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Chapter 3 Oxybutynin Page 49

3.1. DRUG PROFILE

OXYBUTYNIN

Oxybutynin is an anticholinergic and antispasmodic agent used to relieve

bladder and urinary difficulties, consist of difficult to control urination and

frequent urination, by decreasing muscle spasms of the bladder and urinary tract [38]. It is also used in treatment of excessive sweating condition known as

hyperhidrosis [39-41].

Figure 3.A Structure of Oxybutynin

Oxybutynin chloride is administered as a racemate of R- and S-

enantiomers. It competitively antagonizes the muscarinic acetylcholine receptors

including the subtypes M1, M2, and M3. It has a direct effect which is spasmolytic

in nature on the smooth muscles of urinary bladder by acting as local anesthetic

and calcium antagonist, but at doses far higher than those used therapeutically.

Oxybutynin contains one stereocenter. Among the recemate of two

enantiomers R and S enantiomers, R enantiomer is considered to have more

potent anticholinergic effect when compared to S enantiomer, which does not

have anticholinergic activity at the dose levels used regularly[42,43]. Dry mouth,

difficulty breathing, swelling of face, lips, tongue, or throat difficulty, dizziness,

urination, blurred vision, constipation, and drowsiness are the common adverse

effects associated with Oxybutynin [44]


Physicochemical Properties: Oxybutynin chloride is a white crystalline solid,

readily soluble in water and acids, but relatively insoluble in alkalis. The list of

brand names of Oxybutynin are listed in the Table 3.1

Table 3.1: List of Brand Names of Oxybutynin

S. No. Brand Name FormulationAvailable

Strength (mg) Manufacturer

1 OXYSPAS Tablet 2.5 & 5.0 Cipla

2 TROPAN Tablet 2.5 & 5.0 Sunpharma

3 DITROPAN XL Tablet 5.0,10.0 & 15 NDC

3.2 LITERATURE SURVEY

Many analytical methods are published for the determination of Oxybutynin in

pure drug, pharmaceutical dosage forms and in biological samples by using analytical

methods such as Spectrophotometry, High Pressure Liquid Chromatography and Mass

Spectrometry either in single or in combined forms.

Q. Wen et al [45] have developed a method for HPLC-MS determination of

Oxybutynin in plasma and tissue of rats. Blood, bladder and liver samples were

collected and assayed by LC-MS. A ZORBAX extend-C18 column was used to

separate Oxybutynin in plasma and tissue with a mobile phase of a mixture of

Acetonitrile: 0.1% - pH 5.4 acetic acid buffer (50: 50) at a flow rate of 0.5 mL min–1..

It is suitable for pharmacokinetic studies of Oxybutynin gel.

Alaa El-Gindy et al [46] have developed a method for HPLC determination of

Oxybutynin hydrochloride and their degradation products. The proposed method was

based on HPLC separation of Oxybutynin from its degradation product using a VP-

ODS C18 column with a mobile phase consisting of Acetonitrile: 0.01 M Potassium

Dihydrogen Phosphate: Diethylamine (60:40:0.2). Quantitation was achieved with

UV detection at 220 nm based on peak area.

Renato Massoud et al [47] have been developed a method for extraction and

determination of Oxybutynin in human bladder samples by RP-HPLC.

Chromatographic separation was performed with a mobile phase of Acetonitrile:


Water: 1 M Ammonium Acetate (85:13:2, v/v/v) and double (electrochemical and

UV) detection was applied. The retention time of Oxybutynin eluting peak was

around 18 min. The method was applied to compare the Oxybutynin levels into

bladder wall tissue samples after passive diffusion by electromotive drug

administration (EMDA).

K. Srikanth et al [48] have described a method for spectrophotometric

determination of Oxybutynin chloride using UV-Visible Spectrophotometer by

forming ion-association complex with acidic dyes TPOOO or ARS showing

absorption maximum at 480 nm and 430 nm respectively.

