Determination of gentamicin and related impurities in gentamicin sulfate

Significance of the topic

Gentamicin is a critical aminoglycoside antibiotic widely used against serious gram-negative infections. Because of its narrow therapeutic index and potential nephrotoxicity and ototoxicity, accurate characterization of gentamicin composition and related impurities is essential for drug safety, efficacy, and regulatory compliance. The United States Pharmacopeia (USP) and European Pharmacopeia (EP) monographs define stringent criteria for gentamicin purity, requiring sensitive, reproducible analytical methods.

Objectives and overview of the study

The goal of this work was to validate the USP gentamicin sulfate monograph method for both content of gentamicins and organic impurity testing, using a Thermo Scientific Dionex IonPac AmG-3 µm C18 column coupled to pulsed amperometric detection (PAD). The study compared the official 3-potential waveform with an optimized 4-potential waveform for carbohydrate detection, assessed key performance metrics, and applied the method to two commercial gentamicin samples.

Used Instrumentation

• Thermo Scientific Dionex ICS-5000+ HPIC system with DP pump, DC detector/chromatography module, and ED electrochemical detector
• Dionex AS-AP autosampler with 250 µL sample syringe and buffer line
• Dionex IonPac AmG-3 µm C18 guard (4 × 30 mm) and separation (4 × 150 mm) columns
• Gold conventional working electrode (3 mm) and Ag/AgCl reference electrode
• Knitted reaction coil, PEEK mixing tee, nitrogen supply for eluent blanketing

Methodology

Gentamicin components were separated by ion-pair reversed-phase HPLC using TFA/pentafluoropropionic acid mobile phase (pH 2.6) with 30 % acetonitrile, at 0.8 mL/min, 35 °C. A post-column addition of 0.5 M NaOH (0.3 mL/min) enabled PAD. Two electrochemical waveforms were evaluated: the USP’s 3-potential and an extended 4-potential waveform designed to reduce electrode wear. Calibration standards (10–200 µg/mL) and system suitability solutions (100 µg/mL gentamicin + 20 µg/mL sisomicin) were prepared in eluent. Two commercial samples (one within USP spec, one not) were analyzed for content of gentamicins (0.2 mg/mL) and impurities (1 mg/mL). Key metrics: resolution, signal-to-noise, linearity, LOD/LOQ, precision.

Main results and discussion

• Resolution and sensitivity: Using the 4-potential waveform, resolution between gentamicin C2/C2b was 4.2 (>1.5 USP req.), and sisomicin/C1a was 3.75 (>1.2 EP req.). S/N for 10 µg/mL sisomicin was 233 (>20 req.).
• Linearity: R² > 0.997 for all congeners over 10–200 µg/mL.
• Detection limits: LOD and LOQ for sisomicin in powder were 173 µg/g and 577 µg/g, respectively.
• Precision: Five injections of sample #2 yielded RSD < 0.5 % for all congeners.
• Content assay: Both samples met USP criteria for individual components (C1a, C2, C2a, C2b, C1) and C2+C2a (25–55 %) and C2b+C1 (25–50 %).
• Impurity assay: Sample #1 complied with USP/EP impurity limits (sisomicin < 3 %, each individual impurity < 3 %, total < 10 %). Sample #2 exceeded the total impurity limit (14.1 %).
• Waveform comparison: The 3-potential waveform produced higher peak response but the 4-potential waveform significantly improved electrode longevity and long-term reproducibility by avoiding oxidative cleaning potentials.

Benefits and practical applications of the method

This validated HPLC-PAD approach offers high sensitivity without derivatization, robust separation of gentamicin congeners and impurities, and compliance with USP/EP monographs. The optimized 4-potential waveform extends electrode lifespan, reducing maintenance and downtime, making this method well suited for routine quality control in pharmaceutical laboratories.

Future trends and possibilities

• Integration of ultrahigh-performance HPLC and shorter columns to reduce analysis time.
• Expansion to other aminoglycoside antibiotics and their degradants.
• Coupling PAD with mass spectrometry for structural confirmation of unknown impurities.
• Automation of sample preparation and data processing for high-throughput environments.

Conclusion

The Dionex IonPac AmG-3 µm C18 column method with pulsed amperometric detection meets or exceeds USP/EP requirements for gentamicin content and impurity assays. The enhanced 4-potential waveform provides durable, reproducible detection suitable for routine pharmaceutical analysis.

References

1. Cabanes A. et al. J. Liquid Chromatogr. 1991, 14, 1989.
2. Gentamicin Sulfate, USP40-NF35, 2017, p. 4391.
3. Gentamicin Sulfate, EP 8.0, 2012, 2326–2328.
4. Thermo Scientific IonPac AmG-3 µm C18 Columns Product Manual P/N 065728, 2017.
5. Thermo Scientific Technical Note 21: Optimal PAD Settings, ED40 Detector.
6. Gentamicin Sulfate, USP43(3) In-process Revision Monograph.
7. Thermo Scientific ICS-5000+ System Operator’s Manual P/N 065446, 2014.
8. Thermo Scientific Electrochemical Detection User’s Compendium P/N 065340-02, 2013.
9. Thermo Scientific ED40 Detector Operator’s Manual.
10. USP <1225> Validation of Compendial Methods, USP 40 NF 35, 2018.
11. USP <621> Chromatography, USP 37, 2014.