Practical Implication of Stability Indicating Chromatographic Method for Estimation of Clomipramine Hydrochloride and its Related Substances from Capsule Dosage Form

 

Jinal N.Tandel1*, Radhika V. Bhatt1, Nilam M. Patel1, Samir K.Shah2

1Department of Quality Assurance, Sardar Patel College of Pharmacy, Vidyanagar-Vadtal Road, Bakrol,(388315) Gujarat, India

2Department of Pharmacology, Sardar Patel College of Pharmacy, Vidyanagar-Vadtal Road, Bakrol,(388315) Gujarat, India

*Corresponding Author E-mail: jinaltandel1202@gmail.com

 

ABSTRACT:

Clomipramine Hydrochloride is Tricyclic antidepressants (TCAs) drug which cause increased concentration of monoamines in the synaptic cleft, ultimately resulting in antidepressant effect. Literature reveals that all the reported methods are developed for estimation of Clomipramine Hydrochloride (CLH) in bulk and solid dosage form. My research is based on estimation of CLH and its related substances from capsule dosage form by HPLC method. A rapid, precise, selective and sensitive and accurate stability-indicating High Performance Liquid Chromatographic (HPLC) method for estimation of Clomipramine Hydrochloride and its related substances from capsule dosage form was developed and validated in the present work. The chromatographic separation was performed by using BDS Hypersil C18, 250 X 4.6 mm, 5 μ with 2 gm /L Tetra Butyl Ammonium Hydrogen Sulphate (TBAS) in Water (pH 2.5) : Methanol (40:60 v/v) mobile phase, at a flow rate of 1.2 ml/min. The method was validated according to ICH guidelines. The effluent was monitored using a UV detector set at 252nm. The retention time for CLH was observed at 6.2 min. Impurity spiked A, B, C, are retained at 7.8, and 9.4, 3.6, min respectively. The linearity of CLH and impurities was in the range of 1050 – 1800 μg/ml and 0.7 – 4.5μg/ml respectively. The developed method was successfully applied to the analysis of the drugs in their commercial dosage form.

 

KEYWORDS: Clomipramine Hydrochloride (CLH), Impurity, HPLC, Stability, Validation.

 

 


1. INTRODUCTION:

Depression is an extremely common psychiatric condition. Tricyclic Antidepressant is the agent of choice for endogenous depression. Tricyclic antidepressants (TCAs) are chemically related to Phenothiazine, and some have similar non- selective receptor-blocking action. TCAs are potent inhibitors of the neuronal reuptake of norepinephrine and serotonin into presynaptic nerve terminals.

 

At therapeutic concentration, they do not block dopamine transporters. By blocking the major route of neurotransmitter removal, the TCAs cause increased concentration of monoamines in the synaptic cleft, ultimately resulting in antidepressant effect(1-3).

 

Clomipramine Hydrochloride (Fig.1) is chemically 3-(3-chloro-10, 11-dihydro-5H-dibenzo [b,f] azepine-5-yl)- N,N-dimethylpropen-1-amine hydrochloride acting as an Antidepressant. (4-6)

 

Figure 1: Chemical Structure of Clomipramine Hydrochloride

 

Literature survey reveals that many analytical methods have been reported for determination of Clomipramine Hydrochloride (CLH) individually (7-12) and in combination with other drug (13) No single method was reported for the Stability indicating assay method with related substances. Related substances are structurally related to a drug substance and control the degradation impurities. Some related substances like, Imipramine (Fig. 2), Desipramine (Fig. 3) and N – Desemethyl Clomipramine Hydrochloride (Fig. 4) The main purpose of a test for related substances is to control degradation impurities.

 

 

 

Figure 2. Structure of Imipramine (IMP – A)

Figure 3 Structure of Desipramine (IMP – B)

 

 

Figure 4 Structure of N–Desemethyl Clomipramine Hydrochloride (IMP – C)

 

The present work describes the development of a stability indicating RP-HPLC method, which can quantify this component simultaneously with related substances and also separate this component from its degradation products. The International Conference on Harmonization (ICH) guideline entitled “Stability testing of new drug substances and products” requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance. (14) An ideal stability-indicating method is one that resolves the drug and its degradation products efficiently. Consequently, the implementation of an analytical methodology to determine Clomipramine Hydrochloride (CLH) in presence of its degradation products is rather a challenge for pharmaceutical analyst. Therefore, it was thought to study the stability of Clomipramine Hydrochloride (CLH) under acidic, alkaline, oxidative, Photo and Thermal conditions. This paper reports validated stability-indicating HPLC method for simultaneous determination of Clomipramine Hydrochloride (CLH) with their related substances. Method is validated acoording to ICH Q2R1 guidline.(15) The proposed method is simple, accurate, reproducible, stability indicating and suitable for routine determination of Clomipramine Hydrochloride (CLH) inpharmaceutical dosage form.

