7.1 introduction of tadalafil and survey of analytical...

7.1 Introduction of Tadalafil and survey of analytical methods:

Tadalafil (Cialis®) (Fig: 7.1) is used in oral treatment for erectile

dysfunction. Tadalafil is chemically (6R, 12aR)-2,3,6,7,12,12a-hexahydro-

2-methyl-6-(3,4-methylene dioxyphenyl) pyrazino (1′, 2′: 1,6) pyrido- (3,4-

b) indole-1, 4-dione. Tadalafil is a white crystalline solid that melts at

approximately 301-302°C. It is practically insoluble in water and very

slightly soluble in ethanol. The empirical formula of tadalafil is C22H19N3O4.

The molecular weight of Tadalafil is 389.4.

Fig: 7.1 Chemical structure of Tadalafil

NH

N

N

O

O

O

O

Molecular formula: C22H19N3O4

Molecular weight : 389.4

(6R, 12aR)-2, 3, 6, 7, 12, 12a-hexahydro-2-methyl-6-(3, 4-methylene

dioxyphenyl) pyrazino (1′, 2′: 1, 6) pyrido- (3, 4-b) indole-1, 4-Dione

It is widely recognized that enantiomers have distinict biological

interactions and thus potentially different pharmacokinetic,

pharmacological and /or toxicological effects (1, 2). To assure patient

safety and clinical efficacy, the pharmacological evaluation of stereo

isomers is an integral part of new drug development (3, 4). Analytical

169

methods to determine the enantiomeric purity of new investigational drugs

are often attained through a series of generic or screening methodologies

(5-9). Although many analytical techniques can be employed to achieve

this, the most widely used is liquid chromatography (LC) employing a

chiral stationary phase (CSP) (10-15).

Tadalafil is used in oral treatment for erectile dysfunction, is a selective

inhibitor of cyclic guanosine monophosphate (cGMP)-specific

phosphodiesterase type-5 (PDE-5). Through the inhibition on PDE-5, (R,

R)-tadalafil increases the concentration of cyclic guanosine

monophosphate (cGMP), producing smooth muscle relaxation and

increased blood flow to the corpus cavernosum, thereby enhancing erectile

response following appropriate sexual stimulation (1).

Few analytical methods have been reported for the estimation of (R, R)-

tadalafil in formulation using HPLC (17-19) and capillary electrophoresis

with UV detection (20, 21). A high throughput validated analytical method

for the quantitation of (R, R) - tadalafil in human plasma using LC–MS-MS

has been published (22). So far there are no analytical methods available

for separation and quantification of the (R, R)-tadalafil and its enantiomer

in bulk drugs and in pharmaceutical dosage forms. The novelty of this

work is, first time (R, R)-tadalafil and its enantiomer was separated and

quantified in both drug substance and in drug product by HPLC. In this

chapter a brief description on development and validation of a novel LC

170

method for separation and quantitative estimation of (R, R)-tadalafil from

its enantiomer.

7.2 Development and validation of a novel chiral RP-LC method for

the separation of (R, R)-Tadalafil and its enantiomer:

7.2.1 Materials:

Samples of (R, R)-tadalafil and its enantiomer (Fig: 7.2) was obtained

from Dr.Reddy’s Laboratories Ltd, Hyderabad, India. The received samples

were certified and of purity greater than 99.5%. Commercially available

Cialis, 20mg Tadalafil tablets were purchased. HPLC grade acetonitrile

and methanol were purchased from Merck, Darmstadt, Germany. High

pure water was prepared by using millipore Milli-Q plus water purification

system.

7.2.2 Equipment:

The LC system used for method development, forced degradation

studies and method validation was Agilent 1100 series system (Agilent

Technologies, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, United

States) and Waters 2996 PDA system. (Waters Corporation, 34 Maple

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

processed using Empower software (Waters Corporation) on Pentium

computer (Digital equipment Co). The mass studies for Tadalafil and its

enantiomer were carried out on a Quattro LC-MS/MS (Micromass,

Manchester, UK) using Masslynx software.

171

Fig: 7.2 Chemical structure of the enantiomer of (R, R)-tadalafil

NH

N

N

O

O

O

O

Molecular formula: C22H19N3O4

Molecular weight : 389.4

(6S, 12aS)-2, 3, 6, 7, 12, 12a-hexahydro-2-methyl-6-(3, 4-methylene

dioxyphenyl) pyrazino (1′, 2′: 1, 6) pyrido- (3, 4-b) indole-1, 4-Dione.

7.2.3 Sample preparation:

Stock solutions of (R, R)-tadalafil and its enantiomer (400 µg mL-1) were

prepared individually by dissolving appropriate amount in the diluent

(mobile phase was used as diluent - the mobile phase contains a mixture

of water, methanol and acetonitrile in the ratio of 55:40:5 (v/v). A Stock

solution of sample and impurity mixture was prepared (400 µg mL-1) in

diluent.

