Determination of nitrite and nitrate from lactose by ion chromatography using the NGES-A suppressor

Importance of the topic

Determination of trace nitrite (NO2-) and nitrate (NO3-) in pharmaceutical excipients is critical because these anions can act as precursors to carcinogenic N-nitrosamines when secondary or tertiary amines are present in active pharmaceutical ingredients or in formulation processes. Accurate, sensitive, and robust measurement of nitrite and nitrate supports nitrosamine risk assessment, supplier quality control, and regulatory compliance for drug products.

Objectives and overview of the study

This application note demonstrates a suppressed-conductivity ion chromatography (IC) method for simultaneous quantification of nitrite and nitrate in lactose monohydrate and colloidal silicon dioxide. The method uses a Thermo Scientific Dionex IonPac AS19-4µm column and the Dionex NGES Next Generation Electrolytic Suppressor for anion analysis (NGES-A). The aim was to meet USP analytical note performance requirements while improving baseline stability and lowering noise relative to previous suppressor generations.

Methodology

• Chromatography: Dionex IonPac AS19-4µm analytical column (4 × 250 mm) with AG19 guard (4 × 50 mm) using a potassium hydroxide (KOH) gradient eluent generated by an RFIC-KOH cartridge.
• Gradient overview: initial KOH ~5 mM, ramp to ~50 mM around 30 min, hold briefly, then return to 5 mM; total run time 55 min. This gradient provides adequate retention and resolution of nitrite and nitrate from other common anions such as chloride.
• Detection: suppressed conductivity with NGES-A suppressor (4 mm) operated in Recycle mode; suppressor current ~124 mA.
• System parameters: flow rate 1.0 mL/min; injection volume 200 µL (full loop); column oven 40 °C; detector compartment 35 °C; autosampler vial temperature 15 °C; typical system backpressure ~3000 psi.
• Sample preparation: dissolve 250 mg sample in 10 mL diluent (5 mM KOH), sonicate, dilute, and filter through 0.2 µm nylon syringe filter to produce a 25 mg/mL solution. Calibration standards were prepared from combined nitrite and nitrate stock solutions with dilution into 5 mM KOH diluent.
• Quantitation: calibration curves were built across broad ranges and used for sample calculation following the USP-style equation comparing sample peak responses to standards.

Used instrumentation

• Thermo Scientific Dionex Integrion HPIC System (or equivalent Integrion RFIC / ICS-6000 HPIC systems).
• Dionex EGC KOH 500 eluent generator cartridge (RFIC-KOH).
• Dionex IonPac AS19-4µm analytical column and AG19 guard column.
• Dionex NGES-A (Next Generation Electrolytic Suppressor) 4 mm.
• Autosampler (Dionex AS-AP), thermostatted column oven, conductivity detector, and Chromeleon CDS for data acquisition.
• Miscellaneous: 0.2 µm nylon syringe filters, continuously regenerated anion trap column (CR-ATC 600) for sample cleanup/conditioning as used in the workflow.

Main results and discussion

• Sensitivity and LOQ: Based on a 25 mg/mL sample concentration, method LOQs were demonstrated at 0.1 µg/g for nitrite and 0.2 µg/g for nitrate. Signal-to-noise at LOQ was high (S/N ~57 for nitrite and ~77 for nitrate), indicating robust detectability well below regulatory concern levels for many risk assessments.
• Linearity: Calibration was linear across wide concentration ranges (nitrite: 2.5 µg/L to 1000 µg/L; nitrate: 5 µg/L to 2000 µg/L) with correlation coefficients >0.9995 (nitrite r=0.9999, nitrate r=0.9996).
• Precision: LOQ precision (%RSD of replicate injections) was low (nitrite 1.71% RSD; nitrate 0.96% RSD). System precision for spiked sample injections also showed very low %RSD values (typically <2%).
• Accuracy / recovery: Recovery studies at three spiking levels for each analyte gave average recoveries within a broad acceptance range; nitrite recoveries ranged from ~79% (lower level) to ~109% (upper level), while nitrate recoveries ranged from ~98% (middle) to ~118% (lower level). The lower-level nitrite recovery indicates potential matrix effects or extraction inefficiency at the lowest spike that may require attention in some cases.
• Specificity and resolution: Nitrite and nitrate were baseline-resolved from each other and from matrix anions (resolution >1.5). Blank injections showed no interfering peaks at nitrite/nitrate retention times.
• Practical sample data: In analyzed batches of lactose monohydrate, nitrite concentrations were ~0.18 µg/g and nitrate ~0.34 µg/g. A tested colloidal silicon dioxide sample showed similar nitrite (~0.19 µg/g) and slightly higher nitrate (~0.39 µg/g). These values are within trace levels detectable by the method and illustrate its applicability to excipient screening.
• Suppressor performance: The NGES-A suppressor reduced baseline noise and shortened equilibration times relative to earlier electrolytic suppressors, improving LOQs and system robustness for routine testing.

