A High-Throughput Method for the Analysis of 13 Antiretroviral Compounds Using a UHPLC Column and System Combination

Significance of the Topic

The analysis of multiple antiretroviral drugs in a single run is critical for improving throughput and reducing costs in pharmaceutical and clinical laboratories. Ultra high-performance liquid chromatography (UHPLC) offers high resolution and rapid analysis, enabling timely monitoring of drug combinations used in HIV therapy.

Goals and Study Overview

This study demonstrates the performance advantages of the Thermo Scientific Hypersil GOLD VANQUISH 1.9 µm column coupled with the Vanquish UHPLC system for the fast separation of 13 antiretroviral compounds. The work also explores dedicated methods for commonly co-administered fixed-dose combinations to further enhance throughput.

Methodology and Instrumentation

Sample Preparation:

• Primary solutions of 13 antiretrovirals at 1 mg/mL in methanol or water
• Final working solutions at 25 µg/mL (75 µg/mL for cobicistat and abacavir) in 75:25 methanol/water

UHPLC Conditions:

• Column: Hypersil GOLD VANQUISH, 1.9 µm, 50×2.1 mm
• Mobile phases: A – water + 0.1% formic acid; B – methanol + 0.1% formic acid
• Column temperature: 40 °C with pre-heating; injection volume: 1 µL; detection at 254 nm
• Binary gradient methods A–D with flow rates of 1.0–1.2 mL/min and run times from 0.7 to 2.4 min

Main Results and Discussion

The optimized method achieved baseline separation of all 13 compounds within a 2-minute detection window (total cycle 2.4 minutes) at a maximum pressure of 820 bar. By increasing flow rate to operational limits, the detection window for fixed-dose combinations was reduced to approximately 0.7 minutes without loss of resolution. Excellent retention time reproducibility (%RSD < 0.1) and peak symmetry (asymmetry factors ~1.1–1.4) were observed across single-drug and combination assays.

Benefits and Practical Applications

This UHPLC approach delivers:

• High-throughput screening of 13 antiretrovirals in under 2 minutes
• Rapid analysis of 11 fixed-dose combinations in under 1 minute
• Robust retention time precision and consistent peak shapes
• Enhanced laboratory efficiency for drug quality control and clinical monitoring

Future Trends and Opportunities

Emerging UHPLC column chemistries and lower dispersion system designs promise further reductions in analysis time and solvent usage. Integration with mass spectrometry and automated sample preparation workflows can expand method sensitivity and applicability. Data analytics and machine learning may optimize gradient profiles and predict column performance.

Conclusion

By leveraging the high-pressure capability and low dispersion of the Hypersil GOLD VANQUISH column and Vanquish UHPLC system, a rapid and robust UHPLC method was established for 13 antiretroviral drugs and multiple fixed-dose combinations, significantly improving throughput and analytical performance.

Instrumentation Used

• Vanquish UHPLC System (System Base, Binary Pump H, Split Sampler HT, Column Compartment H, Active Pre-heater, Diode Array Detector HL)
• Hypersil GOLD VANQUISH 1.9 µm, 50×2.1 mm column
• LightPipe flow cell, Virtuoso Vial Identification System

References

No references were provided in the original text.