 

2. MATERIALS AND METHODS:

2.1 Method Development:

2.1.1 Reagent and Material:

Clomipramine Hydrochloride and Impurities were kindly gifted by Zydus Cadila Health Care, Changodar, Moraiya. Methanol, tetra butyl ammonium hydrogen sulphate, and Distilled water, all reagents of HPLC grade and Hydrochloric acid, Sodium hydroxide and hydrogen peroxide of AR grade ware procured from Merck (India).

 

2.1.2. Equipments and Instruments:

A double beam UV- visible spectrophotometer (Systronic, UV-2203) was used. ODS C18 column (250mm × 4.6mm, 5µm) with Phosphate buffer (pH-3.5): Methanol (60:40 %v/v) mobile phase. Hamiltone syringe (25 µl) and Systronic- model no. 335 pH meter was used. 1.0 mL min-1 flow rate was maintained. An injection volume of 20 µL was injected by means of Rheodyne syringe or injector and UV detection was done at 273 nm. S 1122 HPLC (Analytical Technologies) with Alchrome A 2000 chromatographic software was employed.

 

2.2 Preparation of Standard Solutions:

2.2.1 Preparation of standard solution (1500 μg/ml):

Accurately Weighed and transferred 150 mg of CLH standard into 100 ml of volumetric flask, Added 70 ml of diluent into it and sonicated to dissolved, cool and diluted up to mark with diluent and mixed well.

 

2.2.2 Diluted standard preparation for related substances (3.0 μg/ml):

From the standard solution diluted 1 ml of solution to 50 ml with diluent. Further diluted 1 ml of the solution to 10 ml with diluent.

 

2.2.3 Preparation of Imipramine Impurity (3.0 μg/ml):

About 5 mg of Imipramine dissolved in 100 ml of diluent (50 μg/ml). Further diluted 3 ml of this solution to 50 ml with diluent. (3.0 μg/ml)

 

2.2.4 Preparation of Desipramine Impurity (3.0 μg/ml):

About 5 mg of Desipramine Impurity dissolved in 100 ml of diluent (50 μg/ml). Further diluted 3 ml of this solution to 50 ml with diluent. (3.0 μg/ml)

 

2.2.5 Preparation of N-Desemethyl Clomipramine Impurity (3.0 μg/ml):

About 5 mg of N-Desemethyl Clomipramine Impurity dissolved in 100 ml of diluent (50 μg/ml). Further diluted 3 ml of this solution to 50 ml with diluent. (3.0 μg/ml)

 

2.2.6 Impurity stock solution (50 μg/ml):

About 5 mg of each Imipramine, Desipramine and N-Desemethyl Clomipramine Hydrochloride impurities dissolved in 100 ml of diluent.

 

2.2.7 Sample preparation – 1500 μg/ml:

Accurately weighed 10 filled capsules and carefully empty all capsules and then weigh 10 empty capsules shells and calculated filled average net content.

Average Net Content: about 350 mg

 

Weighed and transferred capsule content equivalent to 150 mg of CLH in to 100 ml volumetric flask carefully, added about 70 ml of diluent in to it, sonicated for 30 minutes with intermittent shaking, cooled to attain room temperature and made up to volume with diluent and mixed well. It was filtered through 0.45μ syringe filter and filtrate was used as a sample.

 

2.2.8 Sample Preparation – Spike: (1500 μg/ml of Clomipramine and 3.0 μg/ml of each impurity):

Weigh and transferred capsule content equivalent to 150 mg of CLH in to 100 ml volumetric flask carefully, added about 70 ml of diluent and 6 ml of impurity stock solution in to it, sonicated for 30 minutes with intermittent shaking, cooled to attain room temperature and made up to volume with diluent and mixed well. It was filtered through 0.45μ syringe filter and filtrate was used as a sample.

