Development of Stability Indicating RP-HPLC Method for Estimation of Clemastine Fumarate and its Pharmaceutical Dosage Form

 

Ingole L. R.

Ravi Institute of Diploma in Pharmacy, Koradi, Nagpur

 

ABSTRACT:

A simple, fast and precise stability indicating RP-HPLC method was developed for quantification of Clemastine Fumarate in pure and pharmaceutical dosage form. The quantification was carried out using LiChrospher® 100 RP-C₈ endcapped column (5µm,150 x 4.6mm) and mobile phase comprised of methanol and water (0.05% triethyamine) in proportion of ratio 90:10 and degassed under ultrasonication. The flow rate was 0.8ml/min and effluent was monitored at 220nm. The retention time of Clemastine Fumarate was found to be 7.49±0.039 min and peak was separated. The method was validated in terms of linearity, precision, accuracy, specificity, robustness, limit of detection and limit of quantitation in accordance with ICH guideline. Linearity of Clemastine Fumarate was in the range 15-50µg/mL. The percentage recovery of Clemastine Fumarate were 98.46 to 99.99% from tablet formulation. The stability –indicating capability was established by forced degradation experiments. The proposed method suitable for determination of Clemastine Fumarate in pharmaceutical dosage form.

 

 

 

INTRODUCTION:

Clemastine fumarate is a selective histamine H₁ antagonist used for relief of symptoms associated with allergic rhinitis such as sneezing, rhinorrhea, pruritus, lacrimation and multiple sclerosis. It competitively bind to histamine receptor sites, thus reducing the neurotransmitter’s effects. It also inhibit both vasoconstrictor and vasodilator effect of histamine. Clemastine is an antihistamine with anticholinergic and sedative effects. It also act as FIASMA (functional inhibitor of acid sphingomyelinase.²³

 

 

 

 

It is official in USP 32/NF 27and represented by following structure^20,21,22

 

 

Fig 1: Structure of Clemastine Fumarate

 

Since, Clemastine Fumarate is widely used in antihistamine therapy, it is important to develop and validate analytical method for its determination, in pharmaceutical doses form. Several chromatographic techniques have been used for the determination of Clemastine Fumarate, including LC-MS²³, GC-Nitrogen-Phosphorous detection²⁴, HPLC-MS²⁸. Since, stability indicating assay method (SIAM) could not been traced in scientific literature for selective/ specific estimation of Clemastine Fumarate and its degradation products, it was felt necessary to develop a stability –indicating method for determination of Clemastine Fumarate in bulk drug and pharmaceutical dosage form. Hence, it was proposed to develop Validated Stability Indicating Assay Methods for assay of Clemastine Fumarate in bulk and pharmaceutical formulations as per ICH guideline. For this purpose HPLC techniques were thought to be exploited as they are known to be more specific and sensitive for quantitation of analyte. HPLC is the most suitable technique for estimation of drugs and degradation products in very low quantities.

 

Hence, to develop a precise, accurate, reliable, rapid, simple, and specific SIAM for Clemastine Fumarate and its degradation products adopting HPLC method.

 

MATERIALS AND METHOD:

Chemicals and reagents:

Pharmaceutical grade Clemastine fumarate was procured as a gift sample from Wanbury Ltd, Mumbai (India). The tablet formulation of Clemastine Fumarate (Clamist Tablet 1 mg, Wanbury Ltd, Mumbai) was purchased from the local market. Methanol and water used in analysis were of HPLC grade. Triethylamine, hydrochloric acid, sodium hydroxide, hydrogen peroxide used were of analytical grade. Milli Q water and Whatmann filter paper Grade-I were used throughout the experimental work. The solvents were further filtered through nylon membrane filter (0.45 μ, 0.20 μ) and vacuum degassed. The 0.45 μ and 0.20μ Nylon filter papers were purchased from Pall India Pvt. Ltd, Mumbai (India).

