INTRODUCTION TO MOUTHWASH:

Pazopanib (PZP) is an antineoplastic agent used in the treatment of advanced renal cell cancer and advanced soft tissue sarcoma in patients with prior chemotherapy. It is a second-generation multitargeted tyrosine kinase inhibitor.

Pazopanib is chemically 5-({4-[(2,3-dimethyl-2H-indazol-6-yl)(methyl)aminopyrimidin-2-yl}amino)-2-methylbenzene-1-sulfonamide¹and structure of Pazopanib is shown in fig.(1).Pazopanib is a synthetic indazolyl pyrimidine and reaches steady state concentrations of >15μg/ml.

This concentration is high enough to observe maximal inhibition of VEGFR2 phosphorylation and some anti-tumour activity (concentration required to inhibit receptors is 0.01 - 0.084μmol/L)². The oral dose formulation containing Pazopanib 200mg and 400mg is available in the market with a brand name Votrient.

Literature survey revealed that very few methods are available for the analysis of Pazopanib in bulk and in pharmaceutical formulations. We could find HPLC papers^3-4 on stability also spectrophotometric method⁵but no single method for HPTLC hence a stability indicating method was proposed for analysis of Pazopanib. Thus, proves necessity to develop a method which proves to be operative. The present work focused on development of simple, economic and fast stability indicating High-Performance Thin-Layer Chromatographic method for determination of Pazopanib. The HPTLC method was optimized and stability indicating studies were performed as per ICH Q1A(R2) and ICH Q1B guidelines. The developed method was validated as per ICH Q2(R1) guidelines.

Fig 1- Structure of Pazopanib hydrochloride

MATERIAL AND METHOD:

Materials:

Working standard of Pazopanib was received as a gift sample from NATCO Pharma Limited, Kothur, India. All reagents used in the chromatography such as methanol, chloroform, HCl, NaOH, H₂O₂were of AR grade and Aluminium TLC plate precoated with silica gel 60 F₂₅₄were purchased from E. Merck Ltd., Mumbai India.

Methods:

HPTLC System: CAMAG TLC system (Muttenz, Switzerland)

Software: winCAT’s version (1.4.3)

Syringe for Sample Application: 100μl syringe (Hamilton, Bonaduz, Switzerland).

TLC Plate: Aluminium plates precoated with silica gel 60 F₂₅₄ plates (E.Merck, Germany)

Sample Applicator: CAMAG Linomat-V

Gas for Sample Application: Nitrogen

Development Chamber: CAMAG twin trough chamber (10 X 10cm)

TLC Scanner: CAMAG TLC Scanner 3

Chromatographic condition:

Stationary Phase: Aluminium TLC plate precoated with silica gel 60 F₂₅₄ (10 x 10cm)

Mobile Phase: Chloroform: Methanol (9:1v/v)

Saturation Time of Mobile Phase: 15min

Development Time (Run): 10min

Scanning wavelength: 271nm

Rf: 0.55±0.01

Selection of detection wavelength:

λ_maxis selected from UV spectrum of solution 10µg/ml. It was observed that Pazopanib showed good absorbance at 271nm wavelength which is shown in fig (2).

Preparation of stock solution:

Standard stock solution of drug was prepared by weighing 10mg of drug and transferred to a 10ml volumetric flask and volume was made up to10ml with the methanol to get a concentration of 1000µg/ml. The solution was sonicated for drug to dissolve completely. The solution was further diluted as per requirement.

Fig 2: UV spectrum of Pazopanib HCl (λ_max= 271nm)

Preparation of sample solution:

From the standard stock solution 1ml is diluted with methanol to make final volume of 10ml (Concentration 100µl/ml of API).

Stress degradation studies of bulk drug:

To confirm the stability indicating nature of the developed HPTLC method. Pazopanib is subjected to stress conditions as per ICH Q1A (R2)⁶and photo stability as per Q1B¹⁹guidelines.

The stress conditions were Acid hydrolysis (0.5N HCl), Base hydrolysis (0.5N NaOH), Oxidation (30% H₂O₂), Thermal degradation (60˚C for 6hrs) and Photolytic studies as UV illumination (up to 200-watt hours/square meter) and Cool white fluorescent light (up to 1.2 million lux hours) with the objective of getting only 10 to 30% degradation⁷.

For stress degradation, six samples were prepared by transferring 10mg of Pazopanib in 10ml volumetric flasks and added 1ml of 0.5N HCl, 0.1N NaOH and 30% H₂O₂ in each flask. Similarly thermal and photolytic study was performed as per the protocol given in ICH guideline. We had referred to other chromatographic methods in literature to execute the work^8-10

Validation of HPTLC method:

The proposed HPTLC method was developed and validated as per the International Conference on Harmonization ICH Q2 (R1) guidelines¹¹.