3.2.1 MOTIVATION FOR THE METHOD DEVELOPMENT

The primary purpose of this research project was to develop and to validate a

simple HPLC method for determination of Oxybutynin in the APIs, finished product

and its applicability in the estimation of drug in human plasma. Oxybutynin is an

anticholinergic and antispasmodic agent used to relieve bladder and urinary

difficulties as smooth muscle relaxant. Clearly, it is highly important to accurately

measure its concentration alone or in combination with other compounds. A high

speed method was sought to measure the concentration of this compound within a

short span of time. This is beneficial in any pharmaceutical analysis/clinical

environment where the concentration of Oxybutynin is needed to understand any

patient issues along with the pharmaceutical industry to design the multiple steps that

may be needed to prepare the APIs for production. The high speed method will

eliminate/reduce any waste or costs that are required with the preparation of the APIs.

There are no analytical methods that have been reported for the determination

of Oxybutynin in API and one method was reported in formulations at the time of

commencement of research work. Q. Wen et al have developed a HPLC-MS method

for the estimation of Oxybutynin in plasma and tissue of rats and they have used

ZORBAX extend-C18 column to separate Oxybutynin from plasma and tissue, the

column is comparatively expensive with the column that have been used in the current

method, moreover this method is limited only for the analysis of animal samples. Alaa

El-Gindy et al have developed a method meant for only pKa studies using rarely

available buffer solutions and columns. Renato Massoud et al have developed a

Chapter 3 Oxybutynin 2

method using electromotive drug administration in rats and this method is limited

only for analysis of samples from tissue of a rat and electromotive drug administration

device is developed in their own laboratory. These methods report mainly on the

determination of Oxybutynin in plasma/ blood samples and in human bladder

samples. Such methods may not be suitable for regular/routine analysis for

Oxybutynin in pharmaceutical industry because of diversity and complexity in sample

matrix. K Srikanth et al have developed spectrophotometric method for the estimation

of Oxybutynin and spectrophotometric methods are inferior to the HPLC methods and

have limited usage in pharmaceutical industry. The spectrophotometric method is

reported for the estimation of Oxybutynin in tablet dosage form by using the acidic

dyes which may cause chemical interference with metal ions and causes precipitation

may effect the quantification of the drug. Analytical methods based on UV-Visible

Spectrophotometry have major disadvantage that they are time consuming,

cumbersome and laborious. At the same time such methods require more quantity of

active pharmaceutical ingredients, i.e., they are not much sensitive at microgram

level. Also they are not suitable for regular/routine analysis in pharmaceutical

industry where sample size is more.

The determination of Oxybutynin in APIs sample is yet to be found. In

addition, stability-indicating methods are not found for Oxybutynin formulations in

fixed dosage forms. Complete validation parameters were not found in any of the

methods completed in the past.

Hence, by considering all these factors, attempts were made hoping to fill this

gap and succeeded in developing analytical method using HPLC with UV detection.

3.3. EXPERIMENTAL

3.3.1. Instrumentation

Shimadzu VP series HPLC instrument on an Inertsil C18 column (250 mm x

4.6 mm, 5μm) are used to develop the quantitative estimation of Oxybutynin. The

instrument is equipped with a LC 20AT pump and variable wavelength programmable

UV-Visible detector SPD-10AVP. A 20μL Hamilton syringe, Elico SL159 UV-


Visible spectrophotometer, Loba ultrasonic bath sonicator and Denwar weighing

balance are used.

3.3.2. Chemicals and Solvents

The reference sample of Oxybutynin (API) obtained from Sun Pharma,

Ahmedabad. The formulation procured from the local market. HPLC grade of

Acetonitrile, Methanol and Water purchased from Merck Specialities Private Limited,

Mumbai, India. Orthophosphoric acid AR grade is purchased from Qualigens.

3.3.3. The Buffer Solution

10mL of Orthophosphoric acid was diluted to 1000 mL with Water to obtain

1% ortho phosphoric acid solution.

3.3.4. The Mobile Phase

A mixture of Methanol: Acetonitrile: 1% Orthophosphoric acid in the ratio of

15:45:40%, v/v/v was prepared and used as mobile phase. The resultant solution was

filtered through 0.45μm filter.

3.3.5. Standard Solution of the Drug

100 ppm standard solution was prepared for analysis in mobile phase, further

required concentrations were obtained from 100 ppm solution by proper dilution

using mobile phase.

3.3.6. Sample Solution

The tablets of Oxybutynin (OXYSPAS – 5mg) were crushed to give fine

powder. 8ppm solution in mobile phase was prepared from the finely crushed tablet

powder and then filtered through Ultrapore membrane sample filter paper.

