Analytical Quality by Design guided RP-HPLC Method Development and Validation for estimation of Palbocilib employing Box-Behnken optimisation

Analytical Quality by Design guided RP-HPLC Method Development and Validation for estimation of Palbocilib employing Box-Behnken optimisation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Analytical Quality by Design guided RP-HPLC Method Development and Validation for estimation of Palbocilib employing Box-Behnken optimisation Tanvi Painginkar, Mihir Medhansh Doneparthi, Roshan Barwa, Muddukrishna BS, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8901646/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Conventional empirical approaches (trial and error) to HPLC method design tend to yield fragile, non-consistent methods and lack regulatory flexibility, while Analytical Quality by Design offers a structured framework ensuring reliability. In this study a AQbD guided HPLC method was developed and optimised for Palbociclib, a Cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) approved for the treatment of hormone receptor-positive breast cancer. Risk assessment (Ishikawa diagram) tool guided factor selection while Box-Behnken Design enabled optimisation through Design Expert (v.13) software. The optimised method employed a Shimpack C18 column (250 mm x 4.6 mm, 5 µm); mobile phase comprising Buffer (ammonium formate of pH 4.2 adjusted with glacial acetic acid) and Acetonitrile in the ratio 35:65; flow rate of 0.8 mL/min; injection volume of 10 µL; column oven temperature of 35°C and detection wavelength of 357 nm. Validation was performed in accordance with the ICH Q2 (R2) guidelines. The peak was eluted at 4.23 minutes, and the method demonstrated excellent linearity across 3–50 µg/mL with a correlation coefficient (R 2 ) of 0.9999, LOD of 0.75 µg/mL and LOQ of 2.27 µg/mL. Accuracy studies demonstrated recoveries between 98.91-100.88% while precision was evaluated through both intra-day and inter-day studies, consistently showing RSD deviations below 2%. Biological sciences/Cancer Physical sciences/Chemistry Biological sciences/Drug discovery Analytical-QbD Design of experiments Palbociclib CDK4/6i Ishikawa diagram Box-Behnken Design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 INTRODUCTION Analytical methods are fundamental to pharmaceutical research, quality control, and regulatory compliance. Among them, High-Performance Liquid Chromatography (HPLC) is widely used for its sensitivity, reproducibility, and versatility. However, conventional trial-and-error approaches to method development are resource-intensive and often fail to ensure robustness across the method lifecycle. To address these limitations, the Analytical-Quality by Design (AQbD) provides a systematic framework that applies scientific principles and risk assessment to method development. The AQbD approach was developed in alignment with the principles outlined in ICH guidelines (Q9 and Q14), ensuring a structured and guideline‑driven framework for method implementation 1 , 2 . Within the AQbD framework, method development is guided by defining analytical target profile (ATP), Critical Analytical attributes (CAAs), identifying Critical Method Parameters (CMPs) that influence them, and applying risk‑based and statistical tools to optimise performance (Design of Experiment) 3 . This structured approach enhances method reliability under routine variations and facilitates regulatory acceptance throughout the product lifecycle. (Fig. 1) depicts the process flow of the AQbD approach in method development 4 . Palbociclib (marketed as Ibrance) is a selective CDK4/6 inhibitor that is clinically important in the treatment of hormone receptor-positive (HR+), HER2-negative advanced breast cancer in combination with an aromatase inhibitor or fulvestrant. Its pyridopyrimidinone structure (Fig. 2) underlies its kinase inhibitory activity 5 , 6 . Most of the available literature focuses on conventional RP‑HPLC methods for its quantification in bulk drugs and formulations, including stability‑indicating assays and simultaneous estimation with other anticancer agents (Table 1), but no AQbD-based RP-HPLC method has been reported currently for the estimation of Palbociclib 7 – 11 . Table 1 Summary of previously reported methods and the current RP-HPLC method for Palbociclib Chromatographic conditions Column used Linearity (µg/mL) Remarks Reference 0.02 M sodium dihydrogen phosphate buffer (pH 5.5): ACN: Methanol (80:10:10 v/v/v); 1 ml/min flow rate; at 254 nm Intersil C 8 (250 mm x 4.6 mm, 5 µm) 5–50 Simultaneous estimation of palbociclib and letrozole 8 Ammonium acetate (pH 8) adjusted with TEA: ACN (38:62 v/v); 1 ml/min flow rate; at 263 nm Inertsil ODS-3V (250 mm x 4.6 mm, 5 µm) 5 − 1,000 Stability-indicating HPLC method for palbociclib 9 ACN: sodium acetate buffer (30:70, 0.5% TEA); 1 ml/min flow rate; at 260 nm Kromasil C18 (250 mm x 4.6 mm, 5 µm) 4.04–20.19 Content determination of palbociclib in capsules 10 Ammonium formate buffer (pH 4.2) adjusted with glacial acetic acid: ACN (35:65 v/v), 0.8 ml/min flow rate; at 357 nm Shimadzu Shim-pack C18 (250 mm x 4.6 mm, 5 µm) 3–50 AQbD-based RP-HPLC method for quantification of palbociclib in bulk Proposed-present study This study focuses on the development, optimisation and validation of a reliable RP-HPLC method for quantifying Palbociclib in bulk, employing an AQbD framework to ensure method reliability, efficiency and regulatory compliance. MATERIALS AND METHODS Chemicals and Reagents: The pure API (Palbociclib) was provided by MSN Laboratories Pvt. Ltd (Telangana, India) as a gift sample, with a purity 99.60%. HPLC-grade acetonitrile and AR-grade ammonium formate were procured from Finar. Glacial acetic acid (Emplura) was procured from Merck, and ultrapure water was prepared using a Milli-Q purification system for buffer preparation. Instrumentation: A UV-Visible spectrophotometer (Shimadzu UV-1800) was used to determine the maximum absorbance wavelength (λ max ) of Palbociclib, which served as the basis for selecting the detection wavelength. Chromatographic separation was carried out using an HPLC system with a UV-Visible detector (Shimadzu LC20-series), equipped with a Shimadzu Shim-pack C18 Column consisting of 250 mm of length, 4.6 mm of internal diameter and 5 µm particle size. Optimised Chromatographic conditions: The mobile phase was prepared using an ammonium formate buffer (pH 4.2, adjusted with glacial acetic acid) and acetonitrile in the ratio 35:65 (v/v). It was filtered through a membrane filter (0.45 µm) and degassed by sonication. Chromatographic separation was achieved at 35°C using a flow rate of 0.8 ml/min with a 10 µL injection volume. The detection was carried out at 357 nm. Procedure for sample preparation: Diluent A : Acetonitrile and Water in a 50:50 ratio. (The solubility of the drug was high in this mixture) 13 Diluent B : Mobile phase (Buffer: Acetonitrile, 35:65 ratio) Preparation of stock solution (200 µg/mL): A precisely weighed quantity (5 mg) of Palbociclib was dissolved in 20 mL of diluent A. The solution was sonicated for 10 minutes to ensure complete solubilisation (clear solution), followed by dilution with Diluent A to a final volume of 25 mL. The resulting solution was filtered through a 0.45 µm syringe filter prior to analysis. Preparation of calibration standards: The stock solution was diluted using diluent B (Mobile Phase) to prepare calibration standard solutions in the range of 0.05–50 µg/mL. Selection of detection wavelength: A 10 µg/mL solution of Palbociclib was prepared from the stock solution using diluent B and was scanned in the range of 200–400 nm on a UV-Visible Spectrophotometer. The λmax of 357 nm was obtained and was selected as a detection wavelength. RP-HPLC Method Development by AQbD approach: Establishment of ATP, CAAs and CMPS 1 : The ATP, CAAs and CMPS were established systematically through risk assessment and experimental design to ensure method robustness and regulatory compliance. Risk Assessment using Ishikawa Diagram 2 : An Ishikawa diagram (fishbone analysis) was employed to identify potential sources of variability in HPLC performance. Critical parameters such as sample preparation, mobile phase pH and composition, injection volume, flow rate, column oven temperature, selection of column and detection wavelength were examined for their impact on CAAs like retention time, peak area, efficiency (theoretical plates) and peak asymmetry. This qualitative assessment provided a foundation for prioritising CMPs. Preliminary Screening using the one-factor-at-a-time (OFAT) method: Before implementing statistical optimisation, the influence of individual CMPs on CAAs was evaluated using the OFAT approach. This ensured that only the most impactful CMPs were carried forward into the DoE, thereby improving efficiency and reducing experimental burden. Statistical optimisation using Box- Behnken Design (BBD): Following OFAT screening, BBD was applied to systematically study the interactions among four CMPs (buffer concentration, pH of the buffer, flow rate and column oven temperature) and four CAAs (retention time, peak area, peak tailing and net theoretical plates). BBD enabled the development of predictive models and the establishment of a statistically validated method operable design region (MODR), ensuring robustness and reproducibility of the method. Design of Experiment (DoE) Software: Design Expert (v.13), a statistical software, was used to apply DoE approaches such as Box-Behnken Design to optimise chromatographic conditions in HPLC. Analytical Method Validation 14 : Validation of the developed method was performed as per ICH Q2 (R2) guideline, which includes the following parameters: Specificity: To establish specificity, the chromatogram of the blank was compared with the sample (10 µg/ml), which confirmed that the analyte peak was well resolved without any interference from the diluent. System suitability studies: The suitability of the developed chromatographic method was verified through six replicate injections of Palbociclib standard solution (10 µg/ml). The evaluation included determination of retention time, net theoretical plates and tailing factor. The acceptance criteria include retention times showing %RSD values below 2%, theoretical plates exceeding 2000, and tailing factor remaining within the limit of ≤ 2. Linearity and Calibration Curve: The linearity was evaluated across the concentration range of 0.05–50 µg/mL with replicates (n = 3) at each concentration level. The calibration curve was obtained by plotting the mean peak area on the y-axis and the analyte concentration on the x-axis. The regression equation of the calibration curve and the correlation coefficient were determined. Limit of Detection (LOD) and Limit of Quantification (LOQ): The LOD and LOQ were established using two approaches: the chromatographic signal-to-noise ratio method (practical), where LOD and LOQ were defined at S/N values of approximately 3 and 10, respectively; and the calibration curve method (statistical), where values were calculated based on the standard deviation of the response and the slope of the regression line. In the calibration curve method, the following equations were used to determine the LOD and LOQ: LOD = 3.3 x \(\:\frac{\sigma\:}{Slope}\) LOQ = 10 x \(\:\frac{\sigma\:}{Slope}\) Where σ represents the standard deviation of the response. Range: As the LOQ represents the lowest concentration at which the analyte could be quantified with acceptable precision and accuracy, the working range of the method was defined from the LOQ (obtained from the calibration curve method) up to the highest concentration validated within the