Specificity:

The specificity of the method was ascertained by peak purity profiling studies.

Assay:

A blend of commonly used excipients was spiked using Pazopanib. The amount equal to average weight of tablet was taken and dissolved in methanol to obtain a concentration equivalent to 100µg/ml and it was filtered through whatmann filter paper. It was analyzed by developed HPTLC method.

Accuracy:

To check accuracy of the method, recovery studies were carried out by adding standard drug to sample at three different levels 50, 100 and 150% these solutions were applied on TLC plates in triplicates to obtain the densitogram. The drug concentration of Pazopanib was calculated by using linearity equation.

Precision:

The precision of the method was demonstrated by intra-day and inter-day variation studies. In intra-day precision, solution containing 100µg/ml of Pazopanib was prepared and six replicates of 6µl were spotted. Peak area was noted for all. % RSD was calculated.

In the inter-day variation studies, from the same above solution containing 100µg/ml of Pazopanib, six replicates of 6µl were spotted. This procedure was repeated on three consecutive days. Peak area was noted for all and % RSD was calculated.

Limit of detection and Limit of quantification:

The sensitivity of this method was estimated in terms of LOD and LOQ. The SD of the lowest responses and slopes of calibration curves were used for the determination of LOD and LOQ during HPTLC method validation. The LOD and LOQ are calculated using equations LOD = 3.3*SD/S and LOQ = 10*SD/S Where, SD of peak areas of the drug and S is slope calibration curve.

Linearity:

From the standard stock solution (1000µg/ml) of Pazopanib, futher dilutions were prepared. The different volumes 2 to 10µl were spotted. The linearity (relationship between peak area and amount spotted) was determined by analyzing over the range of 200-1000 ng/band for Pazopanib.

Robustness:

Robustness of the method was determined by carrying out the analysis under conditions during which mobile phase ratio and chamber saturation time were altered and the effects on the Rf values and area were noted.

RESULT:

Selection and optimization of mobile phase composition:

To get acceptable peak parameters, different mobile phase compositions were tried. After different trials the suitable mobile phase was found to be Chloroform: Methanol (9:1v/v). The TLC plate was scanned at 271 nm wavelength. A symmetrical and well resolved peak was obtained for Pazopanib at Rf of 0.55(±0.01). The representative HPTLC densitogram of Pazopanib.

Forced degradation study:

The degradation was found maximum in photolytic condition (Fluorescence) (32.85%), minimum in acidic condition (4.90%) and no degradation found in oxidative condition^3-4.

Fig 3: Representative densitogram of Pazopanib (Rf 0.55)

Fig 4: 3Ddensitogram of standard linearity and photolytic degradation(fluorescence) (track 1- blank, 7- stress sample, 2 to 6- standard linearity)

Method validation:

The method validation was done using ICH Q2(R1) guideline¹¹.

Specificity:

The peak purity values for drug peak under stress conditions were found to be more than 0.995, indicating the non-interference of any other peak of degradation product or impurity.

Assay:

Results for assay were found to be within limits and are depicted in Table 2

Table 2: Assay results of pazopanib

Sr. No.	Amount (ng/band)	Mean peak area of Pazopanib	Mean amount recovered	Mean % recovery
1.	400	7725.9	396.63	99.15%
2.	400	7693.1	394.38	98.59%
3.	400	7720.3	396.24	99.06%

Accuracy (Recovery studies):

To examine the accuracy of developed method, recovery studies were performed by standard addition method and the results are expressed as percent recovery. The mean percentage recovery was calculated at each concentration level.

Table 3: Result of accuracy (recovery study) for pazopanib

Sr. No	Level of recovery	Amount of sample taken (ng/band)	Amount standard spiked (ng/band)	% Recovery
1.	50%	400	200	101.81%
2.	100%	400	400	99.97%
3.	150%	400	600	101.23%

Above data obtained from three replicates (n=3) from each concentration.

Precision:

Repeatability, Intra-day and inter-day precisions were determined using six repetitive measurements of a solution (100µg/ml). The application volume of Pazopanib solution was 6µl for the HPTLC method validation. The precision of developed method was expressed in terms of SD and %RSD of the peak area

Table 4. Result of precision study of pazopanib

Replicates	Intra-day area	Inter-day area
Replicates	(600 ng/band)	(600 ng/band)
1	10789.3	10569.8
2	10832.7	10805.2
3	10648.9	10946.9
4	11019.7	11019.7
5	10650.3	10710.5
6	10902.4	10690
Std. Dev.	144.821	168.771
%RSD	1.340	1.564

LOD and LOQ:

The sensitivity of developed method was estimated in terms of a Limit of Detection (LOD) and Limit of Quantification (LOQ). The LOD and LOQ were found to be 19.38ng/band and 58.74ng/band, respectively.