3.3.7 Calculations in Validation Studies

Percentage recovery and area ratio were calculated using the following equation:

% Recovery = ([Peak Area] sample / [Peak Area] standard) × 100


For a set of “n” replicate measurements, percentage relative standard deviation

was calculated as follow: % RSD=SD/Average × 100

The detector sensitivity was determined by calculating the signal to noise ratio using

the following equation: Sensitivity = S/N = Signal/ Noise

Signal = Amount of detector response to the peak from the middle of

the noise to the summit of the peak.

Noise = Amount of noise resulting from the detector that is taken from

a portion of the baseline without any distortions.

3.4 METHOD DEVELOPMENT

The goal of this research was to develop a reversed-phase HPLC method that

can be used to determine the active pharmaceutical ingredient in Oxybutynin APIs

with an adequate resolution and in a minimum analysis time. The intention was to

separate the degradants (if any) and process impurities (if present) from the active

ingredient. To begin method development, important structural information such as

chemical structure, molecular weight, UV spectrum and sample solubility were

reviewed.

Method development [12] consists of selecting the appropriate wave length,

stationary and mobile phases. The following studies were conducted for this purpose.

Systematic studies on various conditions or factors have been done for developing a

method by studying each parameter and keeping all other parameters in constant.

3.4.1. Wavelength Detection

The proper wavelength was needed to determine maximum detector response.

The first step was to run a UV-VIS spectrum (from 190-400 nm) using an HPLC

system equipped with the Photo Diode Array Detector, from the spectrum it is clear

that Oxybutynin absorbs maximum light between 200 nm to 210 nm. The longer

wavelength of 205 nm was selected since it produces less noise, which minimizes

problems that may exhibit around the active ingredient when attempting to quantify

Oxybutynin.


The UV absorption spectrum of diluted solution of the Oxybutynin in

Methanol (at 8ppm) was recorded on a UV spectrophotometer. The spectrum of

Oxybutynin showed absorption maximum at 205 nm, which is selected as detection

wave length.

3.4.2 Selection of Stationary Phase

An aged HPLC column should be used to develop the initial HPLC conditions.

Usually it is more difficult to achieve the required resolution with an aged column

(e.g., column with about 200 injections). This will reflect the worst case scenario

likely to be encountered in actual method uses, and help the long-term method

robustness. In general, all methods developed by HPLC columns were for the same

vendor. The preferred brand of HPLC column should be selected primarily based on

the long term stability and lot-to-lot reproducibility.

Preliminary development trials have performed on Inertsil and Chromosil C18

(250 X 4.6mm, 5µm) columns. Peak tailing factor was found to be low on Inertsil

column compared to the Chromosil column and also less retention time was observed

in Inertsil column without any interferences in formulation samples. Based on which

Inertsil C18 (250 mm x 4.6 mm, 5μm), column was selected as stationary phase.

3.4.3 Selection of the Mobile Phase

The solubility of Oxybutynin was studied in order to determine the proper

ratio of solvents to use as mobile phase to the drug substance for analysis. Referring

to chemical structure, the compound is a base due to the presence of tertiary amine

group and is able to accept proton(s), therefore polarity of the dissolved solvent will

affect the solubility. Different ratios of Methanol and Acetonitrile were tested with 2

minutes vortexing. 50 mg of Oxybutynin reference standard was dissolved in water

and tested, Oxybutynin appears to dissolve completely resulting in a clear solution.

On the other hand, when 100% Acetonitrile was used, the compound dissolved

instantly. Furthermore, when a ratio of 1:1 Water: Acetonitrile was used, the

compound did not dissolve with low viscosity in resultant solution.

In order to get sharp peak and base line separation of the components, carried

out a number of experiments by varying the composition of solvents in mobile phase


and its flow rate. To have an ideal separation of the drug under isocratic conditions,

mixtures of solvents like Methanol, Water and Acetonitrile with or without different

buffers in different combinations were tested as mobile phase. The pH of the mobile

phase will greatly affect its retention time as it interacts with the stationary phase. To

start off, the pH of the mobile phase, which consisted of Orthophosphoric acid, was

adjusted to 2.5. The reason for starting with low pH is to protonate all free silanol

groups in the stationary phase and reduce their chromatographic activities. In

practical, this should avoid any secondary interactions and also reduce tailing. Also, a

mobile phase at pH of 6.5 is a good starting point for most pharmaceutical

applications because it suppresses the ionization of most acidic analytes resulting in

their higher retention.