linearity study. Precision: Intra-day (Repeatability): Intraday precision was assessed by preparing six independent analyte solutions at a concentration of 10 μg/ml and analysing them under optimised chromatographic conditions on the same day. Inter-day: Intermediate precision was determined by analysing six independently prepared analyte solutions at a concentration of 10 μg/ml over three consecutive days to determine the variability between days. Accuracy: Accuracy of the analytical procedure was evaluated through recovery experiments conducted across the validated concentration range. Three levels were selected to represent the extremes and midpoint of the linearity interval, namely 3 µg/mL, 10 µg/mL, and 50 µg/mL. Triplicate analyses were performed at each concentration, and the mean recovery values were derived to assess the method performance. The study was considered acceptable when recoveries fell within 98–102%, and the relative standard deviation did not exceed 2%, in accordance with established validation criteria. The following equation was used to calculate the % Recovery for each level. % Recovery = X 100 Robustness: It was evaluated by introducing minor changes in the analytical method parameters, such as composition of mobile phase (± 2% of the buffer), column oven temperature (± 2 °C) and detection wavelength (±2 nm). The method is considered robust if these changes do not significantly influence the system suitability parameters (retention time, peak tailing and net theoretical plate) and all factors remain within the predefined acceptance limits (%RSD ≤ 2, tailing factor ≤ 2 and theoretical plates > 2000). Benchtop solution stability: Freshly prepared stock solution and working solutions were kept at an ambient laboratory condition (25 o C ±2), exposed to normal light and were analysed at different time intervals (0, 4, 16, 24, 48 hours). At each timepoint, replicate injections (n=3) were performed, and chromatographic parameters, including peak area, retention time, tailing factor and theoretical plates, were compared with the reference solution. RESULTS AND DISCUSSION Initial method trials using acetonitrile-water mixtures in varying ratios resulted in peaks with retention times exceeding 25 minutes and poor peak shape. Subsequent experiments employing different buffer systems across a pH range of 2.5–6.5 were evaluated. Among these, ammonium formate provided optimal performance, yielding a well-defined peak that eluted within 8 minutes. Hence, ammonium formate was selected as the buffer for the final method development. Wavelength selection: A working standard solution of Palbociclib (10 µg/ml) showed a λmax of 357 nm. Additional peaks were observed at 220nm, 264nm, and 302 nm, as shown in Fig. 3. Method Development by an AQbD approach: Risk assessment: The possible factors, as described in Fig. 4, were studied for their influence on the method's performance using an Ishikawa diagram. Each factor was grouped into one of the following categories: Method Parameters, Sample preparation, Materials, Instrumentation, Human Factors, and Environment. After mapping the potential cause, the most impactful parameters were selected based on the risk assessment score. The Key parameters identified were - mobile phase composition and its pH, flow rate, column oven temperature, type of column (C18), diluent for sample preparation and wavelength for detection. Optimisation of the method using BBD: The BBD experimental design comprised 27 runs, incorporating two factor levels (-1,+1) along with a central point (0) to assess the curvature effects. The coded values of the independent variables and their corresponding experimental runs are summarised in Table 2 and Table 3, respectively. Table 2 Coded values for CMPs in Box-Behnken Design Factors Levels -1 0 1 A: pH of Buffer 2.8 3.5 4.2 B: Buffer concentration (%) 30 35 40 C: Flow Rate (mL/min) 0.8 0.9 1 D: Column temperature ( o C) 30 35 40 BBD optimisation yielded the following equations explaining how each CMP influences the CAAs: Retention time = +12.57450 - 1.00369A + 0.031433B - 11.38583C - 0.080967D - 0.003357AB + 0.185714AC – 0.002357AD - 0.072500BC + 0.000050BD + 0.198500CD + 0.188350 A 2 + 0.000807B 2 + 1.32917C 2 -0.001413D 2 Interpretation: The coded equation represents a quadratic polynomial model for retention time and suggests that there is a strong negative effect of pH (-1.00369A) and flow rate (11.3858C) on Retention time. Which implies that increasing Factor A and C reduces the retention time as shown in Figure 5. Peak Area = + 738994.666 – 3152.75A -12876.25B – 72343.58C-362.08D + 3043.50AB + 5068.75AC + 336.00AD – 365.00BC – 2390.75BD + 2979.50CD + 47348.87A 2 + 195.87B 2 + 7446.63C 2 – 2655.37D 2 Interpretation: The coded equation represents a quadratic polynomial model for peak area, while suggesting that there is a very strong negative effect of flow rate (-72343.58C) on peak area, implying that faster flow significantly reduces the area, as shown in Figure 6. Table 3: Experimental Run of Box-Behnken Design (Sample concentration: 20 µg/mL) Run Factor A Factor B Factor C Factor D Retention time Peak Area Theoretical Plates Tailing Factor 1 3.5 35 0.8 30 4.173 821691 2770 1.441 2 3.5 35 0.9 35 3.638 737803 2721 1.458 3 4.2 35 1 35 3.563 716183 3710 1.331 4 3.5 40 0.8 35 4.204 807909 4002 1.34 5 2.8 30 0.9 35 3.454 806391 10422 1.469 6 3.5 30 1 35 3.205 686924 4297 2.268 7 3.5 35 1 40 3.236 674981 2617 1.471 8 3.5 30 0.9 40 3.563 750896 5496 2.383 9 3.5 35 0.8 40 3.752 806951 2734 1.533 10 3.5 40 0.9 40 3.663 723208 3782 1.321 11 2.8 35 1 35 3.134 718375 6303 1.671 12 4.2 35 0.9 40 3.874 783057 3921 1.353 13 2.8 35 0.9 40 3.539 786753 7256 1.655 14 4.2 30 0.9 35 3.907 798085 3616 1.318 15 3.5 30 0.8 35 3.968 833274 5401 2.493 16 2.8 35 0.9 30 3.493 785820 6608 1.747 17 2.8 35 0.8 35 3.97 877565 7911 1.764 18 3.5 30 0.9 30 3.605 741111 3843 2.287 19 3.5 35 0.9 35 3.639 739428 2694 1.461 20 4.2 35 0.9 30 3.861 780780 3797 1.355 21 3.5 35 0.9 35 3.633 739753 2718 1.453 22 3.5 40 0.9 30 3.7 722986 3652 1.319 23 4.2 35 0.8 35 4.347 855098 4082 1.357 24 3.5 35 1 30 3.26 677803 2522 1.383 25 3.5 40 1 35 3.296 660099 3484 1.312 26 4.2 40 0.9 35 4.002 775916 4172 1.362 27 2.8 40 0.9 35 3.596 772048 4631 1.47 Tailing Factor = + 4.94243 – 0.202381A – 0.068233B – 0.41C + 0.003067D Interpretation: The coded equation represents a linear model for the tailing factor. It suggests that there is a negative effect of pH (-0.202381A) on the tailing factor, implying that as the pH increases, the tailing is reduced and the peak symmetry is improved, as shown in Figure 7. Theoretical plates = + 215254.5 – 54061.66A – 4603.783B – 75838.333C + 248.15D + 453.35714AB + 4414.28AC – 37.42857AD + 293.00BC – 15.23BD + 65.50CD + 4738.60544A 2 +44.72167B 2 +24741.667C 2 + 5.72167D 2 Interpretation: The coded equation represents a quadratic second-order polynomial model for theoretical plates. It suggests that there is a strong negative effect of flow rate (-75838.333C), implying that higher flow reduces efficiency, as shown in Figure 8. Method Operable Design Region (MODR): The MODR was established by overlaying contour plots of all critical responses, identifying the 3D design space where method performance consistently met the acceptance criteria. The two critical parameters that were optimised are pH of the buffer (A) and the buffer concentration (B), while the other two parameters, flow rate (C) and column oven temperature (D), were kept constant during the overlay analysis. The yellow area shown in Figure 9 represents the optimised operable design space for the developed method. This region was derived by overlaying contour plots of multiple responses (retention time, peak area, net theoretical plates and tailing factor). The red dots represent the experimental runs. Optimised Chromatographic conditions: The selection of optimised chromatographic conditions was guided by desirability solutions derived from BBD, ensuring robustness and compliance with ICH guidelines. The conditions are shown in Figure 10 with a desirability of 1.0000. The final chromatographic conditions, as shown in Table 4, were further used for method validation. Control strategy: A control strategy is planned for the developed method to deliver reliable and robust results within the obtained MODR. The elements identified as controls are as follows: Mobile phase pH: To obtain reproducible retention time and analyte stability, the pH should be controlled within 2.8-4.2. Buffer concentration in the mobile phase (%): The buffer concentration can be varied in the range 33 - 40%. Flow rate: The flow is set to 0.8 mL/min, with a variation of ±0.05mL. Column Oven Temperature: The temperature is maintained at 35 o C± 2. Table 4: Optimised Chromatographic conditions for Palbociclib estimation: Parameters Chromatographic Conditions Stationary Phase Shimadzu Shim-pack C18 Column (250 mm of length; 4.6 mm of internal diameter and 5 μm particle size) Mobile Phase Ammonium formate buffer (pH 4.2, adjusted with glacial acetic acid) and Acetonitrile ratio 35:65 (v/v) Flow rate 0.8 mL/min Injection Volume 10 µL Column oven temperature 35 o C (±2 o C) Elution Isocratic mode Run time 8 minutes Detector and Wavelength UV-Visible, 357 nm Retention time 4.236 minutes Analytical Method Validation: Specificity: Specificity was assessed by analysing a blank solution and a sample prepared at a concentration of 10 µg/mL, where the chromatogram of the blank showed no interfering peaks Figure 11A, and the analyte peak in the sample was clearly resolved with a retention time of 4.236 minutes Figure B, confirming the absence of solvent interference. System suitability studies: System suitability testing demonstrated consistent retention time, acceptable peak symmetry (≤ 2), and adequate theoretical plates, confirming the reliability of the chromatographic method. The results are summarised in Table 5. Table 5 Summary of system suitability parameters for Palbociclib Replicate No. Retention Time (minutes) Peak Area (mV) Tailing Factor Theoretical Plate 1 4.29 403617 1.374 3870 2 4.28 417189 1.354 3766 3 4.28 409646 1.359 3760 4 4.24 419608 1.361 3843 5 4.28 410679 1.368 3865 6 4.30 413617 1.367 3846 Mean 4.279 412392.7 1.364 3825 SD 0.020 5728.141 0.007 44.900 %RSD 0.4773 1.3890 0.5276 1.1739 Linearity: Linearity study confirmed direct proportionality between concentration and peak area over the range of 0.05–50 µg/ml, with a correlation coefficient (R 2 ) of 0.9999. The results are summarised in Table 6: Table 6 Calibration curve and Linearity Results for Palbociclib method validation Concentration (µg/mL) Mean Peak area (mV) % RSD Intercept Slope Corelation coefficient (R2) 0.05 2428 0.7946 2709.7 40496 0.9999 0.1 5773 1.0415 0.5 25963 1.5387 1 44789 1.1917 5 207718 1.3682 10 412793 1.0761 30 1198567 0.8434 50 2037597 1.5311 LOD and LOQ: The LOD and LOQ derived from the S/N approach were found to be 0.05 µg/ml and 0.5 µg/ml, respectively. By applying the calibration curve method, it yielded higher LOD and LOQ of 0.7 µg/ml and 2.2 µg/ml, respectively. Results of both methods are given in Table 7, with the significance of each method. Range: Based on the method’s LOQ value (2.2 µg/mL), the working range for Palbociclib quantification was set between 3–50 µg/mL, ensuring accuracy and precision throughout. Table 7 LOD and LOQ results obtained by two approaches with their analytical significance Method LOD (µg/ml) LOQ (µg/ml) Significance Signal-to-noise 0.05 0.5 Provides the lowest possible values, showing the method's sensitivity (Qualitative) Calibration curve 0.7 2.2 Defines the lowest concentration that can be quantified with acceptable accuracy and precision (Quantitative) Intra-day