Determination of Streptomycin, Dihydrostreptomycin and Impurities Using Anion-Exchange Chromatography with Electrochemical Detection

Mar 02, 2007

Streptomycin is a water-soluble aminoglycoside antibiotic purified from the fermentation of the actinomycete Streptomyces griseus and used in a variety of pharmaceutical applications. The catalytic hydrogenation of the one carbonyl group of streptomycin produces dihydrostreptomycin, a more stable derivative reported to have fewer side effects.1 These aminoglycosides, like most carbohydrates, lack a good chromophore and, therefore, require high concentrations to be detected by UV absorbance. Carbohydrates can be oxidized on a gold working electrode and, therefore, directly detected by amperometry. Pulsed amperometric detection (PAD) is ideally suited for aminoglycoside antibiotics and their impurities.2–6

High-performance anion-exchange chromatography (HPAE) can separate streptomycin, dihydrostreptomycin and their impurities.6 The CarboPac PA1 (Dionex, Sunnyvale, California, USA) anion-exchange column is the recommended column for use in the USP compendial method for the assay of streptomycin sulphate using HPAE-PAD.6 In this application note, we present the results of our evaluation of the USP method using current Dionex instrumentation, an ICS-3000 ion chromatography system with an electrochemical detector and the electrochemical cell outfitted with a disposable gold working electrode.


Chromatographic conditions conform to the USP Compendial Method6 using the Dionex ICS-3000 consisting of a DP dual gradient pump, DC detector compartment with electrochemical detector, AS autosampler and Chromeleon Chromatography Management Software.

Figure 1
Streptomycin sulphate (reference standard, cat. # 1623003; USP, Rockville, Maryland, USA), dihydroxystreptomycin sesquisulphate (cat. # D-7253; Sigma, St Louis, Missouri, USA) and a second commercial source streptomycin sulphate (cat. # S-6501; Sigma) were dried, dissolved in water and diluted to required concentrations.

Results and Discussion

Figure 1(a) shows the separation of streptomycin and dihydrostreptomycin using the CarboPac PA1. At least 14 streptomycin impurities [Figure 1(b)] are resolved, including the system suitability peak [peak #10, Figure 1(b)] produced from thermal degradation. Mean ± SD intraday (within 24 h) retention time for streptomycin was 12.01 ± 0.07 min (n = 34 injections), peak area was 8.89 ± 0.15 nC·min (5 µM), and resolution (USP definition) from the system suitability peak was 5.8 ± 0.2 min. Intraday peak retention time for streptomycin was 11.76 ± 0.29 min (n = 3 days) and peak area was 7.90 ± 0.71 nC·min. The linear range 4–5 for streptomycin was 1.5–106 µM (20 µL), with r2 = 0.9976. Estimated LOD and LOQ were 0.6 and 2 pmol streptomycin, respectively. The spike recovery of the streptomycin reference standard from a simulated fermentation broth (YPD) ranged from 83% to 93%. We found these results to meet or exceed the system suitability requirements defined in the USP method for streptomycin sulphate. This application is another example of the many capabilities possible using HPAE-PAD for the direct detection and rapid analysis of aminoglycoside antibiotics and their impurities.

Valoran P. Hanko and Jeffrey S. Rohrer, Dionex Corporation, Sunnyvale, California, USA.


1. S.A. Waksman, Streptomycin: Nature and Practical Applications (The Williams & Wilkins Co., Baltimore, Maryland, USA, 1949).

2. V.P. Hanko and J.S. Rohrer, J. Pharm. Biomed. Anal., 40, 1006–1012 (2006).

3. V.P. Hanko and J.S. Rohrer, J. Pharm. Biomed. Anal., 43, 131–141 (2007).

4. Dionex Corporation, Determination of Tobramycin and Impurities Using HPAE-PAD, Application Note 61, LPN 1626, Sunnyvale, California, USA, November (2004).

5. Dionex Corporation, Determination of Neomycin and Impurities Using HPAE-PAD, Application Note 66, LPN 1828, Sunnyvale, California, USA, July (2006).

6. United States Pharmacopeia, The National Formulary, Official Monographs, Streptomycin, USP 29, NF 24, 2008–2009 (2006).

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