Determination of etimicin and related impurities in etimicin sulfate

Determination of Etimicin and Related Impurities in Etimicin Sulfate

Significance of the Topic

Etimicin is a semi-synthetic aminoglycoside antibiotic widely used to combat serious gram-negative and select gram-positive infections. Ensuring its quality and safety in pharmaceutical formulations is critical due to its lack of UV-absorbing chromophores and the potential formation of structurally related impurities during synthesis. Precise control of impurity levels is mandated by pharmacopeial standards to guarantee therapeutic efficacy and minimize toxicity.

Objectives and Study Overview

This study aimed to validate the China Pharmacopoeia monograph method for determining etimicin content and related impurities in etimicin sulfate. The method was implemented using a Thermo Scientific Dionex IonPac AmG-3 µm C18 column coupled to an ICS-5000+ HPIC system with pulsed amperometric detection. Key performance parameters assessed included system suitability, linearity, sensitivity (LOD/LOQ), precision, assay accuracy, impurity quantification, and method robustness.

Instrumental Setup

The analysis employed a Thermo Scientific Dionex ICS-5000+ HPIC system comprising:

• Dionex ICS-5000+ DP Pump and DC Detector modules
• ED Electrochemical Detector with gold working electrode (3 mm) and Ag/AgCl reference
• Dionex IonPac AmG-3 µm C18 guard (4 × 30 mm) and analytical columns (4 × 150 mm)
• AS-AP autosampler with cooling and buffer line
• Chromeleon CDS Software version 7.2.5

Post-column addition of 0.76 M NaOH at 0.3 mL/min enabled pulsed amperometric detection (Carbohydrates, 4-potential waveform).

Methodology and Reagent Preparation

A volatile ion-pairing mobile phase consisting of 0.2 M trifluoroacetic acid, 0.05% pentafluoropropionic acid, 1.5 g/L sodium sulfate, and 4% acetonitrile (pH 3.5) was prepared and degassed by helium sparging. The column temperature was set to 35 °C, flow rate to 0.8 mL/min, injection volume to 20 µL, and run time to 50 min. Standards and samples were dissolved and diluted in the same eluent to achieve appropriate concentrations for assay (25 µg/mL) and impurity analysis (0.25 and 2.5 µg/mL).

Main Results and Discussion

System Suitability
Resolution between netilmicin and etimicin exceeded 5.43 (vs. requirement >4) and the signal-to-noise ratio at 2.5 µg/mL was 77.1 (vs. requirement >10).
Linearity
Calibration over 0.25–25 µg/mL yielded a coefficient of determination (r2) of 0.9993, confirming a broad linear response.
Sensitivity (LOD/LOQ)
LOD and LOQ in solution were 0.0571 and 0.190 µg/mL (S/N of 3 and 10), corresponding to 228 and 761 µg/g in solid powder.
Precision
Six injections of a 25 µg/mL standard over 27 h produced a peak-area RSD of 0.37%, demonstrating high reproducibility.
Assay and Impurities
The test sample contained 89% etimicin. Netilmicin impurity in standard and sample was below 0.0677% and 2.33%, respectively (acceptance <2.5%). Total impurities in the sample (6.65%) exceeded the pharmacopeial limit of 5%.
Robustness
Retention time variations were <2% over extended injections, and peak-area drift was <1% across three days. Proper degassing, helium blanketing, and minimal electrode fouling were critical to maintain stable response.

Benefits and Practical Applications

This validated method meets or exceeds China Pharmacopoeia criteria for etimicin sulfate assay and impurity profiling. It leverages ion-pair HPLC and pulsed amperometric detection without derivatization, simplifying routine quality control workflows in pharmaceutical laboratories.

Future Trends and Applications

Advancements may include higher-throughput ultrahigh-performance liquid chromatography (UHPLC) formats, integration with mass spectrometry for peak identification, and automated data processing via advanced chromatography software. Further method transfer to integrated QbD platforms could enhance regulatory compliance and process understanding.

Conclusion

The application note demonstrates a robust, sensitive, and precise approach for quantifying etimicin and related impurities using Thermo Scientific Dionex IonPac AmG-3 µm C18 columns on ICS-5000+ systems with pulsed amperometric detection. The method fulfills pharmacopeial requirements and is suitable for routine quality assessment of aminoglycoside antibiotics.

References

1. Fan J. et al. Synthesis and structure determination of semisynthetic antibiotic 89-07. Chin. J. Antibiotics. 1995;20:401–406.
2. Etimicin Sulfate, China Pharmacopoeia 2015, pp. 1343–1344.
3. Thermo Scientific Dionex IonPac AmG-3 µm C18 Columns Product Manual. May 2017.
4. Thermo Scientific Application Note 72647: Determination of gentamicin and related impurities in gentamicin sulfate.
5. USP General Chapter <1225> Validation of Compendial Methods. USP 40–NF 35, 2018.
6. USP General Chapter <621> Chromatography. USP 40–NF 35, 2018.