5.1 introduction of tolterodine tartrate and survey of...

5.1 Introduction of Tolterodine tartrate and survey of analytical

methods:

Tolterodine tartrate is chemically designated as (R)-N,N-diisopropyl-

3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamine L-hydrogen

tartrate (Fig: 5.1) is a new muscarinic receptor antagonist intended for

the treatment of urinary urge incontinence and other symptoms related

to unstable bladder (1, 2). The empirical formula of Tolterodine tartrate

is C26H37NO7 and its molecular weight is 475.6. Tolterodine tartrate is a

white, crystalline powder. It is soluble in methanol, slightly soluble in

ethanol, and practically insoluble in toluene.

Fig: 5.1 Chemical structure of Tolterodine tartrate

(R)-N, N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-

phenylpropanamine L-hydrogen tartrate

Molecular formula: C26H37NO7

Molecular weight : 475.6

Detrol reduces spasms of the bladder muscles. Detrol is used to

treat overactive bladder with symptoms of urinary frequency, urgency,

and incontinence. The active moiety in Detrol is Tolterodine.

Tolterodine is a muscarinic receptor antagonist. Both urinary bladder

-

46

OH

H3CN

H.

HO H

OHHCOOH

COOH

contraction and salivation are mediated via cholinergic muscarinic

receptors. After oral administration, Tolterodine is metabolized in the

liver, resulting in the formation of the 5-hydroxymethyl derivative, a

major pharmacologically active metabolite. The 5-hydroxymethyl

metabolite, which exhibits an antimuscarinic activity similar to that of

Tolterodine, contributes significantly to the therapeutic effect. Both

Tolterodine and the 5-hydroxymethyl metabolite exhibit a high

specificity for muscarinic receptors, since both show negligible activity

or affinity for other neurotransmitter receptors and other potential

cellular targets, such as calcium channels. Tolterodine has a

pronounced effect on bladder function. Effects on urodynamic

parameters before 1 and 5 hours after a single 6.4-mg dose of

Tolterodine immediate release were determined in healthy volunteers.

The main effects of Tolterodine at 1 and 5 hours were an increase in

residual urine, reflecting an incomplete emptying of the bladder, and a

decrease in detrusor pressure. These findings are consistent with an

antimuscarinic action on the lower urinary tract. A convenient

synthesis of Tolterodine tartrate was cited in the literature (3) starting

from 1-[(2-hydroxy-5-methyl)phenyl] ethylene accessible in high yield

by aluminia promoted ortho alkenylation of p-cresol with phenyl

acetylene.

-

47

Detrol has Tolterodine tartrate as the main active pharmaceutical

ingredient and the following inactive ingredients: colloidal anhydrous

silica, calcium hydrogen phosphate dihydrate, cellulose

microcrystalline, hypromellose, magnesium stearate, sodium starch

glycolate (pH 3.0 to 5.0), stearic acid, and titanium dioxide. Detrol is

available in Tablets form 1 mg and 2 mg round, white, film- coated

tablets) and in Capsules form (2 mg - blue-green capsules, 4 mg - blue

capsules)

Accordingly the aim of the present study was to establish inherent

stability of Tolterodine tartarate through stress studies under a variety

of ICH recommended test conditions (4-6) to develop a stability

indicating HPLC assay method for the determination of Tolterodine

tartrate and its potential impurities (7-9). Literature survey did not

reveal any simple, sensitive and stability indicating method for the

determination of related components of Tolterodine tartrate in drug

substance and drug product. Among the reported liquid

chromatographic methods, the use of UV detection for quantification of

enantiomer of Tolterodine using chiral HPLC (10) is included. The other

reported methods are applicable for biological matrices using complex

analytical instruments such as mass spectrophotometer (11-15). So far,

to our present knowledge there is no stability indicating LC method for

-

48

assay and related impurities estimation of Tolterodine tartrate in both

bulk drug and pharmaceutical dosage forms.

The target is to develop a suitable stability-indicating HPLC assay

method for Tolterodine tartrate. In the present study, stress studies

were carried out in a conventional reflux method, moreover a fast and

effective microwave based degradation technique was designed and

employed for forced degradation study. Both the studies were carried

out as per ICH. In this chapter a detailed description about stability

indicating LC method for the determination of Tolterodine tartrate and

its potential impurities along with method validation was given.

5.2 Development and validation of a novel HPLC method for the

determination of Related Components in Tolterodine tartrate:

5.2.1 Materials:

Samples of Tolterodine tartrate and its impurities (namely impurity-

1, impurity-2 and impurity-3 shown in Fig: 5.2) were received from

Gensen Laboratories Limited; Mumbai, India. The received samples

were certified and their purity is greater than 99.5. Detrol 2 mg tablets

(Tolterodine tartrate formulation) were purchased from the market.

HPLC grade acetonitrile, analytical reagent grade sodium dihydrogen

phosphate monohydrate, triethylamine and phosphoric acid were

purchased from Merck, Darmstadt, Germany; high pure water was

prepared by using Millipore Milli-Q plus water purification system.

-

49

5.2.2 Equipment:

The LC system used was Waters 2695 quaternary pump plus auto

sampler and a 2996 photo diode array detector (Waters Corporation, 34

Maple Street, Milford, MA, 01757 USA). The output signal was

monitored and processed using empower software on Pentium

computer (Digital equipment Co), water baths equipped with MV

controller (Julabo, Seelabach, Germany) were used for hydrolytic

studies. Stability studies were carried out in humidity chamber

(Thermo lab humidity chamber, India) and photo stability studies were

carried out in a photo stability chamber (Sanyo photo stability

chamber, Leicestershire, UK). Thermal stability studies were performed

in a dry air oven (MACK Pharmatech, Hyderabad, India). The

microwave degradations were performed in MS-2347 (15S/cycle,

2.45GHz, 300w) microwave oven. The LC-MS analysis was performed

on waters quatramicro TM API system equipped with triple quadrapole

(Mass Lynx 4.1).

Fig: 5.2 Chemical structure of impurities of Tolterodine tartrate

Impurity-1

NH

OH

O

OH

OH

OOH

-

50

N-Isopropyl-3-(2-hydroxy-5- methyl phenyl)-3-phenyl propyl amine-L-

hydrogen tartrate

Molecular formula: C23H31NO7 Molecular weight : 433.50

Impurity-2

CH-CH2-CH2-N(CH2-CH2-CH3)2

OCH2C6H5H3C

N, N-di isopropyl- 3-(2-benzyloxy-5-methylphenyl)-3-

phenylpropylamine

Molecular formula: C29H37NO Molecular weight : 415.61

Impurity-3

N

OH HO

N-Isopropyl-Bis-(3-(2-hydroxy-5-methyl) phenyl)-3-phenyl propyl amine

Molecular formula: C38H51NO2 Molecular weight : 553.82

5.2.3 Sample preparation:

Stock solutions of Tolterodine tartrate standard and sample (5.0 mg

mL-1) were prepared by dissolving appropriate amounts in diluent.

Acetonitrile: water (1:1, v/v) was used as diluent. Working solutions of

500 and 100 µg mL-1 were prepared from above stock solutions for

-

51

related components determination and assay determination

respectively. A stock solution of impurities (mixture of impurity-1,

impurity-2 and impurity-3) at 0.5 mg mL-1 was also prepared in diluent.

5.2.4 Preparation of tablets sample solution:

Twenty five tablets were weighed and the content was transferred

into a clean and dry mortar, grinded well. Then an equivalent to 50 mg

of drug was transferred to 100 mL volumetric flask, 30 mL of diluent

was added and kept on rotatory shaker for 10 min to disperse the

material completely, sonicated for 10 min and diluted to 50 mL (500 µg

mL-1). The resulting solution was centrifuged at 3,000 rpm for 25 min

(supernatant solution was used for purity evaluation). 10 mL of

supernatant solution was taken and diluted to 100 mL with diluent

(100 µg mL-1). This solution was filtered using 0.45 µm nylon

membrane filter and used for the assay analysis.

5.2.5 Method development and optimization of chromatographic

conditions:

Forced degradation studies were performed to determine the related

components in Tolterodine tartrate drug substance and drug product to

develop a stability indicating HPLC method for the quantitative

determination of and purity evaluation of Tolterodine tartrate. Stressed

samples obtained during forced degradation studies including the

-

52

samples of impurity-1, impurity-2 and impurity-3 were utilized in the

HPLC method development.

5.2.5.1 Selection of wavelength:

Fig: 5.3 Typical overlaid UV spectrums of Tolterodine tartrate and

its impurities.

4.300 Imp-14.933 Toltirodine15.150 Imp-221.483 Imp-3

294.7 318.4 337.5369.4

392.3279.3

368.2 392.3

207.5

282.8284.0

355.4

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00

Wavelength (nm)

Impurities and Tolterodine tartrate solutions were prepared in

diluent at a concentration of 100 ppm and scanned in UV spectro-

photometer; impurities and Tolterodine tartrate were having UV

maxima at around 210nm (Fig: 5.3). Hence detection at 210 nm was

selected for method development purpose.