A mixture of Methanol: Acetonitrile: 1% Ortho phosphoric acid in the ratio of

15:45:40%, v/v/v with pH-6.5 of the mobile phase was proved to be the most suitable

of all combinations, with better a defined and well resolved peak, free from tailing

and also producing zero noise in blank chromatogram.

3.4.4. Flow Rate

Flow rate of the mobile phase was varied from 0.5 – 1.5 mL/min for optimum

separation. A minimum flow rate as well as minimum run time gives the maximum

saving on the usage of solvents. It was found from the experiments that 1.0 mL/min

flow rate was ideal for the successful elution of the analyte.

3.4.5. Optimized Chromatographic Conditions

Chromatographic conditions as optimized above are shown in Table 3.2.

These optimized conditions were followed for the determination of Oxybutynin in

APIs samples, tablet formulations and plasma samples. The chromatograms of

standard, blank, tablet sample, plasma blank and plasma samples are shown in Graph

3.A, 3.B, 3.C, 3.D and 3.E respectively.


Table 3.2: Optimized Chromatographic Conditions for Estimation of Oxybutynin

Mobile phase Methanol: Acetonitrile : 1% OPA

15:45:40%, v/v/v

Mobile Phase pH 6.5

Pump mode Isocratic

Diluent Mobile phase

Column Inertsil C18 column (250 mm x 4.6 mm,

5μm)

Column Temp Ambient

Wavelength 205 nm

Injection Volume 20 μL

Flow rate 1.0 mL/min

Run time 6 min

Retention Time 2.4 min

Graph – 3.A: Chromatogram of Standard Solution


Graph - 3.B: Chromatogram of Blank Solution

Graph - 3.C: Chromatogram of Sample Solution

Graph - 3.D: Chromatogram of Plasma Sample Blank


Graph 3.E: Chromatogram of Plasma Sample Solution HPLC Report

3.5 VALIDATION OF THE PROPOSED METHOD

After the method was developed, various validation studies were conducted in

a systematic way to determine reliability of the developed method. In other words,

the developed method is suitable for its intended use. Validation parameters studied,

as required by the Food and Drug Administration (FDA) and International Conference

on Harmonization (ICH). The proposed method was validated [17-32] as per ICH

guidelines. The parameters studied for validation were specificity, linearity, precision,

accuracy, robustness, system suitability, limit of detection, limit of quantification and

solution stability.

3.5.1. Specificity

Specificity of the developed method was tested to determine its ability to

measure accurately concentration of Oxybutynin in the presence of potential

degradation products.

Acceptance Criteria:

1. No interferences of the peak of interest in the control or the degraded sample

from the potential degradation products. In other words, the minimum resolution

between Oxybutynin HCl peak and the nearest eluting peak should be NLT 1.5.

2. Peak purity of Oxybutynin active ingredient should be NLT 0.990.


The specificity of method was performed by comparing the chromatograms of

blank, standard and sample. It was found that there is no interference due to

excipients in the tablet formulation and also found good correlation between the

retention times of standard and sample. The specificity results are shown in Table

3.3.

Table 3.3: Specificity Study Results

Name of the Solution Retention Time in Minutes

Blank No Peaks

Standard 2.4

Sample 2.5

3.5.2 Linearity and Range

Linearity is the ability of the method to elicit test results that are directly

proportional to analyte concentration within a given range. Range is the interval

between the upper and lower levels of analyte that have been demonstrated to be

determined with precision, accuracy and linearity using the method as written. The

ICH guidelines specify a minimum specified range from 80 – 120% of the target

concentration.

Linearity was performed by preparing standard solutions of Oxybutynin at

different concentration levels including working concentration mentioned in

experimental condition i.e. 8ppm. Twenty micro liters of each concentration was

injected in duplicate into the HPLC system. The peak responses were read at 205 nm

and the corresponding chromatograms were recorded. The mean peak areas were

calculated and linearity plots of concentration over the mean peak areas were

constructed individually from the developed chromatograms. The regressions of the

plots were computed by least square regression method. Linearity results are

presented in Table 3.4 and calibration plot was provided in Graph 3.F.


