INTRODUCTION:

Breast cancer is the most frequent cancer among women and the second most prevalent cancer in the general population. Despite being treatable, metastatic breast cancer (MBC) is a practically incurable disease, with a median overall survival (OS) of only three years¹ and a 5-year OS of only 25%. Palbociclib, ribociclib, and abemaciclib² were recently approved for the treatment of endocrine-resistant MBC in combination with ET due to their efficacy in extending progression-free survival, increasing clinical benefit rate (CBR), and increasing response rate in a variety of clinical contexts and treatment lines³. Palbociclib is a [6-Acetyl-8-cyclopentyl-5-methyl-2-[5-(piperazin- 1-yl) pyrindin-2-l] amino pyrido [2, 3-d] that is chemically [6-Acetyl-8-cyclopentyl-5-methyl-2-[5-(piperazin- 1-yl) pyrindin-2-l] amino pyrido [2, 3-d] -pyrimidin-7(8H)-one]⁴

Palbociclib (Figure 1) is a medication developed by Pfizer for the treatment of ER-positive and HER2(Human epidermal growth factor receptor 2)-negative breast cancer patients.⁵ A thorough review of the literature found that few HPLC studies^6,7,8,9,10 for the determination of palbociclib alone or in combination with other anticancer drugs are available. Palbociclib can be measured simultaneously with other anticancer medicines such as ribociclib, letrozole, abemaciclib, carbozantinib, pazopanib, olapertib, sorafenib, and sunitinib using LC-MS/MS technologies^11,12. According to our literature review, there are a few UPLC and LCMS/MS methods available, but no UV methods have been described for Palbociclib alone. Though the UV approach was once thought to be a simple, cost-effective, and precise procedure ¹³. The benefits of ultra-performance liquid chromatography over high-performance liquid chromatography in terms of turnaround time, process dependability, method sensitivity, and drug specificity encourage the application of LC techniques to a wide range of drug active chemical groups¹⁴. As a result, it is required to create accurate UV spectrophotometric and UPLC methods in accordance with ICH recommendations that can be used for the analysis of marketed tablet dosage forms as well as routine Palbociclib analysis.

Figure 1: Chemical structure of the Palbociclib

MARTIALS AND METHOD:

Instruments and chemicals:

Analytical technologies Ltd.'s Spectro 2060 plus UV–Visible double beam spectrophotometer with UV–Visible double beam spectrophotometer, 1cm matching Quartz cell Wensar wuc-2L Ultra Sonicator. UPLC column Waters Acquity BEH C18 (2.1 x 50mm, 1.7m). Empower 2 was employed, which is a software suite that comprises an auto sampler and a PDA detector. Sensitivity of 0.1mg. Hetero Drugs Ltd. in Hyderabad, India, provided Palbociclib API. Pfizer Limited, Mumbai, Maharashtra, Ibrance (Palbociclib 75mg). Loba chemicals, Mumbai, India, provided the UPLC grade Methanol and acetonitrile. Analytical grade dipotassium hydrogen phosphate, orthophosphoric acid, and hydrochloric acid were obtained from SD-fine chemicals in Mumbai. Millipore provided HPLC grade distilled water.

Preparation of standard and working solution of Palbociclib in methanol:

Palbociclib was properly weighed at 100mg, transferred to a 100ml Standard flask, dissolved, and brought up to volume with methanol. Palbociclib was present in this solution at a concentration of 1mg per mL. Pipette 10 mL of the aforementioned solution into a 100mL standard flask, then add methanol to bring the volume up to 100mL. Palbociclib was present at a concentration of 100µg/ml in the resultant solution. Pipette 2ml of solution from the standard stock solution into a 10ml standard flask and top up with methanol to make the volume up to 10ml. Palbociclib was present at a concentration of 20µg/ml in the resultant solution.

Study of spectral characteristic of Palbociclib:

The solution was scanned independently in the UV area ranging from 200nm to 350nm after enabling the first adjustments and blank correction using methanol. Palbociclib exhibited a broad absorption spectrum, with maximum absorption at 220nm.

