Determination of Sulfate Counter Ion and Anionic Impurities in Aminoglycoside Drug Substances by Ion Chromatography with Suppressed Conductivity Detection

Significance of the Topic

Accurate quantification of sulfate counter ions and trace anionic impurities in aminoglycoside antibiotics is essential for correct molecular weight determination, stoichiometric balance, and regulatory compliance. These antibiotics, often formulated in sulfate salt forms to improve solubility and stability, may also retain residual inorganic and organic ions from fermentation or purification. Reliable, high-throughput assays are needed to ensure product quality, safety, and adherence to ICH impurity limits.

Objectives and Study Overview

This work evaluates two hydroxide-selective anion exchange columns coupled with suppressed conductivity detection and an electrolytically generated KOH eluent for:

• Determination of sulfate counter ions in ten aminoglycoside sulfate drug substances.
• Screening and quantification of anionic impurities such as chloride, phosphate, acetate, and pyrophosphate.
• Comparing performance of the IonPac AS18 column for routine screening with the AS11-HC column for complex matrices.
• Demonstrating method linearity, detection limits, precision, and recovery in both pharmaceutical standards and commercially available capsules (Humatin®).

Methodology and Instrumentation

All analyses employed a Dionex ICS-3000 RFIC system with on-line KOH eluent generation (EluGen EGC II KOH) and a CR-ATC trap column, coupled to suppressed conductivity detection (ASRS ULTRA II).

Method 1 (AS18 column): Gradient of 22–40 mM KOH at 0.25 mL/min on an IonPac AG18 guard (2×50 mm) and AS18 analytical column (2×250 mm). Run time: 20 min.

Method 2 (AS11-HC column): Multi-step gradient from 1 to 60 mM KOH at 0.38 mL/min on an AG11-HC guard (2×50 mm) and AS11-HC analytical (2×250 mm). Run time: 30 min.

Sample prep: Aminoglycoside solids were dried to determine moisture, dissolved to 100 mg/mL, then diluted to 0.05 mg/mL. Humatin® capsules were dissolved, centrifuged, and assayed at 2.5 mg/mL or 0.25 mg/mL for sulfate.

Used Instrumentation

• Dionex ICS-3000 RFIC system (dual pump, dual eluent generation)
• EGC II KOH eluent generator cartridge and CR-ATC trap column
• IonPac AS18 and AS11-HC anion exchange columns with corresponding AG18 and AG11-HC guards
• Suppressed conductivity detector (ASRS ULTRA II, 2 mm, recycle mode)
• Chromeleon 6.8 Chromatography Workstation

Main Results and Discussion

Method 1 on AS18 provided baseline separation of chloride, sulfate, and phosphate within 16 min, with detection limits of 3 µg/L (Cl⁻), 7.7 mg/L (SO₄²⁻), and 9.3 mg/L (PO₄³⁻). Ten aminoglycoside sulfate compounds exhibited sulfate contents of 13.7–30.2% versus theoretical 16.5–34.6%, and total anionic impurities <0.13%. Recoveries ranged from 92–112% for chloride and 97–101% for sulfate.
Method 2 on AS11-HC resolved additional anions (acetate, pyrophosphate) and quantified impurities in Humatin® capsules. The measured sulfate percentage was 24.7% (theoretical 23.7%), while acetate, chloride, phosphate, and pyrophosphate ranged from 0.025–0.23%. Precision (RSD <3%) and recoveries (93–111%) met pharmaceutical requirements.

Benefits and Practical Applications

• Reagent-free IC streamlines KOH eluent handling and eliminates CO₂ absorption issues.
• AS18 column enables rapid screening of common anions in well-characterized samples.
• AS11-HC column offers broad selectivity for unknown or complex matrices.
• Dual-channel RFIC systems allow simultaneous assay of free base (via IPAD) and counter ion, improving lab efficiency.
• Highly sensitive and robust, suitable for QA/QC, R&D, and regulatory compliance.

Future Trends and Potential Uses

• Integration with pulsed amperometric detection for concurrent aminoglycoside and counter ion analysis in a single workflow.
• Expansion to other antibiotic classes and biopharmaceutical molecules requiring precise ion quantification.
• Implementation of miniaturized IC systems and on-line sample prep for higher throughput in automated production environments.
• Coupling with mass spectrometry to identify unknown anionic contaminants and degradation products.

Conclusion

Reagent-free ion chromatography employing hydroxide-selective columns and suppressed conductivity detection reliably quantifies sulfate counter ions and anionic impurities in aminoglycoside drug substances. The AS18 method provides fast, accurate screening, while the AS11-HC approach handles complex or unknown matrices. The demonstrated precision, recovery, and automation capabilities make these protocols ideal for pharmaceutical quality control and regulatory compliance.

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