and Inter-day precision: The intra-day precision, assessed by analysing replicates within a single day, demonstrated a % RSD of less than 2% across all levels, as shown in Table 8, confirming excellent repeatability of the developed method. Similarly, analysis repeated over three days (inter-day) showed minimal %RSD, supporting method’s reproducibility as shown in Table 9. Table 8 Intra-day precision results (repeatability) Intra-day Precision Concentration (µg/mL) Peak Area (mV) Mean (n = 3) SD % RSD 3 121589 122917.33 1664.8617 1.3544564 3 122378 3 124785 10 416591 420735.67 5983.232 1.4220881 10 418021 10 427595 50 2033798 2034263.3 9211.8191 0.4528332 50 2025293 50 2043699 Table 9 Inter-day (Intermediate precision) results: Inter-day Precision Concentration (µg/mL) Mean Peak Area (mV) (n = 3) Mean SD %RSD Day 1 Day 2 Day 3 3 121255 123157 124768 123060 1758.50761 1.4289839 10 418542 419608 413028 417059.333 3531.68874 0.8468073 50 2063659 2100199 2083877 2082578.33 18304.5842 0.8789386 Accuracy (% Recovery study) : Accuracy testing demonstrated satisfactory recovery of Palbociclib, with mean percentage values between 98.91% to 100.88% at concentrations of 3 µg/ml, 10 µg/mL and 50 µg/mL, as summarised in Table 10. Robustness : The robustness testing was performed by changing mobile phase composition, column temperature and detection wavelength. The system suitability results consistently met the acceptance criteria, indicating the method’s consistency under slight variations in the chromatographic conditions. The result of robustness is summarised in Table 11. Benchtop solution stability : Solution stability was monitored under ambient laboratory conditions for 24 hours, with assay values remaining consistent and replicate injections showing an RSD below 2%. The data confirms that the solutions are stable for a minimum of 24 hours under benchtop storage. Table 10 Accuracy study showing recovery results for Palbociclib Concentration (µg/mL) Peak Area (mV) Mean (%RSD) (% Recovery) 3 121389 122880.667 98.91 3 122478 3 124775 10 416000 412793.667 100.88 10 414658 10 407723 50 2003798 2037596.67 100.49 50 2065293 50 2043699 Table 11 Robustness data for a sample concentration of 10 µg/mL Parameters Variation (± 2) %RSD (n = 3) System Suitability Parameters (n = 3) Retention Time Area Tailing factor (range) Theoretical Plates (range) Buffer concentration (%) 33 1.26 0.59 1.481–1.498 5257–5277 37 1.45 1.14 1.465–1.484 4899–4966 Column Temperature ( o C) 33 0.604 1.14 1.491–1.503 5059–5121 37 0.987 0.605 1.483–1.491 5227– 5263 Detection wavelength (nm) 355 0.856 0.588 1.478–1.486 5079–5106 359 0.70 0.590 1.465–1.484 5080–5099 Life-cycle management and continuous improvement : Managing the life cycle of an AQbD-based HPLC method ensures its reliability and reproducibility while maintaining regulatory standards. Post-validation, the method will be continually monitored, documented and controlled modifications within the design space, ensuring its continued suitability for routine analysis of Palbociclib. Table 12 ANOVA data for all the CMPs and CAAs using BBD Statistical Terms Retention Time (R1) Peak Area (R2) Tailing Factor (R3) Theoretical Plates (R4) F-value 43.66 228.55 5.70 15.19 p-value < 0.0001 < 0.0001 0.0026 < 0.0001 Model Quadratic Quadratic Linear Quadratic A-Buffer conc. 0.4673 1.19x10 8 0.2408 3.27x10 7 B-Buffer pH 0.0480 1.99x10 9 1.40 7.28x10 6 C- Flow rate 1.86 6.28x10 10 0.0202 1.31x10 6 D-Column temp 0.0180 1.57x10 6 0.0028 5.69x10 5 AB 0.0006 3.705x10 7 - 1.00x10 7 AC 0.007 1.02x10 8 - 3.81x10 5 AD 0.003 4.51x10 5 - 68644 BC 0.0053 5.32x10 5 - 85849 BD 6.250 x 10 − 6 2.28x10 7 - 5.79x10 5 CD 0.0394 3.55x10 7 - 4290.25 A 2 0.0454 1.19x10 10 - 2.87x10 7 B 2 0.0022 2.04x10 5 - 6.66x10 6 C 2 0.0009 2.95x10 8 - 3.26x10 5 D 2 0.0067 3.76x10 7 - 1.09x10 5 Residual Analysis Lack of fit F-value p-value Significant Significant Significant Significant 476.31 27.25 4906.01 2250.69 0.0021 0.0359 0.0002 0.0004 R 2 0.9807 0.9963 0.5089 0.9466 Adjusted R 2 0.9583 0.9919 0.4196 0.8843 Conclusion The integration of Ishikawa-based risk assessment, OFAT screening, and BBD optimisation highlights the strength of the AQbD framework in delivering a reliable and reproducible RP-HPLC method. This structured workflow ensures scientific rigour, regulatory flexibility, and lifecycle management, representing a significant advancement over conventional trial-and-error strategies for Palbociclib quantification. Declarations Author’s Contributions All authors contributed to the study conception and design. Tanvi Painginkar carried out the experimental work of HPLC method optimization, DoE analysis, data interpretation/analysis, and drafting the paper. Dr Gundawar Ravi was involved in conception of research work, supervising the experimental work, data analysis, resources, and drafting and reviewing the paper. All authors have made a significant contribution to the research in the manuscript, approved its claims, and agreed to be an author. Competing interests: The author(s) declares no competing interests Acknowledgement: The authors are thankful to (i) Manipal Academy of Higher Education, Manipal, India, for providing Dr TMA Pai Doctoral Scholarship to Tanvi Painginkar, (ii) Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, for providing the necessary facilities and the authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University, Abha, for funding this work through Large Research Project under grant no. RGP.2/728/46. Data Availability All data generated during this study are included in this published article. References ICH guideline for. Analytical Procedure Development Q14. International Council For Harmonisation of Technical Requirements for Pharmaceuticals for Human Use ICH Harmonised Guideline. Qual. Risk Manage. Q9 (R1 ). Beg, S. et al. Introduction to analytical quality by design. in Handbook of Analytical Quality by Design 1–14 (Elsevier, doi: 10.1016/B978-0-12-820332-3.00009-1 . (2021). Kanthiah, S., Joysa Ruby, J., Sgb, H. & Kannappan, V. Navigating the AQbD Landscape: Enhancing Quality Management in Liquid Chromatography Method Development. Biomedical Chromatography vol. 39 Preprint at (2025). https://doi.org/10.1002/bmc.70031 Palbociclib Uses, Dosage, Side Effects, Warnings - Drugs.com. https://www.drugs.com/palbociclib.html DeMichele, A. et al. CDK 4/6 Inhibitor palbociclib (PD0332991) in Rb+ advanced breast cancer: Phase II activity, safety, and predictive biomarker assessment. Clin. Cancer Res. 21 , 995–1001 (2015). Rathore, S. S., Jenita, J. J. L. & Manjula, D. A. Comprehensive Review of Analytical Techniques for Quantifying Cyclin-dependent Kinase 4 and 6 Inhibitors in Biological Samples, Bulk, and Pharmaceutical Samples. Separation Science Plus vol. 8 Preprint at (2025). https://doi.org/10.1002/sscp.202400211 Dange, Y., Bhinge, S. & Salunkhe, V. Optimization and validation of RP-HPLC method for simultaneous estimation of palbociclib and letrozole. Toxicol. Mech. Methods . 28 , 187–194 (2018). Kallepalli, P. & Annapurna, M. M. New Stability-Indicating Liquid Chromatographic Method for Determination of Palbociclib (an Anti-Breast Cancer Drug) . International J. Green. Pharmacy 12 . Seemaladinne, R. A. Review on Analytical Method Development and Validation of Palbociclib. J. Integr. Sci. 36–41 10.37022/jis.v6i1.54 (2023). Turković, L. et al. Three sample preparation methods for clinical determination of CDK4/6 inhibitors with endocrine therapy in breast cancer patient plasma using LC-MS: Cross-validation (red), ecological (green) and economical (blue) assessment. J Pharm. Biomed. Anal 255 , (2025). MolDraw. https://www.moldraw.com/ Liu, W. dong. Thermodynamic modelling, Hansen solubility parameter and solvent effect of palbociclib in fourteen pure solvents at different temperatures. J. Mol. Liq. 356, (2022). ICH Guidelines for. Validation of Analytical Procedures Q2(R2). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8901646","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":599242737,"identity":"537947f5-fb82-4dae-aafa-bb4d3e64d31a","order_by":0,"name":"Tanvi Painginkar","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Tanvi","middleName":"","lastName":"Painginkar","suffix":""},{"id":599242739,"identity":"f12075af-fd3c-4025-a316-2863b2f77f9d","order_by":1,"name":"Mihir Medhansh Doneparthi","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Mihir","middleName":"Medhansh","lastName":"Doneparthi","suffix":""},{"id":599242741,"identity":"b7059cf2-3d21-470e-ad0d-445a2f1c4abd","order_by":2,"name":"Roshan Barwa","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Roshan","middleName":"","lastName":"Barwa","suffix":""},{"id":599242742,"identity":"1384b408-a732-483f-a3c0-2882c7245efd","order_by":3,"name":"Muddukrishna BS","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Muddukrishna","middleName":"","lastName":"BS","suffix":""},{"id":599242744,"identity":"d2038487-72b2-4173-ac67-daf3eae0dd02","order_by":4,"name":"Namdev Dhas","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Namdev","middleName":"","lastName":"Dhas","suffix":""},{"id":599242746,"identity":"d7248e45-1575-4b13-8e46-329da11fb04b","order_by":5,"name":"Riyaz Ali M. Osmani","email":"","orcid":"","institution":"King Khalid University","correspondingAuthor":false,"prefix":"","firstName":"Riyaz","middleName":"Ali M.","lastName":"Osmani","suffix":""},{"id":599242748,"identity":"213eac1a-eb31-4bff-bbc6-2b5b9fe677b9","order_by":6,"name":"Ravi Gundawar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYJACCTgroQJIMAPxAwYGxgbitJyBakkgWgtjG0wvHi3yEbkHb/xss8tnkD78TOLhvG3y5uy8Bz8kMNjIbjiAXYvhjbxky962ZMsGvjQzicRttw13NvMlSyQwpBnj1DIjx0yCdxuzAQMPg7EBUAvjhsM8BkAthxPxaZH8u60eqIX9s0HinNv2QC3GPxIY/uPUIi+RYybNu+0wUAuP4YPEhtuJQC1mQFsO4NRiwPPG2Fr233EDNh6ewgcJx24nbzjMl2aRYJBsPBOXLe05hjffnKk24Odh33DwR81t2w3nzx6+8aHCTrYPly0wcTaEGA9IHLtysC0NmGI8uJWPglEwCkbBiAQA1XJdLdC8JJYAAAAASUVORK5CYII=","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":true,"prefix":"","firstName":"Ravi","middleName":"","lastName":"Gundawar","suffix":""}],"badges":[],"createdAt":"2026-02-17 13:39:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8901646/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8901646/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103943912,"identity":"9ca35716-a9fb-4153-852e-5f2624396e22","added_by":"auto","created_at":"2026-03-04 20:24:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":89780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalytical QbD process flow in HPLC method development and validation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/c06a3a5e50fafaaa5915125b.png"},{"id":103943921,"identity":"5f8b7179-31ce-4d32-9778-b5bc98f5aafb","added_by":"auto","created_at":"2026-03-04 20:24:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31667,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular structure of Palbociclib\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/af08dc9074dc34beee730e54.png"},{"id":103943913,"identity":"d4c27529-b547-4c44-be02-120eceb65ac5","added_by":"auto","created_at":"2026-03-04 20:24:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":88657,"visible":true,"origin":"","legend":"\u003cp\u003eRepresents\u003cstrong\u003e \u003c/strong\u003ethe\u003cstrong\u003e \u003c/strong\u003eUV absorption spectrum of Palbociclib (10µg/ml)\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/223a1d9132d4d6fd88543d37.png"},{"id":103943919,"identity":"1571043e-399f-489b-aeac-08cb8cccc3af","added_by":"auto","created_at":"2026-03-04 20:24:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71926,"visible":true,"origin":"","legend":"\u003cp\u003eSystematic cause-and-effect mapping for development of a robust HPLC method using Ishikawa (fishbone) diagram\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/185aec4fd31fd1498a742125.png"},{"id":103943914,"identity":"c6636bd3-1a0a-4ded-a1b4-63466f1b6498","added_by":"auto","created_at":"2026-03-04 20:24:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":212659,"visible":true,"origin":"","legend":"\u003cp\u003e3D-surface plot depicting the effect of other factors on Retention time using BBD.