 

HPLC Instrumentation and Chromatographic condition:

The analytical separation were carried out on a Shimadzu (LC-20AB prominence with LC solution software) HPLC system equipped with Photo Diode Array detector (SPD-20A) and output signal was monitored. The analytical column was LiChrospher® 100 RP-C8 endcapped column (5µm, 150 x 4.6mm). Mobile phase consisted of Methanol:Water (0.05% triethylamine) in the ratio 90:10. Mobile phase was mixed, filtered through 0.45μ membrane filter and degassed under ultrasonication. The flow rate was 0.8mL/min and run time was 10 minutes. The column was maintained at ambient temperature. UV detection was measured at 220 and the volume of sample injected was 20μl.

 

Standard preparation:

Stock Standard solution (Solution A):

Accurately weighed quantity of Clemastine Fumarate (10.0 mg) was dissolved in methanol to make 10.0 ml solution. (1.0 mg/mL).

 

Working Standard Solution (Solution B):

Accurately measured 1.0 ml of standard stock solution A was diluted to 10.0 ml with methanol (100.0 mg/mL).

Working standard solution (Solution C):

Accurately measured 2.0 ml of working standard solution B was diluted to 10.0 ml with mobile phase (20.0 mg/mL) and used for analysis.

 

 

(a)

 

(b)

Figure 2 : (a) Standard Chromatogram (b) in situ UV spectrum of Clemastine Fumarate

 

RESULTS AND DISCUSSION:

HPLC method development and optimization:

To optimize the chromatographic conditions, different column, mobile phase, flow rate etc., were tested. Methanol: Water (0.05% triethylamine) in the ratio (90: 10 v/v) was preferred as mobile phase at flow rate 0.8 ml/min and gave a retention time of 7.79± 0.039 min. with sharp peak, good peak symmetry and minimum tailing factor. The optimized chromatographic parameters were listed :

 

Elution	:	Isocratic
Colum	:	LiChrospher® 100 RP-C8 endcapped column (5µm, 150 x 4.6mm)
Mobile phase	:	Methanol :Water (0.05% triethylamine) (90:10 v/v)
Detection Wavelength	:	220 nm
Injection volume	:	20 µL (Rheodyne injector)
Flow rate	:	0.8 mL/min
Temperature	:	25°C (Room Temperature)
Retention time	:	7.49 min

 

 

Forced degradation study (Stress studies):

The stress studies were initiated at an initial concentration of 1 mg/mL solutions of Clemastine Fumarate (API) and the periodically withdrawn stressed samples on appropriate dilution conc.20 µg/mL with methanol and exposing it to various stress condition to study the effect over wide range of pH, heat, oxidation and sunlight. All the stability samples were analyzed to study the extent of degradation and if the reasonable degradation (5-20%) with respect to parent drug has seen, the stress testing was stopped at that point. However, no degradation was observed under photo and heat stress conditions.

 

 

A) Acid (0.1M HCl, 1 hr RT)                                                                        B) Base (0.1M NaOH, 1 hr RT)

 

C) Oxidation (3% H₂O₂, 24 hr at R.T)                                                 D) Neutral Stress, 8hr reflux

 

 

E) Photolytic Stress, (5 days)                                                      F) Thermal Stress, (dry heat, 5 days at 100⁰C)

 

Figure 3: HPLC Chromatograms of Forced Degraded Samples (A-F)

 

Table 1: Summary of forced degradation studies by HPLC

 

 

Table 2: Study of System Suitability Parameters

 

The HPLC methods were optimized to resolve the degradants and the parent drug from one another. The results of Forced degradation studies were shown in table 1.

 

Validation of the method:

When method development and optimization are complete, it is necessary to accomplish method validation. The validation studies include linearity, accuracy, precision, sensitivity studies (LOD and LOQ), specificity, robustness and ruggedness.