5.2.5.2 Column Selection:

The main target of the chromatographic method was to get the

separation among impurities namely impurity-1, impurity-2 and

-

53

Absorbance

impurity-3 from Tolterodine and symmetry of Tolterodine peak.

Preliminary experiments were carried in reverse phase using different

commercially available C18 and C8 columns.

Initial trial was carried out on Inertsil ODS (250 x 4.6) mm column

with 5 µm particles using phosphate buffer (pH adjusted to 3.5 with

diluted phosphoric acid) and methanol in 50:50 v/v ratio at 1.0 mL

min-1 flow rate. Tolterodine tartrate and impurity-1 were separated with

a resolution of 0.8. The retention of impurity-2 and impurity-3 was very

high (~ 35 min, ~ 60 min) and tailing factor of the Tolterodine was very

high (~4.0). Similar results were obtained on Waters C18, Inertsil C8

columns (250 x 4.6) mm columns with 5 µm particles.

When Inertsil C8 (250 x 4.6) mm with 5 µm particles, Zorbax Extend

C18 (250 x 4.6) mm with 5 µm particles, Inertsil ODS (250 x 4.6) mm

with 5 µm particles and Waters Symmetry C18 (250 x 4.6) mm with 5

µm particles columns were used as stationary phases, the retention of

impurity-3 was very late (retention time of about 35 min) and the

tailing factor of the Tolterodine is very high (4.0) with 1.0 mL min-1 flow

rate.

Late elution of impurity-2 and impurity-3 could be due to the ability

of impurity-2 and impurity-3 to form lipophilicity with the stationary

phase of C18/C8 Column. To decrease the lipophilicity of impurity-2

and impurity-3 with column, column length was decreased to 150 mm

instead of 250 mm and polarity of the column was also increased with

-

54

less carbon loading C8 stationary phase. In general, in reverse phase

mechanism the retention is based upon the relative hydrophobicities of

analyte molecules and carbon loading of the column. The higher the

carbon load, the more the retention. Carbon load can be proportional to

the chain length, the longer the chain length, the more carbon would

be present and therefore retention would be proportional to chain

length and carbon loading. Hence less carbon loading C8 stationary

(Zorbax C8) with 150mm length was finalized for further method

development. Relative hydrophobicities of general purpose analytical

packings were given in Fig: 5.4.

Fig: 5.4 Relative hydrophobicities of general purpose analytical

packings.

Waters

Symmetry C-18 19.5%

Inertsil ODS

-3V 15%

Zorbax Extend C18 12.5%

Inertsil C8 10.5%

YMC Pack C-8 10%

Zorbax C8 5.5%

5 10 15 20 % Carbon loading

-

55

To improve the tailing factor of the Tolterodine peak particle size of

the column was decreased to 3.5 µ. The purity of silica support is of

strong concern in separating and getting symmetry of polar

compounds. As illustrated at the Fig 5.5, some silica are contaminated

with certain metals (Fe, Al, Ni, Zn, etc).

Fig: 5.5 Surface of Silica Supports for HPLC

These metal contaminants can complex with chelating solutes,

causing asymmetrical or tailing peaks. Other metals in the silica lattice

(especially aluminium) activate surface silanol groups so that they are

highly acidic. Therefore highly purified silica is needed for controlling

tailing in basic and highly polar compounds. Zorbax C8 column which

contains high pure silica was selected for controlling the tailing of

Tolterodine peak. The contents of various metals in Zorbax silica were

given in Table: 5.1.

Table: 5.1 Typical trace metal analysis of Zorbax Silica by ICP, AAS/MS

____________________________________________________________________________Element Content a (PPM)

____________________________________________________________________________

-

56

Na 10Ca 2K < 3Al 1.5Fe 3Mg 4Zn 1

____________________________________________________________________________

a Total :< 35 ppm (no other elements detected, < 1ppm) ; 99.995%

silica [16].

The stationary phase was found to have a great influence on the

retention time and peak shape. Hence the Zorbax C8 (150 x 4.6) mm,

3.5 microns particle size column was chosen for further method

development. Experimental results on different stationary phases were

given in Table: 5.2.

Table: 5.2 Experimental results on different stationary phases

Column Parameter Imp-1 Tolterodine Imp-2 Imp-3

Waters Symmetry C18

(250 x 4.6) mm 5 µm particlesRetention time 12.6 14.5 34.8 60.5

USP Tailing 1.1 2.8 1.1 1.1

Inertsil ODS (250 x 4.6) mm

5 µm particlesRetention time 9.8 11.3 38.1 51.4

USP Tailing 1.0 2.5 1.1 1.1

Zorbax Extend C18


USP Tailing 1.0 2.0 1.1 1.1

Inertsil C8


-

57

USP Tailing 1.0 1.8 1.1 1.1

YMC pack pro C8


USP Tailing 1.0 1.7 1.1 1.1

Zorbax C8

(150 x 4.6) mm, 3.5 microns

particle size

Retention time 4.3 5.0 15.2 21.5

USP Tailing 1.1 1.3 1.1 1.2

5.2.5.3 Effect of Buffer:

Ionic samples especially basic compounds can interact with the

silanols of the silica based columns, which can lead to increased

retention and band tailing. These silanol interactions can be decreased

by applying appropriate experimental conditions. Usually the most

important silanol sample interaction is caused by ion exchange. A

protonated base in the sample exchanges with sodium or potassium or

other cation that attached to an ionized silanol in the column packing.

Silanol effects can be reduced by using higher buffer concentration.

This can be achieved by increasing the buffer concentration. Keeping

this point in view, buffer concentration was increased from 10 mM to

-

58

50 mM (NaH2PO4). Because of this, the interaction of sodium ions in

mobile phase with silanols will be more, therefore it reduces the

interaction of sample with residual silanols, which resulted in improved

peak shape of Tolterodine (tailing 2.2).

5.2.5.4 Effect of additives (Triethylamine):

To further improve the symmetry of Tolterodine peak, triethyl amine

was added to the mobile phase as triethyl ammonium+ can block

residual silanols more strongly than sodium.

Na+ < K+ < NH4+ < Triethyl ammonium + < dimethyl octyl

ammonium +

When 1mL of triethyl amine was used in the mobile phase the

symmetry of Tolterodine peak was improved to 1.8. To further improve

the symmetry of Tolterodine peak, concentration of triethyl amine was

increased. When the concentration of triethyl amine was increased from

1 mL to 5 mL, symmetry of Tolterodine peak improved to 1.3.

5.2.5.5 Effect of Organic solvent:

When methanol was used as a solvent in the mobile phase (buffer:

methanol: 50:50, v/v) the retention time of impurity-3 was very high

(60 min) due to the high interaction of imp-3 with column (lipophilicity)

and so the tailing factor of Tolterodine peak was also high (~1.5). To

improve the retention time of impurity-3 and symmetry of Tolterodine,

-

59

the solvent strength has been increased by replacing methanol with

acetonitrile. Different mixtures of buffer and acetonitrile were applied to

improve the retention time and symmetry of Tolterodine. When 40%

acetonitrile was used as a solvent in the mobile phase (buffer:

acetonitrile 60:40, v/v) the retention time of impurity-3 and the

symmetry of Tolterodine peak was improved with satisfied resolution

between all the impurities. Acetonitrile played a major role to control

the retention time of impurity-3.

5.2.5.6 Effect of pH:

When pH of the phosphate buffer adjusted to 7.5 with diluted

sodium hydroxide solution (buffer: acetonitrile: 60:40, v/v), due to the

ionization of –OH group in impurity-1 and Tolterodine, the polarity

increased and interactions with stationary phase decreased resulted in

poor resolution between impurity-1 and Tolterodine (Rs ~1.1). When pH

of the phosphate buffer adjusted to 2.5 the resolution between

impurity-1 and Tolterodine increased and satisfactory results obtained.

Compared to basic pH, at acidic pH the symmetry of Tolterodine peak is

good. At acidic pH the concentration of ionization of silanols got

minimized and a symmetrical Tolterodine peak was achieved. The pH of

the buffer has played a significant role in achieving the separation

between impurity-1 and Tolterodine peak and symmetry of Tolterodine

-

60

peak (tailing factor of the Tolterodine is 1.3). In the optimized

chromatographic conditions, the resolution between Tolterodine and its

potential impurities namely impurity-1, impurity-2 and impurity-3, was

found to be greater than 2. The typical retentions times were

approximately 4.2, 4.9, 15.1 and 21.5 min for impurity-1, Tolterodine,

impurity-2 and impurity-3 respectively.

