1. INTRODUCTION:

Ensuring the quality of pharmaceutical products is essential for patient safety and therapeutic efficacy, and analytical method development plays a crucial role in achieving this goal. Among various analytical tools, High-Performance Liquid Chromatography (HPLC), especially in reversed-phase mode (RP-HPLC), is widely preferred because of its accuracy, sensitivity, and reproducibility in routine quality control^1,2. Palbociclib, a selective CDK 4/6 inhibitor used in hormone receptor–positive, HER2-negative breast cancer therapy, requires highly reliable analytical methods due to its therapeutic significance and narrow safety margin³. Although several analytical methods for Palbociclib and other pharmaceutical drugs have been reported, many suffer from drawbacks such as long run times, poor peak symmetry, limited sensitivity, non-optimized chromatographic conditions, or the absence of robust stability-indicating features^4,5,7–10. These gaps highlight the need for a faster, more sensitive, and stability-indicating RP-HPLC method.

The present study was therefore undertaken to develop and validate a simple, accurate, and stability-indicating RP-HPLC method capable of overcoming these limitations. A mobile phase consisting of 0.1% sodium hydrogen phosphate buffer and acetonitrile (55:45, v/v) was selected after evaluating several solvent systems, as this combination provided optimal polarity balance, improved peak shape, and consistent retention time. The detection wavelength of 225nm was chosen based on λmax obtained from UV spectral studies, where Palbociclib showed maximum absorbance, ensuring good sensitivity and reliable quantification.

Fig. No.1: Structure of Palbociclib

To ensure that it is suitable for routine quality control and regulatory compliance, the approach was verified in accordance with ICH criteria and was designed to be straightforward, accurate, and reliable⁶.

2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

Palbociclib tablets (Ibrance®) and Palbociclib pure drug (API) were obtained from an authorized supplier. Acetonitrile (HPLC grade), methanol (HPLC grade), potassium dihydrogen phosphate (analytical grade), sodium hydrogen phosphate (analytical grade), and ortho-phosphoric acid (analytical grade) were procured from Merck (India). Freshly prepared double-distilled water was filtered through a 0.45µm membrane filter before use.

2.2 Instrumentation:

All analyses were carried out on calibrated instruments. A Shimadzu LT-1800 UV–Visible spectrophotometer was used for wavelength selection. Chromatographic analysis was performed using Waters Alliance 2695 and Agilent 1260 HPLC systems equipped with a quaternary pump, auto-sampler, column oven, and UV detector, controlled using EZ Chrome Elite software. Separation was achieved on a BDS C18 column (250 × 4.6mm, 5µm).

Weighing was performed using Sartorius, Scaletec BSA224S-CW, and Citizen CX220 analytical balances. Sonication was carried out using Labman and Athena Technologies ultrasonic baths, and pH was adjusted using a Labman/SS-3812 digital pH meter.

2.3 Preparation of Standard and Sample Solutions:

2.3.1 Diluent:

The diluent was a 50:50 v/v mixture of acetonitrile and water, based on solubility studies.

2.3.2 Standard Stock Solution:

A 50ml volumetric flask was filled with precisely weighed 15mg of Palbociclib, which was then dissolved in diluent, sonicated for 10minutes, and the volume was adjusted to achieve a concentration of 300µg/ml.

2.3.3 Working Standard Solution:

From the stock, 1ml was transferred to a 10ml volumetric flask and diluted with diluent to obtain a working standard of 30µg/ml.

2.3.4 Sample Stock Solution:

The average tablet weight was calculated by weighing twenty tablets. One tablet's worth of material was precisely placed into a 100ml volumetric flask, diluted with 50ml of diluent, and sonicated for 25minutes. A 750µg/ml stock solution was obtained by diluting the contents to 100ml and filtering it using a 0.45µm membrane filter.

2.3.5 Working Sample Solution:

From the sample stock, A 10ml volumetric flask was filled with 0.4ml, which was then diluted. to obtain a 30 µg/ml sample solution.

2.4 Chromatographic Conditions:

A mobile phase comprising 0.1% Na₂HPO₄ buffer and acetonitrile (55:45, v/v) at a flow rate of 1.0ml/min was used to accomplish the separation. The injection volume was 20µL, the column temperature was kept at 30°C, and the detection was done at 225nm. Each injection had a total run time of about five minutes.

2.5 Method Validation:

The following parameters were used to validate the developed RP-HPLC method in compliance with ICH Q2(R1) guidelines:

2.5.1 System Suitability:

Six replicas of the standard solution (30µg/ml) were injected to assess the system's suitability.

Acceptance criteria:

· %RSD of peak area: < 2%

· Theoretical plates (N): > 2000

· Tailing factor (T): < 2.0

· Retention time consistency: %RSD < 1%

2.5.2 Specificity:

For the purpose of to verify that there are no interfering peaks at the Palbociclib retention duration, specificity was evaluated by comparing the chromatograms of blank, placebo, and sample solutions.

2.5.3 Linearity:

Six concentration levels, ranging from 7.5–45µg/ml (25–150% of test concentration), were applied to examine linearity. Regression analysis was carried out after calibration curves between peak area and concentration were drawn.

2.5.4 Precision:

Repeatability (intraday): Six replicate injections of standard solution were analyzed on the same day. Intermediate precision (inter-day): Analysis was repeated on different days with different analysts. Precision was expressed as %RSD of peak areas.

2.5.5 Accuracy:

Recovery experiments at 50%, 100%, and 150% of target concentration were used to assess accuracy by adding known concentrations of standard medication to pre-analyzed samples. RSD and mean percentage recovery were computed.

