Method validation based on ICH guidelines of a USP assay method of acetaminophen

Importance of the topic

Analytical method validation underpins the reliability of pharmaceutical quality control by ensuring assays deliver accurate, precise and specific results. Compliance with ICH guidelines and pharmacopeial requirements is essential to meet regulatory standards and to guarantee patient safety.

Objectives and study overview

This study demonstrates the implementation of a full method validation for the USP acetaminophen assay using Thermo Scientific Chromeleon CDS eWorkflows. The goal is to validate accuracy, precision, specificity, linearity, detection and quantification limits, range and robustness, while automating sequence setup, data processing and reporting.

Methodology

A 1 mg/mL acetaminophen stock solution was prepared in methanol. Working standards ranging from 0.02 to 150 µg/mL were prepared to address LOD/LOQ, linearity, accuracy and robustness tests. Chromeleon eWorkflow templates provided predefined injection lists, processing methods with Cobra Wizard peak integration and component tables to set acceptance limits for recovery, RSD and calibration levels. All data evaluation and report generation were executed within Chromeleon CDS to eliminate manual data transfer.

Used Instrumentation

• Thermo Scientific Vanquish Core Quaternary HPLC system: System Base Core (VC-S01-A), Quaternary Pump C (VC-P20-A), Sampler CT (VC-A12-A), Column Compartment C (VC-C10-A-03), Diode Array Detector CG (VC-D11-A) with 13 µL flow cell
• Column: Hypersil GOLD C8, 100 × 4.6 mm, 3 µm
• Mobile phase: A – 1.7 g/L KH2PO4 and 1.8 g/L Na2HPO4 in water; B – Methanol; gradient 1% to 81% B over 7 min; flow rate 1.0 mL/min
• Column temperature 35 °C; sampler temperature 8 °C; detection at 230 nm with spectral scan 210–350 nm

Main results and discussion

Accuracy recoveries fell within predefined limits. Repeatability and intermediate precision met ICH criteria, with one analyst sequence flagged for review. Specificity was confirmed by peak purity analysis. Linearity over 25–150 µg/mL yielded R² > 0.999. Robustness evaluated with ±10% flow rate and ±10 °C temperature shifts passed acceptance. LOD and LOQ, determined by signal-to-noise ratio, were 13.66 ng/mL (0.014%) and 45.52 ng/mL (0.045%), respectively. Automated pass/fail flags and graphical calibration and purity plots accelerated data interpretation.

Benefits and practical applications

• Significant reduction in sequence setup and report generation time
• Elimination of transcription errors through integrated processing
• Full regulatory compliance and audit readiness
• Rapid method transfer to new HPLC platforms with minimal reconfiguration

Future trends and opportunities

Advances in AI-driven peak detection and real-time data quality monitoring will enhance validation workflows. Cloud-based CDS platforms can enable distributed collaborations and remote audit capabilities. Expanding eWorkflows to additional compound classes and regulatory regions will further streamline laboratory operations.

Conclusion

Thermo Scientific Chromeleon CDS eWorkflows provide a complete, automated solution for ICH-based method validation. Applied to the USP acetaminophen assay, this approach delivered robust validation, reduced manual effort and ensured regulatory compliance.

Reference

1. European Directorate for the Quality of Medicines & HealthCare. Validation of Analytical Procedures, PA/PH/OMCL (13) 82 2R, 2014.
2. United States Pharmacopeia. Chapter <1225>, Validation of Compendial Procedures.
3. International Conference on Harmonisation (ICH). Q2(R1) Guidance on Validation of Analytical Procedures, CPMP/ICH/381/95, 1995.
4. United States Pharmacopeia. USP40-NF35 S1, Acetaminophen assay method, 2017.
5. Thermo Scientific AppsLab Library. Method validation application note resources.