5.2.5.7 Optimized chromatographic conditions for the

determination of related components and assay of Tolterodine

tartrate:

Column : Zorbax SB C8, (150x 4.6) mm,

3.5µm particle size

Mobile phase : Buffer: acetonitrile: 60:40 (v/v)

Flow rate : 0.8 mL min-1

Column temperature : 27 ± 2°C

Wavelength of detection : 210 nm

Injection volume : 10µL

Run time : 30 min

Diluent : Acetonitrile: water (1:1, v/v)

Buffer : 50mM sodium dihydrogen phosphate

monohydrate and 5 mL of triethylamine,

pH adjusted to 2.5 using diluted phosphoric

-

61

acid (1mL in 10 mL of Milli-Q water).

Relative Retention Time : Impurity-1 - about 0.87

Impurity-2 - about 3.07

Impurity-3 - about 4.35

The interference of excipients colloidal anhydrous silica, calcium

hydrogen phosphate dehydrate, cellulose microcrystalline,

hypromellose, magnesium stearate, sodium starch glycolate (pH 3.0 to

5.0), stearic acid and titanium dioxide were checked by injecting

sample solutions of excipients. There was no interference of excipients

with impurities (impurity-1, impurity-2 and impurity-3) and Tolterodine

peak.

5.2.5.8 Optimized chromatographic conditions for the

determination of degradation products of Tolterodine tartrate by

LCMS:

Column : Zorbax C8, (150x 4.6)mm,

3.5µm particle size

Flow rate : 0.8 mL min-1

Column temperature : 25 ± 2°C

Injection volume : 10µL

Run time : 30 min

Diluent : Acetonitrile.

-

62

Mobile phase : Water and acetonitrile 60: 40 (v/v).

Quattro micro Tune Parameters:

Source (ES+)

Capillary Voltage : 3.6 (kV)

Cone Voltage : 65.0 (v)

Extractor : 2.00 (v)

RF Lens : 0.1 (v)

Source Temp : 120° C

Desolvation Temp : 250° C

Cone gas flow : 250mL min-1

Analyser

LM1 Resolution : 15.0

HM1 Resolution : 15.0

Ion energy 1 : 0.5

Entrance : 50

Collision : 2

Exit : 50

Multiplier : 650 (v)

Mass analysis was performed for Tolterodine tartrate and its

impurities. Mass Lynx 4.1 software (waters) was used to calculate the

-

63

molecular weight of the protonated molecules according to the accurate

mass data.

Table: 5.3 Mass spectral data of Tolterodine and its impurities

Compound Name Molecular weight (M) (M+H) value

Tolterodine 325.5 326.3

Impurity-1 283.4 284.4

Impurity-2 415.6 416.3

Impurity-3 507. 508.4

Molecular weight of Tolterodine tartrate is 475.6, but molecular

weight of Tolterodine is 325.51. In the LC-MS analysis of Tolterodine

tartrate, the molecular ion of 326.3 is protonated molecular ion (M+H)

of Tolterodine (Molecular weight 325.51). The ion corresponds to

327.35 is M+2H ion (Fig: 5.6).

Impurity-1 is a tartrate salt, it molecular weight is 433.50. Impurity-

1 base molecular weight is 283.41. In the LC-MS analysis of impurity-

1, the molecular ion of 284.4 is protonated molecular ion (M+H) of

impurity-1 base (Molecular weight 283.4). The ion corresponds to 286.3

is M+2H ion (Fig: 5.7).

-

64

Molecular weight of Impurity-2 is 415.6. In the LC-MS analysis of

impurity-2, the molecular ion of 416.3 is protonated molecular ion

(M+H) of impurity-2 (Molecular weight 415.6). The ion corresponds to

417.3 is M+2H ion (Fig: 5.8).

Molecular weight of Impurity-3 is 507.7. In the LC-MS analysis of

impurity-3, the molecular ion of 508.4 is protonated molecular ion

(M+H) of impurity-3 (Molecular weight 507.7). The ion corresponds to

509.47is M+2H ion (Fig: 5.9).

Fig: 5.6 Typical LC-Mass spectrum of Tolterodine tartrate

-

65

TOT113:29:51

07-Jul-2008USP India (P) Ltd.,

m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

%

0

100TOT SST 59 (1.181) Cm (53:69-3:31) 1: Scan ES+

1.09e7326.330

284.331

239.307225.268

285.320

327.385

765.610

Fig: 5.7 Typical LC-Mass spectrum of impurity-1

-

66

TOT113:29:51


m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

%

0

100TOT SST 47 (0.938) Cm (37:50-4:30) 1: Scan ES+

3.61e6284.331

220.259 238.187

239.241

284.463

285.320

567.610

326.330286.309

681.619568.468 682.608


-

67

TOT113:29:51


m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

%

0

100TOT SST 182 (3.663) Cm (171:199) 1: Scan ES+

1.75e7416.349

310.308

224.279283.342

282.287237.264

311.297

374.337325.341

375.326

417.338

508.367418.394

509.357


-

68

TOT113:29:51


m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

%

0

100TOT SST 163 (3.280) Cm (150:167) 1: Scan ES+

3.46e7508.367

284.331466.347

509.423

510.413

5.2.6 Stress Degradation studies/Specificity:

-

69

Specificity of the developed method was assessed by performing

forced degradation studies. Specificity is the ability of the method to

measure the analyte response in the presence of its potential impurities

[11]. The specificity of the developed LC method for Tolterodine tartrate

was determined in the presence of its impurities namely impurity-1,

impurity-2, impurity-3 and degradation products. Forced degradation

studies were performed on Tolterodine tartrate to provide an indication

of the stability indicating property and specificity of the proposed

method [14, 15]. In the present study stress studies were carried out in

a conventional reflux method, moreover a fast and effective microwave

assisted degradation technique was designed and employed for forced

degradation study. Both the studies were carried out as per ICH. The

detailed studies using both the techniques were addressed below.

5.2.6.1 Generation of stress samples:

One lot of Tolterodine tartrate drug substance was selected for

stress testing. From the ICH Stability guideline (Q1A (R2)), “stress

testing is likely to be carried out on a single batch of material [12]”.

Different kinds of stress conditions (i.e., heat, humidity, hydrolysis

(acid, base), oxidative and light) were employed on one lot of Tolterodine

tartrate drug substance. The details of the stress conditions applied

are as follows.

-

70

All stress decomposition studies were performed at an initial drug

concentration of 0.5 mg mL-1 (500 µg mL-1). The stress conditions

employed for degradation study includes light (carried out as per ICH

Q1B), heat (60°C), acid hydrolysis (1N HCl, 80°C for 12h), base

hydrolysis (1N NaOH, 80°C 12h), water hydrolysis (80°C 12h) and

oxidation (6% H2O2, RT 48h). For heat and light studies, study period

was 10 days. Photolytic degradation was carried out by exposing the

Tolterodine tartrate samples in solid state to light providing an overall

illumination of not less than 1.2 million lux hours and an integrated

near ultraviolet energy of not less than 200 W h/m2, which took about

10 days period in photo stability chamber [13]. The above stress

conditions were also applied on Detrol 2 mg tablets to check the

applicability of the developed method.

Peak purity of stressed samples of Tolterodine tartrate was checked

by using 2996 photo diode array detector of waters (PDA). The purity

angle within the purity threshold limit obtained in all stressed samples

demonstrates the analyte peak homogeneity. All stressed samples of

Tolterodine tartrate were analysed for extended run time to check the

late eluting degradants.

Assay studies were carried out for stress samples against qualified

reference standard and the mass balance (% assay+% of impurities + %

-

71

of degradation products) was calculated. Assay was also calculated for

bulk samples and drug product by spiking all three impurities

(impurity-1, impurity-2 and impurity-3) at the specification level (i.e.

0.15% of analyte concentration which is 500 µg mL-1).

5.2.6.2 Microwave assisted Degradations:

A new, fast and effective microwave assisted degradation technique

was designed and employed for hydrolytic forced degradation study. All

stress decomposition studies were performed at an initial drug

concentration of 0.5 mg mL-1 (500 µg mL-1). The stress conditions

employed for degradation study includes, acid hydrolysis (1N HCl, 5

min), base hydrolysis (1N NaOH, 5 min), water hydrolysis (5 min) and

oxidation (6% H2O2, 5 min).

For hydrolytic treatments, 2mL of either 1N HCl, 1N NaOH were

added in each flask containing 5 mg of drug. Prepared solutions were

vortex mixed and subjected to microwave radiation (15s/cycle,

2.45GHz, 300w) for 5 min. After the treatment, samples were allowed to

cool and neutralized.

Assay studies were carried out for stress samples against qualified

reference standard and the mass balance (% assay+% of impurities + %

of degradation products) was calculated. Assay was also calculated for

bulk samples and drug product by spiking all three impurities

-

72

(impurity-1, impurity-2 and impurity-3) at the specification level (i.e.

