new hplc methods for the determination...

CHAPTER-III

NEW HPLC METHODS FOR THE

DETERMINATION OF DAPIPRAZOLE

HYDROCHLORIDE IN BULK AND RELATED

SUBSTANCES

PART-A

New HPLC method for the

determination of Dapiprazole

hydrochloride in bulk drug

3.1.1 INTRODUCTION

3.1.2 LITERATURE SURVEY

3.1.3 EXPERIMENTAL

3.1.4 RESULTS AND DISCUSSIONS

3.1.5 CONCLUSION

Dapiprazole hydrochloride Chapter – III Part A

102

3.1.1 INTRODUCTION

Dapiprazole hydrochloride is an alpha-adrenergic blocking agent1. It produces

miosis by blocking the alpha-adrenergic receptors on the dilator muscle of the iris

without significant action on ciliary muscle contraction, thus there are no changes

indepth of the anterior chamber thickness of the lens 2,3

. Dapiprazole hydrochloride

does not alter the intra-ocular pressure in normal eyes or in eyes with elevated intra

ocular pressure. The structural features of Dapiprazole hydrochloride is shown in the

Table 3.1.1 and physic chemical properties are shown in the Table 3.1.2.

Table 3.1.1 Structural features of Dapiprazope hydrochloride

Official name IUPAC name(s) Structure

Dapiprazole

hydrochloride

5,6,7,8-tetrahydro-3-(2-(4-o-tolyl-1-

piperazinyl)ethyl)-s-triazolo(4,3-

a)pyridine hydrochloride. (Or)

5,6,7,8-tetrahydro-3-(2-(4-(2-methyl

phenyl)-1-piperazinyl)ethyl)-1,2,4-

triazolo(4,3-a)pyridine

hydrochloride.

N

N

N N

N

HCl

Table 3.1.2 Physico-chemical properties of dapiprazole hydrochloride

Molecular formula C19H27N5·HCl

Molecular weight 361.93

Color/Physical state White to off white, amorphous powder

Solubility Freely soluble in water, methanol and in acetonitrile.

CAS Number 72822-12-9


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Mechanism of action: Dapiprazole hydrochloride acts through blocking the alpha-

adrenergic receptors in smooth muscle. Dapiprazole hydrochloride has demonstrated

safe and rapid reversal of mydriasis produced by Phenylephrine and a lesser degree to

Tropicamide 4,5

. In patients with decreased accommodative amplitude due to treatment

with Tropicamide the miotic effect of Dapiprazole hydrochloride may partially increase

the accommodative amplitude.

Dosage and administration: Two drops followed by an additional 2 drops after 5

minutes administered topically to the conjunctiva of each eye after the ophthalmic

examination to reverse the diagnostic mydriasis. Dapiprazole hydrochloride ophthalmic

solution should not be used in the same patient more frequently than once per week.

Indications: Dapiprazole hydrochloride is indicated in the treatment of iatrogenically

induced mydriasis produced by adrenergic (phenylephrine) or parasympatholytic

(tropicamide) agents. Dapiprazole hydrochloride is not indicated for the reduction of

intraocular pressure or in the treatment of open angle glaucoma. The pharmaceutical

formulations of Dapiprazole hydrochloride is shown in the Table 3.1.3.

Table 3.1.3 Pharmaceutical formulations of Dapiprazole hydrochloride

S.No Proprietary

name

Company Formulations

1

Rev-eyes®

Bosch and

Lomb

Ophthalmic eyedrops, 0.5% is supplied in a

kit consisting of one vial of Dapiprazole

hydrochloride (25 mg), one vial of diluents (5

mL) and one dropper for dispensing.


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Monti et al6 have studied the in vitro permeation rate of Dapiprazole

hydrochloride base (DAP-B) through hairless mouse skin as a preliminary step towards

the development of a transdermal therapeutic system. The study involved the evaluation

of the permeability coefficient of the drug applied to the skin in a series of liquid and

semi solid vehicles both in the absence and in the presence of different penetration

enhancers. Orna Geyer et al7

have reported the additive miotic effects of Dapiprazole

hydrochloride and pilocarpine. This study includes the additive miotic actions of

pilocarpine 2% and Dapiprazole hydrochloride 0.5% were evaluated by comparing the

effects of two drugs given together and alone on the reversal of mydriasis induced by

tropicamide (0.5%) and phenylephrine (10%) in one eye each of 60 healthy

volunteers. Sussanne et al8 have compared the Brimonidine versus Dapiprazole

hydrochloride influence on pupil size at various illumination levels. In randomized

prospective study, 19 healthy volunteers received 2 ophthalmic solutions, Dapiprazole

hydrochloride and Brimonidine one in each eye for intra-individual comparison.