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/e067ff9084272b1ee705e0dd.png"},{"id":104401573,"identity":"caba4934-c34f-49b0-b7eb-88de17899725","added_by":"auto","created_at":"2026-03-11 12:13:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":173175,"visible":true,"origin":"","legend":"\u003cp\u003e3D-surface plot depicting the effect of other factors on Area using BBD.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/c3ca53ebbf5d881e1b7b382e.png"},{"id":104402087,"identity":"0011b048-09a6-4d7d-9b57-c0a5bffa08b3","added_by":"auto","created_at":"2026-03-11 12:14:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":231655,"visible":true,"origin":"","legend":"\u003cp\u003e3D-surface plot depicting the effect of other factors on the tailing factor using BBD\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/7fd29019e01a0fb5c30c67eb.png"},{"id":103943922,"identity":"9b2f95e0-0f1b-4d9a-9b2f-03a19ee30af2","added_by":"auto","created_at":"2026-03-04 20:24:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":250992,"visible":true,"origin":"","legend":"\u003cp\u003e3D-surface plot depicting the effect of other factors on theoretical plates using BBD\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/9cce18070fc4446fe834097b.png"},{"id":103943917,"identity":"aa66481a-729f-4052-8baf-e842e4064a6a","added_by":"auto","created_at":"2026-03-04 20:24:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":96225,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of MODR derived from BBD\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/e53f3b14b4d7ec578b0576db.png"},{"id":104402671,"identity":"ef35e054-fac0-46c2-a720-ac9475126136","added_by":"auto","created_at":"2026-03-11 12:16:04","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":45825,"visible":true,"origin":"","legend":"\u003cp\u003eNumerically optimised plots showing ideal chromatographic conditions and responses with maximum desirability (1.000)\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/7fbcf59eff628280af800d2f.png"},{"id":103943923,"identity":"3a0fdbd2-924a-4d91-9bf3-14b941c78fae","added_by":"auto","created_at":"2026-03-04 20:24:54","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":52719,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of (a) Blank and (b) Palbociclib (10 µg/ml)\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/c5620821480f05ca44093253.png"},{"id":104401601,"identity":"feb63fa8-d49f-478b-9e38-c1c7a9406578","added_by":"auto","created_at":"2026-03-11 12:13:05","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":33328,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration Curve for Palbociclib (0.05 – 50 µg/mL)\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/add0d47b8512c84feb2accda.png"},{"id":106093923,"identity":"29ae62e1-1cc0-4e40-b437-0753d2e1acb9","added_by":"auto","created_at":"2026-04-03 11:40:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3282895,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8901646/v1/6ad57057-1dd1-4e06-9e0f-45a34fa7668d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analytical Quality by Design guided RP-HPLC Method Development and Validation for estimation of Palbocilib employing Box-Behnken optimisation","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAnalytical methods are fundamental to pharmaceutical research, quality control, and regulatory compliance. Among them, High-Performance Liquid Chromatography (HPLC) is widely used for its sensitivity, reproducibility, and versatility. However, conventional trial-and-error approaches to method development are resource-intensive and often fail to ensure robustness across the method lifecycle. To address these limitations, the Analytical-Quality by Design (AQbD) provides a systematic framework that applies scientific principles and risk assessment to method development. The AQbD approach was developed in alignment with the principles outlined in ICH guidelines (Q9 and Q14), ensuring a structured and guideline‑driven framework for method implementation \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Within the AQbD framework, method development is guided by defining analytical target profile (ATP), Critical Analytical attributes (CAAs), identifying Critical Method Parameters (CMPs) that influence them, and applying risk‑based and statistical tools to optimise performance (Design of Experiment)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This structured approach enhances method reliability under routine variations and facilitates regulatory acceptance throughout the product lifecycle. (Fig.\u0026nbsp;1) depicts the process flow of the AQbD approach in method development\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePalbociclib (marketed as Ibrance) is a selective CDK4/6 inhibitor that is clinically important in the treatment of hormone receptor-positive (HR+), HER2-negative advanced breast cancer in combination with an aromatase inhibitor or fulvestrant. Its pyridopyrimidinone structure (Fig.\u0026nbsp;2) underlies its kinase inhibitory activity \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Most of the available literature focuses on conventional RP‑HPLC methods for its quantification in bulk drugs and formulations, including stability‑indicating assays and simultaneous estimation with other anticancer agents (Table\u0026nbsp;1), but no AQbD-based RP-HPLC method has been reported currently for the estimation of Palbociclib \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of previously reported methods and the current RP-HPLC method for Palbociclib\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e Chromatographic\u003c/p\u003e \u003cp\u003econditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColumn used\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLinearity (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRemarks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.02 M sodium dihydrogen phosphate buffer (pH 5.5): ACN: Methanol (80:10:10 v/v/v); 1 ml/min flow rate; at 254 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntersil C\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(250 mm x 4.6 mm, 5 \u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSimultaneous estimation of palbociclib and letrozole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmmonium acetate (pH 8) adjusted with TEA: ACN (38:62 v/v); 1 ml/min flow rate; at 263 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInertsil ODS-3V\u003c/p\u003e \u003cp\u003e(250 mm x 4.6 mm, 5 \u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026thinsp;\u0026minus;\u0026thinsp;1,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStability-indicating HPLC method for palbociclib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACN: sodium acetate buffer (30:70, 0.5% TEA); 1 ml/min flow rate; at 260 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKromasil C18 (250 mm x 4.6 mm, 5 \u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.04\u0026ndash;20.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContent determination of palbociclib in capsules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmmonium formate buffer (pH 4.2) adjusted with glacial acetic acid: ACN (35:65 v/v), 0.8 ml/min flow rate; at 357 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShimadzu Shim-pack C18 (250 mm x 4.6 mm, 5 \u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026ndash;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAQbD-based RP-HPLC method for quantification of palbociclib in bulk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProposed-present study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThis study focuses on the development, optimisation and validation of a reliable RP-HPLC method for quantifying Palbociclib in bulk, employing an AQbD framework to ensure method reliability, efficiency and regulatory compliance.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Reagents:\u003c/h2\u003e \u003cp\u003eThe pure API (Palbociclib) was provided by MSN Laboratories Pvt. Ltd (Telangana, India) as a gift sample, with a purity 99.60%. HPLC-grade acetonitrile and AR-grade ammonium formate were procured from Finar. Glacial acetic acid (Emplura) was procured from Merck, and ultrapure water was prepared using a Milli-Q purification system for buffer preparation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInstrumentation:\u003c/h3\u003e\n\u003cp\u003eA UV-Visible spectrophotometer (Shimadzu UV-1800) was used to determine the maximum absorbance wavelength (λ\u003csub\u003emax\u003c/sub\u003e) of Palbociclib, which served as the basis for selecting the detection wavelength. Chromatographic separation was carried out using an HPLC system with a UV-Visible detector (Shimadzu LC20-series), equipped with a Shimadzu Shim-pack C18 Column consisting of 250 mm of length, 4.6 mm of internal diameter and 5 \u0026micro;m particle size.\u003c/p\u003e\n\u003ch3\u003eOptimised Chromatographic conditions:\u003c/h3\u003e\n\u003cp\u003eThe mobile phase was prepared using an ammonium formate buffer (pH 4.2, adjusted with glacial acetic acid) and acetonitrile in the ratio 35:65 (v/v). It was filtered through a membrane filter (0.45 \u0026micro;m) and degassed by sonication. Chromatographic separation was achieved at 35\u0026deg;C using a flow rate of 0.8 ml/min with a 10 \u0026micro;L injection volume. The detection was carried out at 357 nm.\u003c/p\u003e\n\u003ch3\u003eProcedure for sample preparation:\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eDiluent A\u003c/b\u003e: Acetonitrile and Water in a 50:50 ratio.\u003c/p\u003e \u003cp\u003e(The solubility of the drug was high in this mixture)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiluent B\u003c/b\u003e: Mobile phase (Buffer: Acetonitrile, 35:65 ratio)\u003c/p\u003e\n\u003ch3\u003ePreparation of stock solution (200 µg/mL):\u003c/h3\u003e\n\u003cp\u003eA precisely weighed quantity (5 mg) of Palbociclib was dissolved in 20 mL of diluent A. The solution was sonicated for 10 minutes to ensure complete solubilisation (clear solution), followed by dilution with Diluent A to a final volume of 25 mL.\u003c/p\u003e \u003cp\u003eThe resulting solution was filtered through a 0.45 \u0026micro;m syringe filter prior to analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of calibration standards:\u003c/h2\u003e \u003cp\u003eThe stock solution was diluted using diluent B (Mobile Phase) to prepare calibration standard solutions in the range of 0.05\u0026ndash;50 \u0026micro;g/mL.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSelection of detection wavelength:\u003c/h3\u003e\n\u003cp\u003eA 10 \u0026micro;g/mL solution of Palbociclib was prepared from the stock solution using diluent B and was scanned in the range of 200\u0026ndash;400 nm on a UV-Visible Spectrophotometer. The λmax of 357 nm was obtained and was selected as a detection wavelength.