Table 3.4: Linearity Results

Concentration Level Concentration of

Oxybutynin In ppm Mean Peak Area

Level -1 2.0 62162

Level -2 4.0 130828

Level -3 6.0 198705

Level -4 8.0 274458

Level -5 10.0 343516

Level -6 12.0 402085

Range: 2ppm to 12ppm

Slope

Intercept

Correlation coefficient

34153

-3241

0.999

Graph - 3.F: Calibration Plot for Oxybutynin

3.5.3. Method Precision

Method precision was demonstrated by calculating % RSD of six

independent preparations of 100% target concentration of Oxybutynin. Precision is

the degree of repeatability of an analytical method under normal operational

conditions. Precision of the method was performed as intraday precision and interday

precision.


Acceptance Criteria: The % RSD of NMT 1.5% is recommended for Reference

Standard

3.5.3.1. Intraday Precision: To study the intraday precision, six replicate standard

solutions (8ppm) of Oxybutynin were prepared and injected using the set

chromatographic conditions. The percent relative standard deviation (% RSD) was

calculated and it was found to be 1.8%, which is well within the acceptance criteria of

not more than 2.0%. Results of intraday system precision studies are shown in Table

3.5.

Table 3.5: Intraday Precision Results for Oxybutynin

Sample Concentration.

(ppm) Injection

No. Peak Areas

RSD (Acceptance

criteria ≤ 2.0%)

Oxybutynin 8

1 270260

1.8

2 278671

3 269119

4 272053

5 279117

6 268246

3.5.3.2. Interday Precision: To study the interday precision, six replicates of

standard solutions (8ppm) of Oxybutynin were injected on third day of sample

preparation. The percent relative standard deviation (% RSD) was calculated for peak

responses and it was found to be 1.7%, which is well within the acceptance criteria of

not more than 2.0%. Results of system precision studies are shown in Table 3.6.


Table 3.6: Interday Precision Results for Oxybutynin

Sample Concentration

(ppm) Injection No. Peak Areas

RSD (Acceptance criteria ≤ 2.0%)

Oxybutynin 8

1 271584

1.7

2 276218

3 281095

4 270764

5 269287

6 269546

3.5.4 Accuracy

The accuracy of the developed method was tested to determine closeness of

the measured value to the true value. Accuracy of the developed method was studied

by evaluating the recovery of Oxybutynin HCl from spike solutions.

The accuracy of the method was determined by standard addition method. A

known amount of standard drug was added to the fixed amount of pre-analyzed tablet

solution. Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. The standard addition method was performed at 50%,

100% and 150% level of 8ppm. The solutions were analyzed in triplicate at each level

as per the proposed method. The percent recovery and % RSD was calculated and

results are presented in Table 3.7. Satisfactory recoveries ranging from 98.3 to 100.5

were obtained by the proposed method. This indicates that the proposed method was

accurate.


Table 3.7: Accuracy Results

Level Amount of Oxybutynin ( in ppm)

% Recovery %RSD Spiked Recovered

50 %

4 3.94 98.5

1.2 4 3.93 98.3

4 4.02 100.5

100%

8 7.96 99.5

0.6 8 8.01 100.1

8 7.92 99.0

150%

12 11.98 99.8

0.4 12 11.95 99.6

12 11.89 99.1

Mean 99.4 0.7

3.5.5 Robustness

Robustness of the developed method was tested by making slight deliberate

changes to the separation parameters, chromatographic conditions of the method.

This study was conducted to determine ability of the developed method to remain

unaffected by these small deliberate changes.

The robustness study was performed by slight modification in flow rate of the

mobile phase, pH of the buffer and composition of the mobile phase. Oxybutynin at

8ppm concentration was analyzed under these changed experimental conditions.

Three replicate injections were performed with each of the altered chromatographic

condition and the mean peak area was compared against the mean peak area obtained

with the unaltered conditions. It was observed that there were no marked changes in

chromatography and the %assay when compared with unaltered conditions was within

± 2%, demonstrating that the developed method was robust in nature. In other words,

integrity of the chromatogram was maintained and appeared no interferences from

other peaks. The results of robustness study are shown in Table 3.8.