7.2.4 Preparation of Tablets Sample Solution:

Twenty tablets were individually weighed to get the average weight of

the tablets and powdered in mortar. A sample of the powdered tablets,

equivalent to 40 mg of (R, R)-tadalafil was transferred to 100 mL

volumetric flask. About 75 mL of mobile phase was added and kept on

rotatory shaker for 10 min to disperse the material completely and

172

sonicated for 10 min and diluted to 100 mL. The content was centrifuged

for 10 min at 3,000 rpm. The supernatant solution was collected and

filtered using 0.45 µm nylon 66-membrane filter.

7.2.5 Method development and optimization of chromatographic

conditions:

7.2.5.1 Selection of wavelength:

(R, R)-tadalafil and its enantiomer solutions were prepared in diluent at

a concentration of 100 ppm and scanned in UV spectro photometer; both

(R, R)-tadalafil and its enantiomer were having UV maxima at around 220

nm (Fig: 7.3). Hence detection at 220 nm was selected for method

development purpose.

Fig: 7.3 Typical UV spectrums of (R, R)-tadalafil and its enantiomer

and overlaid spectrum of both (R, R)-tadalafil and its enantiomer

220.2

285.1

AU

0.00

0.20

0.40

0.60

220.2

285.1

AU

0.00

0.20

0.40

0.60

0.80

1.00

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00

Wavelength (nm)

173

Absorbance

UV spectrum of (R, R)-tadalafil

UV spectrum of enantiomer of (R, R)-tadalafil

220.2

284.0

323.2 337.5 356.6360.7 368.3 382.7 394.7

220.2

285.1

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00

Wavelength (nm)

7.2.5.2 Selection of column and mobile phase:

A mixture of (R, R)-tadalafil and its enantiomer was used during the

method development study. After a brief survey of existing analytical

methods it was thought to start the development activity initially on chiral

columns both in normal phase mode and reverse phase modes.

Accordingly, initial trials were carried in normal phase mode using

chiral columns. The selection of chiral columns was made based on

existing literature (1-7).

Different stationary phases were selected for method development

purpose. Chiralcel and Chiralpak columns were chosen for method

development purpose in normal phase mode. In these columns, the

stationary phase was derivatives of Polysaccharides such as cellulose and

amylase. These columns are suitable for mobile phase compositions

containing organic solvents and for normal phase type solvents.

174

Absorbance

Overlaid UV spectrum of both (R, R)-tadalafil & its enantiomer

1. Chiralcel OD-H (cellulose tris (3, 5-dimethylphenylcarbamate) on silica-

gel).

2. Chiralpak AD-H (amylose tris (3, 5-dimethylphenylcarbamate) on silica-

gel).

Prior to the analysis the entire HPLC system including the injector and

the injection loop was flushed with methanol, isopropyl alcohol, ethanol

and n-hexane solvent in series. As the HPLC system was equipped with

auto-sampler, this unit also flushed with above series of solvents. The

mobile phase chosen for initial method development purpose was a pre

sonicated mixture of n-hexane: isopropyl alcohol (90:10, v/v). The

chiralcel OD-H column was washed initially with n-hexane for about one

hour. After column flushing, column was equilibrated with the mobile

phase for one hour until a stable base line was achieved. No separation

was observed on chiralcel OD-H column. Another trial was carried on

polysaccharide type chiral column namely chiralpak AD-H. The above

column washing and stabilization steps were same as that of chiralcel OD-

H column.

The mobile phase chosen for initial method development purpose was a

pre sonicated mixture of n-hexane: isopropyl alcohol (90:10, v/v) and

there was no indication of separation. Elution of a single peak without any

resolution was observed. Different combinations of n-hexane, n-heptane

methanol, ethanol and isopropyl alcohol were tried in both the columns.

Resolution (Rs ~ 0.9) was observed on Chiral AD-H column with the

175

mobile phase composition of n-heptane: 2-Propanol: Ethanol: 50:30:20

(v/v/v). To improve resolution trifluoroacetic acid (TFA), triethylamine and

diethyl amine were introduced to the above mobile phase system and

trials were made. No fruitful results were obtained. Since the analysis on

normal phase columns is expensive and the life time of these columns is

short due to coated stationary phase and also these columns are

compatible to limited solvents only (limited solvent compatibility). Keeping

all these points in view gave up the further optimization trails on coated

type polysaccharide columns. Trails were taken in reverse phase to

minimize the cost of analysis.