 

System suitability studies:

The chromatographic conditions were set as per the optimized parameters and mobile phase was allowed to equilibrate with stationary phase to get steady baseline. Five replicate injections of working standard solution C were made separately and the chromatograms were recorded. One of the standard chromatogram is depicted in Fig- 2. and the results of system suitability parameters are given in Table-2

 

Linearity of Response:

Aliquot portions of standard solution B (1.5, 2, 2.5, 3, 4, 5 ml) were diluted to 10.0 ml with mobile phase to get concentration 15-50 µg/mL. The chromatographic conditions were set as per the optimized parameters and mobile phase was allowed to equilibrate with stationary phase to get the steady baseline. Prepared standard solutions of different concentration were injected separately and the chromatograms were recorded. A graph was plotted as peak area vs. concentration of drug (µg/mL) and is depicted in Fig- 3

 

The excellent correlation between Clemastine peak area and concentration was observed with R²=0.999.The regression equation was found to be Y=41049x + 32877. Statistical data are presented in table 3.

 

Figure 3: Calibration graph of Linearity studies

 

Table 3: Results of Linearity Studies

Parameter	Result
Concentration range(µg/mL)	15-50
Equation for straight line	Y= 41049x + 32877
Slope	41049
Y-intercept	+32877
Correlation coefficient	0.999

 

Estimation of Clemastine fumarate in tablet by proposed HPLC method:

(Assay of pharmaceutical formulation):

Standard solution:

Working standard solution (Solution C) was prepared.

 

Sample solution:

Twenty tablets were weighed and average weight was calculated. Tablets were crushed to a fine powder. An accurately weighed quantity of tablet powder equivalent to about 10 mg of Clemastine Fumarate was shaken with about 40.0 ml of methanol for 20 minutes, the volume was made up to 100.0 ml with methanol, and solution was filtered through Whatman Grade I filter paper. 2.0 ml of the filtrate was diluted to 10.0 ml with mobile phase to get concentration of 20.0 µg/mL (on labelled claim basis). Six replicate sample solutions were prepared in similar manner.

Table 4 : Result of Estimation of Clemastine fumarate in Tablet

Procedure:

After equilibration of column with mobile phase, replicate injection of standard and each of six sample solutions were made separately and chromatograms were recorded.

 

The amount of drug present in average weight of tablet (as % of labelled claim) was calculated using following formula:

                                    A_SMP× W_STD × A_WT × 100

 % Labelled Claim = -------------------------------------

                                          A_STD × W_SMP × LC

Where,

A_SMP = Peak area of unknown sample

A_STD = Peak area of standard

W_STD = Weight of standard (µg/ml)

W_SMP = Weight (mg) of sampled tablet powder

 

LC = Labelled claim (mg/ tablet) of the drug

A_WT = Average weight (mg) of tablet

The results are shown in table 4.

 

Accuracy:

Accuracy of the proposed method was ascertained on the basis of recovery studies performed by standard addition method.

 

Standard solution:

Working standard solution C was prepared (20.0 mg/mL) as described under preparation of standard solution.

 

Sample solution:

Accurately weighed quantities of pre-analyzed tablet powder equivalent to about 1.072 (80%) mg of Clemastine Fumarate were transferred to five different 10.0 ml volumetric flasks and accurately known amount of standard Clemastine Fumarate were added to 2^nd, 3^rd, 4^th and 5^th flask respectively (flasks representing 80- 120 % of labelled claim). The drug contents were shaken manually for 20 minutes. Sufficient methanol was added to each flask to adjust the volume to 10.0 ml mark and filtered. 2.0 ml of each of the filtrate was diluted to 10.0 ml with mobile phase.

 

Procedure :

Same as described under estimation of Clemastine Fumarate in tablet.