\u003c/p\u003e\n\u003ch3\u003eRP-HPLC Method Development by AQbD approach:\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of ATP, CAAs and CMPS\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e:\u003c/h2\u003e \u003cp\u003eThe ATP, CAAs and CMPS were established systematically through risk assessment and experimental design to ensure method robustness and regulatory compliance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRisk Assessment using Ishikawa Diagram\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e:\u003c/h2\u003e \u003cp\u003eAn Ishikawa diagram (fishbone analysis) was employed to identify potential sources of variability in HPLC performance. Critical parameters such as sample preparation, mobile phase pH and composition, injection volume, flow rate, column oven temperature, selection of column and detection wavelength were examined for their impact on CAAs like retention time, peak area, efficiency (theoretical plates) and peak asymmetry. This qualitative assessment provided a foundation for prioritising CMPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePreliminary Screening using the one-factor-at-a-time (OFAT) method:\u003c/h2\u003e \u003cp\u003eBefore implementing statistical optimisation, the influence of individual CMPs on CAAs was evaluated using the OFAT approach. This ensured that only the most impactful CMPs were carried forward into the DoE, thereby improving efficiency and reducing experimental burden.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical optimisation using Box- Behnken Design (BBD):\u003c/h2\u003e \u003cp\u003eFollowing OFAT screening, BBD was applied to systematically study the interactions among four CMPs (buffer concentration, pH of the buffer, flow rate and column oven temperature) and four CAAs (retention time, peak area, peak tailing and net theoretical plates). BBD enabled the development of predictive models and the establishment of a statistically validated method operable design region (MODR), ensuring robustness and reproducibility of the method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDesign of Experiment (DoE) Software:\u003c/h2\u003e \u003cp\u003eDesign Expert (v.13), a statistical software, was used to apply DoE approaches such as Box-Behnken Design to optimise chromatographic conditions in HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical Method Validation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e:\u003c/h2\u003e \u003cp\u003eValidation of the developed method was performed as per ICH Q2 (R2) guideline, which includes the following parameters:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSpecificity:\u003c/h2\u003e \u003cp\u003eTo establish specificity, the chromatogram of the blank was compared with the sample (10 \u0026micro;g/ml), which confirmed that the analyte peak was well resolved without any interference from the diluent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSystem suitability studies:\u003c/h2\u003e \u003cp\u003eThe suitability of the developed chromatographic method was verified through six replicate injections of Palbociclib standard solution (10 \u0026micro;g/ml). The evaluation included determination of retention time, net theoretical plates and tailing factor. The acceptance criteria include retention times showing %RSD values below 2%, theoretical plates exceeding 2000, and tailing factor remaining within the limit of \u0026le;\u0026thinsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLinearity and Calibration Curve:\u003c/h2\u003e \u003cp\u003eThe linearity was evaluated across the concentration range of 0.05\u0026ndash;50 \u0026micro;g/mL with replicates (n\u0026thinsp;=\u0026thinsp;3) at each concentration level. The calibration curve was obtained by plotting the mean peak area on the y-axis and the analyte concentration on the x-axis. The regression equation of the calibration curve and the correlation coefficient were determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLimit of Detection (LOD) and Limit of Quantification (LOQ):\u003c/h2\u003e \u003cp\u003eThe LOD and LOQ were established using two approaches: the chromatographic signal-to-noise ratio method (practical), where LOD and LOQ were defined at S/N values of approximately 3 and 10, respectively; and the calibration curve method (statistical), where values were calculated based on the standard deviation of the response and the slope of the regression line.\u003c/p\u003e \u003cp\u003eIn the calibration curve method, the following equations were used to determine the LOD and LOQ:\u003c/p\u003e \u003cp\u003eLOD\u0026thinsp;=\u0026thinsp;3.3 x \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\sigma\\:}{Slope}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eLOQ\u0026thinsp;=\u0026thinsp;10 x \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\sigma\\:}{Slope}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWhere σ represents the standard deviation of the response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRange:\u003c/h2\u003e \u003cp\u003eAs the LOQ represents the lowest concentration at which the analyte could be quantified with acceptable precision and accuracy, the working range of the method was defined from the LOQ (obtained from the calibration curve method) up to the highest concentration validated within the linearity study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePrecision:\u003c/h2\u003e \u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eIntra-day (Repeatability):\u0026nbsp;\u003c/strong\u003eIntraday precision was assessed by preparing\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esix independent analyte solutions at a concentration of 10 \u0026mu;g/ml and analysing them under optimised chromatographic conditions on the same day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInter-day:\u0026nbsp;\u003c/strong\u003eIntermediate precision was determined by analysing six independently prepared analyte solutions at a concentration of 10 \u0026mu;g/ml over three consecutive days to determine the variability between days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccuracy of the analytical procedure was evaluated through recovery experiments conducted across the validated concentration range. Three levels were selected to represent the extremes and midpoint of the linearity interval, namely 3 \u0026micro;g/mL, 10 \u0026micro;g/mL, and 50 \u0026micro;g/mL. Triplicate analyses were performed at each concentration, and the mean recovery values were derived to assess the method performance. The study was considered acceptable when recoveries fell within 98\u0026ndash;102%, and the relative standard deviation did not exceed 2%, in accordance with established validation criteria. The following equation was used to calculate the % Recovery for each level.\u003c/p\u003e\n\u003cp\u003e% Recovery =\u0026nbsp;\u003cimg width=\"89\" height=\"29\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u0026nbsp; X 100\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was evaluated by introducing minor changes in the analytical method parameters, such as composition of mobile phase (\u0026plusmn; 2% of the buffer), column oven temperature (\u0026plusmn; 2 \u0026deg;C) and detection wavelength (\u0026plusmn;2 nm). The method is considered robust if these changes do not significantly influence the system suitability parameters (retention time, peak tailing and net theoretical plate) and all factors remain within the predefined acceptance limits (%RSD \u0026le; 2, tailing factor \u0026le; 2 and theoretical plates \u0026gt; 2000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBenchtop solution stability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFreshly prepared stock solution and working solutions were kept at an ambient laboratory condition (25 \u003csup\u003eo\u003c/sup\u003eC \u0026plusmn;2), exposed to normal light and were analysed at different time intervals (0, 4, 16, 24, 48 hours). At each timepoint, replicate injections (n=3) were performed, and chromatographic parameters, including peak area, retention time, tailing factor and theoretical plates, were compared with the reference solution.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eInitial method trials using acetonitrile-water mixtures in varying ratios resulted in peaks with retention times exceeding 25 minutes and poor peak shape. Subsequent experiments employing different buffer systems across a pH range of 2.5\u0026ndash;6.5 were evaluated. Among these, ammonium formate provided optimal performance, yielding a well-defined peak that eluted within 8 minutes. Hence, ammonium formate was selected as the buffer for the final method development.\u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eWavelength selection:\u003c/h2\u003e \u003cp\u003eA working standard solution of Palbociclib (10 \u0026micro;g/ml) showed a λmax of 357 nm. Additional peaks were observed at 220nm, 264nm, and 302 nm, as shown in Fig.\u0026nbsp;3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eMethod Development by an AQbD approach:\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003eRisk assessment:\u003c/h2\u003e \u003cp\u003eThe possible factors, as described in Fig.\u0026nbsp;4, were studied for their influence on the method's performance using an Ishikawa diagram. Each factor was grouped into one of the following categories: Method Parameters, Sample preparation, Materials, Instrumentation, Human Factors, and Environment. After mapping the potential cause, the most impactful parameters were selected based on the risk assessment score. The Key parameters identified were - mobile phase composition and its pH, flow rate, column oven temperature, type of column (C18), diluent for sample preparation and wavelength for detection.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eOptimisation of the method using BBD:\u003c/h3\u003e\n\u003cp\u003eThe BBD experimental design comprised 27 runs, incorporating two factor levels (-1,+1) along with a central point (0) to assess the curvature effects. The coded values of the independent variables and their corresponding experimental runs are summarised in Table\u0026nbsp;2 and Table\u0026nbsp;3, respectively.\u003c/p\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCoded values for CMPs in Box-Behnken Design\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFactors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eLevels\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-1\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA: pH of Buffer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB: Buffer concentration (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC: Flow Rate (mL/min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD: Column temperature (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBBD optimisation yielded the following equations explaining how each CMP influences the CAAs:\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRetention time\u0026nbsp;\u003c/strong\u003e=\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e+12.57450 - 1.00369A + 0.031433B - 11.38583C - 0.080967D - 0.003357AB + 0.185714AC \u0026ndash; 0.002357AD - 0.072500BC + 0.000050BD + 0.198500CD + 0.188350 A\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e+ 0.000807B\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e+ 1.32917C\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e-0.001413D\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInterpretation:\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe coded equation represents a quadratic polynomial model for retention time and suggests that there is a strong negative effect of pH (-1.00369A) and flow rate (11.3858C) on Retention time. Which implies that increasing Factor A and C reduces the retention time as shown in Figure 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeak Area\u0026nbsp;\u003c/strong\u003e=\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e+ 738994.666 \u0026ndash; 3152.75A -12876.25B \u0026ndash; 72343.58C-362.08D + 3043.50AB + 5068.75AC + 336.00AD \u0026ndash; 365.00BC \u0026ndash; 2390.75BD + 2979.50CD + 47348.87A\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e+ 195.87B\u003csup\u003e2 \u0026nbsp;\u003c/sup\u003e+ 7446.63C\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e\u0026ndash; 2655.37D\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInterpretation:\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe coded equation represents a quadratic polynomial model for peak area, while suggesting that there is a very strong negative effect of flow rate (-72343.58C) on peak area, implying that faster flow significantly reduces the area, as shown in Figure 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u0026nbsp;\u003c/strong\u003eExperimental Run of Box-Behnken Design (Sample concentration: 20 \u0026micro;g/mL)\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRun\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFactor A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFactor B\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFactor C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFactor D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRetention time\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak Area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTheoretical Plates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTailing Factor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e4.173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e821691\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e2770\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.441\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e737803\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e2721\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.458\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e716183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e3710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.331\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e4.204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e807909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e4002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e806391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e10422\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.469\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e686924\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e4297\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e2.268\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e674981\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e2617\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.471\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e750896\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e5496\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e2.383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n 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\u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.861\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e780780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e3797\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.355\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e739753\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e2718\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.453\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e722986\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e3652\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.319\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e4.347\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e855098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e4082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.357\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e677803\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e2522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e660099\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e3484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.312\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e4.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e775916\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e4172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.362\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.55752%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.73451%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2212%;\"\u003e\n \u003cp\u003e3.596\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.1504%;\"\u003e\n \u003cp\u003e772048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.6903%;\"\u003e\n \u003cp\u003e4631\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7965%;\"\u003e\n \u003cp\u003e1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTailing Factor\u0026nbsp;\u003c/strong\u003e=\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e+ 4.94243 \u0026ndash; 0.202381A \u0026ndash; 0.068233B \u0026ndash; 0.41C + 0.003067D\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInterpretation:\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe coded equation represents a linear model for the tailing factor. It suggests that there is a negative effect of pH (-0.202381A) on the tailing factor, implying that as the pH increases, the tailing is reduced and the peak symmetry is improved, as shown in Figure 7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTheoretical plates\u0026nbsp;\u003c/strong\u003e=\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e+ 215254.5 \u0026ndash; 54061.66A \u0026ndash; 4603.783B \u0026ndash; 75838.333C + 248.15D + 453.35714AB + 4414.28AC \u0026ndash; 37.42857AD + 293.00BC \u0026ndash; 15.23BD + 65.50CD + 4738.60544A\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e+44.72167B\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e+24741.667C\u003csup\u003e2\u003c/sup\u003e + 5.72167D\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInterpretation:\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe coded equation represents a quadratic second-order polynomial model for theoretical plates. It suggests that there is a strong negative effect of flow rate (-75838.333C), implying that higher flow reduces efficiency, as shown in \u0026nbsp;Figure 8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod Operable Design Region (MODR):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MODR was established by overlaying contour plots of all critical responses, identifying the 3D design space where method performance consistently met the acceptance criteria. The two critical parameters that were optimised are pH of the buffer (A) and the buffer concentration (B), while the other two parameters, flow rate (C) and column oven temperature (D), were kept constant during the overlay analysis. The yellow area shown in Figure 9 represents the optimised operable design space for the developed method. This region was derived by overlaying contour plots of multiple responses (retention time, peak area, net theoretical plates and tailing factor). The red dots represent the experimental runs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimised Chromatographic conditions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selection of optimised chromatographic conditions was guided by desirability solutions derived from BBD, ensuring robustness and compliance with ICH guidelines. The conditions are shown in Figure 10 with a desirability of 1.0000. The final chromatographic conditions, as shown in Table 4, were further used for method validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eControl strategy:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA control strategy is planned for the developed method to deliver reliable and robust results within the obtained MODR. The elements identified as controls are as follows:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMobile phase pH:\u003c/strong\u003e To obtain reproducible retention time and analyte stability, the pH should be controlled within 2.8-4.2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBuffer concentration in the mobile phase (%):\u0026nbsp;\u003c/strong\u003eThe buffer concentration can be varied in the range 33 - 40%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow rate:\u0026nbsp;\u003c/strong\u003eThe flow is set to 0.8 mL/min, with a variation of \u0026plusmn;0.05mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColumn Oven Temperature:\u0026nbsp;\u003c/strong\u003eThe temperature is maintained at 35 \u003csup\u003eo\u003c/sup\u003eC\u0026plusmn; 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Optimised Chromatographic conditions for Palbociclib estimation:\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"517\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003eChromatographic \u003cstrong\u003eConditions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStationary Phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003eShimadzu Shim-pack C18 Column\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(250 mm of length; 4.6 mm of internal diameter and 5 \u0026mu;m particle size)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMobile Phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003eAmmonium formate buffer (pH 4.2, adjusted with glacial acetic acid) and Acetonitrile ratio 35:65 (v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlow rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003e0.8 mL/min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInjection Volume\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003e10 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eColumn oven temperature\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003e35 \u003csup\u003eo\u003c/sup\u003eC (\u0026plusmn;2 \u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eElution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003eIsocratic mode\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRun time\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003e8 minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDetector and Wavelength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003eUV-Visible, 357 nm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 40.0387%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRetention time\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59.9613%;\"\u003e\n \u003cp\u003e4.236 minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalytical Method Validation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecificity was assessed by analysing a blank solution and a sample prepared at a concentration of 10 \u0026micro;g/mL, where the chromatogram of the blank showed no interfering peaks Figure 11A, and the analyte peak in the sample was clearly resolved with a retention time of 4.236 minutes Figure B, confirming the absence of solvent interference.\u003c/p\u003e\n\u003ch3\u003eSystem suitability studies:\u003c/h3\u003e\n\u003cp\u003eSystem suitability testing demonstrated consistent retention time, acceptable peak symmetry (\u0026le;\u0026thinsp;2), and adequate theoretical plates, confirming the reliability of the chromatographic method. The results are summarised in Table\u0026nbsp;5.