To achieve separation between the enantiomers of Tadalafil a new

stationary phase which is feasible for amino acids separations was

selected, in which macrocyclic glycopeptides were linked through five

covalent bonds to a silica surface. CHIROBIOTIC-T (Fig: 7.4) was the

column selected for this purpose. In this column the macro cyclic

glycopeptide was teicoplanin. The separation mechanism in this column

was may be due to complex chiral environment, possibility for π - π

interactions, chiral hydrogen bonding sites, peptide binding sites and

multi-modal possibilities. This column was selected because of its unique

selectivity for underivatised α, β, γ or cyclic amino acids, N-derivatised

amino acids, alpha hydroxyl – carboxylic acids, acidic compounds

including carboxylic acids, phenols, neutral aromatic analytes and cyclic

aromatic and aliphatic amines. The column has a pH range from 3 to 7.

176

The chirobiotic T column used in this study was a previously used

column (not a brand new column) in reverse phase mode and stored in

15% ethanol solution. This column was chosen for method development

purpose in reverse phase mode since the column was compatible with

aqueous mobile phases with buffers containing a limited percentage of

organic modifiers. Initially the entire chromatographic system was flushed

with methanol and then with warm milli-Q water. Before starting the

analysis flushed the column with milli-Q water and then with ethanol for

30 min at a flow rate of 0.5 mL min-1. After completion of analysis the

column was flushed with ethanol and stored in 15% ethanol (15 mL

ethanol in 85 mL water).

Phosphate buffers were not selected for method development on

chirobiotic T, as per literature suggestion. Initial trial was made with a

degassed mixture of water: methanol (60:40 v/v), elution of a single peak

was observed; no separation was observed between the enantiomers.

Second trial was made with water: methanol (50:50, v/v) as mobile phase.

A resolution (Rs) of about 1.2 was observed between the enantiomer of (R,

R)-tadalafil and (R, R)-tadalafil, but tailing of (R, R) - tadalafil was greater

than 2. One more trial was made with water: methanol (55:45 v/v)

composition. A resolution (Rs) of about 2.5 was observed between the

enantiomer of (R, R)-tadalafil and (R, R)-tadalafil, but tailing of (R, R)-

tadalafil was greater than 1.6. Since resolution was obtained with this

composition, this composition (water: methanol (55:45 v/v)) and

177

CHIROBIOTIC-T (250x4.6) mm with 5µm particle size column was taken

as base and optimisation of the method was carried out.

Fig.7.4 Proposed Structures of CHIROBIOTIC-T

(Macrocyclic Glycopeptide Teicoplanin):

A, B, C, D are inclusion pockets (weak)

7.2.5.3 Optimisation of the method:

In the optimization of the method trials were made by changing the

organic ratio. To decrease the tailing factor (R, R)-tadalafil, acetonitrile was

introduced into the mobile phase. When water: methanol: acetonitrile

(50:45:30, v/v/v) was used as mobile phase, elution of a single peak was

observed, no separation was observed between enantiomers, this might be

due to the higher concentration of acetonitrile. (Here higher organic

modifier (acetonitrile) concentration reduced the retention and enantio

178

OH

NHR

CH

2OH

HO

O

HNCOCH

3

HO

O

NH

2

O

HO

Cl

HHO

H

O

HN

NH

O

H

B

A

OCl

N

O

N

HO

HHO

C

OHOH

O

NHH

NH

O

HOOC

HD

HO

OH

CH

2OHO

HOH

O

CH2OH

HO

HO

OH

selectivity). Further trials were carried with lower concentration of organic

modifier. When water: methanol: acetonitrile (50:40:10, v/v/v) was used

as mobile phase, a resolution (Rs) of about 1.7 was observed between (R,

R)-tadalafil and its enantiomer. Tailing factor of (R, R)-tadalafil was 1.0. To

further improve the resolution, acetonitrile content was decreased in the

mobile phase to 5 % from 10%. When water: methanol: acetonitrile

(55:40:5, v/v/v) was used as mobile phase satisfactory resolution between

(R, R)-tadalafil and its enantiomer (Rs ~2.4) and tailing factor of (R, R)-

tadalafil (~1.1) was observed (Fig: 7.5).

The stationary phase and % of organic phase (both methanol and

acetonitrile) found to be crucial in getting the separation between (R, R)-

tadalafil and its enantiomer. Acetonitrile played a major role in controlling

the tailing of (R, R) Tadalafil.

The interference of excipients (hydroxy propyl cellulose, sodium lauryl

sulphate, micro crystalline cellulose, lactose monohydrate, and

croscarmellose cellulose and magnesium sterate) was also checked by

injecting sample solutions of excipients. There was no interference of

excipients with (R,R) Tadalafil peak and its enantiomer.

