AQbD-Based RP-HPLC Method Development for Simultaneous Estimation of Antineoplastic Agents in Combined Dosage Forms: A Critical Review

Combination antineoplastic regimens — whether co-formulated or co-administered — are now central to oncology practice because they allow simultaneous engagement of multiple resistance pathways while improving dosing convenience. This trend has direct analytical consequences: quality control, stability testing, and regulatory submission all require RP-HPLC methods capable of resolving two or more actives of differing polarity, pKa, and UV response within a single run, while remaining stability-indicating under ICH-mandated forced-degradation conditions.

Analytical Quality by Design (AQbD) reframes method development as a risk-based design exercise rather than a trial-and-error optimization. Building on the QbD principles of ICH Q8(R2) and the risk-management framework of ICH Q9(R1), and now formalized for analytical procedures in ICH Q14 (Analytical Procedure Development), AQbD begins with an Analytical Target Profile (ATP), proceeds through risk assessment of critical method parameters (CMPs), uses Design of Experiments (DoE) — commonly Box-Behnken or central composite designs — to map a design space against critical analytical attributes (CAAs), and concludes with a defined control strategy. This structured approach is progressively displacing empirical, one-factor-at-a-time method development in the pharmaceutical analytical literature.

2. Regulatory and Clinical Relevance

The urgency of this review is underscored by regulatory activity in the antineoplastic combination space. On 20 December 2024, the US FDA granted accelerated approval to the three-drug combination of encorafenib (Braftovi), cetuximab (Erbitux), and mFOLFOX6 chemotherapy for adult patients with BRAF V600E-mutant metastatic colorectal cancer — one of several combination-regimen oncology approvals granted in that approval cycle. Encorafenib alone, and in its combined form with cetuximab, has already been the subject of a dedicated stability-indicating RP-HPLC simultaneous-estimation method (Sriramula & Shivaraj, 2025), illustrating how quickly analytical method development follows clinical approval in this space. Rising global incidence of colorectal, prostate, and BRAF-mutant cancers — alongside the accompanying growth in fixed-dose and co-administered antineoplastic regimens — creates sustained downstream demand for validated, AQbD-compliant RP-HPLC methods capable of supporting batch release, stability studies, and regulatory dossiers for these products.

3. AQbD Workflow for Antineoplastic Combinations

Across the methods surveyed in Section 4, a broadly consistent AQbD workflow recurs: (i) definition of the Analytical Target Profile specifying the analytes, matrix, and required specificity/sensitivity; (ii) an Ishikawa or risk-ranking exercise to shortlist critical method parameters — typically mobile-phase organic ratio, buffer pH, flow rate, and column temperature; (iii) a Plackett-Burman or similar screening design to reduce the parameter list, followed by a Box-Behnken or central composite design to model the design space against retention time, resolution, tailing factor, and peak area as critical analytical attributes; (iv) generation of a design space and normal operating region using response-surface modelling (commonly in Design-Expert software); and (v) definition of a control strategy and full ICH Q2(R1) validation — specificity, linearity, accuracy, precision, robustness, and forced-degradation stability-indication.

4. Literature Review

A representative body of recent AQbD- and DoE-driven RP-HPLC work on antineoplastic agents is summarised below.

Sriramula and Shivaraj (2025) developed and validated a stability-indicating RP-HPLC method for the simultaneous quantification of encorafenib and cetuximab in bulk and pharmaceutical dosage forms, using a Waters X-Terra RP-18 column with an acetonitrile/0.1% TEA buffer mobile phase and UV detection at 240 nm, reporting linearity, accuracy, and precision within ICH-acceptable limits — directly relevant to the December 2024 FDA-approved encorafenib-cetuximab-chemotherapy regimen.

A stability-indicating RP-HPLC method for the antineoplastic combination of niraparib and abiraterone in synthetic mixtures and combined tablet formulations was reported in Discover Applied Sciences (2025), using a Zodiac C18 column with an acetonitrile/sodium phosphate buffer mobile phase, achieving baseline resolution with retention times of 2.2 and 2.7 minutes for the two actives.

Venkatachari and Ramasamy (2025) applied a Box-Behnken design to RP-HPLC method development for irinotecan hydrochloride trihydrate, using QbD risk-assessment tools to shorten retention time while maintaining robustness — illustrating the single-analyte AQbD workflow that underpins the simultaneous-estimation methods discussed above.

An AQbD-driven RP-HPLC method for abiraterone acetate, quantifying the drug in the presence of its degradants, used a Box-Behnken design with three critical method parameters (mobile-phase composition, flow rate, and buffer pH) across seventeen experimental runs, with retention time and peak area modelled as critical analytical attributes using Design-Expert software (PMC, 2022).

A QbD-based, Central Composite Design-optimized stability-indicating RP-HPLC method was reported for larotrectinib, combining Plackett-Burman screening with CCD optimization and an integrated white/green/blue analytical-sustainability assessment — an increasingly common addition to AQbD antineoplastic method papers (Journal of Applied Pharmaceutical Research, 2025).

Outside strict oncology indications, Box-Behnken- and CCD-based AQbD RP-HPLC methods for other simultaneous-estimation combinations — for example rutin and ciprofloxacin in a bilosomal nanoformulation (PMC, 2023) and linagliptin and empagliflozin (Research Square, 2025) — provide useful methodological precedent for design-of-experiments strategy applied to structurally dissimilar co-formulated actives, even though the drugs themselves are not antineoplastic.

5. Identified Gaps and Novelty Statement

Three gaps recur across the literature surveyed. First, published AQbD-RP-HPLC methods for antineoplastic combinations are almost entirely single-method reports; no review to date has consolidated AQbD-specific (ATP-to-control-strategy) workflows across multiple antineoplastic combination methods to identify shared design-space and robustness patterns. Second, integration of green/white/blue analytical-chemistry sustainability metrics remains inconsistent, appearing in only a minority of the antineoplastic-focused papers reviewed. Third, several antineoplastic combinations receiving FDA approval in 2023–2025 (including the December 2024 encorafenib-cetuximab-chemotherapy approval) do not yet have a corresponding AQbD-optimized, stability-indicating RP-HPLC method in the peer-reviewed literature, representing an active method-development opportunity rather than a reviewable gap at present.

6. CONCLUSION

AQbD-driven RP-HPLC method development offers a regulatory-aligned, risk-based route to robust, stability-indicating methods for antineoplastic combination therapies at a time when clinical approvals of such combinations are accelerating. The literature reviewed here demonstrates consistent uptake of Box-Behnken and central composite designs for individual and combined antineoplastic actives, but reveals an emerging lag between newly approved combination regimens and the publication of corresponding AQbD-optimized analytical methods. Closing this lag, and standardizing incorporation of green-chemistry metrics into the AQbD control strategy, represent the principal opportunities for future analytical method development in this therapeutic area.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

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