Amount of C.F. (mg/mL) obtained by using following formula-

             A_SMP  x  W_STD  x  A_WT

T= ------------------------------------------

             A_STD x  10 x  W_SMP  x  LC

 

The percent recovery was then calculated using the formula:

                                  T-B

% Recovery= ------------------- X 100

                                   C

Where,

T =Total drug estimated (mg)

Ew = Weight (µg) of drug calculated from instrument in Vs

Vs = Volume (µL) of sample solution applied

B = Amount of drug contributed by pre-analyzed tablet powder (mg)

C =weight of pure drug added (mg)

 

The results of accuracy (recovery) studies are shown in Table- 5

 

Precision:

Precision of analytical method is expressed as SD or % RSD of series of replicate measurements. Precision of estimation of CF by proposed HPLC method was ascertained by replicate analysis of homogeneous samples of tablet powder. The results are as shown in table 4.

 

Linearity and Range:

Linearity of response:

Chromatographic response (peak area) as a function of concentration was studied and results are depicted in Fig. 4.8 and tabulated in table- 3.

 

Range of method:

From the data obtained under the accuracy studies, a graph was plotted as % labelled Claim vs peak area, which was observed to be linier over the range of 80 – 120 % of labelled claim, (Fig. 4) and result of study is given in tabular form in Table- 6.

 

 

Table 5: Result of Recovery Studies

 

 

Fig 4: Range of method by area

 

Table 6: Results of Range of Method

Parameters	Results
Range	80- 120% of labelled claim
Slope	18076
Y-intercept	- 15680
Correlation coefficient	0.975
Equation for straight line	y = 18076x – 15680

 

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

LOD and LOQ were determined by the method based on standard deviation of the response and the slope of calibration curve as per ICH guidelines and are as follows:

              3.3σ                                        10σ

LOD = ------           and           LOQ = --------

               S                                               S

Signal to noise ratio (k) = 3.3 and 10 for LOD and LOQ respectively

s = Standard deviation of response (Estimated by measuring the response in terms of peak area of standard solution of conc. 15.0 µg/mL for five times and s was calculated) = 39716.25

S = Slope of calibration curve (obtained from calibration curve) = 41049

The results are shown in Table- 7.

 

 

Table 7: Results of LOD and LOQ Studies

Sr. No	Parameters	Values
1.	LOD (µg/mL)	3.19
2.	LOQ (µg/mL)	9.68

 

Specificity:

Standard solution:

Working standard solution was prepared (20.0 mg/mL) as described under preparation of standard solution.

 

Sample solution:

Accurately weighed quantities of tablet powder equivalent to about 1 mg of Clemastine Fumarate were transferred to six different 10.0 ml volumetric flasks. The samples were then exposed to stress conditions as follows:

a.     Normal (control) for 24 hr

b.     Room temperature at 1 hr after addition of 0.1M HCl

c.     Room temperature at 1 hr after addition of 0.1M NaOH

d.     At room temperature in dark after addition of 3 % H₂O₂ for 24 hr.

e.     At 80⁰ C (dry heat) for 24 hr

f.      Sunlight for 24 hr

 

After stipulated time of each stress conditions, flasks were shaken for 20 minutes and filtered. 5.0 ml each of filtrates was diluted to 10.0 ml with mobile phase and analyzed in similar manner as described under estimation of Clemastine Fumarate in tablets. The results of specificity study are shown in Table 8. The chromatograms of specificity study are shown in Fig- 5.

 

 

Table 8: Results of Specificity Studies

Sr. No.	Sample	% of Labelled claim
1	Normal	100.79
2	Acid	87.01
3	Alkali	81.03
4	Oxide	92.04
5	Neutral	82.14
6	Heat	99.18
7	Sunlight	99.06

 

 

A) Normal                                                                                            B) 0.1 M NaOH

 

 

C) 0.1 N HCl                                                                                   D) 3% H₂O₂

 

 

E) Neutral                                                                                              F) Sunlight

 

 

G) Thermal

Figure 5: HPLC Chromatograms of Specificity Studies

 

Robustness:

The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The studies were carried out by changing the wavelength of detection.