Motivation for the method development

The primary purpose of this research work is to develop and validate a new,

simple, precise and accurate RP-HPLC method for determination of Dapiprazole

hydrochloride in the bulk sample.

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3.1.3. EXPERIMENTAL

Apparatus/Instruments

Quantitative validation was performed on Schimadzu liquid chromatograph,

with UV detector module equipped with automatic injector with an injection volume of

20µL and 2693 model pump. The HPLC system was equipped with LC software. Elico,

model LI 120; pH meter and Thermo-hypersil ODS column (250x4.6 mm i.d; particle

size 5 μm) were used.

Chemicals and Reagents: Dapiprazole hydrochloride reference sample is obtained as

a gift sample from M/s Bioleo Analytical Labs India Pvt. Ltd, Hyderabad. Methanol and

acetonitrile HPLC grade (Qualigens), water HPLC grade (Milli-Q) and orho-Phosphoric

acid (Rankem) AR grade were used in present study.

Method Optimization

Determination of the optimum wave length by spectrophotometer: The UV

spectrum of Dapiprazole hydrochloride in water indicates the maximum absorbance at

212nm. For the RP-HPLC analysis, 243 nm was found to be highly suitable wavelength

with a maximum detector response.

Effect of mobile phase composition on retention time: The optimum composition of

mobile phase was determined by comparing the influence of different binary mixtures

such as acetonitrile- methanol and acetonitrile-water and methanol-water in different

proportions. Finally ternary mixtures of methanol, acetonitrile and ortho-phosphoric


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acid in the ratio of 89: 9: 1 (v/v/v) at pH 5.8 has been used for the estimation of

Dapiprazole hydrochloride in bulk drug.

Choice of the stationary phase: To explore the possibility of better separation cyano

(CN) column was tested with the above mobile phase The retention times were long

with polar columns, retention of the analytes on the CN columns was much weaker

resulting in an unacceptable k values (<1) for Dapiprazole hydrochloride. In other

instances, the applicability of C3 silica column was also failed to get proper peak shape

with adequate USP theoretical plates for the analyte of interest. The results with

Thermo-hypersil ODS C-18 column was the best choice because it resulted in

acceptable k values (k > 1), less peak tailing and greater theoretical plate numbers.

Flow rate: Flow rate of the mobile phase was tested from 0.8 – 1.5 mL/min for

optimum separation and it was found that 0.8 mL/min flow rate was ideal for the

successful elution of the analyte.

Chromatographic conditions

Column : Thermo-hypersil ODS C-18 column (250 x4.6 mm, 5 μ).

Mobile phase : Methanol:acetonitrile:o-phosphoricacid 89: 9: 1 (v/v/v) at pH 5.8

Flow rate : 0.8 mL/min.

Run time : 8 min

Temperature : Ambient.

Injection volume : 20 μL

Detection wavelength : 243 nm

Retention time : 3.725min


107

Preparation of Mobile phase: The contents of the mobile phase were methanol,

acetonitrile and ortho-phosphoric acid in the ratio of 89: 9: 1 (v/v/v) at pH 5.8. They

were filtered before use through a 0.45 μm membrane filter and degassed by sonication.

Preparation of standard drug solution: A standard stock solution of the drug was

prepared by dissolving 10 mg of Dapiprazole hydrochloride in 10 mL volumetric flask

containing 5 mL of methanol, sonicated for about 15 min and then made upto 10 mL

with mobile phase to obtained drug solution of 1000 µg/mL.

Working standard solution

The primary standard solution was further diluted by taking 2 mL of the stock

solution in 25 mL volumetric flask and then made upto the mark with mobile phase to

get the concentration of 80 µg/mL.


Method validation

The parameters used to validate the method for the estimation of Dapiprazole

hydrochloride in bulk sample were specificity, precision, accuracy, linearity, robustness.

System suitability

The system suitability tests were carried out on freshly prepared standard

stock solution of Dapiprazole hydrochloride. The system was suitable for use, the

tailing factor for Dapiprazole hydrochloride is 1.15 and USP theoretical plates were

found to be significantly high around 21505.


108

Specificity

The specificity of the Dapiprazole hydrochloride is determined by taking the

80 µg/mLof standard and bulk drug synthesized in house. Chromatograms of reference

standard and bulk drug are shown in the Figure 3.1.1

. Fig. 3.1.1 Specificity chromatograms of Dapiprazole hydrochloride

Precision

The HPLC system was set to equilibrate and then injected 20 µl of 80

µg/mL concentration of Dapiprazole hydrochloride standard solution six times and

recorded the response (peak area). The precision was repeated with the in-house

synthesized sample of same concentration and the results are presented in Table 3.1.4

and Table 3.1.5 respectively.


