Title

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR ESTIMATION OF

FESOTERODINE FUMARATEIN BULK AND ITS EXTENDED RELEASE DOSAGE FORM

Digesh Patel and Dr. K.S. Parikh

Abstract

A simple, Precise, Rapid reproducible, selective and stability

indicating reverse phase HPLC method has been developed for the

estimation of Fesoterodine Fumaratein Bulk as well as

Formulation. The formulation was subjected to ICH recommended

stress conditions. Estimation of Fesoterodine Fumarate was

achieved on Hypersil ODS (50*4.6)mm; 5 µm using buffer(0.01M

Potassium dihydrogen orthophosphate buffer , pH 2.8) and

Acetonitrile in ratio of 55:45 as mobile phase, at the flow-rate

of 0.8 ml/min, at 220 nm detection and 25ºc temperature, on

Agilent 1200 make HPLC instrument having UV detector. The method

was validated for Specificity, Linearity, LOQ, LOD, Precision,

Accuracy and Robustness for Fesoterodine Fumarate. The method was

found to be specific against placebo interference and stress

conditions. Stress testing showed degradation products and

impurity were well-seperated from the parent compound, conforming

stability-indicating capacity of the method.

Key Words

Fesoterodine Fumarate, RP-HPLC, Stability Indicating Method,

Forced Degradation

Introduction

Overactive bladder, also called urge incontinence, is caused by

urinary muscle spasms that cause an urgency to urinate. An

overactive bladder is a condition that results from sudden,

involuntary contraction of the muscle in the wall of the urinary

bladder. Overactive bladder causes a sudden and unstoppable need

to urinate, even though the bladder may only contain a small

amount of urine. There are several medications (anticholinergics)

recommended for the treatment of overactive bladder (Darifenacin

HBr, Fesoterodine Fumarate, Oxybutynin, Solifenacin Succinate,

Tolterodine Tartrate and Trospium). Using these medications in

conjunction with behavioral therapies has shown to increase the

success rate for the treatment of overactive bladder.

Fesoterodine Fumarate (FST) is a new antimuscarinic agent

developed for the treatment of overactive bladder1-4.

Fesoterodine itself is inactive and is rapidly and extensively

converted by ubiquitous esterases to its principal active

moiety,5-hydroxymethyl tolterodine (5-HMT)5. Fesoterodine

Fumarate is commercially available under the brand name of

Toviaz[1-5].

Recently, a stability-indicating liquid chromatography (LC)

method was developed and validated for determination of

Fesoterodine in commercial tablet dosage forms using a monolithic

column [6]. Moreover, for the fast determination of the drug in

tablets with very low levels of residues produced, validated a

specific and sensitive liquid chromatography-tandem mass

spectrometry (LC-MS/MS) method [7]. A UV spectrophotometric

method was published for determination of Fesoterodine in

Extended Release Tablets [8]. Stability indicating method was

published for determination of Fesoterodine fumarate in active

pharmaceutical ingredient [9]. Stability indicating method was

published for determination of Fesoterodine fumarate in its

tablet dosage form having a ph 7.0 mobile phase [10]. Available

literature survey revealed that there is no stability indicating

HPLC method for determination of Fesoterodine Fumarate in

Fesoterodine Fumarate active pharmaceutical ingredient and its

tablet dosage form. Therefore it was felt necessary to develop an

accurate, rapid, and specific stability indicating method for the

determination of assay of Fesoterodine Fumarate in its active

pharmaceutical ingredient and its tablet dosage form.

The present ICH drug stability test guideline suggests that

stress studies should be carried out on a drug substance to

establish its inherent stability characteristics, leading to

separation of degradation impurities and hence supporting the

suitability of the proposed analytical procedure, which must be

fully validated [9].

To our present knowledge we have developed a new accurate and

stability indicating HPLC assay method for determination of

Fesoterodine Fumarate in Bulk drugs and its pharmaceutical

formulations. The main advantage of this method is simple and

accurate having shorter analysis time which leads to decrease

analysis cost and increase productivity.

The empirical formula is C30H41NO7 and its molecular weight is

527.66 and the chemical structure of Fesoterodine Fumarate as

shown in figure 1. Fesoterodine is a prodrug. It is broken down

into its active metabolite, 5-hydroxymethyl tolterodine, by

plasma esterases.

Material

Fesoterodine Fumarate was provided by Cadila healthcare limited,

Dabhasa. Acetonitrile and methanol were of HPLC grade and were

purchased from Lab Scan, Di-ammonium hydrogen phosphate (Merck),

Phosphoric acid (Spectrochem) were collected from local market.

Water was deionised and double distilled. The commercially

available samples of Tablet formulation Toviaz (Pfizer)

containing 4mg and 8mg of Fesoterodine fumarate was used.

Instrumentation

The liquid chromatographic system consisted of an Agilent 1200

series containing quaternary gradient pump, variable wave length

UV detector and Rheodyne injector with 100µl fixed loop.

