Stability Demonstrating RP-HPLC Method Development of Pazopanib Hydrochloride for Rapid and Sensitive Quantification

As an anti-cancer drug, GlaxoSmithKline created pazopanib, a multi-targeted tyrosine kinase inhibitor (TKI) with anti-tumor effects. For its effectiveness in treating advanced soft tissue sarcoma (STS) and advanced renal cell carcinoma (RCC), this drug has received approval from the European Union (EU) and the US Food and Drug Administration (USFDA). The main way that pazopanib works is through its antiangiogenic qualities, which show up as an inhibition of intracellular tyrosine kinase activity linked to the platelet-derived growth factor receptor (PDGFR) and the vascular endothelial growth factor receptor (VEGFR). [1] Pazopanib hydrochloride, a white to slightly yellow solid powder with poor solubility that belongs to BCS class II, is the active pharmaceutical ingredient (API). For oral use, PZB tablets (marketed by Novartis as VOTRIENT) come in 200 and 400 mg dosages; a daily maximum of 800 mg is advised. Along with inactive substances including magnesium stearate, microcrystalline cellulose, povidone, sodium starch glycolate, and particular film coatings based on the tablet's strength, the active ingredient in the tablets is PZB. Hepatotoxicity, hypertension, hypothyroidism, arterial thrombosis, and genotoxicity are among the side effects of pazopanib that can be linked to contaminants in the medication's ingredients and byproducts. Therefore, in order to preserve product integrity and protect patient health, it is now crucial in pharmaceutical development to detect, identify, measure, and control such contaminants. [2-4] Over the years, liquid chromatography has evolved from a preferred analytical method into a cost-effective and nearly essential tool for drug analysis. RP-HPLC, in particular, has played a crucial role in advancing analytical research due to its wide-ranging applications across pharmaceuticals, food, polymers, plastics, environmental studies, and clinical settings. Its key advantages include high speed, enhanced sensitivity, and the capability for simultaneous detection with automated operation. [5] Notably, the integration of tandem mass spectrometry (HPLC-MS) has revolutionized the precise quantification of drugs in biological matrices, as well as in stability testing and impurity profiling, outperforming traditional analytical techniques. [6]

Fig. 1.: Structure of pazopanib hydrochloride

A substantial amount of research has been conducted on developing QbD-based methods for analyzing PZB in biological samples and complex formulations. In contrast, this study presents a straightforward analytical method for PZB using same framework, incorporating a novel mobile phase (MP) composition that has not been previously reported. As a result, the developed method is suitable for determining drug content in various non-biological samples and alternative dosage forms, offering improved reliability and a reduced likelihood of failure. [7-11]

1. REAGENTS AND INSTRUMENTATION:
   1. Reagents:

Pazopanib (PZB) was generously supplied as a free sample by Natco Pharma Pvt. Ltd. (Hyderabad, India), and commercial tablets containing 400 mg of PZB were also obtained from the same source. O-phosphoric acid and HPLC-grade methanol were sourced from Merck Specialties Pvt. Ltd. (Mumbai, India). Milli-Q HPLC-grade water, filtered using a Milli-Q system (Millipore GmBH, Germany), was used throughout the study. All solvents, chemicals, and reagents employed in the development and validation of the analytical method were of HPLC-grade analytical quality.

1. 2. Instrumentation

A high-performance reverse-phase HPLC system (Agilent Technologies Model 1100, Canada) was utilized, featuring a quaternary gradient pump (G130A) and a photodiode array (PDA) detector (G13148 DAD), along with an autosampler for automated sample injection. Instrument parameters were controlled using CHEMSTATION software (version 10.1). The column was maintained at ambient room temperature throughout the analysis. For accuracy and precision, all standard substances were carefully weighed using a calibrated analytical balance (ME204/AD4, Mettler Toledo, Switzerland).

1. 3. Selection of wavelength:

A primary standard solution of PZB at a concentration of 20?µg/mL was prepared by dissolving the drug in methanol, intended for generating a UV-visible (UV-Vis) absorption spectrum. An aliquot of this solution was placed in a quartz cuvette and scanned using a UV-2600 double-beam UV-Vis spectrophotometer (Shimadzu, Japan) over the 200–800?nm wavelength range, with methanol serving as the blank. The UV absorption spectrum was then recorded to identify the isosbestic point and determine the wavelength of maximum absorption (λ max) for the drug.