109

Table 3.1.4 Precision of standard drug with statistics

Injection

No.

Name of the drug

& conc. (80µg/mL)

Retention

time in min

Peak area

1 Dapiprazole hydrochloride 3.724 153345






Mean 3.724 153345

% RSD. 0.9999 0.583

Table 3.1.5 Precision values of sample solution (Bulk drug)

Injection

No.

Name of the drug & conc.

(80µg/mL).

Retention

time in min.

Peak area







Mean 3.724 154678

% RSD. 0.9999 0.566


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Linearity

Aliquots of standard Dapiprazole hydrochloride stock solution were taken in

different 10 mL volumetric flasks and diluted upto the mark with the mobile phase such

that the final concentrations of Dapiprazole hydrochloride are in the range of 20 to120

μg/mL. Each of these drug solutions (20 μL) was injected three times into the HPLC

system, the peak areas and retention times were recorded. Calibration graph was

obtained by plotting peak area versus concentration of Dapiprazole hydrochloride and it

is shown in the Figure 3.1.2. The Chromatograms of the linearity studies are presented

in Fig 3.1.3. The linearity values are shown in the Table 3.1.5.

Table 3.1.5 Standard calibration values of Dapiprazole hydrochloride

Concentration of

drug (µg/mL)

Retention time Peak area

20 3.722 38525

40 3.793 86933

60 3.630 123328

80 3.724 162282

100 3.705 206169

120 3.705 228316

Recovery studies

Recovery studies were conducted by analyzing in-house synthesized bulk

sample in 50%, 100% and 150% by the proposed method. Each concentration was

injected 3 times and the peak areas were recorded. Known amounts of pure drug [100%


111

of the working standard solution contains 80 µg/mL of Dapiprazole hydrochloride] was

then added to each three previously analyzed bulk sample and the total amount of the

drug was once again determined by the proposed method (each concentration was again

injected 3 times). The recovery values are shown in the Table 3.1.6.

Table 3.1.6 Recovery peak areas of Dapiprazole hydrochloride

% of

solution

Conc. in

µg/mL

Bulk drug

Peak area

Average

peak

area

Standard

peak

area

Average

peak area

% of

Recovery

50 40 86846 81901 84168 84305 95.74

100 80 159078 158700 158110 158629 97.73

150 120 222195 226928 219742 222955 101.55

Fig 3.1.2 Calibration graph of Dapiprazole hydrochloroide


112

Fig. 3.1.3 Linearity chromatograms of Dapiprazole hydrochloride


113

Robustness

Variation of the detection wavelength by ±2 nm (245 nm and 241 nm), the

amount of acetonitrile in the mobile phase was varied by ±10%), and mobile phase pH

(± 0.2 pH-units) had no significant effect on the retention time and chromatographic

response of the method indicating that the method was robust. The results of the study

proved the robust nature of the method.

Limit of Detection [LOD] and Limit of Quantification [LOQ]

The limit of detection (LOD) and limit of quantification (LOQ) for Dapiprazole

hydrochloride were determined based on the standard deviation (SD) of the response

and slope (S) of regression line as per ICH guidelines. The LOD and LOQ were found

to be 0.5µg/mL and 1.6µg/mLrespectively. Chromatograms illustrating the LOD and

LOQ are shown in Figure 3.1.4.

Fig. 3.1.4 Typical LOD & LOQ chromatograms of Dapiprazole hydrochloride


114

Optical and regression characteristics of HPLC method and performance &

detection characteristics of HPLC method are shown in the Tables 3.1.7 and 3.1.8

respectively.

Table 3.1.7 Optical and regression characteristics of HPLC method

Parameter Results of the proposed HPLC

method

Detection wavelength (nm) 243

Linearity range (µg/mL) 20-120

Slope (m) 1956.4

Intercept (c ) 3409

Correlation coefficient 0.9974

%Relative standard deviation 0.05

% error in bulk samples 0.13%

Table 3.1.8 Performance & detection characteristics of HPLC method

Parameter Results of the proposed HPLC

method

Retention time (min) 3.725

Theoretical plates (n) 21505

Plates per meter (N) 86020

HETP 1.16x10-5

Peak asymmetry (T) 1.15

Linearity range (µg/mL) 20-120

Limit of Detection (µg/mL ) 0.5

Limit of Quantification

(µg/mL) 1.6


115

3.1.5 CONCLUSION

There are no reports on the HPLC determination of Dapiprazole

hydrochloride in in-house synthesized bulk sample in the literature prior to

commencement of this work. The author has developed a sensitive, accurate and precise

HPLC method for the estimation of Dapiprazole hydrochloride in bulk drug.

