“development and validation of analytical treatment of

1

A Synopsis

On

“Development and Validation of Analytical Methods for the Estimation of Drugs Used in theTreatment of Arthritis”

Submitted to:

Gujarat Technological University

For the Degree ofDoctor of Philosophy

In

Pharmacy

By:Jignasha Maheshkumar Derasari

Registration No: 9050Enrollment No: 119997290014

Under the Guidance ofDr. Vandana B. Patel

Principal & Professor, Babaria Institute of Pharmacy, Varnama, Vadodara

2

A. Title of the thesis and Abstract

Title: “Development and Validation of Analytical Methods for the Estimation of drugs

used in the Treatment of Arthritis”

Abstract

Present research investigation deals with the development of analytical methods and their validation

of drugs used in the treatment of arthritis. Four combinations i.e. 1) Diclofenac potassium and

Febuxostat 2) Febuxostat and Ketorolac tromethamine 3) Febuxostat and Naproxen 4) Ibuprofen and

Tramadol hydrochloride have been chosen for analytical study by stability indicting RP-HPLC and/or

UV spectrophotometric methods.

Two UV spectrophotometric methods viz .simultaneous equation method and first order derivative

method and stability indicating RP-HPLC method have been developed and validated for

combination (1) and (3). These methods (two UV and one stability RP-HPLC) of these two

combinations were compared statistically by one-way ANOVA study with students’t-test. Simple,

accurate, precise, robust and reproducible stability indicating RP-HPLC method has been conducted

for combined dosage form (2) and (4). Stability of all four pharmaceutical dosage forms was assessed

in various degradation conditions like hydrolysis (acid- alkali), oxidation, thermal and photolytic

conditions.

DoE (Design of Experiments) technique, an integral part of QbD has been applied to check the effect

of different variables on acid degradation pattern of both the drugs via Full Factorial Design (FFD) –

23(three factors at two levels) and kinetic study was also carried out to obtain degradation rate

constant, half-life (t0.5), shelf life (t0.9), activation energy (Ea) and Arrhenius energy factor for both the

drugs of combination (1). Moreover, same degraded samples were spiked into LC-MS/MS for

analytical study. CCD (Central Composite Design) has been instigated on robustness study for

simultaneous estimation of ibuprofen and tramadol hydrochloride. 33 (three factors at three levels)

CCD has been planned to evaluate the effect of pH, flow rate and mobile phase composition on

resolution, RT and asymmetric factor of both the drugs.

The calibration plots of all developed methods were linear over the selected concentration range with

a correlation coefficient value nearer 0.999. % assay values and % recoveries of drug of combined

dosage forms were obtained within the limit specified in ICH guidelines i.e. 98-102%. One-way

ANOVA study suggested stability indicating RP-HPLC method is more accurate and precise than UV

spectrophotometric methods for combination 1 and 3. Results of stability studies in different

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conditions for combined dosage forms indicated all the drugs were susceptible to hydrolysis (acidic

and alkaline) whereas comparatively stable towards other degradation conditions.

All the developed methods were able to detect and/or separate the drugs from their potential related

substances. Hence, the proposed methods were found to be simple, sensitive, economical, precise and

robust and can be applicable to the routine analysis of the selected combinations.

Keywords

UV spectrophotometric methods, stability indicating RP-HPLC, QbD, DoE, Kinetic study, ANOVA,

Validation

B. Brief description on the state of the art of the research topic:

Analytical Method Development [1-3]:

Method development usually requires selecting the method requirements and deciding on what type

of instrumentation to utilize and why to utilize. The development of any new or improved method

usually tailors existing approaches and instrumentation to the current analyte as well as to the final

needs or requirements of it.

Need for Analytical Method Development:

Any official method is not reported in IP, BP and USP for estimation of drug or combination

of drugs.

Due to patent regulations, a systemic analytical method may not be available for the drug and

drug combinations in the literature

Analytical methods may not be available for the drug in the form of a formulation due to the

interference caused by the formulation excipient.

Bio analytical methods for the quantitation of the drug may not be available.

Reported analytical procedures may require expensive reagents and solvents. It may also

involve cumbersome extraction and separation procedures and these may not be reliable.

ANALYTICAL METHOD VALIDATION [4-5]

Principle purpose of the method validation is to ensure that a selected analytical method will give

reproducible and reliable results that are adequate for the intended purpose. Validation of analytical

method is to be done according to ICH guidelines

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Various validation parameters are; accuracy, precision (repeatability, intermediate precision and

reproducibility), linearity and range, limit of detection (LOD)/ limit of quantitation (LOQ),

robustness, specificity.

STRESS TESTING [6-8]

According to a FDA guidance document, a stability-indicating method is “A validated quantitative

analytical procedure that can detect the changes with time in the pertinent properties of the drug

substance and drug product. It measures the active ingredients without interference from degradation

products, process impurities, excipients or other potential impurities.”

Stability testing represents an often overlooked area by many pharmaceutical and biotechnology

organizations. The purpose of stability testing is to provide evidence regarding the quality of a drug

substance or drug product and how each may vary with time under the influence of a variety of

environmental factors such as temperature, humidity, light etc. and to establish both a retest period

for the drug substance and a shelf life of drug product.