1. 4. Chromatographic conditions:

Pazopanib was effectively separated on a chromatogram using an Agilent reverse-phase C18 column (4.6 × 250 mm, 5 µm particle size) that had been pre-equilibrated with the mobile phase mixture. The analysis was performed under isocratic conditions with a mobile phase consisting of HPLC-grade 0.1% o-phosphoric acid and methanol in a 59:41 v/v ratio. Prior to use, the mobile phase was filtered through a 0.45 µm membrane filter using a vacuum filtration system and sonicated for 20 minutes. The flow rate was maintained at 1.1 mL/min, the column temperature was set at 25°C, and the injection volume was 20 µL.

1. 5. Standard and sample solutions preparation:

A standard stock solution of PZB with a concentration of 500?µg/mL was prepared using the mobile phase and stored at room temperature, protected from light with aluminum foil. From this primary stock, secondary standard solutions with concentrations of 5, 10, 15, 20, and 25?µg/mL were prepared by appropriately diluting the stock solution with the same mobile phase.

1. 6. Assay validation:

The developed RP-HPLC method for the identification and quantification of pazopanib was validated in accordance with the technical guidelines set by the International Council for Harmonisation (ICH) for pharmaceuticals intended for human use. As part of the validation, key parameters such as specificity, linearity, range, precision, accuracy, sensitivity, and robustness were thoroughly assessed. Additionally, system suitability testing was performed following the standards outlined by the United States Pharmacopeia (USP). [12,13]

1. 1. 1. Specificity:

The specificity of the developed method was evaluated by measuring PZB in tablet formulations. The chromatogram was examined to check for any interfering peaks at the retention time of the analyte. Additionally, the method’s specificity was further tested by exposing PZB to forced degradation to identify any possible interference from degradation products at PZB’s retention time.

1. 1. 2. Linearity and range:

An exact amount of PZB was dissolved in 20 mL of the mobile phase to prepare the primary stock solution with a concentration of 0.5 mg/mL. Working standard solutions were then prepared by diluting this stock to concentrations of 5, 10, 15, and 20 µg/mL. To evaluate linearity and range, each working standard solution was injected twice. The peak area (Pa) for each concentration was recorded, and a plot of concentration versus peak area response was created. By plotting both the peak area and response factor against concentration for each calibration standard, the method’s range for PZB was established.

1. 1. 3. Accuracy:

Accuracy was determined by calculating the percentage recovery of PZB in quality control (QC) samples at low (LQC), middle (MQC), and high (HQC) concentration levels. Duplicate samples were prepared for each level, containing known PZB concentrations of 4, 5, and 6 µg/mL for LQC, MQC, and HQC, respectively. The percentage recovery and relative standard deviation (RSD) were then computed.

1. 1. 4. Precision:

Precision of the analytical method was evaluated by assessing intra-day (repeatability) and inter-day (intermediate) precision using the percentage relative standard deviation (%RSD). Duplicate quality control (QC) samples at three different concentrations were analyzed to investigate both types of precision. For repeatability, LQC (5 µg/mL), MQC (15 µg/mL), and HQC (25 µg/mL) samples were prepared and tested on the same day. To assess inter-day precision, three separate QC samples were prepared and analyzed over three consecutive days under consistent experimental conditions.

1. 1. 5. Sensitivity:

The limit of detection (LOD) assesses the sensitivity of the analytical method through a visual determination approach. The LOD and limit of quantification (LOQ) were calculated using the formulas 3.3 × standard deviation divided by the slope and 10 × standard deviation divided by the slope, respectively.

1. 1. 6. Robustness: 

The robustness of the method was evaluated by deliberately changing chromatographic parameters such as flow rate (FR), wavelength, and mobile phase (MP) composition. The MP composition was varied by ±1%, the flow rate by ±0.04 mL/min, and the wavelength by 2 nm. The effects of these changes on retention time (Rt) and peak area (Pa) were examined to assess the method’s robustness.

1. 1. 7. System suitability:

Suitability testing was conducted in accordance with USP guidelines to verify that the chromatographic system was appropriate for the intended analysis. The test involved three injections of a standard stock solution containing 15 µg/mL of PZB to determine parameters such as theoretical plates (Tp), peak area (Pa), retention time (Rt), and height equivalent to a theoretical plate.

1. 1. 8. Stress studies:

To evaluate the degradation behavior of PZB, it was subjected to various stress conditions. Acid hydrolysis was performed by treating 20 µg of PZB with 5 mL of 0.1N HCl, basic hydrolysis with 5 mL of 0.1N NaOH, and neutral hydrolysis with 5 mL of water, all at room temperature for 2 and 5 hours. Oxidative degradation was carried out using 3% H?O? under the same conditions and durations. Prior to HPLC analysis, the samples were neutralized, diluted with the mobile phase as needed, and then injected for analysis.