PART-B

Stability indicating HPLC method for

determination of Dapiprazole

hydrochloride drug and its related

substance in API

3.2.1 Introduction

3.2.2 Literature survey

3.2.3 Experimental

3.2.4 Results and Discussion

3.2.5 Forced degradation study

3.2.6 Conclusion

Dapiprazole hydrochloride Chapter – III Part B

116

3.2.1 INTRODUCTION

Dapiprazole hydrochloride (DAP) is an alpha-adrenergic blocking agent. The

novel synthetic method 9-13

has been developed and reported to synthesize Dapiprazole

hydrochloride starting from stable reactants such as ethyl 3-chloro propionate and o-

tolyl piperzine, which appears as a impurity in the final product. The novel synthetic

route9-13

for Dapiprazole hydrochloride was shown in the Figure 3.2.1 and its impurity

is shown in the Figure 3.2.2.

CH3 O Cl

O

Ethyl 3-chloropropionate

+

NH

N

1-Phenylpiperazine

Na2Co3

CH3Cl/FeCl3 CH3 O N

ON CH3

Ethyl 3-[4-(o-tolyl-1-piperazinyl]propionate

N2H4.H2O

C2H5OH

H2N

HN N

ON CH3

3-[4-(o-tolyl)-1-piperazinyl]propionicacid hydrazide

N

OCH3

6-methyl-2,3,4,5-tetrahydropyridine

N

N CH3

N

N

N

DapiprazolehydrochlorideHCl

.HCl

NH

N CH3

1-o-tolylpiperazine

Impurity-A

Fig. 3.2.1 Synthesis of Dapiprazole hydrochloride


117

HN

NCH3

Impurity-A

1-O- tolyl piperazine

Fig 3.2.2 Chemical structure of Dapiprazole hydrochloride impurity (DP)


Very few articles are published for the Dapiorazole hydrochloride in pure drug,

pharmaceutical dosage forms focused on pharmacodynamic properties and its clinical

applications. To the best of our knowledge there are no chromatographic methods

reported for the estimation of Dapiprazole hydrochloride and its process related

impurity. A brief account of literature survey for Dapiprazole hydrochloride is as

follows.

Valeri et al14

have reported a research article focused on ocular

pharmacokinetics of Dapiprazole hydrochloride. Following topical instillation on the

eye, it crosses the corneal epithelium reaching high concentrations in the ocular tissue

and producing a prompt miotic and hypotensive effect. Palmery et al15

have studied the

distribution and excretion of 3H-Dapiprazole hydrochloride in the rat. Tissue

distribution as well as biliary, urinary and fecal excretion of 3H-Dapiprazole

hydrochloride was studied in the rat. The product is found in many tissues, including the

brain. About 23 and 57% of the dose is excreted in the urine and feces and about 65% is


118

eliminated in the bile. Bianchi et al16

have studied the ocular pharmacokinetics of

radioactive Dapiprazole hydrochloride after topical and parenteral administration.

Objectives of the present research work

To characterize the process related impurity by using modern spectroscopic

techniques viz., UV, FT-IR, 1H NMR and MS-MS, optimize the chromatographic

conditions for separation and validation of process-related and forced degradation study

of Dapiprazole hydrochloride bulk drug.

3.2.3 EXPERIMENTAL

Materials and reagents: All the reagents are of analytical-reagent grade unless stated

other wise. Glass-distilled and deionized water (Nanopure, Barnstead, USA); HPLC-

grade acetonitrile, potassium dihydrogen orthophosphate, sodium hydroxide,

hydrochloric acid, hydrogen peroxide and ortho-phosphoric acid (S.D.Fine chem,

Mumbai, India) were used. Samples of Dapiprazole hydrochloride and its impurity-A

(1-o-tolyl piperazine) were a kind of gift from Bio-leo analytical laboratories Pvt. Ltd,

Hyderabad.

Apparatus/Instruments

Liquid chromatograph: A liquid chromatograph from Waters alliance was used.

Waters alliance 2695 separation module (Waters Corporation, Milford, USA) equipped

with 2998 PDA detector with Empower 2 software was used for the analysis. The

chromatographic data and the integrated data were recorded using HP-+Vectra (Hewlett

Packard, Waldbronn, Germany) computer system. Elico, model LI 120; pH meter and

an Inertsil ODS C-18-3V column were used.


119

Charecterization of drug and impurity were carried on UV, FT-IR, 1H NMR and

MS/MS as discussed in the Chapter II page no 58.