According to ICH and FDA, stress testing fulfills 3 main purposes:

I. To provide a stability assessment of the drug substance or the drug product.

II. To elucidate the possible degradation pathway of the drug substance or API in drug Product.

III. To investigate the stability indicating power of the analytical procedure applied for the drug

substance and drug product.

For new drug products or drug products containing more than one active ingredient or determination

of drug and its metabolites, development of analytical method (Spectrophotometric, High

performance liquid chromatography (HPLC) & High performance thin layer chromatography

(HPTLC)), LC-MS, LC-MS/MS) is essential. The official test methods that result from these

processes are used by quality control laboratories to ensure the identity, purity, potency, and

performance of drug products.

Pathology and management of rheumatoid arthritis (RA) has evolved significantly over last two

decades. Since the 1990's it was known that irreversible joint damage occurred early in the disease

and hence early use of DMARDs was recommended [9]. Despite the available knowledge managing

patients of rheumatoid arthritis was always a challenge. The textbook criteria for diagnosing the

disease hardly seemed helpful in identifying early disease [10]. The use of sequential monotherapy

resulted in relatively poor remission rates [11]. The evaluation of patients was mainly focused on

musculoskeletal manifestations and so other systemic complications which were often missed [12].

The last decade has seen radical change in every domain of management of RA.

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Non-Steroidal Anti Inflammatory Drugs (NSAIDs) are most frequently and commonly used to

combat with the joint problems associated with Arthritis. Considering all the aspects of arthritis

treatment, extensive literature review was carried out on the drugs (alone and in combination with

other drugs) used in the treatment of arthritis [13-34]. Four combinations i.e. 1. Diclofenac potassium

and Febuxostat 2) Febuxostat and Ketorolac tromethamine 3) Febuxostat and Naproxen and 4)

Ibuprofen and Tramadol hydrochloride were selected for analytic method development by UV &

stability RP-HPLC; QbD based statistical analysis, ANOVA study and degradation product analysis

by LC-MS/MS.

C. Definition of the problem:

Literature review revealed few analytical methods i.e., RP-HPLC, stability indicating RP-HPLC and

UV spectrophotometric for an individual drug and in combination with other drugs. Besides these,

few methods were reported for these combinations also. However, they are not adequate to monitor a

change in chemical properties over the time under various stressed conditions and the effects of

critical parameters on drug response and to demonstrate the degradation pattern of the drugs in their

combined dosage forms. Moreover, use of novel techniques (QbD) and sophisticated instrumentation

(LC-MS/MS) is lacking.

D. Objective and Scope of work:

Based on extensive literature review, attempt has been made:

1. To develop simple, accurate, precise, economical, sensitive and robust analytical methods by

using UV, RP-HPLC that could separate drug from its potential related substances.

2. To conduct forced degradation study under various stressed conditions like hydrolysis (acid-

alkali), oxidation, thermal and photolysis.

3. To validate developed methods as per ICH guidelines which ensure optimum method

performance over the life cycle of the drugs used in the treatment of arthritis.

4. Implementation of a QbD approach by using DoE techniques to measure the effect of critical

factors on drugs response.

5. Qualitative analysis of forced degradation products of Diclofenac potassium and Febuxostat

by LC-MS/MS.

Scope of research work

Present research work involves development and validation of analytical methods i.e., stability

indicating RP-HPLC and UV spectrophotometric methods, statistical analysis by QbD and ANOVA.

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Conducted analytical study suggests following possible research outcomes:

1. Degradation products identification could be done by LC-MS/MS and their structural

elucidation with novel techniques i.e. IR, NMR and MS can be possible.

2. Stability indicating RP-HPLC method can be extended in biological fluids for bio analysis of

drugs combination, their degradation behavior in different biological medium, BA/BE studies.

3. QbD based refinement of method can be done.

E. Original contribution by the thesis

Whole analytical studies conducted have not yet been reported. Thus, thesis will provide a scientific

database for further development of sound research with sophisticated instrumentation and novel

techniques.

F. Methodology of Research, Results/Comparison

Four Pharmaceutical combinations have been selected for analytical study.

1) Diclofenac potassium and Febuxostat 2) Febuxostat and Ketorolac tromethamine

3) Febuxostat and Naproxen 4) Ibuprofen and Tramadol hydrochloride

Detailed methodology and results comparison of methods for those combinations are described as

below;

1. DICLOFENAC POTASSIUM (DP) AND FEBUXOSTAT (FB)

UV spectrophotometric methods: Selection of detection wavelength

20 µg/mL solution of each drug viz. DP and FB was prepared in 0.1 N NaOH. Then

solutions were scanned between 200-400 nm against distilled water as a reagent blank and

λmax selected for DP and FB were 276 nm and 314 nm, respectively. Later on the λmax

were confirmed by linearity study.

1.1. Simultaneous Equation Method:

1.1.1. Preparation of standard of DP

50 µg/ mL of standard solution of DP was prepared in 0.1 N NaOH. This solution was

further diluted with distilled water in order to prepare standard solutions of 2, 3, 4, 5, 6

and 7 µg/ mL of DP.

1.1.2. Calibration curve preparation and absorptivity determination for DP

The above solutions were scanned in UV and their absorbance was measured at 276

nm and 314 nm respectively and linearity study was carried out. The regression

coefficient and absorptivity values of DP were measured.