0.15% of analyte concentration which is 500 µg mL-1). HPLC studies on

Tolterodine tartrate under different stress conditions suggested the

following degradation behavior.

5.2.6.3 Degradation in acidic solution:

No degradation was observed when Tolterodine tartrate was

subjected to stress conditions in 0.1N HCl at room temperature for

48h. Even after reflux for 12 h in 1N HCl also, no major degradation

products were observed. The drug was stable under acidic conditions

(Fig 5.10 (b)). When drug was subjected to microwave assisted acid

degradation (1N HCl) for 5 min, no major degradation products were

observed. The drug was stable under acidic conditions Fig 5.11 (a).

Fig: 5.10 Typical HPLC chromatograms of acid hydrolysis

3.05

1

AU

0.00

0.02

0.04

0.06

0.08

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

-

73

(a) Blank chromatogram of acid hydrolysis (1N HCl)

3.703

4.018

4.145 4.3

204.5

80To

ltirod

ine - 5

.202

6.835

10.75

6

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Tolti

rodi

ne -

5.22

2

PurityAuto Threshold

AU

Deg

rees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60

X axis - Time in minutes Y axis - Absorbance AU

Purity Angle Purity Threshold Purity Flag Peak Purity

0.075 0.269 No Pass

-

74

(b) Tolterodine tartarate under 1N HClreflux for 12h

(c) Peak purity plot of acid hydrolysis

Fig: 5.11 Tolterodine tartrate stressed with 1N HCl in micro wave

oven for 5 min.

3.713

4.145 4.321 4.5

834.7

24To

ltirodin

e - 5.

183

6.309

6.846

10.774

19.730

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Toltir

odine

- 5.

212


AU

Degr

ees

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes4.95 5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65 5.70 5.75


0.098 0.354 No Pass


5.2.6.4 Degradation in basic solution:

No degradation was observed when Tolterodine tartrate was

subjected to stress conditions in 0.1N NaOH at room temperature for

48h. But when Tolterodine tartrate was refluxed for 12h in 1N NaOH at

-

75

(b) Peak purity plot of acid hydrolysis

(a) Tolterodine tartrate stressed with 1N HClin microwave oven for 5 min.

80°C, drug showed significant sensitivity towards base treatment and

Tolterodine underwent degradation with time and degraded into

unknown impurity (~32%) at RRT ~ 0.61 (Fig 5.12 (b)).

Tolterodine tartrate showed significant sensitivity towards base

treatment under microwave conditions. The drug gradually underwent

degradation with in 5 min under microwave conditions and degraded

into unknown impurity (~32%) at RRT ~ 0.61. (Fig 5.13 (a))

Fig: 5.12 Typical HPLC chromatograms of base hydrolysis

3.12

9

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

3.267

3.267

3.585

3.801

4.471

4.972

Toltiro

dine -

5.216

9.648

15.167

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

-

76

(b) Tolterodine tartrate stressed with 1N NaOH for 12h at 80°C

(a) Blank chromatogram of base hydrolysis (1N NaOH)

Toltir

odine

- 5.2

34


AU

Degr

ees

0.00

0.05

0.10

0.15

0.20

0.25

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60


Fig: 5.13 Typical HPLC chromatograms of base hydrolysis in

Microwave oven

3.255

3.590

4.061 Imp

-1 - 4.4

71Tol

tirodin

e - 5.1

58

15.163

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00


0.052 0.235 No Pass

-

77

(a) Tolterodine tartarate stressed with 1N NaOH in microwave oven for 5 min

(c) Peak purity plot of base hydrolysis

Toltir

odine

- 5.

234


AU

Degr

ees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60


5.2.6.5 Oxidative conditions (Stress degradation using peroxide):

The drug was exposed for 48h with 6% hydrogen peroxide.

Tolterodine tartrate was stable towards the hydrogen peroxide

treatment. A little degradation (negligible) was observed (~0.6%)

(Fig 5.14 (b)).

The drug was exposed for 5 min with 6% hydrogen peroxide under

microwave conditions. Tolterodine tartrate was stable towards the

hydrogen peroxide treatment. A little degradation (negligible) was

observed (~0.5) (Fig 5.15 (a)).

Fig: 5.14 Typical HPLC chromatograms of oxidative degradation


0.066 0.266 No No

-

78

(b) Peak purity plot of base hydrolysis

3.27

8

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

4.023

4.484

Toltiro

dine -

5.212

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Tolti

rodi

ne -

5.22

1


AU

Deg

rees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65



0.131 0.242 No Pass

-

79

(a) Blank chromatogram of oxidative degradation (6%H2O2)

(b) Tolterodine tartrate stressed with 6%H2O2 for 48h

(c) Peak purity plot of oxidative degradation

Fig: 5.15 Typical HPLC chromatograms of oxidative degradation in

microwave oven

4.029

Imp-1 -

4.482

4.652

Toltiro

dine -

5.182

7.518

9.995

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Toltir

odine

- 5.

226


AU

Degr

ees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65 5.70


5.2.6.6 Degradation in neutral (water) solution:


0.116 0.230 No Pass

-

80

(a) Tolterodine tartrate stressed with 6% H2O2in microwave oven for 5 min

(b) Peak purity plot of oxidative degradation

The drug was refluxed at 80°C for 10h in neutral (water) solution.

No degradation was observed. The drug was stable towards water

hydrolysis (Fig 5.16(b)).

The drug was exposed for 5 min with water under microwave

conditions. Tolterodine tartrate was stable towards the water hydrolysis

(Fig 5.17(a)).

Fig: 5.16 Typical HPLC chromatograms of water degradation

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

3.749

3.998

4.283

4.544

Toltiro

dine -

5.206

15.217

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

-

81

(a) Blank

(b) Tolterodine tartrate refluxed with water for 12h

Tolti

rodi

ne -

5.21

7


AU

Deg

rees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65

Purity Angle Purity Threshold Purity Flag Peak Purity0.092 0.277 No No


Fig: 5.17 Typical HPLC chromatograms of water degradation in

Microwave oven

3.665

4.318

4.436

Imp-1

- 4.55

7To

ltirodin

e - 5.

220

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Tolti

rodin

e - 5

.220


AU

Degr

ees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65


0.118 0.158 No Yes -

82

(a) Tolterodine tartrate stressed with water in micro wave oven for 5 min

(c) Peak purity plot of water degradation

(b) Peak purity plot of water degradation


5.2.6.7 Photolytic conditions:

The drug was stable to the effect of photolysis. When the drug

powder was exposed to light for an overall illumination of 1.2 million

lux hours and an integrated near ultraviolet energy of 200-watt

hours/square meter (w/mhr) (in photo stability chamber), no

degradation of the drug was observed (Fig: 5.18(a)).

Fig: 5.18 Typical HPLC chromatograms of photolytic degradation

Toltiro

dine -

5.228

AU

0.00

0.05

0.10

0.15

0.20

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Toltir

odine

- 5.2

28


AU

Degr

ees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65

-

83

(a) Tolterodine tartrate under photolytic degradation for 10 days

(b) Peak purity plot of photolytic degradation


5.2.6.8 Thermal Degradation:

The drug was stable to the effect of temperature. When the drug

powder was exposed to dry heat at 60°C for 10 days, no degradation of

the drug was observed (Fig: 5.19(a)).

Fig: 5.19 Typical HPLC chromatograms of thermal degradation

Toltir

odine

- 5.22

1

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Toltir

odine

- 5.

221


AU

Degr

ees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65


0.083 0.279 No No

-

84

(a) Tolterodine tartrate under thermal conditions for 10 days

(b) Peak purity plot of thermal degradation


5.2.6.9 Degradation of Detrol tablet in acidic solution:

No degradation was observed when Detrol (Tolterodine tartrate) was

subjected to stress conditions in 0.1N HCl at room temperature for

48h. Even after reflux for 12h in 1N HCl also, no major degradation

products were observed. The drug was stable under acidic conditions

(Fig 5.20 (b)).

Fig: 5.20 Acid degradation of Detrol Tablets

3.05

1

AU

0.00

0.02

0.04

0.06

0.08

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00


0.078 0.257 No Pass

-

85

(a) Blank chromatogram of acid hydrolysis of Detrol tablet

3.504

3.683

4.310

4.568

Toltir

odine

- 5.22

2

20.05

7

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Toltir

odine

- 5.

222


AU

Degr

ees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60


Peak purity test performed for the Tolterodine peak using

photodiode array (PDA) detector confirmed the homogenity of the peak

for all the stressed samples. Assay of all stressed samples were

calculated using qualified standard of Tolterodine tartrate. Considering


0.075 0.269 No Pass

-

86

(b) Detrol tablets refluxed with 1N HCl for 12h

(c) Peak purity plot of acid degradation

the purities from the respective chromatograms of stressed samples,

mass balance (%assay + % degradents + % impurities) was calculated

for each stress sample. The mass balance of stressed samples was

close to 99.4%. This clearly demonstrates that the developed HPLC

method was found to be specific for Tolterodine tartrate in presence of

its impurities (impurity-1, impurity-2 and impurity-3) and degradation

products. No degradants were observed after 15 min in the extended

runtime of 30 min for all the Tolterodine tartrate stressed samples.

