Repeatability and Quantitation in a Method Using the Alliance HPLC System with the 2998 Photodiode Array Detector and the ACQUITY QDa Detector

Importance of the topic

High performance liquid chromatography is a cornerstone technique for separating complex mixtures, but reliable peak identification and quantitation often require complementary detection modes. Combining photodiode array and mass detection enhances confidence in compound identity and purity while enabling precise quantitation in a single analysis.

Objectives and study overview

This study evaluated the repeatability and quantitation of irbesartan and a related impurity (compound A) using an Alliance HPLC system equipped with a 2998 PDA detector and an ACQUITY QDa mass detector. The goal was to align detector dynamic ranges and confirm performance for pharmaceutical analysis.

Methodology and instrumentation

Standard solutions of irbesartan (0.100 mg/mL) and six levels of related compound A (0.0005–0.250 mg/mL) were prepared in methanol. Gradient separation was performed on an XSelect HSS T3 column (4.6×250 mm, 5 μm) at 40 °C over 25 minutes. Flow splitting and dilution were achieved using an isocratic solvent manager with a restrictor and splitter kit. Detection and data processing were handled by Empower 3 FR2 CDS.

• HPLC: Alliance system with 2998 PDA and ACQUITY QDa
• Column: Waters XSelect HSS T3, 4.6×250 mm, 5 μm
• Mobile phase: Water, acetonitrile and 1% formic acid gradient
• Flow rate: 300 µL/min split to 1000 µL/min wash
• PDA: 210–400 nm scan, 254 nm quantitation
• MS: ESI+ mode, SIR m/z 429.4 (API) and 447.3 (impurity)

Main results and discussion

Retention time reproducibility was under 0.1% RSD for both analytes. Area repeatability was <0.15% on the PDA and <1.5% on the QDa for the impurity. Calibration on the PDA was linear from 0.0005 to 0.250 mg/mL (r2=0.9999), while the mass detector showed linearity from 0.0005 to 0.050 mg/mL (r2=0.995). Flow splitting successfully aligned detector sensitivities, enabling equivalent quantitative performance across both channels.

Benefits and practical applications of the method

• Orthogonal detection improves peak identity confirmation and purity assessment
• Aligned dynamic ranges allow simultaneous UV and MS quantitation
• Single-software integration simplifies data processing and reporting

Future trends and potential applications

Advances may include integration with high-resolution mass spectrometry for detailed structural elucidation, automation for higher throughput, miniaturized flow splitting for reduced solvent consumption, and AI-driven data analysis to further enhance method robustness and decision support.

Conclusion

• Mass detection added to PDA-equipped LC enhances identification confidence
• Dynamic range alignment via isocratic solvent manager enables dual-mode quantitation
• Both detectors exhibited excellent linearity and repeatability for irbesartan and impurity
• Empower software facilitated unified acquisition and analysis

Reference

• Chavali A, Wheat TE, McConville P. Repeatability and Quantitation in a Method Using the Alliance HPLC System with the 2998 Photodiode Array Detector and the ACQUITY QDa Detector. Waters Corporation; 2014.