1.1.3. Preparation of standard of FB

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100 µg/ mL of standard solution of FB was prepared in 0.1 N NaOH solution. This

solution was further diluted with distilled water in order to prepare standard solutions

of 4, 8, 12, 16, 20 and 24 µg/ mL of FB.

1.1.4. Calibration curve preparation and absorptivity determination for FB

The above solutions were scanned in UV and their absorbance was measured at 276

and 314 nm respectively and linearity study was carried out. The regression coefficient

and absorptivity values of FB were measured.

1.1.5. Application of simultaneous equation method on tablet formulation of DP

and FB.

20 tablets were accurately weighed and quantity equivalent to 100 mg of DP and 40

mg of FB dissolved in 100 mL of 0.1N NaOH. 10 mL of above solution was diluted

up to 100 mL with distilled water. Sonication was done for 10 mins. It was mixed and

filtered the resulting solution with Whatman filter paper. 1.5 mL of resulting solution

was taken and diluted to 10 mL with distilled water. The absorbance of the resulting

solution was measured at λ1 276 nm and λ2 314nm and calculate the content by

simultaneous equations

Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1)

Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1)

The absorptivity of DP at 276 and 314, aX1 = .0292 and aX2 = 0.0021 respectively.

The absorptivity of FB at 276 and 314, aY1 = 0. 0156 and aY2= 0.0641 respectively

The absorbance of the diluted sample at 276 and 314, A1 and A2 was found to be

1.768 & 0.8994 respectively.

1.2. 1st Order Derivative Method:

1.2.1. Selection of detection wavelength and calibration curve preparations for

DP and FB

All absorption spectra recorded for simultaneous equation method were converted into

1st order derivative form. They were traced with smoothing factor (Δλ) =2 and scaling

factor 10. Derivative spectra of DP and FB were overlaid and the Zero crossing point

of DP and FB were measured. The calibration plots were constructed and linearity was

performed.

1.2.2. Application of 1st order derivative method for simultaneous estimation DP

and FB in sample mixture.

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Standard solution of mixture was prepared by the same procedure explained in section

1.1.5. The absorbance of the resulting solution was measured at λ1 260.77 nm for the

determination of DP and λ2 275.96 nm for FB and calculate the content by the straight

line equation, Y=mX+C, where; Y= absorbance value, m=slope and C= intercept.

Both m and C values were taken from straight line equation of calibration curve.

1.3.Stability indicating RP-HPLC:

1.3.1. Preparation of standard solutions of DP and FB:

100 µg/mL of solution was prepared for each-DP and FB in methanol. These

solutions were further diluted to get a series of concentrations for DP (25-65

µg/mL) and for FB (10-26 µg/mL). 293 nm wavelength was selected for

detection from the overlain spectra of DP (45 µg/mL) and FB (18 µg/mL).

1.3.2. Assay procedure for tablet formulation of DP and FB.

Twenty tablets were crushed and powder equivalent to 100 mg of DP and 40

mg of FB was weighed and was transferred to 100 mL of volumetric flask.

Methanol was used as a diluent and volume was made up to the mark to get a

sample solution of 1000 µg/mL of DP and 400 µg/mL of FB. 0.45 mL was

pipetted out and diluted up to 10 mL to get final concentration of DP (45

µg/mL) and FB (18 µg/mL). This solution was spiked into the system and

chromatogram was recorded.

1.3.3. Finally optimized chromatographic conditions for method development:

Parameters ConditionsStationary phase Agilent eclipus C18 column, 250×4.6mm,

5 µm particle sizeMobile phase Ammonium acetate Buffer: Acetonitrile (47:53),

pH=4 adjusted with ortho phosphoric acidPump mode IsocraticFlow rate (mL/min) 0.9 mL/minRun time (min) 20Volume of injection (µm) 20Detection wavelength (nm) 293Retention time (min) 12.01in for DP and 7.61 min for FB

1.3.4. Forced degradation studyWorking solutions of DP (45 µg/mL) and FB (18 µg/mL) were used for the study of variousvalidation parameter and stability study of both the drugs in different forced degradation conditions.

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Typeof

degradation

Degradation conditions

Acidic 0.1 N HCl, 30 minutes at RTAlkaline 0.1 N NaOH, 30 minutes at RT

Oxidative 3 mL 3% H2O2, 2 hours at 60°CThermal 500mg tablet powder was heated in oven at 70ºC

for 24 hrsPhotolytic UV radiation for 24 hours

1.1.FFD and kinetic study on acid degradation of DP and FB:

1.1.1. FFD (Full Factorial Design)

Three critical factors i.e. concentration of HCl (0.05 M & 0.5 M), time (15 minutes &

60 minutes), temperature (25°C & 60°C) were identified and their lower level (-1) and

higher level (+1) values were selected for Full Factorial Design.

Stat-Ease, inc., Design-Expert ®, version 7.0.0. software was used for FFD analysis.

All these data were entered in design setup given for 23 full factorial design and then

analyze the statistical results.

Based on the available results, optimization was done via numerical settings to get a

set of optimum conditions which would give maximum degradation of DP closer to

FB.

From these suggested optimum conditions, any two conditions were randomly selected

and exercise was performed again to find out % error.