Method development

Detection of wavelength: The proper wavelength was needed to determine maximum

detector response. The first step was to run a UV spectrum (from 190-400 nm) using an

HPLC system equipped with the photo diode array detector. From the spectrum it is

clear that Dapiprazole hydrochloride absorbs maximum light between 200nm to 210

nm. The medium wavelength of 205 nm was selected since it produces less noise,

which minimizes problems that may arise while validation of Dapiprazole

hydrochloride and its process related impurity-A.

Selection of stationary phase: Preliminary development trials have performed with

various octadecyl columns (C18 columns) of different types and dimensions from

different manufacturers were tested for the peak shape and the number of theoretical

plates of Dapiprazole hydrochloride. Finally by switching to Inertsil ODS-C18-3V

column (250 mm x 4.6 mm, 5μ) there was a substantial increase in the theoretical plates

(~50000) with a significant improvement in the peak shapes with 1.14 tailing factor. It

also produced adequate resolution between Dapiprazole hydrochloride and its process

related impurity. As a result, it was selected as an optimum one and used throughout

this investigation.

Selection of the mobile phase: To optimize the chromatographic conditions, different

combinations of acetonitrile-water (80:20, 40:60) and acetonitrile-potassium dihydrogen

ortho phosphate buffer (30:70 and 20:80) were tested. Potassium dihydrogen ortho


120

phosphate buffer with acetonitrile 85:15 (v/v) at pH 3.2 was preferred because it

resulted in a greater response to Dapiprazole hydrochloride and its impurity with good

resolution and free from tailing factor.

Flow rate: Flow rate of the mobile phase was tested from 0.6-1.5 mL/min for optimum

separation and it was found that 1.0 mL/min flow rate was ideal for the successful

elution of the analytes.

Optimized chromatographic conditions

The optimized conditions followed for the determination of Dapiprazole

hydrochloride and its process related impurity A.

Chromatographic conditions

Mobile phase : Buffer : acetonitrile [85:15 (v/v)] at pH-3.2

Column : Inertsil ODS C-18-3V (250x4.6mm, 5µ particle size)

Flow rate : 1.0 mL/min

Injection volume : 20 L

Detector : Photo diode array (PDA)

Wavelength (λmax) : 205nm

Column temperature : Ambient

Run time : 30 minutes

Diluent : Mobile phase


121

Analytical procedures

Mobile phase: Solvent-A: Buffer solution: 4.0 g of potassium dihydrogen ortho

phosphate is dissolved in 100mL of HPLC grade water and adjusted to pH-3.2 with

dilute ortho-phosphoric acid 10% (v/v) in water.

Solvent-B: Acetonitrile (HPLC Grade)

Preparation of mobile phase: Buffer solution and acetonitrile were mixed in the ratio

of 85:15 (v/v). The resultant solution was thoroughly mixed and filtered through a poly

tetra fluoro ethylene (PTFE) filter of 0.45 µm pore size using vacuum pump and

degassed by sonication to expel the dissolved gases in solvent system. It was used as a

mobile phase for analysis.

Preparation of standard solution: Standards of Dapiprazole hydrochloride and its

process related impurity (30 mg each) were accurately weighed and transferred into 100

mL volumetric flasks, dissolved in mobile phase and made upto the mark with the

mobile phase to get 300 µg/mL each of Dapiprazole hydrochloride and its process

related impurity solution.

Preparation of working standard solution: 1.0 mL of the above stock solution was

transferred and adequately diluted upto 100 mL in volumetric flask with mobile phase

to get the concentration of 3 µg/ mL (0.15% level).


The present study was aimed at developing a chromatographic method for

separation and quantitative determination of Dapiprazole hydrochloride and its process


122

related impurity. The characterization of Dapiprazole hydrochloride and impurity- A

were performed by spectroscopic data shown in Figure 3.2.3 (a) to (h).

Fig. 3.2.3 (a) 1H-NMR spectra of Dapiprazole hydrochloride

Figure 3.2.3 (b) Mass spectra of Dapiprazole hydrochloride


123

Fig. 3.2.3(c) FT-IR spectra of Dapiprazole hydrochloride

Fig. 3.2.3 (d) UV-Vis Spectra of Dapiprazole Hydrochloride

Fig. 3.2.3 (e) 1H-NMR spectra of Dapiprazole hydrochloride impurity


124

Fig. 3.2.3(f) Mass spectra of Dapiprazole hydrochloride impurity

Fig. 3.2.3(g) FT-IR spectra of Dapiprazole hydrochloride impurity

Fig 3.2.3 (h) UV-Vis Spectra of Dapiprazole hydrochloride impurity


125

Method validation

The developed method was validated with respect to system suitability,

specificity, accuracy, precision, linearity and limit of detection and quantification and

LOQ at precision level.

