Simultaneous quantification of nevirapine and low-level impurities

Significance of the topic

The accurate measurement of active pharmaceutical ingredients and their low‐level impurities is critical for drug safety, regulatory compliance and therapeutic efficacy. High dynamic range detectors reduce sample preparation steps and increase throughput in quality control and drug development laboratories.

Study objectives and overview

This study evaluated the dynamic range and noise performance of the Thermo Scientific Vanquish DAD FG diode array detector in the simultaneous quantification of the HIV reverse transcriptase inhibitor nevirapine and its trace impurities (A, B and C) in a single UHPLC run.

Methodology and instrumentation

The method was based on a previously developed USP HPLC assay adapted to UHPLC with a ballistic gradient. Stock solutions of nevirapine (1 mg/mL) and impurities (100–200 µg/mL) were prepared in acetonitrile and diluted in 10 mM ammonium acetate buffer (pH 5.0)/acetonitrile. Calibration standards ranged from 0.1 to 1000 µg/mL.

Used instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system (Binary Pump H, Split Sampler HT, Column Compartment H)
• Thermo Scientific Vanquish Diode Array Detector FG with semi-micro flow cell (2.5 µL)
• Column: Syncronis C18, 2.1 × 100 mm, 1.7 µm, 100 Å at 50 °C
• Mobile phase A: 10 mM NH4Ac pH 5.0 (85:15 water/acetonitrile); B: acetonitrile; flow rate 0.8 mL/min
• Detection: 240 nm, 100 Hz, 0.05 s response
• Data system: Thermo Scientific Chromeleon 7.2 SR5

Main results and discussion

The detector demonstrated linearity in peak height from 0.1 to 850 µg/mL nevirapine (0.5–3700 mAU) with R² > 0.9996. Weighted peak‐area calibration (1/amount) further improved accuracy over the full range (R² > 0.9998, deviations < 5%). Retention times were reproducible (RSD ≤ 0.15%). In an 850 µg/mL nevirapine standard, all impurities were detected below 0.03% relative area; spiked samples at 0.05% impurity yielded deviations of 6–21% for impurities and < 2% for the API. Quantification down to 0.012% relative area was achieved.

Benefits and practical applications

• Eliminates multiple dilutions by covering API and impurity levels in one run
• Wide dynamic range up to 3.7 AU with low noise for reliable trace‐level detection
• Improves throughput and simplifies impurity profiling workflows in pharmaceutical QA/QC

Future trends and potential applications

Advances in UV-visible detector design, combined with chemometric data analysis and high-resolution separations, will further enhance impurity profiling sensitivity and accuracy. Integration with automated sample preparation and artificial intelligence-driven peak identification may streamline regulatory reporting and accelerate drug development.

Conclusion

The Vanquish DAD FG detector offers exceptional linearity and low baseline noise, enabling simultaneous quantification of nevirapine and low-level impurities in a single UHPLC run. This capability supports stringent ICH impurity thresholds while reducing sample handling and analysis time.

References

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2. International Conference on Harmonisation. Q3A(R2): Impurities in New Drug Substances. 2006.
3. International Conference on Harmonisation. Q3B(R2): Impurities in New Drug Products. 2006.
4. Sichilongo K, Chinyama M, Massele A, Vento S. Comparative chromatography–mass spectrometry studies on nevirapine in human plasma. J Chromatogr B. 2014;945–946:101–109.
5. United States Pharmacopeia. USP40-NF35 S1, Nevirapine method. 2017.
6. Franz H, Fabel S. Fast Nevirapine Impurity Profiling Using UHPLC-DAD. Thermo Fisher Scientific Application Brief 170. 2016.