 

2.2.9 Preparation of mobile phase:

2.0 gm of Tetra Butyl Ammonium Hydrogen Sulphate (TBAS) dissolved in 1000 ml of water and filtered and degassed by sonication. Then take 400 ml of solution and mixed with 600 ml of Methanol in 1000 ml of volumetric flask. (40:60)

 

2.2.10 Selection of wavelength for detection (10 μg/ml):

About 5 mg of CLH, Imipramine impurity, Desipramine Impurity and N- Desemethyl Clomipramine Impurity dissolved in 50 ml of Methanol. Further diluted 5 ml of this solution to 50 ml with Methanol. Each solution was scanned between 200-400 nm using Methanol as a blank. The point at which drug show absorbance was selected as wavelength for determination.

 

2.3 Preparation of calibration curve:

2.3.1 For CLH:

375.0 mg of drug dissolved in 50 ml of diluent (7500 μg/ml stock solution). The ranging of standard CLH from 1050 μg/ml – 1800 μg/ml. The solutions were prepared by pipetting out 3.5 ml, 4 ml, 4.5 ml, 5 ml, 5.5 ml, and 6 ml of the stock solution in to 25 ml of volumetric flasks. Make up volume with diluent. The solution contain 1050 μg/ml, 1200 μg/ml, 1350 μg/ml, 1500 μg/ml, 1650 μg/ml, 1800 μg/ml respectively.

 

2.3.2For impurities:

5.0 mg of impurities dissolved in 100 ml of diluent (50 μg/ml stock solution). The ranging of impurities from 0.7 μg/ml – 4.5 μg/ml. The solutions were prepared by pipetting out 1.5 ml, 3.0 ml, 4.5 ml, 6.0 ml, 7.5 ml and 9.0 ml of the stock solution in to 100 ml of volumetric flasks. Make up volume with diluent. The solutions contain 0.7 μg/ml, 1.5 μg/ml, 2.2 μg/ml, 3.0 μg/ml, 3.7 μg/ml and 4.5 μg/ml respectively.

 

2.4 Validation of HPLC Method For Related Substance:

The different concentrations of Clomipramine impurities solutions ranging from 0.7 – 4.5 μg/ml. The solutions were prepared by pipetting out 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 ml of the stock solution of impurities in 100 ml volumetric flasks and the volume was adjusted to mark with diluent to get 0.7, 1.5, 2.2, 3.0, 3.7 and 4.5 μg/ml solutions respectively. The graph of peak area obtained versus respective concentration was plotted. As per ICH guidelines Q2R1, the method validation parameters studied were specificity, linearity, accuracy, and precision, limit of detection and limit of quantification.

 

2.4.1 Specificity:

Specificity is ability to measure specifically the analyte of interest without any interference of excipients and mobile phase component. For the determination of specificity of the standard solution, Marketed formulation and solution of impurities was injected. All chromatograms were compared and check for any interference of excipients peak. Chromatogram of blank (only mobile phase) was also recorded to check any interference.

 

2.4.2 Linearity (Calibration curve):

The series consisted of different concentrations of CLH solutions ranging from 1050 - 1800 μg/ml. The solutions were prepared by pipetting out 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 ml of the stock solution of CLH in 25 ml volumetric flasks and the volume was adjusted to mark with diluent to get 1050, 1200, 1350, 1500, 1650, and 1800 μg/ml solution respectively.

 

2.4.3 Precision:

Intraday precision for CLH:

Sample of 4, 5 and 6 ml of working standard solution of CLH (7500 μg/ml) was transferred in to a 25 ml volumetric flask. The volume was adjusted up to mark with diluent to get 1200, 1500 and 1800 μg/ml solution of CLH. The area of peaks were measured three different times on the same day and % RSD was calculated.

 

Intraday precision for Impurities:

The precision of the related substance method was checked by injecting three individual sample preparations of CLH (1500 μg/ml) spiked with 1.5, 3.0 and 4.5 μg/ml solution of imp-A, imp-B, and imp-C. The area of peaks were measured three different times on the same day and % RSD was calculated.

 

Interday Precision for CLH:

Sample of 4, 5 and 6 ml of working standard solution of CLH (7500 μg/ml) was transferred in to a 25 ml volumetric flask. The volume was adjusted up to mark with diluent to get 1200, 1500 and 1800 μg/ml solution of CLH. The area of peaks were measured three different times on the three different days and % RSD was calculated.