System suitability

The system suitability was conducted using 1.5% (w/v) of the impurity spiked

to Dapiprazole hydrochloride (3.0 g/mL) and evaluated by making three replicate

injections. The system was suitable for use if the tailing factors for Dapiprazole

hydrochloride and its impurity were 1.55 (observed value is 1.06) and the resolution

was 1.90 (Observed value is 7.33). The quantities of impurity and assay of

Dapiprazole hydrochloride were calculated from their respective peak areas.

Chromatogram of system suitability and data is shown in Figure 3.2.4 and Table 3.2.1

respectively.

Fig. 3.2.4 System suitability chromatogram of Dapiprazole and its impurity


126

Table 3.2.1 System suitability data of Dapiprazole and its impurity

Injection No Dapiprazole hydrochloride

peak area

Impurity

peak area

1 200638 373154

2 203987 375684

3 204905 376229

4 206387 374472

5 205412 372124

6 209302 373105

Average* 205105.2 374128

% RSD 0.15 0.4

Resolution Resolution between Dapiprazole

hydrochloride and its impurity is 7.33

* Average of six determinations

Specificity

The specificity of method will be demonstrated by the ability to analyze

Dapiprazole hydrochloride from finished product sample matrix. A separate solution of

blank, standard and batch samples of Dapiprazole hydrochloride were evaluated along

with impurity solutions. Specificity values are presented in the Table 3.2.2.

Table 3.2.2 Specificity values of Dapiprazole hydrochloride and its impurity

Sample Retention time (min) Retention time in spiked solutions (min)

Dapiprazole

hydrochloride

16.849 16.145

Impurity-A 12.422 12.242


127

The specificity chromatograms of Dapiprazole hydrochloride and its impurity were

shown in Figure 3.2.5.

Fig. 3.2.5 Specificity chromatograms Dapiprazole hydrochloride and its impurity


128

System precision

The system precision was conducted using 3.0µg/mL of the process related

impurity and Dapiprazole hydrochloride (100% level) and evaluated by making six

replicate injections. System precision data is shown in the Table 3.2.3.

Table 3.2.3 System precision study of Dapiprazole and its impurity

Sample

Dapiprazole hydrochloride

working standard

Impurity-A

RT Peak area RT Peak area

Injection-1 16.025 196518 12.255 350951

Injection-2 15.976 202520 11.625 352916

Injection-3 15.899 202576 12.121 350220

Injection-4 15.855 202619 12.138 351093

Injection-5 15.875 202711 12.170 350647

Injection-6 15.930 201854 12.206 352122

Average 15.937 201466 12.0853 351322

Std.Dev. 0.064 2443.5 0.23085 1004.2

% RSD 0.40 1.2 0.62 0.3

Method Precision

The method precision was conducted by using 4.5µg/mL of the impurity spiked

to Dapiprazole hydrochloride (3µg/ mL) at 100% level and evaluated by making six

replicate injections. The method precision data is given in the Table 3.2.4.


129

Table 3.2.4 Method precision study of Dapiprazole and its impurity

Sample

Dapiprazole hydrochloride

working standard

Impurity-A

RT Peak area RT Peak area

Injection-1 15.930 200638 11.620 510526

Injection-2 15.954 203987 11.625 520205

Injection-3 15.960 204905 11.638 530699

Injection-4 15.983 206387 11.655 551231

Injection-5 15.994 205412 11.663 567571

Injection-6 15.947 209302 11.664 580584

Average 15.961 205105 11.6441 543469.3

Std.Dev. 0.023 2849.4 0.019 2759.784

% RSD 0.15 1.4 0.079 1.102

Linearity: The linearity of Dapiprazole hydrochloride impurity is also studied by

preparing standard solutions at seven different levels ranging from 0.75µg/mL to

6µg/mL. The data were subjected to statistical analysis using a linear regression model,

the regression equations and coefficients (r2) are given in Table 3.2.5. The calibration

graphs and linearity chromatograms for Dapiprazole hydrochloride and its impurity

were shown in the Figure 3.2.6 and 3.2.7 respectively.

Range: The standard aliquots are prepared with in the concentrations of 0.75, 1.5, 2.25,

3.0, 3.75, 4.5 and 6.0 µg/mL of with respect to test concentration. The concentration of

impurity at 0.15% (w/v) is 3.0µg/mL. The results obtained from the linearity studies

with reference to concentration of analytes, the range was found to be 0.75µg/mL to

6.0µg/mL.