5.2.7 Analytical method Validation and its results:

The developed and optimized HPLC method was taken up for

validation. The analytical method validation was carried out in

accordance with ICH guidelines [10, 11 and 12].

5.2.7.1 System suitability test:

System suitability testing is an integral part of analytical procedure.

The tests are based on the concept that the equipment, electronics,

analytical operations and samples to be analyzed constitute an integral

system that can be evaluated as such. System suitability test

parameters to be established for a particular procedure depend on the

type of procedure being validated.

To the Tolterodine tartrate standard, impurity-1, impurity-2 and

impurity-3 were spiked at 0.15% level with respect to the concentration

of Tolterodine tartrate and injected for five times into HPLC system.

Resolution between impurities and Tolterodine tartrate, tailing factor,

-

87

theoretical plates for impurities and Tolterodine tartrate, RSD% for the

areas of impurities and Tolterodine tartrate was calculated. System

suitability results were tabulated (Table: 5.4).

Fig: 5.21 Typical Blank, Tolterodine tartrate Sample and SST

chromatograms

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Toltir

odine

- 5.

219

AU

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

-

88

(a) Blank

(b) Tolterodine tartrate sample

Imp-1 -

4.297

Toltiro

dine -

4.936

Imp-2 -

15.15

5

Imp-3 -

21.49

0

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Table: 5.4 System suitability results

Compound (n=5)Resolution

(Rs)USP Tailing

factor (T)No. of theoretical

plates (N)RSD%for area

Impurity-1 -- 1.1 11666 0.5

Tolterodine

tartrate3.1 1.2 14215 1.2

Impurity-2 28.0 1.0 16750 0.3

Impurity-3 11.6 1.0 19945 0.8

n = Number of determinations

5.2.7.2 Precision:

The precision of an analytical procedure expresses the closeness of

agreement (degree of scatter) between a series of measurements

obtained from multiple sampling of the same homogeneous sample

under the prescribed conditions. It was usually expressed as RSD%.

Precision is a measure of the degree of reproducibility of the analytical

-

89

(c) Tolterodine tartrate spiked with imps

method under normal operating circumstances. The precision of the

analytical method is determined by assaying a sufficient number of

aliquots of a homogenous sample to be able to calculate statistically

valid estimates of SD or RSD (CV). Precision includes repeatability,

intermediate precision and reproducibility. Repeatability is the

precision of a method under the same operating conditions over a short

period of time (Determining RSD). Intermediate Precision expresses

within-laboratories variations, different days, different analysts,

different equipment etc. Reproducibility expresses the precision

between laboratories (collaborative studies).

Repeatability of the assay method was studied by performing system

precision (Table: 5.5), then by carrying out six independent assays of

Tolterodine tartrate test sample against qualified reference standard

and RSD of six consecutive assays was 0.16% (Table: 5.6). Results

showed insignificant variation in measured response, which

demonstrated that the method was repeatable with RSDs below 0.2%.

Table: 5.5 System precision results of assay study

-

90

Table: 5.6 Precision results of the assay study

Preparation %Assay

1 99.69

2 99.40

3 99.77

4 99.50

5 99.52

S.No Area of Tolterodine peak

Injection-1 3091029

Injection -2 3133448





Mean 3101396

SD 18239.4

RSD% 0.6

-

91

6 99.35

Mean 99.54

SD 0.16

RSD% 0.16

95% Confidence interval of

mean{ 99.41,99.67 }

Repeatability for related components study was checked initially by

performing system precision and then by injecting six individual

preparations of Tolterodine tartrate (0.5 mg mL-1) spiked with 0.15% of

impurity-1, impurity-2 and impurity-3 with respect to Tolterodine

tartrate analyte concentration. The RSD% for area % of impurity-1,

impurity-2 and impurity-3 for six consecutive determinations was

respectively tabulated as below (Table: 5.7). Results showed

insignificant variation in measured response, which demonstrated that

the developed method was repeatable with RSDs below 0.7% for related

components.

Table: 5.7 System precision results of related components study

-

92

Table: 5.8

Precision results of related components study

Preparation Impurity-1 Impurity-2 Impurity-3

1 0.149 0.152 0.147

2 0.149 0.154 0.146

3 0.148 0.153 0.147

4 0.148 0.151 0.146

5 0.149 0.152 0.147

6 0.148 0.153 0.147

Mean 0.149 0.153 0.147

SD 0.001 0.001 0.001

RSD% 0.37 0.69 0.35

95% Confidence interval

of mean {0.148,0.149} {0.152,0.153} {0.146,0.147}

Intermediate precision for assay and related components study

was performed by carrying out six independent assays of Tolterodine

S.No Area of Tolterodine peak







Mean 15183157

SD 60890.5

RSD% 0.4

-

93

tartrate sample against qualified reference standard and RSD of six

consecutive assays and for related components study was performed by

injecting six individual preparations of Tolterodine tartrate (0.5 mg mL-

1) spiked with 0.15% of impurity-1, impurity-2 and impurity-3 with

respect to Tolterodine tartrate analyte concentration over different days,

different instruments, different columns and with different analysts.

Reproducibility of the method was checked by performing the precision

study in a different laboratory (Table: 5.9).

Table: 5.9 Results of Intermediate precision and reproducibility

S.No Parameter VariationRSD%

for assay

RSD%for related substances

Resolutionbetween

impurity-1 and

Tolterodine

1 Different

System

(a) Waters 2695

Alliance system

(b) Agilent 1100

Series VWD system

0.8

0.4

< 1.1

< 1.1

3.1

3.0

2Different

Column

(a) B.No: 00120701

(b)B.No:00120702

0.7

1.1

< 1.1

< 1.1

3.1

3.0

3Different

Analyst

(a) Analyst-1

(b) Analyst-2

0.6

0.5

< 1.1

< 1.1

3.1

3.0

4Different

Lab

(a) Lab-1

(b) Lab-2

0.3

0.2

< 1.1

< 1.1

3.1

3.2

5.2.7.3 Limit of quantification (LOQ) and limit of detection (LOD)

LOQ and LOD was established for impurity-1, impurity-2 and

impurity-3 based on signal to noise ratio method [9].

5.2.7.4 Limit of quantification (LOQ)

-

94

The quantitation limit of an individual analytical procedure is the

lowest amount of analyte in a sample which can be quantitatively

determined with suitable precision and accuracy. The quantitation limit

is a parameter of quantitative assays for low levels of compounds in

sample matrices and is used particularly for the determination of

impurities and/or degradation products.

To establish limit of quantification for impurity-1, impurity-2 and

impurity-3 a series of solutions with different known concentrations

were prepared and injected into the chromatographic system. For each

injection signal to noise ratio was monitored. Precision and accuracy

studies were carried out at that concentration where the S/N was

about 10. Based on the results the concentration was confirmed as

limit of quantification (LOQ) (Table 5.10).

Table: 5.10 LOQ values of the impurities

S.No Impurity name Concentration Signal to noise Ratio

1 Impurity-1 0.0035 µg mL-1

9.7


10.0


10.0

5.2.7.5 Limit of detection (LOD)

The detection limit of an individual analytical procedure is the

lowest amount of analyte in a sample, which can be detected but not

necessarily quantitated as an exact value.

-

95

To establish limit of detection for impurity-1, impurity-2 and

impurity-3 a series of solutions with different concentrations were

prepared and injected into the chromatographic system. For each

injection signal to noise ratio was monitored. The concentration where

the S/N was about 3 was chosen as limit of detection (LOD) value

(Table: 5.11).

Table: 5.11 LOD values of the impurities

S.No Impurity name Concentration Signal to noise ratio


2.1

2 Impurity-2 0.0018 µg mL-1 2.2

3 Impurity-3 0.0038 µg mL-1 2.2

5.2.7.6 Precision at Limit of quantification level:

The precision at limit of quantification level for related components

was checked by injecting six individual preparations of impurity-1,

impurity-2 and impurity-3 at their LOQ level with respect to Tolterodine

tartrate analyte concentration. Upon repeated injections at

quantification limit, the peak properties (retention time, area) were not

affected. Results showed insignificant variation in measured responses

which demonstrate that the method was repeatable at LOQ level with

RSD below 3.1%. The RSD% of area of impurity-1, impurity-2 and

-

96

impurity-3 for six consecutive determinations was 3.1, 2.0 and 1.2

respectively (Table: 5.12).

Table: 5.12 Precision results for related components at LOQ level.