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of system suitability parameters for Palbociclib\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReplicate No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRetention Time (minutes)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePeak Area (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTailing Factor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTheoretical Plate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e403617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3870\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e417189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3766\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e409646\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3760\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e419608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3843\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e410679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3865\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e413617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3846\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e412392.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3825\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5728.141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.900\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e%RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3890\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.1739\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eLinearity:\u003c/h2\u003e \u003cp\u003eLinearity study confirmed direct proportionality between concentration and peak area over the range of 0.05\u0026ndash;50 \u0026micro;g/ml, with a correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) of 0.9999. The results are summarised in Table\u0026nbsp;6:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCalibration curve and Linearity Results for Palbociclib method validation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003cp\u003ePeak area\u003c/p\u003e \u003cp\u003e(mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e% RSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIntercept\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSlope\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCorelation coefficient\u003c/p\u003e \u003cp\u003e(R2)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.7946\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003e2709.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003e40496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003e0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0415\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.5387\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44789\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.1917\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e207718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3682\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e412793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0761\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1198567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8434\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2037597\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.5311\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eLOD and LOQ:\u003c/h2\u003e \u003cp\u003eThe LOD and LOQ derived from the S/N approach were found to be 0.05 \u0026micro;g/ml and 0.5 \u0026micro;g/ml, respectively. By applying the calibration curve method, it yielded higher LOD and LOQ of 0.7 \u0026micro;g/ml and 2.2 \u0026micro;g/ml, respectively. Results of both methods are given in Table\u0026nbsp;7, with the significance of each method.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eRange:\u003c/h2\u003e \u003cp\u003eBased on the method\u0026rsquo;s LOQ value (2.2 \u0026micro;g/mL), the working range for Palbociclib quantification was set between 3\u0026ndash;50 \u0026micro;g/mL, ensuring accuracy and precision throughout.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLOD and LOQ results obtained by two approaches with their analytical significance\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLOD (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLOQ (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSignificance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSignal-to-noise\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProvides the lowest possible values, showing the method's sensitivity (Qualitative)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCalibration curve\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDefines the lowest concentration that can be quantified with acceptable accuracy and precision (Quantitative)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003eIntra-day and Inter-day precision:\u003c/h2\u003e \u003cp\u003eThe intra-day precision, assessed by analysing replicates within a single day, demonstrated a % RSD of less than 2% across all levels, as shown in Table\u0026nbsp;8, confirming excellent repeatability of the developed method.\u003c/p\u003e \u003cp\u003eSimilarly, analysis repeated over three days (inter-day) showed minimal %RSD, supporting method\u0026rsquo;s reproducibility as shown in Table\u0026nbsp;9.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIntra-day precision results (repeatability)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eIntra-day Precision\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePeak Area\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mV)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e% RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e121589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e122917.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1664.8617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1.3544564\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e122378\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e124785\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e416591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e420735.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e5983.232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1.4220881\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e418021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e427595\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2033798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2034263.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e9211.8191\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.4528332\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2025293\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2043699\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInter-day (Intermediate precision) results:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eInter-day Precision\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConcentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMean Peak Area\u0026nbsp;(mV) (n\u0026thinsp;=\u0026thinsp;3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e%RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDay 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eDay 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eDay 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e121255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e123157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e124768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e123060\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1758.50761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.4289839\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e418542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e419608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e413028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e417059.333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3531.68874\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.8468073\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2063659\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2100199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2083877\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2082578.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18304.5842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.8789386\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAccuracy (% Recovery study)\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eAccuracy testing demonstrated satisfactory recovery of Palbociclib, with mean percentage values between 98.91% to 100.88% at concentrations of 3 \u0026micro;g/ml, 10 \u0026micro;g/mL and 50 \u0026micro;g/mL, as summarised in Table\u0026nbsp;10.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRobustness\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe robustness testing was performed by changing mobile phase composition, column temperature and detection wavelength. The system suitability results consistently met the acceptance criteria, indicating the method\u0026rsquo;s consistency under slight variations in the chromatographic conditions. The result of robustness is summarised in Table\u0026nbsp;11.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBenchtop solution stability\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eSolution stability was monitored under ambient laboratory conditions for 24 hours, with assay values remaining consistent and replicate injections showing an RSD below 2%. The data confirms that the solutions are stable for a minimum of 24 hours under benchtop storage.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab10\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 10\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAccuracy study showing recovery results for Palbociclib\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeak Area\u003c/p\u003e \u003cp\u003e(mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean (%RSD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(% Recovery)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e121389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e122880.667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e98.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e122478\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e124775\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e416000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e412793.667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e100.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e414658\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e407723\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2003798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2037596.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e100.