Benefits and practical applications of the method

• High sensitivity and reproducibility for nitrite and nitrate at low µg/g levels relevant to nitrosamine risk assessment in pharmaceuticals.
• No chemical derivatization required, avoiding additional sample-handling steps, potential contamination, and variability associated with derivatization reactions.
• Robust separation using the AS19 column allows analysis of excipients with potentially high ionic backgrounds (e.g., chloride) with minimal coelution issues.
• Improved suppressor technology (NGES-A) provides lower noise and faster baseline stabilization, reducing analyst intervention and increasing throughput for routine QC labs.
• Method flexibility: applicable to multiple excipient types (demonstrated for lactose monohydrate and colloidal silicon dioxide) and suitable for incorporation into pharmacopoeial testing workflows and supplier qualification programs.

Future trends and possible uses

• Further lowering of quantification limits through hardware improvements, alternative detector combinations (e.g., coupling IC to MS for confirmatory analysis), or enhanced sample concentration/cleanup strategies.
• Wider application across diverse excipient matrices, including those with complex organics that may require orthogonal detection modes (UV or MS) to confirm identities in challenging matrices.
• Automation and integration with laboratory information management systems (LIMS) for routine, high-throughput excipient screening and supplier monitoring.
• Regulatory convergence and adoption of standardized IC workflows for nitrosamine risk assessments across pharmacopeias and industry guidance documents.
• Development of multi-analyte IC methods that simultaneously monitor nitrite/nitrate and other relevant ionic impurities to streamline excipient quality control.

Conclusion

The described suppressed-conductivity IC method using the Dionex IonPac AS19 column and NGES-A suppressor provides a sensitive, precise, and robust approach for quantifying nitrite and nitrate in lactose monohydrate and colloidal silicon dioxide. The method meets USP-style system suitability criteria, demonstrates excellent linearity and precision, and delivers low LOQs suitable for nitrosamine risk assessments without requiring derivatization. The enhanced suppressor performance contributes to lower noise and faster baseline stabilization, making the method practical for routine pharmaceutical quality control and supplier screening.

Reference

1. Ahn I.; Lee S.; et al. Development and Application of Analytical Methods to Quantitate Nitrite in Excipients and Secondary Amines in Metformin API at Trace Levels Using Liquid Chromatography–Tandem Mass Spectrometry. Chemosensors, 2025, 307, 1–21.
2. Baumann M.; Naff K. Quantification of Nitrite in Excipients and Chemicals: A Versatile and Highly Sensitive Method Using Headspace Gas Chromatography Coupled to Mass Spectrometry. Organic Process Research & Development, 2024, 28, 1–12.
3. Mahendra D.; Roy R. Ion Chromatography in Pharmaceutical Analysis: Emerging Trends and Future Directions. Int. J. Pharm. Sci., 2025, 3, 1175–1178.
4. Kissner R.; Koppenol W. H. Qualitative and Quantitative Determination of Nitrite and Nitrate with Ion Chromatography. Methods in Enzymology, 2005, 396, 61–68.
5. Hickert S.; Naf K.; et al. Nitrite Testing in Excipients: Industry Best Practices. European Journal of Pharmaceutical Sciences, 2025, 213, 1–8.
6. Thermo Fisher Scientific Application Note. Determination of Nitrite in Pharmaceuticals: Application Note 73987, (2025).
7. Gerardi H.; Feeley L.; et al. Determining Low ppb Levels of Nitrite in Polymeric Excipients. ResearchGate Technical Note 386567575, 2024, 1–13.
8. USP Analytical Note. Determination of Nitrite and Nitrate in Lactose by Ion Chromatography as Part of Nitrosamine Risk Assessment in Excipients (USP Analytical Note, 2025).
9. USP Analytical Note. Determination of Nitrite and Nitrate in Povidone by Ion Chromatography as Part of Nitrosamine Risk Assessment in Excipients (2025).
10. USP Analytical Note. Quantification of Nitrite and Nitrate in Colloidal Silicon Dioxide by Ion Chromatography (2025).