Table 3.8: Robustness of Oxybutynin

Condition Mean area % assay % difference

Unaltered 275613 100.0 -

Flow rate at 0.8 mL/min Flow rate at 1.2mL/min

276984

271028

100.5

98.3

0.5

1.7

Mobile phase:

MEOH:ACN : 1%OPA

13% 42% 45%

17% 38% 45%

278369

277254

101.0

100.6

1.0

0.6

pH of mobile phase at 6.0 279086 101.3 1.3

pH of mobile phase at 6.8 278637 101.1 1.1

3.5.6. Solution Stability

To perform the solution stability, three replicates of standard and sample

solutions at 8 ppm (stability samples) were prepared and stored separately at ambient

temperature (25±10°C) for two days. After the intended storage period, both the

standard and sample stability solutions were compared against a freshly prepared

standard solution (comparison sample) using the proposed method. It is noticed that

the % stability of Oxybutynin was more than 98%, demonstrating insignificant

degradation in both standard and formulation samples. The results of solution stability

are shown in Table 3.9.

Table 3.9: Stability Results for Oxybutynin

S. No

Concentration

(ppm) Solution

Mean Peak Area

%Stability

1 8 Fresh standard Solution (Comparison sample)

27627 -

2 8 Stored Standard Solution(Stability

sample) 274618 99.4

3 8 Stored Sample Solution

(Stability sample) 274104 99.2


3.5.7. System Suitability Studies

Before performing any validation study, it should be established that the

HPLC system and the developed method are capable of providing data of acceptable

quality. These tests are used to verify that the resolution and repeatability of the

developed method are adequate for the analysis to be performed. System suitability

tests are based on the concepts that the equipment, electronics, analytical operations,

and samples constitute an integral system that can be evaluated as whole parameters

such as plate count, tailing factor, resolution and repeatability (%RSD for retention

times and peak areas) are determined and compared against the specifications set for

the method. Two working standards are prepared for the study of system suitability

System suitability was studied under each validation parameter by injecting

six replicates of the standard solutions at 8ppm concentration. For the method

following limits were considered as acceptance criteria, tailing factor ≤ 2, theoretical

plates > 2000 and %RSD for peak area ≤ 2%. The % drift is also less than 2%. These

results fulfilled the required system suitability acceptance criteria. The results of

system suitability tests are provided in Table 3.10.

Table 3.10: System Suitability Results for Oxybutynin

Parameter Tailing factor Theoretical plates% RSD for peak

response

Specificity study 1.69 4711 1.4

Linearity study 1.16 6813 1.8

Precision study 1.26 7140 1.8

3.5.8. Limit of Detection and Limit of Quantification

To determine the Limit of Detection sample was dissolved by using mobile

phase and injected until peak was disappeared. After 0.1ppm dilution, Peak was not

clearly observed. So it confirms that 0.1ppm is Limit of Detection for Oxybutynin

using the current method and Limit of Quantification is 0.25ppm. For establishing

Limit of Quantification, six replicates of standard at 0.25ppm were prepared and


quantified with a relative standard deviation of 2.0%. The LOD and LOQ of

Oxybutynin are given in Table 3.11.

Table 3.11: Limit of Detection and Limit of Quantification for Oxybutynin

Parameter Measured volume

Limit of Quantification 0.25ppm

Limit of Detection 0.1ppm

3.5.9 Analysis of Commercial Formulation

For assay of Oxybutynin (OXYSPAS – 5mg), 20 tablets were weighed and

calculated the average tablet weight. Accurately weighed and transferred the

powdered sample equivalent to 10mg of Oxybutynin in to a 10mL volumetric flask.

5mL of mobile phase was added and sonicated to completely dissolve the drug and

final volume was made with the diluent. Mixed well and filtered the solution through

0.45µm filter. Further pipetted out 0.2mL of the above solution into a 25mL

volumetric flask and diluted up to mark with mobile phase to get a final concentration

of 8ppm. An aliquot of this solution was injected into HPLC system. Peak area of

Oxybutynin was measured and compared against the standard response; the proposed

method was able to estimate Oxybutynin with an accuracy of 98.8% in tablet

formulation.