2. RESULTS AND DISCUSSION:
   1. Method development

All prepared samples were protected with aluminum foil to avoid contamination and exposure during the experiment. After thorough optimization, the RP-HPLC method for quantifying PZB was initiated using a mobile phase ratio of 59:41 v/v, comprising 0.1% o-phosphoric acid as the aqueous component and methanol as the organic component. The previously established chromatographic conditions were applied. PZB absorbance was measured at a wavelength of 266 nm, as shown in Fig. 2, with a retention time of 3.9 minutes. System suitability parameters—including retention time (Rt), peak area (Pa), theoretical plates (Tp), tailing factor (Tf), and height equivalent to a theoretical plate—were all found to be within acceptable limits.

Fig. 2.: Uv-visible spectrum of Pazopanib.

1. 2. Method validation
      1. Specificity

The chromatogram of pazopanib revealed no interfering peaks at its retention time of 3.9 minutes (Fig. 3). Although degradation products appeared under oxidative stress conditions during forced degradation studies, they did not significantly affect the retention time of PZB. Therefore, the developed and validated RP-HPLC method was confirmed to be specific for pazopanib.

Fig. 3.: RP-HPLC- chromatogram of PZB

1. 1. 2. Linearity and range

The linearity range for PZB was established between 5–20 µg/mL, as shown in Fig. 4. The calibration curve demonstrated an excellent correlation coefficient (R²) of 0.999, indicating a strong linear relationship between PZB concentration and the observed response (y-values). The equation derived from the calibration curve (Equation 1), where y denotes the response and x represents the PZB concentration, confirmed the method’s suitability for accurately quantifying both low and intermediate concentrations of PZB.

* y = 101.85x + 41.588…… Eq. 1.

Fig. 4.: Calibration curve of PZB.

1. 1. 3. Accuracy and Precision

The study evaluated the accuracy and precision of the newly developed RP-HPLC method by performing three replicates at each of the three QC levels. As summarized in Table 1, the %RSD values for both accuracy and precision were below 1%, falling within the acceptable limits set by the FDA. These results confirm the method’s high precision and accuracy.

Table. 1.: Accuracy results of PZB

1. 1. 4. Sensitivity

The analysis yielded a limit of quantification (LOQ) of 0.14 µg/mL and a limit of detection (LOD) of 0.05 µg/mL, highlighting the method’s outstanding sensitivity and reliability. These results confirm the method's capability to accurately detect and quantify the substance even at very low concentrations.

1. 1. 5. Robustness

In the developed method, deliberate modifications were made to the mobile phase composition, flow rate, and wavelength. As shown in Table 2, these changes had no significant effect on the drug’s retention time, indicating that the method is robust for the analysis of PZB.

Table. 2.: Robustness experiment results

1. 1. 6. System suitability

The system suitability parameters were carefully evaluated and found to be within acceptable limits, confirming the appropriateness of both the system and chromatographic conditions for this method. Examination of the results showed consistent peak area (Pa) and retention time (Rt) for PZB across three consecutive injections (Table 3), with %RSD values for all parameters remaining below 1%. This highlights the instrument’s high precision and the method’s ability to deliver reliable results.

Table. 3.: System suitability results for PZB (n=3).

1. 1. 7. Stress studies

A study was conducted to evaluate the percentage degradation of PZB under various stress conditions (Table 4). Overall, minimal degradation was observed across most conditions. However, a significant increase in degradation was noted after 5 hours of exposure to 0.1 N NaOH and oxidative environments. Previous research has indicated that PZB is prone to degradation under strong acidic, alkaline, and oxidative hydrolysis, particularly at elevated temperatures. In oxidative stress studies, two distinct peaks appeared in the chromatogram, indicating the formation of two degradation products with retention times of 2.1 and 4.5 minutes (Fig. 5c). Additionally, results showed more pronounced hydrolysis in alkaline conditions (Fig. 5b). Fragment in PZB samples treated with NaOH and H?O?—likely resulting from the cleavage of the ether bond within the PZB molecule. Importantly, the molecular ion peak was consistently observed under all stress conditions (HCl, NaOH, and H?O?), highlighting the structural resilience of the PZB molecule. Overall, the degradation studies confirm that PZB maintains high stability even when exposed to harsh chemical environments.

Table. 4.: Outcomes of stress studies.

a)

b)

c)

d)

Fig. 5.: Stress studies and their chromatogram: a) Acidic hydrolysis b) Alkaline hydrolysis c) Oxidative degradation d) Neutral hydrolysis