Estimation of Palbociclib in Pharmaceutical dosage forms:

In a glass mortar, twenty pills containing 100mg palbociclib were precisely weighed and finely pulverised. A 50mg palbociclib weight was correctly weighed and transferred to a 50mL standard flask. 10 mL of methanol was added and carefully stirred for 10 minutes. The clear solution was filtered and transferred to the flask using a Whatmann No 1 filter paper, and the volume was increased to 50ml with diluent (Methanol: phosphate buffer, 50:50). The concentration of the resultant solution was 1mg/ml. Pipette 1ml of the aforementioned solution into a 100ml standard flask and dilute to volume with diluent to reach a final concentration of palbociclib of 100µg/ml. Pipette 0.5ml from the 100µg/ ml solution into a 10ml standard flask and fill to the mark with diluent to achieve a palbociclib concentration of 5µg/ml. The final solution's absorbance was measured at 220nm. Palbociclib concentration was calculated.

Validation parameters:

Accuracy:

Recovery studies were used to determine the accuracy of the suggested approach. Pure standard drug at different concentrations levels, i.e. 80 percent, 100 percent, and 120 percent, was added to a fixed amount of drug from a dosage form. The proposed method was used to determine the total concentration. The experiment was repeated three times with each concentration, and the average percent recovery of the additional standard was calculated.

Precision:

When the procedure is used repeatedly to several samplings of homogenous samples, the precision of the analytical method is defined as the degree of agreement among individual test findings. The intra-day and inter-day precision were assessed by evaluating the Palbociclib standard solution six times on the same day (intra-day study) and then repeating the process the next day (inter-day study). The precision values were expressed in terms of standard deviation or relative standard deviation (coefficient of variance).

Preparation of calibration curve and study the linearity of Palbociclib:

A range of sample solution in the range of 1-70g/mL has been prepared for the creation of a calibration curve to explore the linearity. To produce the final concentrations, accurately pipette out 0.1ml to 7ml solution from the standard solution into fourteen 10ml standard flasks. Volumes were created up to 10ml using methanol. Each solution's absorbance was measured at 220nm. The calibration curve was made by graphing the concentrations on the X axis against the absorbance on the Y axis. In addition, the regression coefficient was calculated.

LOD and LOQ:

A Calibration curve was created for 6 times at 220nm using 5µg/mL for the investigation of LOD and LOQ, and the SD of the intercept was computed for the medication. LOD = 3.3 x σ /slope is the formula used to determine LOD. In the case of LOQ, the (σ/slope) was multiplied by ten.

Robustness:

The robustness of an analytical methodology is a measure of its capacity to remain unaffected by small but deliberate changes in method parameters during the usual stage of development. The method's robustness was put to the test in a range of scenarios, including wave length alterations. A palbociclib solution with a concentration of 10µg/mL was utilized in the investigation.

Method Development by Ultra Performance Liquid Chromatography:

Standard Preparation for the Analysis:

Palbociclib standard, 25mg, was transferred to a 25ml volumetric flask, dissolved, and then made up to volume with mobile phase. To reach the final concentrations of 20µg/ml, 0.2ml of the aforementioned solution was transferred into a 10ml volumetric flask and made up to volume with mobile phase.

Preparation of 0.05M Phosphate buffer Solution:

6.8043 grammes potassium dihydrogen orthophosphate was weighed and transported to a 1000ml beaker, where it was dissolved and diluted with purified water to 1000ml. Orthophosphoric acid was used to bring the pH down to 4.

Preparation of Mobile Phase:

The mobile phase in this experiment was made up of a 50:50 mixture of Buffer (pH adjusted to 4 with ortho phosphoric acid) and Acetonitrile. 500mL (50%) acetonitrile and 500mL (50%) of the above-prepared phosphate buffer were thoroughly mixed and degassed in an ultrasonic water bath for 15 minutes. Under vacuum filtration, the fluid was filtered through a 0.45m filter.

Assay of marketed Palbociclib formulation:

In a glass mortar, Ibrance (Palbociclib 75mg) was precisely weighed and finely ground. A 100mg Palbociclib weight was carefully weighed and transferred to a 100mL standard flask. To dissolve entirely, 10ml of methanol was used and gently swirled for 10minutes. A Whatmann No 1 filter paper was used to transfer 10mL of clear supernatant solution to a 100mL standard flask. The residue was extracted twice more with 20mL mobile phase (50:50 methanol: phosphate buffer) and passed through the same filter paper, with the volume increased to 100mL with mobile phase. Pipette 1ml of the resulting 1mg/ml solution into a 100ml standard flask and top up with mobile phase to volume. Palbociclib concentration in the final solution was 100µg/ml. Pipette 2mL of solution into a 10mL standard flask and adjust the volume to the desired level. The chromatographic system was injected with the final solution. The chromatogram that was observed was recorded.