179

Fig: 7.5 Typical chromatograms of (R, R)-tadalafil spiked with its

enantiomer at 0.15% level & racemic mixture of Mixture of

enantiomer of (R, R)-tadalafil & (R, R)-tadalafil

AU

-0.020

-0.015

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

SS

-Tad

alaf

il -

10.7

36

RR

-Tad

alaf

il -

12.7

66

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

SS

-Tad

alaf

il -

10.3

22

RR

-Tad

alaf

il - 1

2.47

6

X axis - Time in minutes Y axis - Absorbance AU

180

(b) Mixture of enantiomer of (R, R)-tadalafil & (R, R)-tadalafil

(a) 100% (R, R)-tadalafil + 0.15% its enantiomer Spiked

7.2.5.4 Optimized chromatographic conditions for the determination

of enantiomeric purity of Tadalafil:

Column : Chirobiotic-T, (250x4.6) mm with 5µm

particle size.

Mobile phase : Water, methanol and acetonitrile (55:40:5)

(v/v/v).

Flow rate : 1.0 mL min-1

Column temperature : 27 °C

Wavelength of detection : 220 nm (UV detection technique)

Injection volume : 20µL

Run time : 30 min

Diluent : Mobile Phase

7.2.5.5 Optimized chromatographic conditions for the determination

of molecular weights of Tadalafil and its enantiomer by LCMS:

Column : Chirobiotic-T, (250x4.6) mm with 5µm

particle size.

Mobile phase : Water, methanol and acetonitrile (55:40:5)

(v/v/v).

Flow rate : 1.0 mL min-1

Column temperature : 27 °C

Injection volume : 5µL

Run time : 20 min

Diluent : Mobile Phase

181

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 Lynx v 4.1 software (waters) was used to calculate the molecular

formulas of the de protonated molecules according to the accurate mass

data.

182

7.2.5.6: Mass spectral data of Tadalafil and its enantiomer:

Mass spectral data (Table: 7.1) was recorded using Electro spray

ionization technique (ES - technique) and the values obtained for (M-H)

confirm the molecular weights (M) of the (R, R)-tadalafil and Enantiomer of

(R, R)-tadalafil (Fig: 7.6-Fig: 7.7).

Table: 7.1 Mass spectral data of (R, R)-tadalafil and its enantiomer

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

(R, R)-tadalafil 389.4 388.3

Enantiomer of

(R, R)-tadalafil389.4 388.3

183

Fig: 7.6 Typical ES (-) Mass spectrum of (R, R)-tadalafil

184

Fig: 7.7Typical ES (-) Mass spectrum of

enantiomer of (R, R)-tadalafil

187

7.2.6 Validation of Analytical method 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 [23, 24].

7.2.6.1 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 (8).

To the (R, R)-tadalafil standard and enantiomer of (R, R)-tadalafil was

spiked at 0.15% level with respect to the concentration of (R, R)-tadalafil

and injected for five times into HPLC system. Resolution between

enantiomer of (R, R)-tadalafil and (R, R)-tadalafil, tailing factor, theoretical

plates for enantiomer of (R, R)-tadalafil and (R, R)-tadalafil, RSD% for the

areas of enantiomer of (R, R)-tadalafil and (R, R)-tadalafil was calculated.

System suitability results were tabulated (Table: 7.2 and Fig: 7.8).

188

Fig: 7.8 Blank, (R, R)-tadalafil Sample and system suitability

chromatograms

AU

-0.020

-0.015

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

189

(a) Blank

(b) (R, R)-tadalafil sample

(c) (R, R)-tadalafil spiked with 0.15% of its enantiomer

AU

-0.020

-0.015

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

RR

-Tad

alaf

il - 1

2.54

4

AU

-0.020

-0.015

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

SS

-Tada

lafil

- 1

0.736

RR

-Tada

lafil -

12.

766


Table: 7.2 System suitability results

190

AU

-0.020

-0.015

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

RR

-Tad

alaf

il - 1

2.54

4

AU

-0.020

-0.015

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

SS

-Tada

lafil

- 1

0.736

RR

-Tada

lafil -

12.

766


Table: 7.2 System suitability results

191

Name Retention time in

min

Resolution (RS )

Tailing factor (T)

No. of Theoretical plates (N)

RSD% forfor area

Enantiomer of (R, R)-tadalafil 10.7 - 1.06 12586 0.4

(R, R)-tadalafil 12.7 2.45 1.01 13427 0.6

7.2.6.2 Limit of quantification (LOQ) and limit of detection (LOD):

LOQ and LOD was established for enantiomer of (R, R)-tadalafil based

on signal to noise ratio method.

(a) Limit of quantification (LOQ):

The limit of quantitation (LOQ) of an 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.

To establish limit of quantification for enantiomer of (R, R)-tadalafil, 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: 7.3).

Table: 7.3 LOQ value of the Enantiomer of (R, R)-tadalafil

192

S.No Impurity name Concentration Signal / Noise ratio

1 Enantiomer of (R, R)-tadalafil 0.032 µg mL-1 10.2

(b) 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.