3.5.10 Plasma Sample Analysis

To evaluate the general applicability of the HPLC method, Oxybutynin was

analyzed in different sample matrices. The proposed HPLC procedure was further

tested using spiked human plasma samples. In all cases, no interferences from the

indigenous plasma components were observed. The simplicity of the proposed

extraction procedure and the high extraction efficiency are among the essential

features of the proposed HPLC method, making this method suitable for routine,

efficient and fast extraction of Oxybutynin from complex matrices. The method is

accurate, precise and specific. The described HPLC assay can be easily applied for the

quantification of the degradation products.


Human fresh frozen plasma containing K2EDTA as anticoagulant was

procured from M/S. Lakshmi sai clinical labs and required concentration of

Oxybutynin spiked samples (in plasma) were prepared using working solutions.

Preparation of Plasma Spiked Samples: Fresh frozen plasma stored at -20°C was

thawed and used for the preparation of Oxybutynin spiked samples. 0.2mL of 400ppm

working solution was pipetted out into a 10mL volumetric flask and further diluted up

to the mark with plasma, to get 8ppm concentration sample. During the preparation of

spiked sample volume of diluent/working solution was kept ≤ 5% of the total plasma

volume, to avoid unnecessary changes in matrix components.

Sample Extraction / Preparation: 0.5mL of plasma spiked sample was taken into a

test tube and added 100µL of 1M NaOH solution and vortexed. Further to the sample,

5mL of Dichloromethane was added and subjected to extraction on a plasma extractor

using 50rpm for 20 min. Later centrifuged the sample for 10min at 3000 rpm,

collected 4mL of the separated organic layer and evaporated to dryness at 50°C. The

residue was reconstituted with 100µL of mobile phase and the sample was injected

into the HPLC system using the proposed chromatographic conditions.

3.6 RESULTS AND DISCUSSIONS

A RP-HPLC method was developed and validated for the determination of

Oxybutynin in APIs and Pharmaceutical dosage forms. The present study is the first

report on assay of Oxybutynin in API. In this method isocratic elution method is

selected for the analysis of Oxybutynin API because it gave better base line separation

and peak width, which is suitable for the routine analysis of Oxybutynin. The

developed method was validated as per ICH guidelines (ICH, 1996) and its updated

international convention (ICH, 2002).

RP-HPLC method was developed for the estimation of Oxybutynin. Literature

survey indicates that, only spectrophotometric method was available, which is based

on ion-association complexes with TPOOO dye, for the estimation of Oxybutynin in

tablet dosage forms. The sensitivity and specificity of spectrophotometric method

currently in use may not be adequate because it is reported that some substances (eg,

some metal ions), which may present in the biological fluid/placebo/ in excipients,


may cause chemical interference by forming complexes with dye. Therefore, this

method is also criticized because of its low specificity, sensitivity and reproducibility.

Currently developed HPLC method is more sensitive, specific and reproducible in

determination of Oxybutynin. HPLC can be applied without a complex formation and

any other initial process and it is not affected by chemical interference. HPLC method

is always preferred due to its analytical sensitivity when compared to

spectrophotometric method. On the other hand, repeatability of experimental assays is

an important qualification for its safety.

Liquid chromatography method development began with the optimizing

mobile phase composition and column type. The feasibility of several mixtures of

solvent such as Acetonitrile and Methanol using different buffers such as Ammonium

acetate, Ammonium formate, Acetic acid and Formic acid with variable pH range 3–6

was tested for complete chromatographic resolution of Oxybutynin from interfering

tablet matrix. The versatility, suitability and robustness of the method were checked

with several C18 and Cyano (CN) columns.

Different mobile phases were tested to develop precise, accurate and suitable

RP- HPLC method for the estimation of Oxybutynin and the proposed

chromatographic conditions were found to be appropriate for the quantitative

determination. The report obtained by the assay of Oxybutynin standard was

summarized in Fig: 3.A. System suitability tests were carried out with each validation

experiment and the results are summarized in Table 3.10. In all cases, the relative

standard deviation (RSD) for the analytic peak area for two consecutive injections

was < 2.0%.

Chromatograms of blank solution, standard and samples of tablet and plasma

contained no co-eluting peaks of analyte at the respective retention time.

Representative chromatograms of blank plasma, blank fortified with Oxybutynin are

shown in Graph 3.D and Graph 3.F respectively. The retention times of the analyte

shows less variability with a % RSD well within the acceptable limit of ±2%. This

results indicates the proposed method is highly specific and selective in both tablet

and plasma samples.