 

Intraday precision for Impurities:

The precision of the related substance method was checked by injecting three individual sample preparations of CLH (1500 μg/ml) spiked with 1.5, 3.0 and 4.5 μg/ml solution of imp-A, imp-B, and imp-C. The areas of peaks were measured three different times on the three different days and % RSD was calculated.

 

Repeatability:

Repeatability was performed by analyzing six separate CLH sample solution of concentration 1500 μg/ml that were prepared by spiking the related substances to give 3 μg/ml of each IMP-A, IMP-B, and IMP-C. The % RSD for each related substances was evaluated.

 

2.4.4 LOD and LOQ:

The LOD (Limit of Detection) was estimated from the set of 5 calibration curves used to determine method linearity. The LOD may be calculated as

LOD = 3.3 × σ/S

Where,

σ = Standard deviation of the Y- intercepts of the 5 calibration curves

S = Mean slope of the 5 calibration curves

The LOQ (Limit of Quantitation) was estimated from the set of 5 calibration curves used to determine method linearity. The LOQ may be calculated as

LOQ = 10 × σ/S

Where,

σ = Standard deviation of the Y- intercepts of the 6 calibration curves

S = Mean slope of the 6 calibration curves

 

2.4.5 Accuracy (% Recovery):

It was determined by calculating the recovery of CLH and impurities from formulation by standard addition method. To a fixed amount of 80%, 100% and 120% for CLH and LOQ, 50%, 100%, and 150% for impurities amount of standard was added and calculated using regression equation. Known amount of standard solutions of CLH (1200, 1500 and 1800 μg/ml) and impurities (LOQ, 1.5, 3.0 and 4.5μg/ml) were added to a pre-quantified sample solution of CLH. (1500 μg/ml). Each solution was injected in triplicate and the percentage recovery was calculated by measuring the responses and fitting these values into the regression equations of the respective calibration curves.

 

2.4.6 Solution stability:

Solution stability of CLH in the assay method was carried by both the sample solution and standard solution in tightly capped volumetric flask at room temperature (i.e. 25 şC) up to 24 hours. The solution stability of CLH and its impurities in the related substance method was carried out by leaving spiked sample solution in tightly capped volumetric flasks at room temperature for 24 hours. The change in response of sample solution was evaluated. Solution stability of proposed method was determined by analysis of solution containing 1500 μg/ml of CLH and 3.0 μg/ml for impurities at different time interval and %RSD was calculated.

 

2.4.7 Robustness:

Prepared solutions were analyzed per proposed method with small but deliberate change in chromatographic condition as listed below:

1. Change in Flow rate: 1.0 ml/min, 1.4 ml/min

2. Change in mobile phase composition: Buffer (TBAS, pH 2.5) : Methanol in the ratio (38 : 62 % v/v) and (42 : 58 % v/v)

3. Change in temperature: 30şC, 40şC The mean peak area with its standard deviation and % relative standard deviation were computed at each level.

 

2.4.8 System Suitability parameters:

System suitability is the checking of a system to ensure system performance before or during the analysis of unknowns. Parameters such as Theoretical plates, Tailing factors, Resolution were determined and compared against the specifications set for the method.

 

2.4.9 Analysis of marketed formulation: Twenty capsules were weighed accurately and their average weight was determined. The content of capsules was taken out and accurately weighed powder of CLH and prepared the 1500 μg/ml of solution. Prepared the solution was injected to system with stated chromatographic condition

 

2.5 FORCE DEGRADATION STUDY:

2.5.1 Acid degradation

Transferred about 1050 mg of capsule content (eq. to 150 mg of CLH) in to 100 ml volumetric flask, added about 20 ml of diluent in to it, sonicated for 30 minutes with intermittent shaking, then added 5ml of 1N HCl into it and it was kept for 6 hours at 60°C temperature in water bath, cooled to attain room. Then added 5 ml of 1N NaOH to neutralized it and volume was made up to mark with diluent and mixed well.

 

2.5.2 Base degradation:

Transferred about 1050 mg of capsule content (eq. to 150 mg of Clomipramine Hydrochloride) in to 100 ml volumetric flask, added about 20 ml of diluent in to it, sonicated for 30 minutes with intermittent shaking, then added 5ml of 1N NaOH into it and it was kept for 6 hours at 60°C temperature in water bath, cooled to attain room. Then added 5 ml of 1N HCl to neutralized it and volume was made up to mark with diluent and mixed well.