To establish limit of detection for enantiomer of (R, R)-tadalafil 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. The concentration where the S/N was about 3

was chosen as limit of detection (LOD) value (Table: 7.4).

Table: 7.4 LOD value of the enantiomer of (R, R)-tadalafil

S.No Impurity name Concentration Signal / Noise ratio

1 Enantiomer of(R, R)-tadalafil 0.013 µg mL-1 2.7

7.2.6.3 Precision at Limit of Quantification level:

193

The precision at limit of quantification level was checked by injecting

six individual preparations of enantiomer of (R, R)-tadalafil at its LOQ level

with respect to test concentration (i.e. 400 µg mL-1). The RSD % for area of

enantiomer of (R, R)-tadalafil for six consecutive determinations was 4.5

(Table: 7.5).

Table: 7.5 Precision results of enantiomer of (R, R)-tadalafil at LOQ

level.

Preparation Area of enantiomer of (R, R)-tadalafil

1 1579

2 1542

3 1406

4 1584

5 1511

6 1478

Average 1516.7

SD 67.6

RSD% 4.5

95% Confidence interval of mean {1462.6,1570.8}

7.2.6.4 Accuracy at Limit of Quantification level:

Standard addition and recovery experiments were conducted to

determine accuracy of the developed method for the quantification of

enantiomer of (R, R)-tadalafil in [R, R)-tadalafil sample at LOQ level.

The recovery study for enantiomer of (R, R)-tadalafil was carried out in

triplicate at LOQ level of the (R, R)-tadalafil target analyte concentration

(400 µg mL-1). Prepared three different solutions containing enantiomer of

194

(R, R)-tadalafil at the limit of quantification level and injected each

solution once. Prepared the sample solution for three times from the same

homogeneous sample (at the analyte concentration i.e. 400 µg mL-1) and

injected each solution once. Prepared three different sample solutions

containing enantiomer of (R, R)-tadalafil at the limit of quantification level

and injected each solution once, calculated % Recovery.

The percentage recovery of enantiomer of (R, R)-tadalafil was calculated

(Table: 7.6). The method showed consistent and high absolute recovery at

LOQ level with a mean absolute recovery of 100.2% for drug substance

and 100.8 for drug product. The obtained absolute recovery was normally

distributed around the mean with uniform RSD values. The method was

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

Table: 7.6 Recovery at LOQ level

S.No Impurity nameDrug Substance

Mean recovery (%)(n = 3 )

Drug ProductMean recovery (%)

(n = 3 ) RSD%

1 Enantiomer of(R, R)-tadalafil 100.2 100.8 0.8

7.2.6.5 Precision:

The precision of an analytical procedure expresses the closeness of

agreement (degree of scatter) between a series of measurements obtained

195

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 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).

The precision of the developed method was evaluated initially by

performing system precision (Table: 7.7), then by injecting six individual

preparations of (R, R)-tadalafil (400 µg mL-1) spiked with 0.15% of

enantiomer of (R, R)-tadalafil with respect to Nateglinide analyte

concentration. The RSD% for % of enantiomer of (R, R)-tadalafil, for six

consecutive determinations was respectively as below (Table: 7.8).

Table: 7.7 Results of System precision study

196

Table: 7.8

Results of precision study

S.No Area of (R, R)-tadalafil peak

Injection-1 19851236

Injection-2 19754982

Injection -3 19646981




Mean 19697867

SD 92586.0

RSD % 0.47

Preparation Retention time % enantiomer of(R, R)-tadalafil

1 10.736 0.148

2 10.736 0.150

3 10..735 0.149

4 10.736 0.151

5 10.736 0.149

6 10.735 0.152

Average 10.736 0.150

SD 0.0004 0.0013

RSD% 0.004 0.897

95% Confidence interval of mean {10.735,10.736} {0.149,0.151}

197

Results showed insignificant variation in measured response which

demonstrated that the developed method was repeatable with RSD s below

1.5%.

Intermediate precision study was performed by injecting six

individual preparations of (R, R)-tadalafil (400 µg mL-1) spiked with 0.15%

of % enantiomer of (R, R)-tadalafil with respect to (R, R)-tadalafil 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: 7.9).