Method validation:

System suitability:

It was evaluated to verify whether the analytical system is working properly and can able to give accurate and precise results. This study was performed by using the solution of 40µg/mL solution of Palbociclib injected six times. Average tailing factor for the peaks due to Palbociclib in Standard solution should not be more than 2.0. Average theoretical plates for the Palbociclib peaks in standard solution should not be less than 2000.

Accuracy:

A recovery study was undertaken at various levels of pure Palbociclib (80%, 100%, and 120%) to justify the accuracy of the suggested approach. To obtain the various levels, different amounts of standard Palbociclib were added to a fixed concentration of Palbociclib tablet sample solution. This study was repeated three times, with the % recovery and percentage mean recovery calculated each time.

Precision:

The method's precision was investigated by determining the Palbociclib solution at 100 percent concentration. The intraday precision was determined by analysing the six Palbociclib sample solutions in triplicate (n=6), and the interday precision was determined by analysing for six times at three distinct concentrations, i.e. 10µg/ml, 20µg/ml, and 40µg/ml. The chromatograms were captured on film. Palbociclib's peak area and retention duration were determined, and the relative standard deviation (RSD) was calculated. For the area of six standard injections results, the percent RSD should not exceed 2%.

Linearity:

To carry out the linearity test, aliquots from the Palbociclib standard solution were diluted with mobile phase in five different concentrations ranging from 1-100µg/ml of Palbociclib. The acquired data was subjected to regression analysis after a calibration curve was generated for the drug under research, taking into account concentration versus peak area. In the prescribed range, the connection between concentration and peak area should be linear, and the correlation coefficient should not be less than 0.99.

Limit of Detection (LOD):

The experiment was carried out by placing a quantity of tablet powder equivalent to 50mg of medicines in a 50 ml volumetric flask, adding 15ml of mobile phase, sonicating for 15 minutes, and then increasing the volume to 25ml with the same solvent. Then, using mobile phase, 0.5ml of the aforementioned solution was diluted to 10ml. To reach dilute concentrations of 2.5µg/ml, 0.5 ml of the aforesaid solution was removed and the volume increased to 10ml. A further 0.5ml of this solution was extracted, and the volume was increased to 10ml to produce the final concentration of 0.125µg/ml. After filtering the solution, it was put into the chromatographic apparatus. For the LOD solution, the signal to noise (S/N Ratio) value must be 3. The LOD solutions were made, injected three times, and the area of each injection was quantified in UPLC. For the area of six replicate injections, the percent RSD was found to be within the prescribed ranges.

Limit of quantitation (LOQ):

An amount of Palbociclib tablet powder equivalent to 25 mg of pharmaceuticals was put to a 25ml volumetric flask, 15ml of mobile phase was added, and the flask was sonicated for 15 minutes before the volume was brought up to 25ml using the same solvent. Then, using mobile phase, 0.5ml of the aforementioned solution was diluted to 10ml. 0.5ml was taken from the aforesaid solution to bring the total volume to 10ml. A 1ml aliquot of the aforesaid solution was transferred to a 10ml flask and volumated to reach dilute concentrations of 0.25 µg/ml. After filtering the solution, it was put into the chromatographic apparatus. For the LOQ solution, the signal to noise (S/N Ratio) value must be 10. The LOQ solutions were produced, injected three times, and the area of each injection was quantified in UPLC. For the area of six replicate injections, the percent RSD was found to be within the prescribed ranges.

Robustness:

This study is used to determine the proposed method's ability to remain unaffected by modest, but deliberate changes in method parameters, as well as its reliability in routine use. By infusing the Palbociclib working standard solution into the UPLC and modifying the flow rate, detection wavelength, and organic solvent composition from the normal chromatographic conditions, robustness was tested.