 

2.5.3 Peroxide degradation:

Transferred about 1050 mg of capsule content (eq. to 150 mg of Clomipramine Hydrochloride) in to 100 ml volumetric flask, added about 20 ml of diluent in to it, sonicated for 30 minutes with intermittent shaking, then added 5ml of 3% H2O2 into it and it was kept for 60 min. at room temperature and volume was made up to mark with diluent and mixed well.

 

2.5.4 Thermal degradation:

Transferred about 1050 mg of capsule content (eq. to 150 mg of Clomipramine Hydrochloride), (which was previously exposed in oven at 80°C for 5 days) in to 100 ml volumetric flask, added about 70 ml of diluent in to it, sonicated for 30 minutes with intermittent shaking, cooled to attain room temperature and made up to volume with diluent and mixed well.

 

2.5.5 Photo Sunlight degradation:

Transferred about 1050 mg of capsule content (eq. to 150 mg of Clomipramine Hydrochloride), (which was previously exposed in sunlight for 5 days) in to 100 ml volumetric flask, added about 70 ml of diluent in to it, sonicated for 30 minutes with intermittent shaking, cooled to attain room temperature and made up to volume with diluent and mixed well.

 

3. RESULT AND DISCUSSION:

Selection of Wavelength:

For the selection of wavelength, working standard solution of CLH was scanned in the range of 200-400 nm. The drug showed reasonably good absorbance at 252 nm. And impurities were also give good absorbance at 252 nm so, 252 nm was selected as the wavelength which is shown in following Fig.5

 

Figure 5 UV spectra of CLH

 

Mobile Phase optimization:

Various mobile phases with different ratios of different solvents, column and flow rate were used. A stability indicating RP-HPLC mobile phase has been developed with 2 gm /L Tetra Butyl Ammonium Hydrogen Sulphate (TBAS) in Water (pH 2.5) : Methanol (40:60). This provides optimum polarity for proper separation and resolution. (table 1) Chromatogram of API and with impurities (fig.6, fig.7), and system suitability parameters (table 2) are shown below.

 

Optimized Chromatographic Condition

Table 1 Optimized Chromatographic conditions for Clomipramine HCl

Mobile phase

2 gm/L Tetra Butyl Ammonium Hydrogen Sulphate (TBAS) in Water (pH 2.5) : Methanol (40:60 %v/v)

Column

ODS-BP Hyperchrome C18 (250 × 4.6 mm, 5 µm)

Column temperature

35°C

Injection volume

20 µL

Flow rate

1.2 ml /min

Wavelength

252 nm

Diluent

Water : Methanol (50:50 %v/v)

Run time

20 min

 

 


Figure 6 Chromatogram of CLH (API)

 

Figure 7 Chromatogram of API spiked with impurities

 

Table 2 System suitability parameters for final condition

No.

Name

RT (Min)

Area

%Area

Tailing factor

Resolution

1

IMP – C

3.6

320452

0.051

1.0

-

2

CLH

6.1

98842925

99.66

1.1

7.8

3

IMP – A

7.8

216158

0.20

1.0

3.1

4

IMP - B

9.4

365856

0.066

1.0

4.1

 

METHOD VALIDATION

Specificity

For specificity chromatogram of blank, placebo, API of CLH, and various impurity (fig 8-14) were taken

Figure 8 Chromatogram of Blank

 

Figure 9Chromatogram of Placebo

 

Figure 10 Chromatogram of CLH

 

Figure 11 Chromatogram of marketed formulation

 

Figure 12 Chromatogram of IMP - A

 

Figure 13 Chromatogram of IMP - B

 

Figure 14 Chromatogram of IMP – C

 

 


Linearity and Range:

The linearity study was carried out for drug and impurities at different concentration levels. The linearity of CLH and impurities was in the range of 1050 – 1800 μg/ml and 0.7 – 4.5 μg/ml respectively.(fig15-16) % RSD of all results was less than 2%.linearity data for CLH and its related substances are shown in Table 3-6. For linearity calibration curve of the drug and its impurity are shown in fig.17-20. From the data its found that Correlation coefficient value for the CLH (concentration ranges 1050 - 1800 μg/ml) was found to be 0.999. Correlation coefficient value for the impurities was found to be (concentration ranges 0.7- 4.5 μg/ml) IMP - A 0.999, IMP – B 0.999 and IMP – C 0.999. Hence, the method is linear within the range.