PreparationImpurity-1

area

Impurity-2

area

Impurity-3

area

1 1152 2623 5423

2 1103 2569 5396

3 1163 2548 5387

4 1109 2496 5569

5 1165 2501 5451

6 1195 2596 5450

Average 1147.8 2555.5 5446

STD 35.5 50.9 65.8

RSD% 3.1 2.0 1.295% Confidence

intervalof mean

{1176.2,1119.4} { 2514.78,2596.22} {5393.30,5498.70}

5.2.7.7 Accuracy at LOQ level:

Standard addition and recovery experiments were conducted to

determine accuracy of the present method for the quantification of

impurities in Tolterodine tartrate sample at LOQ level.

-

97

The recovery studies for impurity-1, impurity-2 and impurity-3

were carried out in triplicate at LOQ level of the Tolterodine tartrate

target analyte concentration (500 µg mL-1). The percentage recovery of

impurity-1, impurity-2 and impurity-3 was calculated (Table: 5.13).

The method showed consistent and high absolute recoveries at LOQ

level with mean absolute recovery ranging from 96.4 % to 102.5%. The

obtained absolute recoveries were normally distributed around the

mean with uniform RSD values. The method was found to be accurate

with low % bias (< 2.0).

Table: 5.13 Recovery at LOQ level

S.No Impurity name Mean recovery (%)(n = 3 )

RSD%

1 Impurity-1 96.4 1.5

2 Impurity-2 102.5 1.8

3 Impurity-3 101.5 1.8

5.2.7.8 Linearity:

5.2.7.8 (a) Linearity study for assay estimation:

The linearity of an analytical procedure is its ability (within a given

range) to obtain test results which are directly proportional to the

concentration (amount) of analyte in the sample. The linearity of the

assay study was established by injecting test sample at 50, 75, 100,

125 and 150% of Tolterodine tartrate assay concentration (i.e.100 µg

-

98

mL-1). Each solution was injected twice (n=2) into HPLC system and the

average area at each concentration was calculated (Table: 5.14).

Calibration curve obtained by least square regression analysis between

average peak area and the concentration showed (Fig: 5.22) linear

relation ship with a regression coefficient of 0.9999. The best fit linear

equation obtained was y= 31114 x conc -57240. At all concentration

levels, standard deviation of peak area was significantly low and RSD

was below 0.7%. Analysis of residuals indicated that the residuals were

normally distributed around the mean with uniform variance across all

concentrations (Fig: 5.23) suggesting the homoscedastic nature of data.

Selected linear model with univariant regression showed minimum %

bias indicating goodness of fit which was further supported by the low

standard error of estimate and mean sum of residual squares.

Table: 5.14 Linearity results of the assay method

Concentration (%)Average Tolterodine

Peak area

50 150905475 2264641100 3056772125 3819590150 4620859

Correlation Coefficient (r) 0.9999Slope 31114.236

Intercept -57240.4

% Y-intercept -0.019

-

99

Fig: 5.22 Linearity plot for Assay study

Linearity of Assay

y = 31114x - 57240

R2 = 0.9999

1000000

2000000

3000000

4000000

40 60 80 100 120 140 160

Concentration

Avg

are

a

Table: 5.15 Residual summary of linearity results of assay study

Con’cin %

Mean area Response achieved

Response calculated from

trend line equation

Residual (response practical -response

theoretical)

Residual square

50 1509054 1498460 -10594 112232836

75 2264641 2276310 11669 136165561

100 3056772 3054160 -2612 6822544

125 3819590 3832010 12420 154256400

150 4620859 4609860 -10999 120978001

Residual sum of squares 530455342

Trend line equation y= 31114 X -57240

-

100

Fig 5.23 Residual plot for the linearity results of assay study

Residual plot

-61135

-41135

-21135

-1135

18865

38865

58865

0 1 2 3 4 5

Order of Residuals

Res

idu

als

5.2.7.8(b) Linearity study for related components:

Linearity experiments were carried out by preparing the Tolterodine

tartrate sample solutions containing impurity-1, impurity-2 and

impurity-3 from LOQ to 200% (i.e. LOQ, 25, 50, 75, 100, 125, 150, 175

and 200%) with respect to their specification limit (0.15%).

Calibration curve was drawn by plotting average area of the

impurity (impurity-1, impurity-2 and impurity-3) on the Y-axis and

concentration on the X-axis (Fig: 5.24, Fig: 5.25, Fig: 5.26) which

showed linear relation ship with a regression coefficient of greater than

0.999 for three impurities. At all concentration levels, standard

-

101

deviation of peak area was significantly low and RSD was below 2.0%.

Analysis of residuals indicated that the residuals were normally

distributed around the mean with uniform variance across all

concentrations (Fig: 5.27, Fig: 5.28, Fig: 5.29) suggesting the

homoscedastic nature of data. Selected linear model with univariant

regression showed minimum % bias indicating goodness of fit which

was further supported by the low standard error of estimate and mean

sum of residual squares.

Table: 5.16 Linearity study for related components

S.No% Level w.r.t

specification limit(i.e. 0.15%)

Impurity-1 (Average

peak area)

Impurity-2 (Average

peak area)

Impurity-3 (Average

peak area)

1 LOQ 1236 2580 5548

2 25 12676 13821 13575

3 50 25525 27678 27176

4 75 35149 39996 40910

5 100 50617 52924 54498

6 125 62386 66566 67340

7 150 75124 80695 81454

-

102

8 175 86101 92729 94339

9 200 100702 107094 107987

Correlation coefficient (r) 0.999 0.999 0.999

Slope 499.46 531.69 539.18

Intercept -127.335 334.53 235.97

% y-intercept -0.003 0.006 0.004

Fig: 5.24 Linearity plot for impurity-1

Linearity plot for impurity-1

y = 499.46x - 127.34

R2 = 0.9991

0

20000

40000

60000

80000

100000

120000

0 50 100 150 200

Concentration

Mea

n A

rea


-

103

Linearity plot for impurity-2

y = 531.69x + 334.53

R2 = 0.9998

2000

22000

42000

62000

82000

102000

0 50 100 150 200

Concentration

Mea

n Ar

ea


Linearity plot for Impurity-3

y = 539.17x + 235.96R2 = 0.99995

0

20000

40000

60000

80000

100000

0 50 100 150 200Concentration

Mea

n A

rea

Table: 5.17 Residual summary of Linearity results of impurity-1

-

104

%Con’cMean area response achieved

Response calculated thru trend

line equation

Residual (Response practical -response

theoretical)

Residual square

2.3 1236 1021 -215 46045

25 12676 12359 -317 100388

50 25525 24846 -679 461503

75 35149 37332 2183 4766188

100 50617 49819 -798 637347

125 62386 62305 -81 6535

150 75124 74792 -332 110450

175 86101 87278 1177 1385706

200 100702 99765 -937 878606


Trend line equation y = 499.46x - 127.34

Fig: 5.27 Residual plot for the Linearity results of impurity-1

Residual plot for impurity-1

-5061

-3061

-1061

939

2939

4939

0 2 4 6 8 10

Order of Residuals

Res

idu

als

-

105


Conc. in %

Mean area response achieved

Response calculated thru trend line

equation


theoretical)Residual square

4.8 2580 2886.6 307 94029

25 13821 13626.8 -194 37721

50 27678 26919.0 -759 576035

75 39996 40211.3 215 46345

100 52924 53503.5 580 335855

125 66566 66795.8 230 52799

150 80695 80088.0 -607 368413

175 92729 93380.3 651 424166

200 107094 106672.5 -421 177637


Trend line equation y = 531.69x + 334.53

Fig: 5.28 Residual plot for the Linearity results of impurity-2


-5292

-3292

-1292

708

2708

4708

0 2 4 6 8 10

Order of Residuals

Res

idu

als

-

106


Con’c in %

Mean area response achieved

Response calculated thru trend line equation


theoretical)

Residual square

10.1 5548 5681.6 134 17843

25 13575 13715.2 140 19659

50 27176 27194.5 18 341

75 40910 40673.7 -236 55833

100 54498 54153.0 -345 119053

125 67340 67632.2 292 85387

150 81454 81111.5 -343 117334

175 94339 94590.7 252 63358

200 107987 108070.0 83 6882


Trend line equation y = 539.17x + 235.96

Fig: 5.29 Residual plot for the linearity results of impurity-3


-5450

-3450

-1450

550

2550

4550

0 2 4 6 8 10

Order of Residuals

Res

idu

als

-

107

5.2.7.9 Accuracy:

The accuracy of an analytical procedure expresses the closeness of

agreement between the value which is accepted either as a conventional

true value or an accepted reference value and the value found which is

sometimes termed trueness.

5.2.7.9(a) Accuracy of the assay method:

Accuracy for assay study was established by injecting three

preparations (n=3) of test sample at 50, 100 and 150% of analyte

concentration (i.e.100µg mL-1). Each solution was injected once into

HPLC system and the mean peak area of Tolterodine peak was

calculated.