Standard curves were constructed using six standard concentrations in the

range of 2 to 12ppm for Oxybutynin. The linearity of peak area responses versus

concentrations was demonstrated by linear least square regression analysis. The linear


regression equation was y = 34153x – 3241 (r2= 0.999). Linearity values were shown

in Table: 3.4. Precision was evaluated by carrying out six independent sample

preparations of standard at 8ppm. Percentage relative standard deviation (%RSD) was

found to be less than 2% for interday (1.8) and intraday (1.7) variations, which proves

that method is precise. Results were shown in Table 3.5 and 3.6 for interday and

intraday precisions respectively.

The precision was defined as the relative standard deviation (RSD) of the

determined concentrations of the same QCs, whereas accuracy was assessed as the

percentage to the nominal concentration (%). To check the degree of accuracy of the

method, recovery studies were performed in triplicate by standard addition method at

50%, 100% and 150%. Known amounts of Oxybutynin standard were added to pre-

analyzed samples and were subjected to the proposed HPLC method. Results of

recovery studies were shown in Table 3.7. The results showed good recoveries

ranging from 98.3 to 100.5 %. The mean recovery data obtained for each level as well

as for all levels combined was 2.0% of the label claim for the active substance with an

R.S.D. < 2.0%, which satisfied the acceptance criteria set for the study.

The sample was dissolved by using mobile phase and injected until peak was

disappeared and there was no dilution peak was observed clearly after 0.1ppm

dilution peak was not clearly observed. So it confirms that 0.1ppm is the Limit of

Detection and the Limit of Quantification is 0.25ppm.To evaluate the robustness of

the developed RP-HPLC method, small deliberate variations in the optimized method

parameters were done. The effect of change in flow rate, pH and mobile phase ratio

on the area, retention time and tailing factor were studied. The method was found to

be unaffected by small changes like ± 0.4 change in pH, ± 0.2 change in flow rate and

± 2 change in mobile phase composition. It was observed that there were no marked

changes in chromatography and the %assay when compared with unaltered conditions

was within ± 2%, demonstrating the robustness of the method.

The stability studies were evaluated by storing the solutions at ambient

temperature (25±100C) and checked in triplicate after two successive days of storage

and the data were compared with freshly prepared samples. In each case, it could be

noticed that solutions were stable for 48 hrs, as during this time the results did not

decrease below 98%. This denotes that Oxybutynin is stable in standard and sample

solutions for at least 2 days at ambient temperature. The validated method was applied


for the assay of commercial tablets OXYSPAS containing of 5mg Oxybutynin.

Sample was analyzed for five times after extracting the drug as mentioned in assay

sample preparation of the experimental section. The results were found to be similar

to that of labeled content

Statistical analysis of all these results has been carried out revealing high

accuracy and precision of the method. The RSD for all parameters was found to be

less than two, which indicates the validity of method and assay results obtained by

this method are in fair agreement. The developed method can be used for routine

quantitative estimation of Oxybutynin in multi component pharmaceutical

preparations and in APIs. The proposed method for the assay of Oxybutynin is

simple, rapid. Therefore, it is suitable for the routine analysis of Oxybutynin for

quality control of pharmaceutical dosage forms.

The developed method was applied to analysis of plasma spiked samples of

Oxybutynin, which showed inadequate selectivity in presence of endogenous plasma

components. Extraction of plasma spiked samples was carried out using liquid-liquid

extraction with Dichloromethane and precipitation technique was deliberately

avoided, as the drug was relatively non polar (log P- 4.3) and also as precipitation

method will produce more unclean samples which may also reduce the column life.

Based on the results in plasma samples, it is concluded that the current method was

not selective for biological samples and further work need to be performed on the

chromatographic separation and also on the sample extraction procedures.

The current validated HPLC method for Oxybutynin offers significant

advantages in terms of sensitivity and selectivity, sample preparation, short run time

(6 min) and lower volume of sample requirements (10 µl). From the results of all the

validation parameters and applicability of the assay, it can be concluded that the

present method can be useful for regular and routine analysis of pharmaceutical

dosage forms and also for the systemic pharmacokinetic studies of Oxybutynin with

desired precision and accuracy along with high-throughput.

chapter - oxybutynin - information and...

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