130

Table 3.2.5 Linearity values of Dapiprazole hydrochloride and its impurity

Fig. 3.2.6 Calibration graphs of Dapiprazole hydrochloride and its impurity

%Level

Concentration (µg/mL) Average area response

Working

standard

Dapiprazole

impurity

Working

standard

Dapiprazole

hydrochloride

impurity

25 0.75 0.75 46594.9 84715.6

50 1.5 1.5 100374.6 173940.2

75 2.25 2.25 152605.2 262723.2

100 3 3 212597.2 355342.0

125 3.75 3.75 262917.8 447581.0

150 4.5 4.5 312560.5 534962.6

200 6 6 416278.9 710569.9

LOQ 0.459 0.192 21643.1 37523.9

Slope 2113 3578

Y–Intercept 3231 2275

Corrélation coefficient 0.9998 1.000


131

Fig. 3.2.7 Linearity chromatograms of Dapiprazole and its impurity


132

Accuracy/Recovery

Accuracy of the method by recovery of the impurity was determined by

analyzing Dapiprazole hydrochloride sample solutions spiked with impurity at five

different concentration levels ranging from 25% (contains 0.75 µg/mL of each of

Dapiprazole hydrochloride and its process related impurity-A) to 150% (contains 4.5

µg/mL of each of Dapiprazole hydrochloride and its process related impurity-A) in

duplicate with respect to specified limit. The percentage recoveries were found to be in

between 99.90 to 103.52% for the impurity A. The results of accuracy studies from

standard solution and process related impurity were shown in Table 3.2.6; recovery

values demonstrated that the method was accurate within the desired range.

Table 3.2.6 Accuracy studies of Dapiprazole hydrochloride impurity

Solutions at

diff. spiked

levels

Dapiproazole process related impurity-A

Amount added

(in mg/mL)

Amount found

(in mg/mL)

%recovery

25% 0.000757 0.000775 102.37

50% 0.001515 0.001535 101.32

75% 0.002272 0.002308 101.58

100% 0.00303 0.003027 99.90

125% 0.003787 0.003805 100.47

150% 0.004545 0.004705 103.52


133

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ for the Dapiprazole hydrochloride impurity were determined

based on the standard deviation of the response and slopeof the regression line. The

LOD and LOQ for the dapiprazole impurity were found to be 1.0% (0.03 µg/ mL) and

2.5 % (0.075µg/ mL) respectively. LOD and LOQ data for impuity were shown in the

Table 3.2.7.

Table 3.2.7 LOD and LOQ data for Dapiprazole impurity

Linearity

Levels

Impurity

(µg/mL)

Peak area/

Response

Impurity

Std.Dev.

Average residual std.

deviation

of impurity-A=2275

Slope=3578

LOD=1.9% (0.057 µg/mL)

LOQ=6.4 % (1.92 µg/mL)

25% 0.75 84715 1330.2

50% 1.5 173940 703.9

75% 2.25 262723 4748.4

100% 3.0 355342 1011.8

125% 3.75 447581 2314.3

150% 4.5 534962 2198.3

200% 6.0 710570 5438.8

Precision at LOQ level

Once the system is suitable for analysis to establish the precision of method at

LOQ level, standard solution of Dapiprazole and its impurity were prepared at about the

predicted LOQ concentration and injected into the sytem six times. Precision at LOQ

level values are given in the Table-3.2.8.Typical chromatogram representing precision

at LOQ level is shown in the Figure 3.2.8.


134

Table 3.2.8 Precision data at LOQ level of Dapiprazole impurity

Sample injected at LOQ level Retention time Peak area

LOQ solution-1 11.995 21964






Average 11.999 21755

Standard deviation 0.035 495

% RSD 0.29 2.3

Fig. 3.2.8 Chromatogram of precision study at LOQ Level

Robustness

The parameters selected for robustness study were flow rate (±10%), column

variation and instrument variation. The above deliberate changes are applied at two

levels of robustness study, i.e at upper and lower levels. This study indicates that there


135

is no effect on the determination of related substance and selectivity for the test method.

The method is sufficiently robust to carry the quantification of impurity in quality

assurance of Dapiprazole hydrochloride. The results of the robustness studies are shown

in Table 3.2.9. Robustness chromatograms are shown in Figure 3.2.9.

Fig. 3.2.9 Robustness chromatograms of Dapiprazole and its impurity


136

Table 3.2.9 Robustness study of Dapiprazole hydrochloride impurity

Parameters

studied

RT Peak

area

Std.dev %RSD Percent

difference

%

limit

Resol-

ution

Flow decrease

(0.9mL/minute)

12.896 69282 706.5 1.3 7.1 33 5.89

Flow increase

(1.1mL/minute)

10.802 49136 349.7 0.7 7.1 33 5.41

Different

column

12.337 67382 888.6 1.3 21.4 33 7.18

HPLC

instrument

variation

12.049 66741 857 1.285 21.4 33 5.688

3.2.5 FORCED DEGRADATION STUDY

Stability-indicating study of Dapiprazole hydrochloride in bulk form was

validated by the proposed method in accordance with ICH guidelines. The drug was

subjected to oxidative, acidic, basic, photolytic and thermal stress conditions at zero &

48 hours.