1.1.2. Acid degradation kinetic study:

Acid degradation kinetic study of both the drugs was performed using 0.12 N HCl

concentration at various temperatures (293K, 313K,333K and 353K– 20°C, 40°C,

60°C and 80°C, respectively) up to 10 hrs (0,2,4,6,8,10) and Log % drug remaining Vs

Time was plotted to check the order of reaction.

Degradation constant (K), half-life (t0.5), shelf life (t0.9) and activation energy Ea were

calculated for each line. Further obtained data were applied on Arrhenius equation for

Arrhenius frequency factor (A), activation energy Ea, degradation rate constant (K),

half-life (t0.5 ) and shelf life (t0.9) at RT by plotting graph of Log k Vs. 1/TX1000.

Slope of the equation was used for the calculation using LogK=Log A-Ea/2.303 RT,

where R= gas constant (1.987 k/cal. mol)

1.2. Quality analysis of degraded impurity by LC-MS/MS

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Degradation samples of DP and FB were spiked into LC-MS/MS for degraded product

peak identifications. Though area was drastically decreased after degradation, there

were no separate peaks identified for degradation products along with API.

RESULTS:

1. All the three methods (two UV and one stability RP-HPLC) were validated as

per ICH guidelines and validation parameters were obtained in specified limits.

% recovery values and assay readings were found to be between 98-102%.

Moreover, stability study indicated both the drugs were more susceptible to

acid hydrolysis and most stable to alkali hydrolysis.

2. 23FFD suggested the chosen model was significant as p-values (0.0280 for DP

and 0.0053 for FB) obtained by ANOVA analysis were less than 0.1000. As

per perturbation plot, pareto chart (t-value and Bonferroni values for individual

factor and 2 FI) and 3D surface plots, concentration of HCl was the most

critical factor responsible for acid degradation.

3. Straight line (linear response) obtained by kinetic study was shown the

degradation of both the drugs was followed FIRST ORDER KINETIC.

2. FEBUXOSTAT (FB) AND KETOROLAC TROMETHAMINE (KT)

2.1.Stability indicating RP-HPLC2.1.1. Preparation of standard solutions and working solutions of FB and KT:

Standard solutions of FB (400 µg/mL) and KT (150 µg/mL) were prepared in

methanol. These solutions were further diluted up to 10 ml to get a series of

concentrations for FB (50-150 µg/mL) and for KT (18.75-56.25 µg/mL) which

were used as working standards. 321 nm wavelength was selected for detection

from the overlain spectra of FB (100 µg/mL) and KT (37.5 µg/mL).

2.1.2. Assay procedure for tablet formulation of FB and KT.

Twenty tablets were crushed and quantity equivalent to 40 mg of FB and 15

mg of KT was transferred to 100 mL of volumetric flask. Volume was made up

to the mark with diluent, methanol. 2.5 ml of sample solution was taken and

diluted up to 10 mL to get final concentration of FB (100 µg/mL) and KT (37.5

µg/mL). This solution was spiked into the system and chromatogram was

recorded.

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5 µm particle sizeMobile phase Buffer(KH

2PO

4): Acetonitrile (60:40)

pH=4 adjusted with ortho phosphoric acidPump mode IsocraticFlow rate (mL/min) 1 mL/minRun time (min) 15Volume of injection (µm) 20Detection wavelength (nm) 321Retention time (min) 7.533 min for FB and 3.797 min for KT

2.1.4. Forced degradation study

Working solutions of FB (100 µg/mL) and KT (37.5 µg/mL) were used for the study of various

validation parameter and stability study of both the drugs in different forced degradation conditions.

Typeof

degradation

Degradation condition & time

Acidic 1 N HCl, reflux 2hoursAlkaline 1 N NaOH, 30 minutes at RT

Oxidative 3 mL 3% w/v H2O2, 8 hoursThermal 500mg tablet powder was heated in oven at 105ºC

for 3 hrsPhotolytic UV radiation for 21 hours

RESULTS:

Developed stability indicating method was validated as per ICH guidelines and validation parameters

were obtained in specified limits. % recovery values and assay readings were found to be between

98-102%. Moreover, stability study indicated FB and KT, both were more susceptible to acid &

alkali hydrolysis whereas stable to oxidation.

3. FEBUXOSTAT AND NAPROXEN:

UV spectrophotometric methods: Selection of detection wavelength

10 µg/mL solution of each drug viz. Febuxostat (FB) and Naproxen (NP) was prepared in methanol

and were scanned between 200-400 nm against methanol as a reagent blank. The λmax selected for

Febuxostat and Naproxen were 314 nm and 271 nm, respectively. Later on the λmax were confirmed

by linearity study.

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3.1.Simultaneous equation method3.1.1. Preparation of standard of FB

100 µg/ mL of standard solution of FB was prepared in methanol. It was

further diluted with diluent in order to get working standards of 2, 3, 4, 5, 6

and 7 µg/mL of FB.

3.1.2. Calibration curve preparation and absorptivity determination for FB

The above solutions were scanned in UV and their absorbance was measured

at 314 and 271 nm respectively and linearity study was carried out. The

regression coefficient and absorptivity values were measured for FB.

3.1.3. Preparation of standard of NP

125 µg/mL of standard solution of NP was prepared in methanol. It was further

diluted with diluent-methanol in order to get working standards of 12.5, 18.75,

25, 31.25, 37.5 and 43.75 µg/mL of NP.