Linearity and Range:

The linearity was found in the range of 200-1000ng/band. The linearity of Pazopanib at 271nm was shown in fig (6). The linearity equation for drug is y=14.58x + 1943 with coefficient of correlation (R²) 0.998 fig. (7).

Fig 6: Linearity of Pazopanib in 3D spectrum (track 1- blank, 200-1000ng/band)

Fig 7: Calibration curve for Pazopanib

Robustness:

As there was no significant change in the Rf value of Pazopanib (standard Rf values 0.55 ± 0.02). Each sample was studied in triplicate (n= 3) and the obtained peak areas are utilized to calculate % RSD, which was found to be less than 2 suggesting method is robust. The result of robustness is depicted in table 5.

Table 5. Result of robustness study of pazopanib

Factor	Level	Peak area (for 200ng/band)	Rf
Mobile phase composition (±0.1ml)
7.9:2.1	-0.1	4969.9	0.53
9:1	0	4897.1	0.55
9.1:0.9	+0.1	4930.2	0.55
	RSD:	0.7389
Duration of chamber saturation (± 5min)
13 min	-5	4958.6	0.54
15 min	0	4899.9	0.55
17 min	+5	4986.7	0.55
	RSD:	0.8950

Summary of validation parameters:

The summary of validation parameters is depicted in table 6.

Table 6: Summary of validation parameters

Sr. No.	Validation parameters		Result
1.	Linearity equation		y = 14.58x + 1943
	R²		0.998
	Range		200-1000ng/band
2.	Precision		% RSD
	a. Intraday		1.61
	b. Interday		1.90
3.	Assay		98.56%
4.	Accuracy	50% level	101.81%
		100% level	99.97%
		150% level	101.23%
5.	LOD		19.38ng/band
6.	LOQ		58.74ng/band
7.	Specificity		Specific
8.	Robustness		Robust

DISCUSSION:

A simple, fast and economical stability indicating HPTLC method was developed and validated for Pazopanib. Since there was no HPTLC method published for the stability studies and literature reported variations in the results of HPLC stress conditions. Thus, proves necessity to develop a method which turn out to be operative.

Shabada et al³ developed HPLC method where no details of degradation were reported. Ashok et al¹²developed HPLC method where all stress conditions as per ICH were not studied and Kishor et al⁴developed HPLC method where no degradation was found under any condition. We have optimized all stress conditions to obtain 5-30% degradation. K Vinodkumar et al¹³developed stability indicating HPLC method where the results of % degradation matched with our results with little variations at thermal and oxidation condition. We had referred to other chromatographic methods in literature to plan the work^14-18.

The photolytic degradation as cool white fluorescent light condition is not reported in any paper mentioned above, but we have done cool white fluorescent condition as per ICH Q1B¹⁹ and the degradation was found to be maximum i.e. 32.84%.

Thus, method proved to be repeatable, precise, accurate and robust. The procedure is simple, rapid and inexpensive in comparison with other chromatographic methods.

ACKNOWLEDGEMENT:

The authors are thankful to the principal and the management AISSMS College of Pharmacy, Pune for providing excellent infrastructure and facilities for undertaking this research work.

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Received on 16.02.2024 Modified on 13.05.2024

Res. J. Pharma. Dosage Forms and Tech.2024; 16(3):210-214.

DOI: 10.52711/0975-4377.2024.00033

Condition	Time	Rf of stressed drug	Mean % recovery	Mean % degradation	Peak purity
Condition	Time	Rf of stressed drug	Mean % recovery	Mean % degradation	r(s,m)	r(m,e)
0.5N HCl	24hrs	0.55	95.10%	4.90%	0.999258	0.998141
0.5N NaOH	24hrs	0.55	91.34%	8.66%	0.999787	0.996571
30% H₂O₂	7 days	0.55	100%	0%	0.999155	0.999875
Thermal	6hrs	0.56	87.56%	12.44%	0.999303	0.997752
UV	Up to 200watt hours/square meter	0.55	94.81%	5.19%	0.999811	0.999656
Fluorescence	Up to 1.2 million lux hours	0.55	67.15%	32.85%	0.999785	0.999746