Assay (%w/w) of test solution was determined against three

injections of qualified Tolterodine tartrate standard (Table 5.20). The

method showed consistency and high absolute recovery at all three

concentration (50,100 and 150%) levels with mean absolute recovery

ranging from 98.3 to 100.3%. The obtained absolute recoveries were

normally distributed around the mean with uniform RSD values. The

method was found to be accurate with low % bias (< 1.0).

Table: 5.20 Assay recovery study for drug substance

S.No Concentration (%) Mean recovery (%)(n = 3)

RSD%

1 50 98.3 0.37

-

108

2 100 99.5 0.38

3 150 100.3 0.07

The method showed consistent and high absolute recoveries at all

three concentration (50,100 and 150%) levels in the placebo spiking

method with mean absolute recovery ranging from 99.2 to 100.7%

(Table 5.21). Placebo spiking method was indicated that the obtained

absolute recoveries were normally distributed around the mean with

uniform RSD across three concentration levels suggesting

homoscedastic nature of the data. Thus it can be summarized that

there was no significant interference of excipients and the method was

found to be accurate with low % of bias values (< 1.0). Recovery study

indicated that the method was suitable for determination of Tolterodine

tartrate from tablets.

Table: 5.21 Assay recovery study for drug product

S.No Concentration (%) Mean recovery (%)(n = 3)

RSD%

1 50 99.2 0.23

2 100 100.7 0.18

3 150 99.5 0.16

5.2.7.9(b) Accuracy study for related components:

-

109

Accuracy study for related components was established at 50, 100

and 150% of the impurities specification limit (0.15%).

Accuracy at 50% impurity specification level:

Test solution prepared in triplicate (n=3) with impurities (impurity-

1, impurity-2 and impurity-3) at 0.075% level with respect to analyte

concentration (i.e. 0.5 mg mL-1). Each solution was injected thrice into

HPLC. Mean % recovery of impurities calculated in the test solution

using the area of impurities standard at 0.15% level with respect to

analyte (Table: 5.22).

Table: 5.22 Recovery at 50% level

S.NoImpurity

Name

Drug substanceMean

Recovery (%)(n = 3 )

RSD%

Drug productMean


RSD%

1 Impurity-1 95.4 0.4 94.8 0.5

2 Impurity -2 99.1 0.6 96.3 0.2

3 Impurity -3 98.5 0.5 98.3 0.6




concentration (i.e. 0.5 mg mL-1). Each solution was injected once into

HPLC. Mean %recovery of impurities calculated in the test solution

using the area of impurities standard at 0.15% level with respect to

analyte (Table: 5.23).

-

110


S.NoImpurity

Name

Drug substanceMean


RSD%

Drug productMean


RSD%

1 Impurity -1 98.9 0.7 95.6 0.8

2 Impurity -2 94.5 0.8 94.5 0.4

3 Impurity -3 96.6 0.6 96.5 0.6




concentration (i.e. 0.5 mg mL-1). Each solution was injected once into

HPLC system. Mean %recovery of impurities calculated in the test

solution using the area of impurities standard at 0.15% level with

respect to analyte (Table: 5.24).


S.NoImpurity

Name

Drug substanceMean


RSD%

Drug productMean


RSD%

1 Impurity -1 100.9 0.3 100.2 0.8

2 Impurity -2 99.9 0.9 99.8 0.2

3 Impurity -3 103.0 0.9 98.8 0.6

The developed method showed consistent and high absolute

recoveries of three impurities at all three concentration (50,100 and

150%) levels in drug substance and the placebo spiking method with

-

111

mean absolute recovery ranging from 94.5 to 103.0% in drug substance

and 94.5 to 100.2% in drug product. Placebo spiking method indicated

that the obtained absolute recoveries were normally distributed around

the mean with uniform RSD across three concentration levels

suggesting homoscedastic nature of the data. Thus it can be

summarized that there was no significant interference of excipients and

the method was found to be accurate with low % of bias values (< 1.0).

Recovery study indicated that the developed method was suitable for

determination of impurities of Tolterodine tartrate from tablets.

5.2.7.10 Solution state stability and mobile phase stability:

The solution stability of Tolterodine tartrate in diluent (i.e.

acetonitrile and water in the ratio of 1:1 v/v) for the assay study was

carried out by leaving the test solutions of sample in tightly capped

volumetric flasks at room temperature for two days. The same sample

solution were assayed for every six hours interval up to the study

period, each time freshly prepared reference standard was used to

estimate the assay of sample. The RSD% of assay of Tolterodine

tartrate during solution stability experiments was with in 1.0.

The solution stability of Tolterodine tartrate and for its related

components was carried out by leaving sample solution in tightly

capped volumetric flask at room temperature for 48 hours. Content of

-

112

impurity-1, impurity-2 and impurity-3 were checked for every six hours

interval up to the study period. No significant change was observed in

the impurity content during solution stability experiments from the

initial values up to the study period. Hence Tolterodine tartrate sample

solution is stable for at least 48 hours in the developed method.

The mobile phase stability of the developed method for Tolterodine

tartrate and for its related components was checked by injecting fresh

sample solutions. Fresh sample solutions were assayed for every six

hours interval up to the study period, each time freshly prepared

reference standard was used to estimate the assay of sample. The RSD

% of assay of Tolterodine tartrate during solution stability experiments

was with in 1.0. Content of impurity-1, impurity-2 and impurity-3 was

checked for every six hours interval up to the study period of 48 hrs.

No significant change was observed in the impurity content during

mobile phase stability experiments from the initial values up to the

study period. Hence Tolterodine tartrate mobile phase is stable for at

least 48 hours in the developed method.

Table 5.25 Summarizes assay content obtained at different

interval, solution stability and mobile phase stability results of the

assay study

S.No IntervalSolution stability

Assay (%w/w)

Mobile phase stability

Assay (%w/w)

1 0h 99.8 99.9

2 6h 99.6 99.8

-

113

3 12h 99.8 99.6

4 18h 99.4 99.8

5 24h 99.2 99.6

6 30h 99.3 99.8

7 36h 99.6 99.6

8 42h 99.2 99.8

9 48h 99.7 99.9

RSD% 0.24 0.12

5.2.7.11 Robustness:

The robustness of an analytical procedure is a measure of its

capacity to remain unaffected by small, but deliberate variations in

method parameters and provides an indication of its reliability during

normal usage. To determine the robustness of the developed method

experimental conditions were purposely altered and the resolution

between impurity-1 and Tolterodine was evaluated. In each of the

deliberately altered chromatographic condition (flow rate 0.6 mL min-1

and 1.0 mL min-1, % of acetonitrile from 100% to 95% and 105% in the

mobile phase, buffer pH from 2.5 to 2.4 and 2.6, column temperature

from 27°C to 22 °C and 32°C) the resolution between impurity-1 and

Tolterodine tartrate was greater than 2.0, illustrating the robustness of

the method (Table: 5.26; Fig: 5.30-Fig: 5.33).

Table: 5.26 Results of robustness study

S.No Parameter VariationResolution (Rs) between

impurity-1 and Tolterodine peak1 Temperature(± 5°C (a) At 22°C 3.09

-

114

of set temperature) (b) At 32°C 3.162 Flow rate (± 20% of

the set flow)

(a) At 0.6 mL min -1

(b) At 1.0 mL min-1

3.16

3.10

3 pH (± 0. 1 unit of set

pH)

(a) At 2.4

(b) At 2.6

3.11

3.144 Organic ratio

(acetonitrile content)

(a) 95%

(b) 105%

3.10

3.171)

Fig: 5.30 Robustness study - Effect of change in temperature

3.163.173.163.143.09

1.5

2

2.5

3

3.5

4

4.5

5

21.0 23.0 25.0 27.0 29.0 31.0 33.0

Temperature of Column in°C

Reso

lutio

n

Fig: 5.31 Robustness study - Effect of change in flow rate

-

115

3.103.153.163.173.16

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0.5 0.6 0.7 0.8 0.9 1.0 1.1

Flow rate mL/min

Reso

lutio

n

Fig: 5.32 Robustness study- Effect of change in mobile phase

buffer pH

3.123.143.163.113.05

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

2.2 2.3 2.4 2.5 2.6 2.7 2.8

Buffer pH

Res

olut

ion

-

116

Fig: 5.33 Robustness study - Effect of change in Mobile phase

organic content composition

3.153.173.163.103.02

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

85 90 95 100 105 110 115% of Acetonitrile composition

Reso

lutio

n

5.2.7.12 Mass balance

Mass balance is a process of adding together the assay value and

the levels of degradation products to see how closely these add up to

100% of the initial value, with due consideration of the margin of

analytical error [10]. Its establishment hence is a regulatory

requirement. The mass balance is very closely linked to the

development of stability indicating assay method as it acts as an

approach to establish its validity. The stressed samples of Tolterodine

tartrate bulk drug were assayed against the qualified reference

standard and the results of mass balance obtained were very close to

-

117

99.4%. Mass balance study results were presented below (Table: 5.27

and Table: 5.28).