Chromatography analysis was performed on a Hypersil ODS column

having 50mm X 4.6mm internal diameter and 5µm particle size. All

the drugs and chemicals were weighed on Mettler Toledo analytical

Balance.

Chromatographic conditions

Column : Hypersil ODS (50X4.6)mm, 5 µm

Wavelength (λ) : 220 nm.

Column Temperature : 25°C.

Flow Rate : 0.8 mL/min.

Injection Volume : 50 μL

Run Time : 5 min.

Approximate Retention time : 2.8 min

Diluent : Water: Acetonitrile in the ratio of

50:50.

Preparation of Buffer

1.36 gm of Potassium di-hydrogen phosphate and 4.0 ml TEA were

dissolved in 1000ml of HPLC grade water and pH was adjusted to

2.8 with the help of ortho-phosphoric acid.

Preparation of Mobile Phase

550 ml of Buffer (pH 2.8) and 450 ml of Acetonitrile were mixed

and filtered through 0.45 μm filter, sonicate for 10minutes to

degas and used as mobile phase. Use mobile phase as diluent.

Preparation of Standard Solution

A 40mg of Fesoterodine Fumarate weighed and transferred to a 100

ml volumetric flask. 50ml of acetonitrile was added and sonicate

for 5min. Volume was made up to the mark with acetonitrile.

Further dilute 5.0ml of this solution to 25ml using diluent.

Preparation of Test Sample for 4mg

Weigh accurately 20 tablets and calculate the average weight.

Weigh and break 10 tablets in to two species and transfer them

into 500 ml volumetric flask. Add 50ml of acetonitrile and

sonicate for 15 min. then 100 ml acetonitrile and sonicate for 15

min. add 200 ml diluent and sonicate for 25 min then made the

volume up to mark with diluent. The solution was centrifuged at

5000 rpm for 10 min. The solution was filtered through 0.45 µ

Millipore PVDF filter; filtrate was collected after discarding

first few ml.

Preparation of Test Sample for 8mg

Weigh accurately 20 tablets and calculate the average weight.

Weigh and break 10 tablets in to two species and transfer them

into 1000 ml volumetric flask. Add 50ml of acetonitrile and

sonicate for 15 min. then 100 ml acetonitrile and sonicate for 15

min. add 200 ml diluent and sonicate for 25 min then made the

volume up to mark with diluent. The solution was centrifuged at

5000 rpm for 10 min. The solution was filtered through 0.45 µ

Millipore PVDF filter; filtrate was collected after discarding

first few ml.

Method Validation Parameters and Procedures

System Suitability

System suitability was performed and system suitability

parameters were calculated at the start of study for each

parameter. The values of system suitability results obtained were

recorded in Table 1.

Specificity

Check for interference from blank and placebo. A blank

preparation, standard preparation and sample preparation were

prepared as per method .Peak purity index for the main peak in

standard preparation and sample preparation were determined and

recorded in Table 2.

Linearity and Range

Linearity was determined at seven levels over the range of 40% to

160% with respect to the test concentration. A standard stock

solution was prepared and further diluted to attain concentration

of about 40%, 60 %, 80%, 100%, 120%, 140% and 160% of sample

concentration. Each standard preparation was injected in six

replicates. The mean area at each level was calculated and a

graph of mean area versus concentration (%) was plotted. The

correlation co-efficient (r), y-intercept, slope of regression

line and residual sum of squares were calculated and recorded in

Table 3.

Accuracy (Recovery)

The accuracy of the analytical method for assay of FESOTERODINE

FUMARATE SUCCCINAT was established at three levels in triplicate,

viz. 50%, 100% and 150% of the test concentration. Standard was

prepared as per method. The sample preparations were done by

mixing known amount of FESOTERODINE FUMARATE SUCCCINAT working

standard with placebo. Amount found, %Recovery and mean recovery

was calculated at each level and recorded in Table 4.

Precision

Method Precision (Repeatability)

Method precision was established by assaying six sample

preparations under same conditions. Individual assay values mean

assay value, %RSD was calculated and recorded in Table 5.

Intermediate Precision

The procedure followed for method precision was repeated on a

different day, by a different analyst, using a different HPLC

system & different column lot using same lot of sample.

Individual assay values mean assay value and %RSD were calculated

and recorded in Table. The mean assay value was compared with the

mean assay value obtained in method precision study and

difference of the mean assay and overall % RSD was calculated and

recorded in Table 6 and Table 7.

Robustness

The robustness of the method was established by making deliberate

minor variations in the following method parameters.

1) Column temperature : 5.0°C

2) pH of buffer : 0.2 units

3) Flow rate : 10.0 %

4) Organic phase ratio : 2.0 %

Blank, standard preparation and sample preparation were prepared

and injected. The effect of changes observed on system

suitability parameters were recorded in Table 8.

Solution Stability

The standard and sample solutions were prepared as per method and

analyzed at regular time intervals.