3.1.4. Calibration curve preparation and absorptivity determination for NP

The above solutions were scanned in UV and their absorbance was measured

at 314 nm and 271 nm respectively and linearity study was performed. The

regression coefficient and absorptivity of NP was measured.

3.1.5. Application of simultaneous equation on synthetic mixture of FB and NP

Quantities equivalent to 40 mg of FB and 250 mg of NP were taken into 100

mL volumetric flask. Methanol was added to dissolve the content and volume

was made up to the mark to get 400 µg/mL of FB and 2500 µg/mL of NP. 10

ml of solution was pipetted out from the stock solution and diluted upto 100

mL to have 40 µg/mL of FB and 250 µg/mL of NP. 1 mL of this solution was

further diluted and measured at λ1 314 and λ2 276 nm. Calculate the content

present in label by simultaneous equations.

3.2. First Order Derivative Method:3.2.1. Determination of wavelength (ZCP-Zero Crossing Point)

Linearity range for FB was found to be 2-7 µg/mL and for NP was 12.5-43.75

µg/mL. All absorption spectra were converted into 1st derivative form. They

were traced with smoothing factor (Δλ) =4 and scaling factor 10. Derivative

spectra of FB and NP were overlaid and ZCP of FB was found to be 314.28

nm which is suitable wavelength for determination of NP. ZCP of NP was

found to be 297.30 nm which is suitable wavelength for determination of FB.

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Calibration curves were plotted on above mentioned range of concentrations

for both the drugs and regression coefficient of FB was found 0.9985 at 297.30

nm and of NP was found 0.9992 at 314.28 nm.

3.2.2. Application of 1st Order Derivative Method on synthetic mixture of FB

and NP

Standard solutions and working solutions containing of FB and NP were

prepared as per the procedure given in section 3.1.5. Working solution of 1 mL

was taken in to 10 mL volumetric flask and volume was made upto the mark to

get 4 µg/mL of FB & 25 µg/mL of NP. Absorbance of it was measured at

297.30 nm for the determination of FB and at 314.28 for the determination of

NP. These values were put in to straight line equations of calibration curves

and % assay was calculated for the proposed method.

3.3.Stability indicating RP-HPLC:

3.3.1. Preparation of standard solutions and working solutions of FB & NP:

100 µg/mL of standard solution was prepared for each-FB and NP in methanol.

Both the solutions (0.6,0.8,1.0,1.2,1.4 mL of FB and

0.375,0.500,0.625,0.750,0.875 mL of NP) were further diluted up to 10 ml to

get a series of concentrations for FB (6-14 µg/mL) and for NP(37.5-87.5

µg/mL) which were used as working standards. 288 nm wavelength was

selected for detection from the overlain spectra of FB (10 µg/mL) and NP

(62.5 µg/mL).

3.3.2. Assay procedure of FB & NP on synthetic mixture

Quantities equivalent to 40 mg of FB and 250 mg of NP was transferred to 100

mL of volumetric flask. Methanol was added to dissolve the content and

volume was made up to the mark. Further, 10 mL of standard solution was

taken and dilute up to 100 mL to obtained working solution of 40 µg/mL of FB

and 250 µg/mL of NP. 2.5 ml of sample solution was pipetted out and diluted

up to 10 mL to get final concentration of FB (10 µg/mL) and NP (62.5 µg/mL).

This solution was spiked into the system and chromatogram was recorded.



5 µm particle size

14

Mobile phase Methanol: Acetonitrile : KH2PO

4

Buffer(35:20:45) pH=5 adjusted with 10 MKOH

Pump mode IsocraticFlow rate (mL/min) 0.9 mL/minRun time (min) 20Volume of injection (µm) 20Detection wavelength (nm) 288Retention time (min) 7.525 min for FB and 5.034 min for NP


Working solutions of FB (10 µg/mL) and NP (62.5 µg/mL) were used for the study of various

validation parameter and stability study of both the drugs in different forced degradation conditions.

Typeof

degradation


Acidic 1 N HCl, 1hour at RTAlkaline 1 N NaOH, 30 minutes at RT

Oxidative 3 mL 30% w/v H2O2, 1 hour at 60°CThermal Working solution was kept in oven at 80ºC for 3

hoursPhotolytic UV radiation for 24 hours

RESULTS:

All the three developed (two UV and one stability RP-HPLC) methods were validated as per ICH

guidelines. The obtained validation parameters were in specified limits. % recovery values and assay

readings were found to be between 98-102%. Moreover, stability study indicated that FB was more

susceptible to alkali hydrolysis and NP was unstable to acid hydrolysis, whereas both were stable to

other stressed conditions.

4. IBUPROFEN (IBU) AND TRAMADOL HYDROCHLORIDE (TRM)

4.1.Stability indicating RP-HPLC method:4.1.1. Preparation of standard solution and selection of detection wavelength

1000 µg/mL of IBU and 100 µg/mL TRM standard solution were prepared in

mobile phase [buffer (0.05M phosphate buffer): methanol, pH=5 (30:70%)].