Stock solution of Dapiprazole hydrochloride: Accurately weighed 25 mg of

Dapiprazole hydrochloride sample and transferred into a 50 mL volumetric flask and

made upto 50mL with mobile phase to get the concentration of 500 µg/mL of

Dapiprazole hydrochloride standard solution.

Preparation of test solutions: 0.1N sodium hydroxide (NaOH), 0.1N Hydrochloric

acid (HCL) and 3% hydrogen peroxide prepared as disussed in the Chapter II, part-B

page no 78.


137

Acid stress studies

About 5 mL of Dapiprazole hydrochloride stock solution was transferred into

25 mL volumetric flask containing 20mL of 0.1N hydrochloric acid. The zero hour

sample solution has been prepared by taking immediately 5 mL of the above primary

working standard solution and neutralize with 5 mL of 0.1N sodium hydroxide solution

and made upto 25 mL with diluents in volumetric flask. The Acid stress study sample

after 48 hours was prepared like zero hr solution. The above solutions inject once after

system suitability solution and evaluate the degradants in chromatogram and compare

with or without acidify (initial) values.

Base stress studies

About 5 mL of Dapiprazole hydrochloride stock solution is transferred into 25

mL volumetric flask containing 20 mL of 0.1N sodium hydroxide solutions. The zero

hour sample solution was prepared by taking immediately 5 mL of the above primary

working standard solution and neutralized with 5 mL of 0.1N hydrochloric acid solution

and made upto 25 mL with diluents in volumetric flask. The base stress study sample

after 48 hours was prepared like zero hr solution. The above solutions were injected into

liquid chromatograph, after system suitability solution and evaluate the degradants in

chromatogram and compare with or without alkalinization (initial) values

Peroxide stress studies

About 5 mL of Dapiprazole hydrochloride stock solution is transferred into

25 mL volumetric flask containing 20 mL of 3% hydrogen peroxide solution. The

48hour sample solution was prepared by taking immediately 5 mL of the above primary


138

working standard solution and diluted upto 25 mL with diluents in volumetric flask and

injected into the system after system suitability solution and evaluates the degradants in

chromatogram and compare with/without oxidized (initial) values. The typical

chromatogram of Dapiprazole hydrochloride in peroxide induced degradation studies

shows that the compound is extremely sensitive in oxidation studies.

Thermal stress studies

To determine stability of Dapiprazole hydrochloride towards heat, 30 mg of

Dapiprazole hydrochloride sample is kept in petri dish and placed in oven at 40°C and

80°C up to 48 hours. After 48 hours, the thermally exposed samples at 400C and 80

0C

are further used to prepare the testing solution at the strength of 3000 µg/mL and

injected in to the liquid chromatograph.

Photo-degradation studies

Photo degradation of the Dapiprazole hydrochloride is deteremined by the

effects of UV irradiation by keeping the 30 mg of sample in open petri dish at lab light

and UV light. Samples were checked for initial degradation after exposure for 30

minutes at 40°C and at 80°C. After 48 hours, the samples are removed from the UV

light cabinet. The light exposed samples are further used to prepare the testing solution

by dilution with mobile phase at the strength of 3000 µg/mL and injected in to the

liquid chromatograph. The degradation chromatograms are shown in Figure 3.2.10 and

the degradation results are given in Table 3.2.10.


139

Fig 3.2.10 Forced degradation chromatograms of Dapiprazole hydrochloride


140

Table 3.2.10 Forced degradation study of Dapiprazole hyd rochloride

Stress

condition

Color of

the

solution

RT of

Dapiprazole

hydrochloride

RT in min. of

predominant

Degradants

Percent of

degradation

Acid 0.1 N

HCl Clear

16.055 7.1, 8.9 and 11.19 0.08

Base 0.1 N

NaOH Clear

16.967 10.4 and 11.5 0.04

Oxidation

3% H202 Clear

16.299 4.1 and 22.3 8.0

UV Light at

254 nm Clear

16.248 No degradants. Negligible

Lab light Clear 16.279 No degradants Negligible

Heat. Clear 16.183 No degradants Negligible

3.2.6 CONCLUSION

The liquid chromatographic method with isocratic elution was developed for

the simultaneous determination of Dapiprazole hydrochloride and its impurity and the

forced degradation study of the bulk drug was completely validated and proved to be

reliable, sensitive, accurate, precise and robust. The method has higher sensitivity

towards the determination of impurity and it is the first time that such method appears

in the literature.


141

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