon G., J. Chrom. A. (1997)

761, 41-51.

[71] Ghost peaks in reversed-phase gradient HPLC: a review and update, Stuart W., J. Chrom.

A (2004)1052, 1-2, 1-11.

[72] Implementation of a rapid and automated high performance liquid chromatography

method development strategy for pharmaceutical drug candidates, Elizabeth F.H., Patrick

L., Sergey G., J. Chrom. A (2006)1107, 1-2, 79-87.

[73] Development of some stationary phases for reversed-phase HPLC, Kirkland J.J.,

J. Chrom. A (2004)1060, 1-2, 9-21.

[74] Simultaneous variation of temperature and gradient steepness for reversed-phase high-

performance liquid chromatography method development, Dolan J.W., Snyder L.R.,

Saunders D.L., Heukelem L.V., J. Chrom. A (1998)803, 1-2, 33–50.



[75] HPLC method development in early phase pharmaceutical development, Henrik T.R.,

Kelly A.S., Sheetal G., Sep. sci. Techn. (2007)8, 353-371.

[76] Validation of analytical procedures: text and Methodology. In: ICH Harmonized

Tripartite Guidelines Q2 (R1). November 2005.

[77] Understanding and Implementing Efficient Analytical Methods Development and

Validation, Jay B., Kevin J., Pierre B., Anal.Chem.Test. (2003)1, 6-13.

[78] Validation of HPLC methods in pharmaceutical analysis, Ashley S. L., Sep. sci. Techn.

(2005)6, 191-192.

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