Working standards of Ibuprofen (48μg/ml) and Tramadol Hydrochloride

(6μg/ml) were prepared by diluting 0.48 mL and 0.6 mL in to 10 mL of

15

diluent, respectively. These solutions were scanned separately in the range of

200-400nm and 215 nm wavelength was selected for analytical study.

4.1.2. Assay procedure of tablet formulation of IBU and TRM.

Twenty tablets were crushed and powder equivalent to 40 mg of IBU and 5 mg

of TRM was taken into 100 mL of volumetric flask. Mobile phase was used as

a diluent and volume was made up to the mark to obtain 400 µg/mL of IBU

and 50 µg/mL of TRM. Sample solutions were taken in different aliquots (0.4,

0.8, 1.2, 1.6, 2.0, 2.4 mL) and were diluted with mobile phase to get a series of

different concentration range for IBU and TRM (2+16, 4+32, 6+48, 8+64,

10+80, 12+96 µg/mL). Working solution of 48 µg/mL of IBU and 6 µg/mL of

TRM was spiked in to system and chromatogram was recorded to calculate the

% content present in the sample mixture.


Parameters ConditionsStationary phase Intercil C18

(150mm X 4.6 mm i.d., 5 μm particle size)Mobile phase Buffer (0.05 M phosphate buffer) :Methanol

(pH-5, adjusted with 1% o-phosphoric acid)(30:70 v/v)

Pump mode IsocraticFlow rate (mL/min) 1 mL/minRun time (min) 10Volume of injection (µm) 20Detection wavelength (nm) 215Retention time (min) 3.770 min for IBU and 2.543 min for TRM


Working solutions of IBU (48 µg/mL) and TRM (6 µg/mL) were used for the study of various

validation parameter and stability study of both the drugs in different elevated forced degradation

conditions.

Typeof

degradation


Acidic 1 N HCl, 3hour at 60°CAlkaline 1 N NaOH, 3hour at 60°C

Oxidative 2 mL 20% w/v H2O2, 3hour at 60°CThermal 500 mg powder was kept in oven at 100ºC for 24

hoursPhotolytic UV radiation for 24 hours

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4.1.5. CCD on robustness study of IBU and TRM

Central Composite Design (CCD) was designed at 3 levels (-1, 0, +1) with

three critical factors (33) i. e. pH of mobile phase, flow rate, mobile phase

composition and effect of them were evaluated on final responses of RT of

both the drugs, resolution and asymmetric factors of both the drugs.

RESULTS:

Developed stability indicating method was validated as per ICH guidelines and validation parameters

were obtained in specified limits. % recovery values and assay readings were found to be between

98-102%. Moreover, stability study in elevated conditions were indicated that IBU and TRM were

unstable to thermal degradation and whereas less susceptible to hydrolytic conditions.

G. Achievement with respect to objectives

1. Total 8 simple, accurate, precise, economical, sensitive and robust analytical methods

(two UV methods and one stability RP-HPLC method for combination 1 and 3, one

stability RP-HPLC method for combination 2 & 4) have been developed that could

separate drug from its potential related substances.

2. All the developed methods have been validated as per ICH guidelines. The obtained

results of each validation parameter was within the standard limit which ensures optimum

method performance over the life cycle of the drugs used in the treatment of arthritis.

Statistical comparison (one way ANOVA/ paired t-test) of methods indicated RP-HPLC

methods were more precise and accurate as compared to UV spectrophotometric methods.

3. FFD on acid degradation of FB and DP was indicated concentration of HCl was the most

critical factor responsible for acid degradation Experimental trials were extended up to

acid degradation kinetic profile to get degradation rate constant, half-life, shelf life and

activation energy for both drugs.

4. CCD was applied on robustness study for combination 4. Study suggested pH was the

critical factor to influence the value of RT. It was also indicated the effect of other factors

like flow rate and mobile phase composition on resolution & asymmetric factor.

H. Conclusion

Analytical study was suggested that all the developed methods were found to be accurate, precise,

economical and robust. Results of validation parameters obtained for each method was mentioned

below:

17

1) Analysis of tablet formulation was found 100.09% and 100.07% for DP and 99.85% and

99.88 % for FB for UV spectrophotometric method 1 and method 2, respectively. % assay of

DP and FB were obtained 100.37 and 101.11 for stability indicating RP-HPLC method.The

obtained validation parameters for all the methods were within the limits (98-102 %) specified

in the ICH guidelines Q2 (R1). ANOVA (students’t-test) study suggested RP-HPLC method

is more precise and selective than UV spectrophotometric methods. Stability study of both the

drugs showed maximum degradation of them in acidic conditions whereas stable towards

other conditions.

Moreover, FFD study on acid degradation of DP and FB shown concentration of HCl is the

most critical factor for responsible for degradation. Both the drugs follow first order kinetic.

LC-MS/MS analysis of degraded samples had shown only API peaks.

2) Stability indicating RP-HPLC method was developed for FB and KT and the obtained %

assay results for FB and KT were 100.39 and 100.24, respectively. The results of forced

degradation study indicated both the drugs were more susceptible to hydrolysis (Acidic and

alkaline.

3) Analysis of validation parameters for two UV spectrophotometric and stability indicating RP-

HPLC method was suggested all three methods were accurate, precise, robust and sensitive.