2.5.6 Limit of Detection (LOD) and Limit of Quantification (LOQ):

The following formulas were used to determine LOD and LOQ based on the calibration curve's slope (S) and response standard deviation (σ):

LOD = \frac {3.3\sigma} {S}, \quad LOQ = \frac {10\ sigma} {S}

2.5.7 Robustness:

Small planned changes in process parameters, including as flow rate (±0.1 ml/min), mobile phase ratio (±2%), and column temperature (±2 °C), were used to test robustness. Assay results and system appropriateness were assessed.

3. RESULT AND DISCUSSION:

3.1 Method Development and Optimization:

The ideal circumstances for chromatography enabled a well resolved, sharp, and symmetrical peak for Palbociclib at 2.19 minutes. The new RP-HPLC method showed consistent system suitability metrics, such as tailing factor (<2) and theoretical plates (>2000), demonstrating peak symmetry and column efficiency.

With a correlation coefficient (r2) of 0.999, the method demonstrated outstanding linearity over the concentration range of 7.5–45µg/ml. High repeatability was confirmed by precision data showing %RSD values below 0.6% for both system and procedure precision. Three amounts of concentration (50%, 100%, and 150%) were used for accuracy studies showed mean recoveries between 98.9–99.9%, verifying the reliability of the method.

The method's high sensitivity was confirmed by the computed LOD and LOQ values, which were 0.08µg/ml and 0.24µg/ml, respectively. Method robustness was demonstrated by robustness testing, which showed that intentional changes in flow rate, mobile phase composition, and column temperature had no discernible impact on assay findings. Also, acetonitrile was selected over methanol because it produced sharper peaks, shorter retention time, and lower backpressure, resulting in better overall chromatographic performance for Palbociclib.

Variations in buffer concentration and different C18 columns were also tested, and the optimized conditions provided the best peak symmetry, retention time, and system suitability.

The developed method offers a significantly shorter run time, higher sensitivity, and improved peak symmetry compared with previously reported RP-HPLC methods for Palbociclib and other pharmaceutical compounds^4,5,11–15. Its simple mobile phase, minimal sample preparation, and excellent system suitability make it highly suitable for routine industrial quality control. The method’s robustness and stability-indicating capability further enhance its applicability for large-scale pharmaceutical analysis and regulatory compliance.

Fig. No. 2: Optimized Chromatogram of Palbociclib

3.2 Linearity Data:

Table No. 1: Linearity table for Palbociclib

Palbociclib
Conc (μg/ml)	Peak area
0	0
7.5	730672
15	1408817
22.5	2177005
30	2838834
37.5	3550339
45	4204425

Fig. No. 3: Calibration curve of Palbociclib

3.3 Limit of Detection (LOD) and Limit of Quantification (LOQ):

The observed standard deviation and the calibration curve's slope were used to calculate the developed method's sensitivity in terms of LOD and LOQ. The table displays the computed LOD and LOQ values. The low values obtained confirm the high sensitivity of the method for Palbociclib.

Table No. 2: LOD and LOQ Data for Palbociclib.

Molecule	LOD	LOQ
Palbociclib	0.08µg/ml	0.24µg/ml

3.4 Robustness:

By purposefully varying chromatographic parameters including flow rate (±0.1ml/min), mobile phase composition (±5%), and column temperature (±5°C), the resilience of the new technique was assessed. The %RSD values for Palbociclib remained within acceptable limits under all altered conditions, indicating the robustness of the method. The results are presented in Table.

Table No. 3: Robustness Data for Palbociclib

Sr. No.	Condition	% RSD of Palbociclib
1	Flow rate (-) 0.9ml/min	0.4
2	Flow rate (+) 1.1ml/min	0.6
3	Mobile phase (-) 50 B:50 A	0.4
4	Mobile phase (+) 60 B:40 A	0.2
5	Temperature (-) 27 °C	0.5
6	Temperature (+) 33 °C	1.0

4. DISCUSSION:

The developed RP-HPLC method demonstrated superior analytical performance compared to previously reported methods for Palbociclib estimation. The optimized mobile phase of 0.1% sodium hydrogen phosphate buffer and acetonitrile (55:45, v/v) provided a balanced polarity that improved peak symmetry and resolution while maintaining a short retention time¹.

The method's suitability for quantitative investigation is confirmed by a linear relationship between peak area and concentration with a correlation coefficient of 0.999. The approach is accurate and repeatable, as evidenced by the high percentage recovery (99.61%) and low %RSD values (<1%) for precision. The low LOD and LOQ values signify that even trace quantities of Palbociclib can be accurately detected, an essential criterion for quality control applications.

Compared with the studies reported by Patil and Shinde (2022) and Srikanth and Reddy (2021), the current method offers reduced analysis time and improved sensitivity, making it advantageous for routine use in pharmaceutical industries. The robustness data further confirm the method’s reliability under small variations in experimental conditions, as suggested by ICH Q2 (R1) guidelines.

Overall, the established approach can be applied with safety to the routine estimate of Palbociclib in dosage forms since it fulfils the requirements of selectivity, accuracy, linearity, precision, and robustness.

5. ACKNOWLEDGEMENT:

The authors would like to thank Channabasweshwar Pharmacy College (Degree), Latur for providing the laboratory space and assistance needed to complete this study. The authors also extend heartfelt thanks to Spectrum Pharma Research Solutions, Hyderabad for their technical assistance and valuable guidance during the analytical study.

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Received on 07.11.2025 Revised on 05.01.2026

Accepted on 12.02.2026 Published on 16.04.2026

Available online from April 18, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(2):123-127.

DOI: 10.52711/2231-5675.2026.00019

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