 

Figure 15 Overlain chromatogram for CLH

 

Figure 16 Overlain chromatogram of impurities

 

 


Table 3 Linearity data of CLH

Concentration (µg/ml)

Mean ± SD

%RSD

1050

67496630 ± 449445

0.6

1200

77234895 ± 652393

0.8

1350

87237689 ± 662214.8

0.7

1500

97121486 ± 715304.7

0.7

1650

104879865 ± 1651290

1.5

1800

115365498 ± 1834596.8

1.5

 

Concentration ug/ml

 
Figure 17 Calibration curve for CLH (1050-1800 ug/ml)

 

 

Table 4 Linearity data for IMP - A

Concentration (µg/ml)

Mean ± SD

%RSD

0.7

54484.4 ± 321.7535

0.5

1.5

107993.6 ± 3835.459

0.3

2.2

158165.6 ± 4503.802

0.2

3.0

215442.8 ± 7183.465

0.3

3.8

270109.4 ± 8613.239

0.3

4.5

322244.8 ± 11781.28

0.3

 

Figure 18 Calibration curve for impurity – A

 

 

Table 5 Linearity data for impurity – B

Concentration (µg/ml)

Mean ± SD

%RSD

0.7

91532.6 ± 274.43

0.2

1.5

183008.2 ± 2821.68

0.1

2.2

265642.8 ± 2341.45

0.8

3.0

364386.6 ± 2916.85

0.8

3.8

456213.8 ± 3438.83

0.7

4.5

545167.6 ± 6200.89

0.1

 

Figure 19 Calibration curve for impurity – B

 

Table 6 Linearity data for impurity – C

Concentration (µg/ml)

Mean ± SD

%RSD

0.7

77712 ± 589.562

0.7

1.5

166085.5 ± 4421.402

0.2

2.2

247159.5 ± 7383.406

0.2

3.0

323842 ± 6424.607

0.1

3.8

397975.5 ± 1982.024

0.4

4.5

479854 ± 8043.283

0.1

 

Figure 20 Calibration curve for impurity – C

 

Precision

For the precision study, intraday and inter day precision three different concentration limits are selected that are lower middle and higher. % R.S.D of the intra-day precision and inter-day precision for the CLH was found in the range of 0.2 – 1.0 % and 0.5- 0.6 % respectively (table-7, 9), for the IMP - A 0.2 – 0.4% and 0.2 – 0.3 % respectively (table-7, 9), for the IMP - B 0.5 – 1.0 % and 0.3 – 1.6 % respectively (table-8,10), for the IMP – C 0.2 – 0.5 % and 0.2 – 1.3 % respectively (table-8,10). Hence, the method is found to be precise.


 

Intraday precision

Table 7 for CLH and IMP A

Clomipramine HCl

IMP - A

Conc. (μg/ml)

Mean area ± SD

% RSD

Conc. (μg/ml)

Mean area ± SD

% RSD

1200

78228774 ± 843655.6

1.0

1.5

110036.7 ± 512.40

0.4

1500

98495465 ± 263510.8

0.2

3.0

221384.7 ± 579.23

0.2

1800

119495863 ± 470554.6

0.3

4.5

333763.3 ± 780.44

0.2

 

Table 8 for IMP B and IMP C

IMP - B

IMP - C

Conc. (μg/ml)

Mean area ± SD

% RSD

Conc. (μg/ml)

Mean area ± SD

% RSD

1.5

185626.7 ± 1970.005

1.0

1.5

161158.3 ± 739.36

0.4

3.0

363397.3 ± 1865.18

0.5

3.0

312696.7 ± 692.92

0.2

4.5

546165 ± 2859.04

0.5

4.5

473648 ± 2676.50

0.5

 

Interday precision

Table 9 for CLH and IMP A

Clomipramine HCl

IMP - A

Conc. (μg/ml)

Mean area ± SD

% RSD

Conc. (μg/ml)

Mean area ± SD

% RSD

1200

78287182 ± 428011.7

0.5

1.5

110954.7 ± 392.23

0.3

1500

98064222 ± 515705.9

0.5

3.0

228998 ± 5620.34

0.2

1800

111293533 ± 757230

0.6

4.5

324877.3 ± 1256.98

0.3

 