Table: 7.9 Results of Intermediate precision and reproducibility

S.No Parameter Variation

RSD % for% enantiomer of (R, R)-tadalafil

Resolutionbetween

enantiomer of(R, R)-tadalafil &(R, R)-tadalafil

198

1 Different System

(a) Waters 2695Alliance system(b) Agilent 1100

series VWD system

1.21.1

2.42.4

2 Different Column

B.No (a): US10017459

B.No (b): US10017562

1.21.0

2.42.5

3 Different Analyst

(a) Analyst-1(b) Analyst-2

1.20.9

2.42.4

4 Different Laboratories

(a) Lab-1(b) Lab-2

1.20.8

2.42.4

7.2.6.6 Linearity:

The linearity of an analytical method is its ability (within a given range)

to obtain results, which are directly proportional to the concentration

(amount) of analyte in the sample (8). Linearity experiments were carried

out by preparing the (R, R)-tadalafil sample solution containing

enantiomer of (R, R)-tadalafil from LOQ to 200% ((LOQ), 0.015, 0.0375,

0.075, 0.1125, 0.15, 0.1875, 0.225 and 0.3%) with respect to their

specification limit (0.15%). Each solution was injected thrice (n=3) into

HPLC and calculated the average area at each concentration.

Calibration curve was drawn by plotting average area of the

enantiomer of (R, R)-tadalafil on Y-axis and concentration on X-axis (Fig:

7.9) which showed linear relation ship with a regression coefficient of

199

greater than 0.999 for enantiomer of (R, R)-tadalafil (Table: 7.10). At all

concentration levels, standard deviation for 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: 7.10) 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: 7.10 Results of the linearity experiments

% Level w.r.t specification limit(i.e. 0.15%)

Enantiomer of (R, R)-tadalafil (Average peak Area)

5.33 1623

10 3200

25 7869

50 15698

75 24125

100 32025

125 39856

150 48265

200 64236

Correlation Coefficient (r) 0.9999

Slope 321.74

Intercept -143.85

200

% Y-intercept -0.004

Fig 7.9: Linearity graph for enantiomer of (R, R)-tadalafil

Linearity plot for Enantiomer of (R, R)-tadalafil

y = 321.737x - 143.846R2 = 0.9999

500

10500

20500

30500

40500

50500

60500

70500

0 50 100 150 200 250

% Concentration

Ave

rage

Are

a

Fig: 7.10 Residual plot for the enantiomer of (R, R)-tadalafil

Residual plot for Enantiomer of (R, R)-tadalafil

-3202.5

-2202.5

-1202.5

-202.5

797.5

1797.5

2797.5

0 2 4 6 8 10

Order of Residuals

Resi

dual

s

Table: 7.11 Residual summary of enantiomer of (R, R)-tadalafil

201

Con (%)Mean area response achieved

Response calculated thru

Trend line equation

Residual (Response practical -Response

theoretical)

Residual square

5.33 1623 1571.0 -52.0 2701.5

10 3200 3073.6 -126.5 15989.6

25 7869 7899.7 30.6 939.4

50 15698 15943.2 245.2 60098.5

75 24125 23986.7 -138.3 19140.7

100 32025 32030.2 5.2 26.5

125 39856 40073.7 217.7 47371.5

150 48265 48117.2 -147.8 21859.6

200 64236 64204.2 -31.8 1014.4

Residual sum of squares 169141.84

Trend line equation y = 321.737x - 143.846

7.2.6.7 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. Standard

addition and recovery experiments were conducted to determine accuracy

of the present method for the quantification of enantiomer of (R, R)-

tadalafil in bulk drug samples and drug product of (R, R)-tadalafil.

Accuracy of the developed method was established at 50, 75, 100 and

150% of the enantiomer of (R, R)-tadalafil specification limit (0.15%) with

respect to analyte concentration (400 µg mL-1).

Quantification of enantiomer of (R, R)-tadalafil:

202

Prepared the sample solution three times with respect to analyte

concentration (400 µg mL-1) and injected into the chromatographic system.

The enantiomer of (R, R)-tadalafil is absent in both bulk sample and drug

product.

a. Accuracy at 50% impurity specification level:

Prepared test solution in triplicate (n=3) containing enantiomer of (R,

R)-tadalafil at 0.075% level to the limit of enantiomer of (R, R)-tadalafil

with respect to (R, R)-tadalafil concentration (400 µg mL-1). Each solution

was injected once into chromatographic system. % Mean recovery of

enantiomer of (R, R)-tadalafil was calculated from this solution using the

area of impurity standard at 0.15% level with respect to analyte (Table:

7.12).

Table: 7.12 Recovery at 50% level

S.No Impurity NameDrug substance

Mean recovery (%)(n = 3 ) RSD%

Drug productMean recovery (%)

(n = 3 ) RSD%

1 enantiomer of(R, R)-tadalafil 98.0 0.7 97.8 0.9

b. Accuracy at 75% impurity specification level:




was injected once into chromatographic system. %Mean recovery of


203


7.13).





(n = 3 ) RSD%


c. Accuracy at 100% impurity specification level:


R)-tadalafil at 0.15% level to the limit of enantiomer of (R, R)-tadalafil with

respect to (R, R)-tadalafil concentration (400 µg mL-1). Each solution was

injected once into chromatographic system. %Mean recovery of



7.14).