 

The results are shown in Table- 9

 

Table 9: Results of Robustness Studies

% Estimation	Change in wavelength (± 2 nm)
	Clamist®
	220.0	218.0	222.0
Mean*	98.51	99.74	99.34
± SD	0.334	0.258	0.236
% RSD	0.339	0.259	0.238

 

Ruggedness:

The studies were carried out for two different parameters i.e. for different time intervals (Intra-day and Inter-day) and different analysts.

 

a.     Intra-day and Inter-day:

The samples were analyzed at different times on same day and on different days. The percent of labelled claim were calculated and the results are shown in Table 10-a

 

b.    Different Analysts:

The samples were analyzed by two different analysts by proposed method. The results are shown in Table 10-b.

 

 

 

10-a: Result of Ruggedness by Intra-day and Inter-day

Sr.No.	Observation	% of labelled claim
		Clamist®
		Intra Day	Inter Day
1.	I	99.51	99.25
2.	II	98.79	98.53
3.	ІІІ	99.18	98.09
Mean*		99.16	98.62
±SD		0.3601	0.5856
%RSD		0.3631	0.5937

 

Table 10-b: Result of Ruggedness By different Analyst

Sr. No.	Obs.	% of labelled claim
		clamist®
		Analyst I	Analyst II
1.	I	98.51	98.04
2.	II	98.79	98.60
3.	III	98.12	98.02
Mean*		98.47	98.22
±SD		0.3365	0.3292
%RSD		0.3417	0.3352

 

CONCLUSION:

A simple, rapid, accurate, and precise RP-HPLC method for analysis of Clemastine Fumarate in pure and tablet dosage form had been developed and validated in accordance with ICH guidelines. The results of assay of Clemastine Fumarate tablet obtained by proposed HPLC method are quite concurrent and reproducible. The recoveries of the drug from tablet matrix were about 100% indicating accuracy and reliability of method and non-interference of excipients. Hence, it may be adopted for routine assay of Clemastine Fumarate free of interferences from its degradation products in tablet formulation. The proposed HPLC method in true sense can be said to be specific Stability Indicating Assay Method for Clemastine Fumarate, due to its capacity to estimate the drug content unequivocally free of interference from its degradation products.

 

Table 11: Summary of validated parameters for proposed method

 

REFERENCES:

1.    ICH, Q1A (R2), “Stability Testing of New Drug Substances and Products”, International Conference on Harmonisation, IFPMA, Geneva, 2003 (2).

2.    FDA, Guidance for Industry; Stability Testing of Drug Substancesand Drug Products (Draft guidance), Food and Drug Administration; Rockville, MD, 1998.

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7.    ICH, Q1B, “Stability Testing: Photo stability testing of New Drug Substances and Products”, In Proceedings of the International Conference on Harmonisation; IFPMA, Geneva, 1996.

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26. W. S. Hassana, Magda M. El-Henaweea, Ayman A. Gouda, ‘‘Spectrophotometric determination of Diphenhydramine hydrochloride, Chlorphenoxamine hydrochloride and Clemastine by Ion- pair complexs”. Spectrochimica Acta Part –A 69,2008 p. 245-255.

27. S. Soad. Abd El-Hay, M. Y. El-Mammli and A. A. Shalaby, ‘‘Spectrophotometric determination of Clemastine hydrogen fumarate, Fexofenadine hydrochloride and Moxepril hydrochloride through Ion-pair formation with chromatrope 2R”. Res. J. Pharm. Bio. Chem. Sci. (RJPBCS) 2011, 2(1), p. 497- 509.

28. V. Horvath, A. Tolokan, A. Egresi, ‘‘High-performance liquid chromatography –Tandem mass spectrometric method for determination of Clemastine in human plasma”. J. chromatogr. B. 816 2005 p. 153-159.

 

 

 

 

 

Received on 09.02.2019         Modified on 20.03.2019

Accepted on 04.04.2019 ©A&V Publications All right reserved