Table 10 for IMP B and IMP C

IMP - B

IMP - C

Conc. (μg/ml)

Mean area ± SD

% RSD

Conc. (μg/ml)

Mean area ± SD

% RSD

1.5

182508.3 ± 722.11

0.3

1.5

159257 ± 459.46

0.2

3.0

361241 ± 5657.19

1.5

3.0

311739.7 ± 667.45

0.2

4.5

541682.3 ± 6848.51

1.6

4.5

469294.3 ± 6364.64

1.3

 

 

Repeatability of sample solution spiked with impurities

Table 11 Repeatability of CLH, IMP- A IMP-B, IMP- C

No.

Area of CLH (1500 µg/ml)

Area of IMP –A (3 µg/ml)

Area of IMP – B (3 µg/ml)

Area of IMP – C (3 µg/ml)

1

98102487

240574

359987

318857

2

97856574

239874

345874

325417

3

99254171

245698

349574

325147

4

98657846

226584

365241

318571

5

97998562

245876

352214

315274

6

99165741

230054

350004

312264

Mean

98505897

238110

353815

319255

SD

609941.9

4060.016

6304.513

5254.229

% RSD

0.6

1.7

1.7

1.6

 

Accuracy:

Accuracy determine over 80%,100%,120% level for CLH And impurity and data found within the limit. (Table12), so concluded that method was accurate.

 

Table 12 Accuracy data for CLH, IMP- A IMP-B, IMP- C

Level

% recovery of CLH (1500 µg/ml)

% recovery of IMP –A (3 µg/ml)

% recovery of IMP – B (3 µg/ml)

% recovery of IMP – C (3 µg/ml)

80%

100 ± 1.039

99.0 ± 0.378

100.8 ± 1.761

100.0 ± 0.251

100%

100.5 ± 1.242

 99.5 ± 1.769

 100.3 ± 0.494

100.0 ± 0.351

120%

100.4 ± 1.081

 99.6 ± 1.069

 100.2 ± 1.550

99.9 ± 1.123

% RSD

1.02

1.2

1.2

0.8

 

Robustness:

Different internal changes like Flow rate (±2), Temperature of column (±5), and Mobile phase composition (±2) done and chromatogram developed data shows that (Table 13) there are no changes in % RSD. Its still within limit so method was found to be robust.

 

Table 13 Robustness data for CLH, IMP- A IMP-B, IMP- C

Robustness parameters

%RSD of CLH (1500 µg/ml)

%RSD of IMP –A (3 µg/ml)

%RSD of IMP – B (3 µg/ml)

%RSD of IMP – C

(3 µg/ml)

Flow rate (1.0)

0.5

1.2

1.3

0.2

Flow rate (1.4)

0.6

1.7

1.8

1.4

Temp. (30)

0.7

0.2

1.5

0.2

Temp. (40)

0.4

0.2

1.6

0.2

Mobile phase 38:62 %v/v

0.4

1.7

1.1

1.7

Mobile phase 42:58 %v/v

0.2

0.2

1.6

1.5

 

Table 15 Summery of Validation Parameter

Parameters

CLH

IMP - A

IMP - B

IMP - C

Linearity (µg/ml)

1050 – 1800

0.7 – 4.5

0.7 – 4.5

0.7 – 4.5

Precision (% RSD)

Intraday (n=3)

0.2 – 1.0

0.2 – 0.4

0.5 – 1.0

0.2 – 0.5

Interday (n=3)

0.5 – 0.6

0.2 – 0.3

0.3 – 1.6

0.2 – 1.3

Repeatability (n=6)

0.6

1.7

1.7

1.6

Accuracy (% Recovery)

100 – 100.5

99.0 – 99.6

100.2-100.8

99.5-100.0

LOD (μg/ml)

3.799

0.169

1.495

2.521

LOQ (μg/ml)

11.51

0.513

4.531

7.641

Robustness (% RSD)

Flow rate (ml)

1.0

0.5

1.2

1.3

0.2

1.4

0.6

1.7

1.8

1.4

Temperature

30°C

0.7

0.2

1.5

0.2

40°C

0.4

0.2

1.6

0.2

Mobile phase ratio

38:62

0.4

1.7

1.1

1.7

42:58

0..2

0.2

1.6

1.5

 