The stressed samples of Tolterodine tartrate tablets (Detrol, 2 mg) were

also checked for % assay against qualified reference standard and the

assay results obtained were satisfactory.

Table: 5.27 Mass balance studies

Forced degradation results under Conventional reflux method

S.No Stress condition Time% Assay of

active substance

Mass balance (% Assay+

% impurities+% degradants)

1Acid hydrolysis(1N HCl,80°C )

12h 99.1 99.6

2Base hydrolysis

(1N NaOH, 80°C )12h 64.7 99.5

3Oxidation (6% H202)

48h 99.0 99.7

4 Water hydrolysis (80°C) 12h 99.5 99.8

5 Thermal (60° C) 10 days 99.6 99.7

6Light (photolytic

degradation)10 days 99.7 99.8

Table 5.28 Microwave assisted forced degradation results

S.No Stress condition Time% Assay of

active substance

Mass balance (% Assay+

% impurities+% degradants)

1Acid hydrolysis

(1N HCl )5 min 99.2 99.5

2Base hydrolysis

(1N NaOH)5 min 67.0 99.4

-

118

3 Oxidation (6% H202) 5 min 99.0 99.4

4 Water hydrolysis 5 min 99.1 99.8

5.3 Analysis of Tolterodine tartrate drug substance stability

samples:

One manufacturing lot of Tolterodine tartrate drug substance was

placed for stability study in chambers maintained at ICH defined

conditions. Long term (25°C+ 2°C/ 60% RH + 5%RH) and accelerated

(40°C+ 2°C/ 75% RH + 5%RH) stability study was carried out for

Tolterodine tartrate bulk drug and the related components were

monitored in the stability samples using the developed HPLC method

conditions. The analysis of stability samples were carried up to 12

months period. The stability data results obtained are presented in

Table: 5.29 and Table: 5.30. The developed HPLC method performed

satisfactorily for the quantitative evaluation of stability samples.

-

119

Table 5.29 Accelerated stability data (storage conditions: 40°C /

75% RH)

Batch No : 01002 Packing and Storage : Each sample packed in a polyethylene bag in a triple laminated bag and kept in a HDPE drum.

Stability study duration: 3 months. Temperature 40 + 2°C Relative Humidity 75+ 5%

Duration Description IdentificationLoss on drying (%w/w)

Assay by HPLC(On dried basis)

Related Substance by HPLC

Imp-1 Imp-2 Imp-3 Total Imp

Specification White powderIRSpectrum should concord with the standard.

NMT 0.5% 98.0-102.0% w/w

NMT 0.15%

NMT 0.15%

NMT 0.15% NMT 0.5%

Initial White powder Complies 0.14% 99.84% 0.08% 0.02% 0.06% 0.28%1st month White powder Complies 0.14% 99.72% 0.09% 0.04% 0.07% 0.32%2nd month White powder Complies 0.15% 99.73% 0.08% 0.04% 0.06% 0.35%3rd month White powder Complies 0.15% 99.76% 0.09% 0.04% 0.07% 0.30%

-

120

Table 5.30 Long term stability data (storage conditions: 25°C /

60% RH)

Batch No : 01002 Packing and Storage : Each sample packed in a polyethylene bag in a triple laminated bag and kept in a HDPE drum.

Stability study duration: 12 months. Temperature 25 + 2°C Relative Humidity 60+ 5%

Duration Description IdentificationLoss on drying (%w/w)

Assay by HPLC(On dried basis)

Related Substance by HPLC

Imp-1 Imp-2 Imp-3 Total Imp

Specification White powderIRSpectrum should concord with the standard.

NMT 0.5% 98.0-102.0% w/w

NMT 0.15%

NMT 0.15%

NMT 0.15% NMT 0.5%

Initial White powder Complies 0.14% 99.84% 0.08% 0.02% 0.06% 0.28%1st month White powder Complies 0.10% 99.78% 0.06% 0.04% 0.04% 0.24%2nd month White powder Complies 0.12% 99.74% 0.08% 0.02% 0.05% 0.25%3rd month White powder Complies 0.12% 99.76% 0.07% 0.03% 0.05% 0.29%6th month White powder Complies 0.14% 99.80% 0.07% 0.03% 0.05% 0.28%12th month White powder Complies 0.12% 99.75% 0.07% 0.03% 0.06% 0.30%

-

121

5.4 Summary and Conclusion:

A novel liquid chromatographic method for the determination of

related components in Tolterodine tartrate was developed for

Tolterodine tartrate after subjecting the samples to stress testing under

ICH recommended conditions. The RP-LC method developed for

quantitative and related components determination of Tolterodine

tartrate is rapid, precise, accurate and selective. The method was

completely validated showing satisfactory data for all the method

validation parameters tested. The developed method was found

“specific” to the drug and for dosage form, as the peaks of the

degradation products did not interfere with the drug peak. Thus the

proposed method can be employed for assessing the stability of

Tolterodine tartrate as bulk drug and also for its dosage form.

-

122

Table: 5.31 Summary of analytical method validation

Testparameter

Related components study Assaystudy

Impurity-1 Impurity-2 Impurity-3 Tolterodine

Precision (RSD%) 0.37 0.69 0.35 0.16

LOD (µg mL-1) 0.0009 0.0018 0.0038 -NA-

LOQ (µg mL-1) 0.0035 0.0072 0.0151 -NA-

Precision at LOQ

(RSD %)3.1 2.0 1.2 -NA-

Linearity

(Corre coefficient)0.999 0.999 0.999 0.9999

Accuracy

% recovery

(Drug stbstance)

95.4-100.9 94.5-99.9 96.6-103.0 98.3-100.3

Accuracy

%recovery

(Drug product)

94.8-100.2 94.5-99.8 96.5-98.8 99.2-100.7

Solution

stability

Stable up to

48h

Stable up to

48h

Stable up to

48hStable up to 48 h

-

123

Mobile phase

stability

Stable up to

48h

Stable up to

48h

Stable up to

48h

Stable up to

48h

Robustness

Resolution between

impurity-1 and

Tolterodine is greater than

3.0

Resolution between

Tolterodine and impurity-2 greater than

5.0

Resolution between

impurity-2 and impurity-3 greater than

5.0

Resolution between

impurity-1 and Tolterodine

greater than 3.0

References:

1. Nilvebrant L, Glas G, Jonsson A, Sparf B. Neurourol Urodyn

13: 1994, pp-433–435.

2. Nilvebrant L, Stahl M, Andersson K-E . Neurourol Urodyn

14: 1995, pp-523–524.

3. Botteghi C, Corrias T, Maschetti M, Paganelli S, Piccolo O.

Org Process Res Dev 6: 2002, pp-379–383.

4. International Conference on Harmonization (2003) Stability testing

of new drug substances and products Q1A (R2). International

Conference on Harmonization, IFPMA, Geneva

5. Singh S, Bakshi M. Pharm Tech Online 24:2000, pp-1–14.

6. International Conference on Harmonization (1996) Photo stability

testing of new drug substances and products Q1B.

International Conference on Harmonization, IFPMA, Geneva

7. Bakshi M, Singh S. J Pharm Biomed Anal 28:2002, pp-1011–1040.

-

124

8. Singh S, Singh B, BahugunaR,WadhwaL, Saxena R. J Pharm

Biomed Anal 41:2006, pp-1037–1040.

9. Carstensen JT, Rhodes CT (eds) (2002) Drug stability principles and

practices, 3rd edn. Marcel Dekker, New York

10.Ravindra Kumar Y, Ramulu G, Vevakanand VV, Vaidyanathan G,

Keesari Srinivas, Kishore Kumar M, Mukkanti K, Satyanarayana

Reddy M, Venkatraman S, Suryanarayana MV J. Pharm

Biomed Anal 35:2004, pp-1279–1285.

11.Zhang B, Zhang Z, Tian Y, Xu F. J Chromatogr B 824: 2005, pp-

92–98.

12.Bjo¨ rkman HT, Edlund P-O, Jacobsson SP. Anal Chim Acta

468: 2002,pp-263–274.

13.Swart R, Koivisto P, Markides KE. J Chromatogr B 736: 1999, pp-

247–253

14.Palme´ r L, Andersson L, Andersson T, Stenberg U. J Pharm

Biomed Anal 16: 1997, pp-155–165.

15.Swart R, Koivisto P, Markides KE. J Chromatogr A 828: 1998,

pp-209–218.

16.J.J. Kirkland, C.H. Dilks, Jr., and J.J. DeStefano, L.Chromatogr.,

635 (1993) p-19.

-

125

5.1 introduction of tolterodine tartrate and survey of...

Documents