RESULTS:

UV Spectrophotometric method optimization and validation study:

To optimized the solvent system for the ideal and linear UV spectrum of Palbociclib, a range of solvents, mixture of solvents with buffer systems with different volume ratio were tested. Finally, the solvent methanol: phosphate buffer was selected. With this solvent UV spectrum was obtained linear at the λ_max220nm as shown in optimized UV spectrum in Figure 2. The solvent methanol and phosphate buffer was used for the subsequent validation studies and the assay ofPalbociclib marketed dosage form.

Figure 2. Optimized UV spectrum of Palbociclibbulk (A) and Marketed dosage form (B)

Method validation study results by UV spectrophotometer.

The percentage of assay of the Palbociclib marketed dosage form “Ibrance”, (each tablet contains 75mg of Palbociclib.) was found 99.845% which is found within the limit i.e. 90-102%. The details of the result shown in Table 1.

Table 1: Assay of marketed dosage from by the UV-Visible spectroscopy

Formulation

Label Claim in mg

*Absorbance at 220.0nm

Amt of Drug found mg

% Label

Claim

Ibrance (Palbociclib 75 mg)

75

0.785

74.13

98.845

In the study of linearity the developed method was found linear in the range of 1-70µg/ml, with a regression coefficient (R²) of 0.998. The recovery study was performed to justify the accuracy of the developed the average percentage recovery of Palbociclib was found 99.18 ±0.352, as shown in Table 2. The average %RSD for the intraday precision study was found 0.98 and found 0.69 for the interday precision. In the study of LOD, the detection limit was found 0.50µg/ml, and quantitation limit was found 1µg/ml. The details are shown in summary of validation parameters as shown in Table 2. In the robustness study of the developed UV method where measured absorbance at different wavelengths were considered,the %RSD value was found 1.34.

Table 2: Summary of the validation Parameters by UV methods

Parameters	Palbociclib
Accuracy (% recovery)	99.18
Linearity (mg/ml)	1-70
Co-relation co-efficient	0.997
LOD mg/ml	0.50
LOQ mg/ml	1.00
Intra day Precision (% RSD)	0.98
Inter day Precision (% RSD)	0.69

UPLC Method optimization:

In the HPLC method development approach, prior the selection of the optimised condition, several preliminary trials has been conducted by utilising different solvent combinations, various buffers, pH, temperature, flow rate, and columns in order to rationalize the retention time, peak shape, and other chromatographic parameters. The mobile phases were prepared by incorporating various buffer system with organic solvents. Numerous solvents (methanol, acetonitrile), buffers (ammonium acetate, orthophosphoric acid, potassium dihydrogen orthophosphate) used in the different volume ratio. Various analytical columns were tested with U.V detection at 268nm. Finally a selected mobile phase consisting of methanol and phosphate buffer in the ratio of 50:50(v/v) using Waters C₁₈, 250mm x 4.6mm .i.d., 5mm Particle size, analytical column was found optimum condition with the isocratic elution at 220nm PDA detection and at the flow rate of 0.6mL/min, In this chromatographic conditions Palbociclib was fully separated at 0.533 minute retention time with an improved sensitivity and excellent peak shape, shown in Figure 3.

Figure 3. Optimized UPLC chromatogram of Palbociclib

Method Validation by developed UPLC method:

The developed and optimized chromatographic condition was utilized for the following validation study and force degradation study as per the ICH guidelines. System Suitability is a test to check that the equipment used for analytical measurements is working adequately. Parameters such as tailing factor, peak area, retention time and theoretical plates were considered. The %RSD of area, tailing factors, theoretical plates and retention time were 0.05, 1.10, 0.46 and 0.36 respectively. No corresponding peak was observed at the retention time of the analyte in the analysis of specificity. The assay of marketed dosage form using developed HPLC method was conducted and the amount of drug in Ibrancetablets (marketed formulation)was found 74.07mg and %assay was 98.76%. The details was given in Table 3 and chromatogram was shown in Figure 3. The calibration curve showed linearity in the range of 1-100µg/ml, for Palbociclib (API) with correlation coefficient (r²) of 0.995.