% assay results were within 98-102%. Forced degradation study of FB and NP in various

degradation conditions showed NP was more degraded in acidic condition whereas FB in

alkaline condition. RP-HPLC method is more accurate and precise than UV methods as per

ANOVA (students’t-test) study.

4) % assay of stability indicating RP-HPLC method for IBU and TRM were 99.85 and 99.99.

CCD method on robustness study showed all three factors i.e. pH, flow rate and mobile phase

compositions were critical to influence the responses like RT, resolution and asymmetric

factor. The obtained results suggested the range selected for each level must be minimized.

18

G. List of all publications

1. Papers published:

i. “Spectrophotometric Determination of Febuxostat and Diclofenac Potassium in

Their Combined Dosage Form by Simultaneous Equation and First Order

Derivative Methods, Derasari, J. M.; Patel, V. B., Asian Journal of Research in

Chemistry; October 2013, Vol. 6 Issue 10, p968-972.

ii. “Development and Validation of Stability indicating LC-PDA method for

Simultaneous Assessment of Febuxostat and Ketorolac Tromethamine in Tablet

Dosage Form”, J. M. Derasari and V. B. Patel, Asian Journal of Chemistry;

Vol.29, No.1 (2017), 69-75, http://dx.doi.org/10.14233/ajchem.2017.20134.

2. Poster presented:

i. Poster entitled “QbD approach in optimization of acid degradation for a validated

stability indicating LC method of Febuxostat and Diclofenac potassium in bulk

and in tablet formulation: Application to acid degradation kinetic study” was

presented at APTICON-2016, Annual National convention organized by

Association of Pharmaceutical Teachers of India (APTI) at Manipal University,

Manipal, Karnataka during 14th to 16th October, 2016. (National)

ii. Poster entitled “Simultaneous RP-HPLC Assessment of Febuxostat and Ketorolac

tromethamine in tablet dosage form for degradation behavior under stressed

conditions’ was presented at NIPICON-2016, 3rd Nirma Institute of Pharmacy

International Conference held during 21st to 23th January, 2016. (International)

iii. Poster entitled “Development and Validation of Stability Indicating LC-PDA

Method For Simultaneous Estimation of Febuxostat and Naproxen in Bulk and in

Combined Dosage Form” was presented at Two Days National Seminar on

Spectral Interpretation: A Systemic approach held on 30th & 31st January, 2017.

(National)

I. References

1. Skoog DA, West DM, and Hollar FJ (2004) Fundamentals of Analytical Chemistry, 8th

Edition; Saunders College Publishing, Philadelphia, pp 973-992.

19

2. Beckett AH, Stenlake JB (2001) UV-visible Spectrophotometry: Practical Pharmaceutical

Chemistry Part-II, 4th Edition; CBS Publishers and Distributors, New Delhi, pp 285-97.

3. Sethi PD (2008) High Performance Liquid Chromatography-Quantitative Analysis of

Pharmaceutical Formulations, reprint, CBS Publishers and Distributors, New Delhi, pp 14-

15, 116-120.

4. ICH, Validation of Analytical Procedures: Text and Methodology. International

Conference on Harmonisation, IFPMA, Geneva 2005.

5. Potdar MA (2009) Current Good Manufacturing Practices for Pharmaceuticals, 1st Edition;

Pharmamed Press, Hyderabad, pp 462-468.

6. Baertschi SW, Raynold DW (2005) Pharmaceutical Stress Testing-Predicting drug

Degradation, special Indian Edn, Eli Lilly and Company, Indianapolis, pp 2-4, 13,14.

7. ICH, Stability Testing of New Drug Substances and Products. International Conference on

Harmonisation, IFPMA, Geneva, 1993.

8. ICH, Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and

New Drug Products: Chemical Substances. International Conference on Harmonisation,

IFPMA, Geneva, 1999.

9. Weinblatt ME (1996) Rheumatoid arthritis: treat now, not later! Ann Intern Med 124:773e4.

10. Suresh E (2004) Diagnosis of early rheumatoid arthritis: what the non-specialist needs to

know. J R Soc. Med, 97:421e4

11. Wolfe F, Hawley DJ, Cathey MA (1990) Termination of slow acting antirheumatic therapy in

rheumatoid arthritis: a 14-year prospective evaluation of 1017 consecutive starts. J

Rheumatology; 17:994e1002.

20

12. Anon. Guidelines for the management of rheumatoid arthritis. American College of

Rheumatology Ad Hoc Committee on Clinical Guidelines. Arthritis Rheum 1996; 39:713e22.

13. Molleti S, Rao V, Jayaveera KN (2013) A stability lndicating RP-UPLC Method for

Estimation of Febuxostat and Its lmpurities in Bulk Drugs and Pharmaceutical Dosaage

Forms, Am. J. Pharm Tech Res.; 3(2), 405-412.

14. Rajyalakshmi CH, Benjamin T, Ram babu C (2013) Stress Degradation Studies and

Validation Method for Quantification of Febuxostat in Formulations by Using RP-HPLC, Int.

J. of Res. in Pharm. and Biomedical Sci, 4(1), 138-144.

15. Hsu YH, Liou YB, Lee JA, et al., (2006) Assay of Naproxen by High-Performance Liquid

Chromatography and Identification of Its Photoproducts by LC-ESI MS, Biomedical

Chromatography, 20(8), 787-793.

