Determination of nitrite impurity in dalteparin sodium using ion chromatography

Significance of the Topic

The quantification of nitrite impurities in low-molecular-weight heparin products such as dalteparin sodium is critical for ensuring patient safety and regulatory compliance. Nitrite can arise during the depolymerization process and must be tightly controlled according to pharmacopeial standards. The availability of sensitive, selective, and robust analytical methods supports quality assurance in pharmaceutical manufacturing and release testing.

Objectives and Study Overview

This study evaluated the United States Pharmacopeia (USP) monograph procedure for nitrite determination in dalteparin sodium using a Thermo Scientific Dionex IonPac AS15 column paired with ion chromatography and electrochemical detection (IC-ED). Key performance parameters—such as chromatographic separation, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and robustness—were assessed and compared against USP acceptance criteria. A real sample of dalteparin sodium was analyzed to demonstrate method applicability.

Instrumentation

• Thermo Scientific Dionex ICS-5000+ HPIC system (pump, conductivity detector, autosampler).
• Electrochemical detector with glassy carbon working electrode and Ag/AgCl reference electrode.
• Dionex IonPac AS15 analytical column (3×150 mm).
• Chromeleon CDS software version 7.2.9.

Methodology

Sodium acetate eluent (8.2 g/L, pH 4.3) was delivered at 0.5 mL/min at 30 °C. The autosampler temperature was set to 5 °C and injection volume was 25 µL. Nitrite standards were prepared at concentrations of 2.5–25 µg/L by serial dilution of a 500 µg/L solution. A dalteparin sample solution (4 mg/mL) was prepared in degassed type I water. Electrochemical detection was carried out at +1.00 V in a 30 °C detection compartment.

Main Results and Discussion

• System Suitability: Theoretical plates ≥5278 (USP ≥4000), tailing factor 0.8–0.95 (USP 0.8–1.2), RSD of peak area 1.7% (USP <2%).
• Linearity: Calibration in the 2.5–25 µg/L range yielded r2 = 0.9998 (USP ≥0.995).
• Detection Limits: LOD 0.292 µg/L (72.9 ng/g), LOQ 0.972 µg/L (243 ng/g).
• Accuracy: Recovery of nitrite spiked into sample ranged from 96.2% to 105%.
• Robustness: Minor variations (±10%) in eluent concentration, flow rate, and temperature had negligible effects on retention time and peak shape.
• Sample Analysis: Nitrite content in dalteparin sodium was determined as 1.76 ppm, passing the USP specification of ≤5 ppm.

Benefits and Practical Applications

This IC-ED method offers high sensitivity without derivatization, selective detection through electrode potential control, and broad linear dynamic range. It is well suited for routine quality control of nitrite impurity in pharmaceutical heparin preparations, ensuring compliance with USP and European Pharmacopoeia requirements.

Future Trends and Opportunities

Emerging directions include integration of mass spectrometric detection for structural confirmation, miniaturized and high-throughput IC systems, automation of sample preparation, and development of greener eluents. Advances in electrode materials may further lower detection limits and expand application to other inorganic impurities.

Conclusion

The described method successfully meets or exceeds USP performance criteria for nitrite determination in dalteparin sodium using a Dionex IonPac AS15 column and IC-ED. It provides a reliable, sensitive, and selective approach for routine impurity monitoring in pharmaceutical quality control.

References

1. United States Pharmacopeia 42/The National Formulary 37, Dalteparin Sodium monograph, 2019.
2. European Pharmacopoeia, Dalteparin Sodium monograph, 2005.
3. Thermo Scientific Dionex ICS-5000+ Ion Chromatography System Operator’s Manual, Part No. 065446, December 2014.
4. Thermo Scientific Electrochemical Detection User’s Compendium, Part No. 065340-02, April 2013.
5. Thermo Scientific Dionex IonPac AS15 Columns Product Manual, Part No. 031362-10, June 2014.
6. United States Pharmacopeia 42/The National Formulary 37, General Chapter <1225>, Validation of Compendial Methods, 2019.
7. United States Pharmacopeia 42/The National Formulary 37, General Chapter <621>, Chromatography, 2019.
8. Thermo Scientific Dionex ICS-6000 Operators Manual, Part No. 22181-97002, Revision 01, February 2018.