(n = 3 ) RSD%


d. Accuracy at 150% impurity specification level:



204


was injected once into chromatographic system. %Mean recovery of



7.15).





(n = 3 ) RSD%


The mean absolute recovery of enantiomer of (R, R)-tadalafil in drug

substance was ranged from 98.0 to 100.8%. The mean absolute recovery

of enantiomer of (R, R)-tadalafil in drug product of (R, R)-tadalafil was

ranged from 97.8% to 102.5%. This recovery study indicated that the

method was suitable for determination of enantiomer of (R, R)-tadalafil in

drug substance and drug product.

7.2.6.8 Solution stability and mobile phase stability:

The solution stability for enantiomer of (R, R)-tadalafil was carried out

by leaving both unspiked and spiked sample solution in tightly capped

volumetric flask at room temperature on a laboratory bench for 48h.

Content of enantiomer of (R, R)-tadalafil was determined for every 6h

interval and compared with freshly prepared solution at each time point.

205

Mobile phase stability was also carried out for 48h by injecting the

freshly prepared sample solutions for every 6h interval. Content of

enantiomer of (R, R)-tadalafil was checked in the test solutions. Mobile

phase prepared was kept constant during the study period.

No significant change was observed in the content of enantiomer of (R,

R)-tadalafil during solution stability and mobile phase stability

experiments. The solution stability and mobile phase stability experiments

data confirms that sample solutions prepared in diluent and mobile phase

used during the study was stable up to the study period of 48h.

7.2.6.9 Robustness:

The capability of the method to remain unaffected by small but

deliberate variations in the method parameters was study in order to

anticipate the problems, which may arise during the regular application of

the developed method (23). To determine the robustness of the developed

method, experimental conditions were deliberately altered and the

resolution between enantiomer of (R, R)-tadalafil and (R, R)-tadalafil was

evaluated.

a. Flow rate:

The flow rate of the mobile phase was 1.0 mL min -1 . To study the effect

of flow rate on the resolution 0.2 units of flow changed from 1.0 mL min-1

(i.e. 0.8 mL min-1 to 1.2 mL min-1 and Fig 7.11).

206

b. Column Temperature:

The effect of change of column temperature on resolution was studied

at 22°C to 32°C instead of 27°C, while the other mobile phase components

were held constant as stated in chromatographic conditions (Fig 7.12).

c. Mobile phase composition:

The effect of change in percent of methanol on resolution was studied

by varying from -10% to +10% of 40% of methanol (i.e. 36 % to 44% of

total volume), while the rest composition was held constant as stated in

chromatographic conditions (Fig 7.13).

In all these conditions (flow rate, column temperature, mobile phase

composition the resolution between the enantiomer of (R, R)-tadalafil and

(R, R)-tadalafil was always greater than 2.2 illustrating the robustness of

the method (Table: 7.16).

Table: 7.16 Robustness results of the method

S.No Parameter VariationResolution between

enantiomer of (R, R)-tadalafil &[R, R)-tadalafil

1 Temperature (a) At 22°C(b) At 32°C

2.312.43

2 Flow rate (a)At 0.8 mL min-1

(b)At 1.2 mL min-12.362.21

3 %Methanol (a) At 90%(b) At 110%

2.412.22

Fig: 7.11 Robustness study - Effect of Temperature

207

2.432.31 2.39

2.44 2.42

1.0

1.5

2.0

2.5

3.0

3.5

4.0

21.0 23.0 25.0 27.0 29.0 31.0 33.0

Temperature of Column in°C

Res

olut

ion

Fig: 7.12 Robustness study - Effect of Flow

2.212.312.442.412.36

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0.7 0.8 0.9 1.0 1.1 1.2 1.3

Flow rate mL/min

Res

olut

ion

Fig: 7.13 Robustness study - Effect of Mobile phase composition

208

2.222.312.41 2.43

2.44

1.0

1.5

2.0

2.5

3.0

3.5

4.0

85 90 95 100 105 110 115

Mobile phase composition (%Methanol)

Reso

lutio

n

7.3 Stability study of (R, R)-tadalafil bulk drug as per ICH Conditions

(Q1AR2):

One manufacturing lot of (R, R)-tadalafil 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 (R, R)-tadalafil bulk drug and

content of enantiomer of (R, R)-tadalafil was monitored in the stability

samples using the developed HPLC method conditions. The analysis of

stability samples was carried up to 12 months period. The enantiomer of

(R, R)-tadalafil was absent in drug substance sample considered for

stability studies. The enantiomer of (R, R)-tadalafil was not observed in

both long term and accelerated stability conditions. The developed HPLC

209

method performed satisfactorily for the quantitative evaluation of stability

samples.