Table 3: Assay of marketed dosage form by the UPLC method

Brand name of tablet	Labelled amount of Drug (mg)	Mean (±SD) (n=6) of the obtained amount	Mean (± SD) Assay (n = 6)
Ibrance (Palbociclib 75 mg)	75	74.07 (±0.03)	99.82 (±0.34)

Figure 3: Optimized UPLC chromatogram of Palbociclib

The Minimum concentration level at which the analyte can be detected (LOD) and quantified (LOQ) were found to be 0.125and1.00µg/ml respectively. In the study of intra and interday precision, the %RSD was found 0.92and 0.63. The accuracy of the developed method was demonstrated by conducted the recovery study. The average recovery of the Palbociclib using UPLC method was 98.53%. The results of all validation parameters was summarised in Table 4.

Table 4: Summary of Validation Parameters by UPLC methods

Parameters	Palbociclib
Accuracy (% recovery)	98.53
Linearity mg/ml	1-100
Co-relation co-efficient	0.998
LOD mg/ml	0.125
LOQ mg/ml	0.25
Intra day Precision (% RSD)	0.92
Inter day Precision (% RSD)	0.63

The method was found robust with the change in flow rates for ±0.1mL/min, the mobile phase methanol and phosphate buffer ratios (53: 47and 43: 53), detection wavelength (222nm and 218nm). The tailing factor was considered for every changed conditions. The calculated percentage RSD for robustness study were found 0.56 and 0.34 for increased and decreased flow rate, %RSD value 0.59 to 0.29 for the decreased and increased mobile phase ratio the average %RSD result was 0.54 shown in Table 5.

Table 5. Robustness study of the Palbociclib by UPLC method

Change in parameter	*% RSD of tailing factor (n=3)**
Flow (1.1 ml/min)	0.53
Flow (0.9 ml/min)	0.88
Less Organic	0.59
More Organic	0.39
Wavelength of Detection (254 nm)	0.58
Wavelength of detection (258 nm)	0.98
Average %RSD	0.54

DISCUSSION:

The solvent methanol and phosphate buffer was considered acceptable for optimising the UV spectrum of Palbociclib at 220nm using the UV spectrophotometric method. The results of the UV Visible spectrophotometric method validation study were found within the limit and verified the method’s suitability. Similarly, the mobile phase methanol and phosphate buffer at 50:50 (v / v) ratio using Waters C18, 250mm x 4.6mm.i.d., 5µm particle size, analytical column, at 220nm PDA detection, under optimised chromatographic conditions in the UPLC method. The 0.6mL/min flow rate was found to be optimum. Within the retention time of 0.533 minutes, Palbociclib was separated with better sensitivity and excellent peak shape. The regression coefficient value is very close to 1, was found in the linearity study for both the UV and HPLC methods, indicating the linearity of the methods developed. For the precision analysis using the UV and UPLC methods, the percentage of RSD values were found to be less than 2, which suggested that the UV and HPLC methods developed were found to be accurate. The average percentage recovery results was 98.00 percent for both the UV method and UPLC method showed the accuracy of the methods produced since the values are within the acceptance limit. The results of the detection and quantitation limit showed that both the methods developed for Palbociclib were found to have been sensitive and effortlessly quantifiable. It was found to be substantially acceptable in the assay of the marketed dosage form of Palbociclib using both the UV and UPLC method. The results of the robustness analysis shows that there are no major changes onthe minor deliberate changes in the column temperature, mobile phase ratio and mobile phase and flow rate, which suggest that the method developed was found robust.

CONCLUSION:

The results of the pharmaceutical dosage form analysis of palbociclib suggested that the spectrophotometric and UPLC method produced is highly reliable, accurate and robust and is in good agreement with the drug's labelled claim. For both the developed method, the empirical evidence from the validation studies showed that the findings are within the acceptance criteria and considered validated according to ICH guidelines. In addition, the authors should indicate that the present developed methods of UV and RP-HPLC has excellent sensitivity, accuracy and reproducibility.

ACKNOWLEDGEMENT:

The authors are thankful to the Institute of pharmacy and technology, salepur for providing the necessary facilities to carry out the research work.

CONFLICT OF INTEREST:

The authors declared no conflict of interest” in the manuscript.

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Received on 03.03.2022 Modified on 14.06.2022

Research J. Pharm. and Tech 2023; 16(6):2759-2764.

DOI: 10.52711/0974-360X.2023.00453