16. Paul W. Elsinghorsta, Kinziga M, Rodamera M, et.al. (2011) An LC–MS/MS procedure for

the Quantification of Naproxen in Human Plasma: Development, Validation, Comparison

with Other Methods, and Application to a Pharmacokinetic Study, J of Chromatography B,

879, 1686–1696.

17. Modi NR, Mothalia CP, Prajapati KR, et al.,(2013) Development and Validation of

Spectrophotometric Method for Simultaneous Estimation of Diclofenac Potassium and

Febuxostat in Tablet Dosage Forms, lnt. J of Pharm. Sci., 4(3), 268-278.

18. Sanghavi V, Mahajan M, Rajput SJ (2013) Validation of RP HPLC Method for Simultaneous

Estimation of Febuxostat and Diclofenac Potassium in Bulk Drug and in Bilayer Tablet

Formulation, 4(3), 103-106.

19. Prathap B, Dey A, Rao GHS, (2014) Analytical Method Development and Validation for

Simultaneous Estimation of Febuxostat and Ketorolac in Bulk and Pharmaceutical Dosage

Form in Rat Plasma by RP-HPLC, Indo American J of Pharm. Res.; 4(4), 1717-1729.

21

20. Derasari JM, Patel VB (2013) Spectrophotometric Determination of Febuxostat and

Diclofenac Potassium in Their Combined Dosage Form by Simultaneous Equation and First

Order Derivative Methods, Asian J of Res. in Chemistry, 6(10), 968-972.

21. C. Sojitra, S. Rajput (2012) Development and validation of RP-HPLC methods for

simultaneous estimation of Naproxen and Esomeprazole magnesium trihydrate in combined

pharmaceutical formulation, Int. J. of Pharmacy and Pharm. Sci. 4(3), 533-537.

22. Thula K, Patel RA, Maheshwari DG (2014) Simultaneous Estimation of Febuxostat and

Naproxen in Synthetic Mixture by Reverse Phase High Performance Liquid Chromatography

Method, Int. J. of Pharmacy and Pharm. Sci. 6(7), 470-474.

23. Tarnowski T, Tsina I, Yuen Ling Tam, et al. (1996) An Indirect (derivatization) and a Direct

HPLC Method for the Determination of the Enantiomers of Ketorolac in Plasma, J of Pharm.

and Biomedical Ana., 15, 403-417.

24. Srihari Molleti, V. Rao, Jayaveera KN, et al. (2013) Stability Indicating RP-UPLC Method

for Estimation of Febuxostat and Its Impurities in Bulk Drugs and Pharmaceutical Dosage

Forms, Am. J. Pharm. Tech. Res. 3 (02), 404-422.

25. Vijaya V, Vrushali Joshi TS, et al. (2013) New Simultaneous UV-Visible Spectrophotometric

Methods for Estimation of Ofloxacin and Ketorolac Tromethamine in Ophthalmic Dosage

Form, Asian J. Pharm. Ana. 3 (02), 53-57.

26. Khairnar DA, Chaudhari CS, Anantwar SP, et al. (2014) Method Development and Validation

of Ketorolac tromethamine in tablet formulation by RP-HPLC method, Int. J of Pharm. Sci.

and Res., 5 (09), 3696-3703.

27. Kumara Swamy G., Kumar JMR, Sheshagiri rao JVLN, (2012) Simultaneous Estimation of

Febuxostat and Ketorolac in Pharmaceutical Formulations by Spectroscopic Method, Int. J.

Chem. Tech Res. 4 (02), 847-850.

22

28. Joshi RS, Pawar NS (2011) Development and Validation of UV Spectrophotometric Method

for Simultaneous Estimation of Paracetamol and Ibuprofen in Pure and Tablet Dosage Form,

Der Pharmacia Sinica, 2(3), 164-171.

29. Nallasivan PK, Saranya KS (2010) Simultaneous Determination of Ibuprofen and Tizanidine

in Bulk Drug and Its Combined Dosage Form by Spectrophotometry, Der Pharmacia Lettre,

2(6), 289-295.

30. Manikanta A, Swathi A (2012) Development and Validation of UV Spectrophotometric

Method for Simultaneous Estimation of Ibuprofen, Paracetamol and Caffeine in

Pharmaceutical Dosage Form, Am. J. PharmTech Res.,2(6), 541-547.

31. Patel M, Patel B (2013) Simultaneous Estimation of Ibuprofen and Phenylephrine

Hydrochloride in Bulk and Combined Dosage Form by First Derivative UV

Spectrophotometry Method, J. of Spectroscopy, 1-5.

32. Rathore GS, Basniwal PK (2009) Spectrophotometric Estimation of Tramadol Hydrochloride

in Pharmaceutical Dosage Forms, Asian J. of Chem., 21(8), 6111-6115.

33. Manikandan K, Kathiravan S (2012) Development and Validation of Tramadol Hydrochloride

in Bulk and Solid Dosage Form by RP-HPLC Method, Int. J of Bio. & Pharm. Res., 3(2),

245-251.

34. Sayed EI, Mohamed KM (2011) Development and Validation of High-Performance Liquid

Chromatography–Diode Array Detector Method for the Determination of Tramadol in Human

Saliva, J Chromatographic Separation Technique, 2-6.

“development and validation of analytical treatment of

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