7.4 Summary and conclusions of the present study:

A new chiral RPLC method was developed for chiral separation of (R,

R)-tadalafil and its enantiomer in drug substance and drug product under

ICH recommended conditions. (R, R)-tadalafil and its enantiomer was

baseline resolved (Rs > 2.2) on Chirobiotic-T column. The method is found

to be linear from LOQ to 200%. The method is precise, accurate, rugged

and robust. Both (R, R)-tadalafil and its enantiomer was stable in sample

solution for a study period of 48h. Mobile phase was stable for a study

period of 48 h. The method was completely validated showing satisfactory

data for all the method validation parameters tested (Table 7.18). Thus the

developed method can be employed for the quantitative determination of

(R, R)-tadalafil and enantiomer of (R, R)-tadalafil in drug substance, drug

product and in-process materials.

Table: 7.17 Summary of the Validation results

Validation parameter Enantiomer of (R,R)-tadalafil

Precision (RSD %)

Retention timeArea

0.0040.89

210

LOD-LOQ

Limit of detection (µg mL-1)Limit of quantification (µg mL-1)

Precision at LOQ (RSD %)Accuracy at LOQ (%Recovery)

Drug substanceDrug product

0.0130.0324.5

100.2100.6

Linearity

Calibration range (%)Calibration points

Correlation coefficientCalibration equation

Slopeintercept

5.33-2009

0.9999321.737x-143.85

321.74-143.85

Accuracy (%Recovery)

Drug substanceDrug product

98.0-100.897.8-102.5

Robustness Resolution betweenenantiomer of (R, R)-tadalafil and[R, R)-tadalafil is greater than 2.2

Solution Stability/ Mobile phase Stability

Stable up to 48h

References:

1. Ariens EJ, Wuis EW, Roy J. Coll Phys Lond 28, 1994, pp395–398

2. Maier NM, Franco P, Lindner W. J Chromatogr A 906:2001, pp-3–33

3. De Camp WH Chirality 1: 1989, pp-2–6

4. De Camp WH. J Pharm Biomed Anal 11, 1993, pp-1167–1172

5. Matthijs N, Perrin C, Maftouh M, Massart DL, Heyden YY. J

Chromatogr A 1041, 2004, pp-119–133

6. Matthijs N, Perrin C, Maftouh M, Massart DL, Vander Heyden Y

J Pharm Biomed Anal 27, 2002, pp-515–529

211

7. Perrin C, Matthijs N, Mangelings D, Granier-Loyaux C, Maftouh M,

Massart DL, Vander Heyden Y. J Chromatogr A 966, 2002, pp-119–

134

8. Perrin C, Vu VA, Matthijs N, Maftouh M, Massart DL, Vander Heyden

Y. J Chromatogr A 947, 2002, pp-69–83.

9. Anderson ME, Aslan D, Clarke A, Roeraade J, Hagman G. J

Chromatogr A 1005, 2003, pp-83–101.

10. Roussel C, Del Rio A, Pierrot-Sanders J, Piras P, Vanthuyne N

J Chromatogr A, 1037, 2004, pp-311–328

11. Okamoto M. J Pharm Biomed Anal 27, 2002, pp-401–407

12. Gasparrini F, Misiti D, Villani C. J Chromatogr A 906, 2001. pp-

35–50

13. Franco P,Senso A, Oliveros L, Minguillon C. J Chromatogr A

906, 2001, pp-155–170

14. Felix G. J Chromatogram A 906, 2001, pp-171–184

15. Ward TJ, Hamberg DM. Anal Chem 76, 2004, pp-4635

16. http://www.cialis.com, NDA 21-468 (2003) 3

17. Aboul-Enein HY, Ali I. Talanta 65, 2005, pp-276

18. Shakya AK, Abu-awwad AN, Arafat TA, Melhim M

J Chromatogr B 852, 2007, pp-403–408

19. Cheng CL, Chou CH. J Chromatogr B, 822, 2005, pp-278

20. Ali I, Aboul-Enein HY. Chromatographia 60, 2004, pp-187

21. Rodrı´guez Flores J, Berzas Nevado JJ, Castanˇ eda Penˇ alvo G,

212

Mora Diez N .J Chromatogr B, 2004, pp-811:231

22. Ramakrishna NVS, Vishwottam KN, Puran S, Koteshwara M, Manoj

S, Santosh M, Chidambara J, Wishu S, Sumatha B

J Chromatogr B 809, 2004, pp-243

23. ICH Draft Guidelines on Validation of Analytical Procedures (1995)

Definitions and Terminology, Federal Register, vol 60, IFPMA,

Switzerland, 11260

24. Validation of compendial methods (2007) The United States

Pharmacopeia, 30th edn. USP30

213

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