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2065293\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2043699\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab11\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 11\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRobustness data for a sample concentration of 10 \u0026micro;g/mL\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariation\u003c/p\u003e \u003cp\u003e(\u0026plusmn;\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eSystem Suitability Parameters\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRetention Time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTailing factor (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTheoretical Plates\u003c/p\u003e \u003cp\u003e(range)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBuffer concentration (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.481\u0026ndash;1.498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5257\u0026ndash;5277\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.465\u0026ndash;1.484\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4899\u0026ndash;4966\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eColumn Temperature (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.604\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.491\u0026ndash;1.503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5059\u0026ndash;5121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.605\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.483\u0026ndash;1.491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5227\u0026ndash; 5263\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDetection wavelength (nm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.478\u0026ndash;1.486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5079\u0026ndash;5106\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.465\u0026ndash;1.484\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5080\u0026ndash;5099\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLife-cycle management and continuous improvement\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eManaging the life cycle of an AQbD-based HPLC method ensures its reliability and reproducibility while maintaining regulatory standards. Post-validation, the method will be continually monitored, documented and controlled modifications within the design space, ensuring its continued suitability for routine analysis of Palbociclib.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab12\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 12\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA data for all the CMPs and CAAs using BBD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStatistical Terms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRetention Time (R1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePeak Area\u003c/p\u003e \u003cp\u003e(R2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTailing Factor (R3)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTheoretical Plates (R4)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF-value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e228.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep-value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eModel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQuadratic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQuadratic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLinear\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eQuadratic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA-Buffer conc.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4673\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.19x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2408\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.27x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB-Buffer pH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.99x10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.28x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC- Flow rate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.28x10\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.31x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eD-Column temp\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.57x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.69x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.705x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.02x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.81x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.51x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e68644\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.32x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85849\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.250 x 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.28x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.79x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.55x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4290.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.19x10\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.87x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.04x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.66x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.95x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.26x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.76x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.09x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eResidual Analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLack of fit\u003c/p\u003e \u003cp\u003eF-value\u003c/p\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSignificant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSignificant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSignificant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSignificant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e476.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4906.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2250.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9807\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9466\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAdjusted R\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9919\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4196\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.8843\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe integration of Ishikawa-based risk assessment, OFAT screening, and BBD optimisation highlights the strength of the AQbD framework in delivering a reliable and reproducible RP-HPLC method. This structured workflow ensures scientific rigour, regulatory flexibility, and lifecycle management, representing a significant advancement over conventional trial-and-error strategies for Palbociclib quantification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor\u0026rsquo;s Contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Tanvi Painginkar carried out the experimental work of HPLC method optimization, DoE analysis, data interpretation/analysis, and drafting the paper. Dr Gundawar Ravi was involved in conception of research work, supervising the experimental work, data analysis, resources, and drafting and reviewing the paper. All authors have made a significant contribution to the research in the manuscript, approved its claims, and agreed to be an author.\u003c/p\u003e\n\u003ch2\u003eCompeting interests:\u003c/h2\u003e\n\u003cp\u003eThe author(s) declares no competing interests\u003c/p\u003e\n\u003ch2\u003eAcknowledgement:\u003c/h2\u003e\n\u003cp\u003eThe authors are thankful to (i) Manipal Academy of Higher Education, Manipal, India, for providing Dr TMA Pai Doctoral Scholarship to Tanvi Painginkar, (ii) Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, for providing the necessary facilities and the authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University, Abha, for funding this work through Large Research Project under grant no. RGP.2/728/46.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eICH guideline for. Analytical Procedure Development Q14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInternational Council For Harmonisation of Technical Requirements for Pharmaceuticals for Human Use ICH Harmonised Guideline. \u003cem\u003eQual. Risk Manage.\u003c/em\u003e \u003cb\u003eQ9\u003c/b\u003e(R1 ).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeg, S. et al. Introduction to analytical quality by design. in Handbook of Analytical Quality by Design 1\u0026ndash;14 (Elsevier, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/B978-0-12-820332-3.00009-1\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-12-820332-3.00009-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanthiah, S., Joysa Ruby, J., Sgb, H. \u0026amp; Kannappan, V. Navigating the AQbD Landscape: Enhancing Quality Management in Liquid Chromatography Method Development. \u003cem\u003eBiomedical Chromatography\u003c/em\u003e vol. 39 Preprint at (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/bmc.70031\u003c/span\u003e\u003cspan address=\"10.1002/bmc.70031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalbociclib Uses, Dosage, Side Effects, Warnings - Drugs.com. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.drugs.com/palbociclib.html\u003c/span\u003e\u003cspan address=\"https://www.drugs.com/palbociclib.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeMichele, A. et al. 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Anal\u003c/em\u003e \u003cb\u003e255\u003c/b\u003e, (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolDraw. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.moldraw.com/\u003c/span\u003e\u003cspan address=\"https://www.moldraw.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, W. dong. Thermodynamic modelling, Hansen solubility parameter and solvent effect of palbociclib in fourteen pure solvents at different temperatures. \u003cem\u003eJ. Mol. Liq.\u003c/em\u003e 356, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eICH Guidelines for. Validation of Analytical Procedures Q2(R2).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Analytical-QbD, Design of experiments, Palbociclib, CDK4/6i, Ishikawa diagram, Box-Behnken Design","lastPublishedDoi":"10.21203/rs.3.rs-8901646/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8901646/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eConventional empirical approaches (trial and error) to HPLC method design tend to yield fragile, non-consistent methods and lack regulatory flexibility, while Analytical Quality by Design offers a structured framework ensuring reliability. In this study a AQbD guided HPLC method was developed and optimised for Palbociclib, a Cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) approved for the treatment of hormone receptor-positive breast cancer. Risk assessment (Ishikawa diagram) tool guided factor selection while Box-Behnken Design enabled optimisation through Design Expert (v.13) software. The optimised method employed a Shimpack C18 column (250 mm x 4.6 mm, 5 \u0026micro;m); mobile phase comprising Buffer (ammonium formate of pH 4.2 adjusted with glacial acetic acid) and Acetonitrile in the ratio 35:65; flow rate of 0.8 mL/min; injection volume of 10 \u0026micro;L; column oven temperature of 35\u0026deg;C and detection wavelength of 357 nm. Validation was performed in accordance with the ICH Q2 (R2) guidelines. The peak was eluted at 4.23 minutes, and the method demonstrated excellent linearity across 3\u0026ndash;50 \u0026micro;g/mL with a correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) of 0.9999, LOD of 0.75 \u0026micro;g/mL and LOQ of 2.27 \u0026micro;g/mL. Accuracy studies demonstrated recoveries between 98.91-100.88% while precision was evaluated through both intra-day and inter-day studies, consistently showing RSD deviations below 2%.\u003c/p\u003e","manuscriptTitle":"Analytical Quality by Design guided RP-HPLC Method Development and Validation for estimation of Palbocilib employing Box-Behnken optimisation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-04 20:24:48","doi":"10.21203/rs.3.rs-8901646/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f8457a53-c89f-40a0-9d6f-27fa69c79046","owner":[],"postedDate":"March 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63765163,"name":"Biological sciences/Cancer"},{"id":63765164,"name":"Physical sciences/Chemistry"},{"id":63765165,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2026-04-01T16:55:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-04 20:24:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8901646","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8901646","identity":"rs-8901646","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}