Result, Discussion and Conclusion

The system suitability parameters are well within acceptance

criteria. Therefore the system and chromatographic conditions

are suitable for use. For Lienarity, the correlation coefficient

value was found to be 0.9998. The areas obtained were directly

proportional to the concentration of analyte in the sample. The

method can, therefore be termed as linear in the specified range.

Based on the linearity results, the working range of the method

can be established as 40% to 160 % of the working concentration.

The % recovery at each level, mean %recovery and % RSD met the

established acceptance criteria. Hence, the method can be termed

accurate at 50 %, 100% and 150%. The results obtained were well

within the acceptance criteria, so the method can therefore be

termed as precise and rugged. The system suitability parameters

and absolute difference between % assay obtained from normal

condition and varied condition were well within acceptance

criteria, hence method can be termed as robust. The solution

stability was checked for the sample preparation and standard

preparation for 36 hours. The standard preparation and sample

preparation were stable in solution form for 36 hours at room

temperature.

References

1. M.Tzefos, C.Dolder,J.L. Olin, Ann Pharmacother, 43, 1992 (2009).

2. M.Vella, L.Cardozo, Expert Opin Drug Saf, 10, 805 (2011)

3. A.Gomelsky, RR.Dmochowski, Drugs Today (Barc), 46, 81 (2010)

4. P.Ellsworth, SJ.Berriman, M.Brodsky, Am J Manag Care ,15, 115

(2009)

5. K.McKeage, GM.Keating, Drugs, 69, 731 (2009)

6. M.S.Sangoi, V.Todeschini, M.Steppe, Talanta, 84, 1068 (2011)

7. M.S.Sangoi, M.Steppe, Eur J. Mass Spectrom (Chichester, Eng), 16, 653

(2010)

8. M.S.Sangoi, Vitor Todeschini and Martin Steppe, Acta Chim. Slov.,

59, 136 (2012)

9. B.V. Rami Reddy, B.S.Reddy, M. Sravan Kumar, C. Rambabu,

Rasayan J. Chem. Vol. 5(2),(2012), 239-245

10. Tentu Nageswara Rao et al. / Int. Res J Pharm. App Sci.,

2012; 2(4): 35-40

11. ICH Guidelines, Stability testing of new drug substances and

drug products: test and methodology Q1 A (R2),

February (2003)

12. ICH, Stability Testing of New Drug Substances and Products

(Q1AR), International Conference on Harmonization, IFPMA,

Geniva; (2000)

List of Tables

Table 1: System Suitability Test Parameters

System SuitabilityParameters

FESOTERODINEFUMARATESUCCCINAT

Theoretical plates (N) 4267Tailing factor (AS) 1.06

% RSD (n=5) 0.12

Table 2: Specificity and Selectivity study

Study Result

Specificity SpecificSelectivity Selective

Table 3: Linearity and Range Data

Parameter FESOTERODINEat 220 nm

Linear Range

(µg/ml)40 - 120µg/ml

Correlation co-

efficient 0.9998

Slope (s) 7.4Intercept 34.1

Table 4: Data derived from Accuracy Experiment

Level

Set

mgAdded

mgAdded(Actual

)

mgRecovered

%Recover

y

Mean%

Recovery

%RSD

50% 1 40 40.1 40.0 99.8100.0 0.2550% 2 40 40.2 40.3 100.3

50% 3 40 40.1 40.1 100

100% 1 80 79.9 80.1 100.3 100.3 0.19

100% 2 80 80.0 80.1 100.1

100% 3 80 79.8 80.2 100.5

150% 1 120 120.2 120.0 99.8

100.1 0.21150% 2 120 120.1 120.2 100.0

150% 3 120 119.9 120.2 100.3

Table 5: Determination of Method Precision (Repeatability)

Sets % Assay Mean %Assay %RSD

1 100.3

100.2 0.4

2 99.43 100.64 100.15 100.36 100.2

Table 6: Determination of Intermediate Precision

Sets % Assay Mean %Assay %RSD

1 100.7 100.3 0.45

2 100.23 99.84 100.65 100.76 99.7

Table 7: Comparison of Intermediate Precision with Method Precision

Parameter Mean Assay (%) AbsoluteDifference (%)

Method Precision(Repeatability) 100.2

0.1Intermediate Precision(Ruggedness) 100.3

Table 8: Data derived from Robustness Experiment

Parameters % RSD Theoretical

plates

Normal Conditions 0.12 4267

Temperature (25°C)

-5°C 0.31 4060

+5°C 0.44 4136

pH (2.8)

-0.2 unit 0.25 4240

+0.2 unit 0.54 4278

Flow Rate (0.8mL/min)

-10% 0.11 3998

+10% 0.36 4129

Organic phase Ratio

-2% 0.19 4006

+2% 0.28 4290

List of Figures

Figure 1: Fesoterodine Fumarate

Figure 2: Diluent

Figure 3: Standard Solution

Figure 4: Sample Solution