Table 14 Analysis of marketed formulation

Label Claim (mg)

Amount found (mg)

% Amount found

50

49.2

100.6

49.6

100.3

50.1

100.6

50.3

99.6

49.8

99.4

Mean

49.8

100.1

SD

0.4301

0.5656

% RSD

0.8

0.5


Analysis of marketed formulation

Different 5 times repeat the procedure and % RSD was found to be <1 (table 14) so can be say that method is applied in marketed preparation

 

 

 

FORCE DEGRADATION STUDY:

Force degradation study was carried out in various conditions like Hydrolytic, Oxidative, Thermal and Photolytic. The different Chromatogram for each condition were shown in figures. The data were described in Tables as follows.


1. Acid Degradation - Chromatogram of blank and sample was developed (Fig 20-21) in acidic medium

 

Figure 20 Chromatogram of blank

 

Figure 21 Chromatogram of sample

 

2. Base Degradation - Chromatogram of blank and sample was developed (Fig 22-23) in basic medium

 

Figure 22 Chromatogram of blank

 

Figure 23 Chromatogram of sample

3. Oxidative degradation - Chromatogram of blank and sample was developed (Fig 24-25) in peroxide medium

 

Figure 24 Chromatogram of blank

 

Figure 25 Chromatogram of sample

 

4. Thermal degradation - Chromatogram of blank and sample was developed (Fig 26-27) in thermal medium

 

Figure 26 Chromatogram of blank

 

Figure 27 Chromatogram of sample

 

5. Photolytic degradation - Chromatogram of blank and sample was developed (Fig 28-29) in photolytic medium

 

Figure 28 Chromatogram of sample

 

Figure 29 Chromatogram of sample

 

Summary data for the degradation study was shown in table16

Table 16. Degradation data summary

Stress type

Stress condition

% Degradation

Remark

Control sample

As such sample

-

-

Acid degradation

1 N HCl 5 ml for 6 hr at 60°C

17.76 %

Degraded to IMP – A and other unknown degradants

Base degradation

1 N NaOH 5 ml for 6 hr at 60°C

16.81 %

Degraded to IMP – A and other unknown degradants

Peroxide degradation

5 ml 3% H2O2 at room temp. For 1 hr

18.74 %

Degraded to IMP – A and other unknown degradants

Thermal degradation

Exposed in oven at 80°C for 5 days

13.78 %

Degraded to IMP – A and other unknown degradants

Sunlight degradation

Exposed in sunlight for 5 days

12.77 %

Degraded to IMP – A and other unknown degradants

 

 


4. REFERENCES:

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2.       Rang HP., Dale MM., Ritter JM., and Flower RJ. Rang and Dale’s Pharmacology. Elsevier Ltd, 2007. pp 557.

3.       Finkel R., Clark M., Cubeddu L., Harvey R., and Champe P. Lippincott’s Illustrated Pharmacology. Published by Walters Kluwer (P) Ltd, New Delhi, 2009. pp 146.

4.       Clomipramine HCl “Drug Profile”. WWW.hmdb.ca/metabolites/HMDB15372

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11.     Mostafavi SA., Tahvilian R., and Rafeepour Z, A Simple Sample Preparation with HPLC-UV Method for Estimation of Clomipramine From Plasma. Iranian Journal of Pharmaceutical Research. 2010; 9(3): 243-250.

12.     Nagavi J., and Dhanesshwar S. Development and Validation of an RP-HPLC Method for Determination Clomipramine Hydrochloride. Indo American Journal of Pharmaceutical Research. 2014; 4(4): 1939-1948.

13.     Elqudaby HM., and Frage YZ. Optimized and Validated Spectrophotometric Methods for the Determination of Clomipramine Hydrochloride and Paroxetine Hydrochloride in Drug Formulation. International Journal of Research in Ayurveda and Pharmacy. 2012; 3(4): 537-546.

14.     ICH (International Conference on Harmonization) Q3B (R2) Impurities in new drug substance. 2008

15.     International Conference Harmonization. ICH Q2 (R1): Validation of Analytical Procedure. Text and Methodology. 2005.

 

 

 

 

 

 

Received on 24.03.2017       Accepted on 18.06.2017     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2017; 7(2): 100-112.

DOI:  10.5958/2231-5675.2017.00017.5