Keywords

C18, high-performance anion-exchange chromatography, pulsed amperometry analysis, derivatization-free, direct detection

The Thermo Scientific™ Dionex™ IonPac™ AmG-3µm C18 column is a high performance silica-based C18 column specifically designed for ion-pairing reversed-phase liquid chromatographic analysis of various aminoglycoside based antibiotics, including drug purity, impurity characterization and quantification, as well as residual control testing in different matrices. The unique column chemistry provides unusual tolerance toward low pH, high temperature, and aqueous mobile phases.

Product Highlights:

• Excellent selectivity for aminoglycosides

• Superior tolerance towards acidic eluents

• High efficiency and throughput

Benefits

• High-efficiency, high-throughput column

• Flexibility to use eluent across a pH range of 1–9 for the analysis of a wider range of aminoglycosides

• Available in PEEK hardware for use with HPAE-PAD or HPLC-PAD methods

Rugged reversed-phase PEEK columns designed for aminoglycoside antibiotic analysis

PRODUCT SPECIFICATIONS

Thermo Scientific™ Dionex™ IonPac™ AmG-3µm C18 column

2

Introduction Aminoglycosides are a group of antibiotics with similar amine-modified sugar structures. They are capable of inhibiting protein synthesis, so are widely used as clinical and veterinary medicines to treat bacterial infections. However, these antibiotics have serious side effects and can cause varying degrees of ototoxicity and nephrotoxicity. Therefore, it is important to develop sensitive and reliable analytical methods to characterize and quantify drug purity to determine and monitor aminoglycoside residues in different matrices, including blood, urine, and different animal-derived foods.

Pulsed amperometric detection (PAD) is a sensitive and selective electrochemical detection method. Table 1 shows the quadruple-potential waveform detection that can be used for detection of aminoglycosides.

Time (s) Potential (V) Integration

0.00 +0.10

0.20 +0.10 Start

0.40 +0.10 End

0.41 -2.00

0.42 -2.00

0.43 +0.60

0.44 -0.10

0.50 -0.10

A waveform is a series of voltage steps that work to charge the electrode, detect the analyte, clean the electrode, and then restore the surface.1 The entire process takes milliseconds and is ongoing throughout the run. This repeated cleaning and detecting is called pulsing; the process is known as pulsed amperometric detection, or PAD. The pulsing waveform ensures the electrode surface is always clean and the results are reproducible.

Applications Analysis of Gentamicin Gentamicin is a widely used broad spectrum antibiotic. It is produced by the fermentation process of Micromonospora purpurea and consists of a mixture of related gentamicin components and fractions. The major components of the gentamicin C complex are: C1, C1a, C2, C2a and C2b. Figure 1 shows an isocratic separation of gentamycin sulfate using a simple mobile phase consisting of 100 mM TFA and 0.025% PFPA/ CH3CN (97:3, pH 2.6). The chromatogram shows the five cogeners C1, C1a, C2, C2a and C2 are well separated.

Figure 1 shows the separation of gentamicin and related impurities using the Dionex IonPac AmG-3µm C18 column.

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Dionex IonPac AmG-3μm C18(4×150 mm, 4×30 mm)35 °C97% (0.1 M TFA) / 3% CH

3CN (pH 2.6)

Quadruple-potential PAD

33. Sisomicin 35. Gentamicin C1a41. Gentamicin C2 42. Miconomicin43. Gentamicin C2a46. Gentamicin C1 All other peaks in the chromatogramare unknown

Column:

Temp.: Eluent:Detection:

Peaks:

Figure 1. Gentamicin analysis using the Dionex IonPac AmG-3µm C18 Column

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Minutes

nC

0.25 mg/mL

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Peaks: 1-N’ethylgaramineRing(A+B) of EtimicinSisomicinGentamicin C1a1,3-N,N-di-ethlgaramineMiconomicinNetilmicinEtimicin6’-hydroxyetimicin1-N-ethylmicomicin6’-N-ethylgentamicin C1a

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Dionex IonPac AmG-3μm C18 Column (4×150 mm, 4×30 mm)35 °C96% (0.2 M TFA, 0.05% PFPA, 1.5 g/L Na

2SO4,

0.8% (V:V) 50% wt. NaOH, pH 3.5) / 4% CH3CN

Quadruple-potential PAD

Column:Column Temp.: Eluent:

Detection:

Figure 2. Analysis of etimicin and related impurities using the Dionex IonPac AmG-3μm C18 column

Figure 3. Analysis of spectinomycin using the Dionex IonPac AmG-3μm C18 column

Analysis of Etimicin Etimicin is an aminoglycoside that is structurally related to gentamicin and is used in the treatment of severe infections, particularly those resistant to gentamicin. Figure 2 shows the separation of etimicin and related impurities using the Dionex IonPac AmG-3μm C18 column.

Analysis of Spectinomycin Spectinomycin is a water-soluble broad-spectrum aminoglycoside antibiotic. It is industrially produced by

fermentation of the bacterium Streptomyces spectabilis. Spectinomycin is also produced in nature by many organisms including cyanobacteria and various plant species. It is used for intravenous administration to treat infections. Spectinomycin must be analyzed and all impurities must meet specified limits before a manufactured lot is used clinically. Figure 3 shows the separation of spectinomycin under isocratic conditions using 100 mM TFA.

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Dionex IonPac AmG-3μm C18 Column (Analytical: 4×150 mm)30 °C0.1 M TFAQuadruple-potential PAD

Column:Column Temp.: Eluent:Detection:

Minutes

nC

5.10.Others.

Peaks: Spectinomycin(4R)-dihydrospectinomycinImpurities

Product Specification

Product Description Part Number

Dionex IonPac AmG-3µm C18 PEEK (4.0 x 150 mm) Analytical Column 302693

Dionex IonPac AmG-3µm C18 PEEK (4.0 x 30 mm) Guard Column 302694

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Ordering Information

Specification Value

Diameter (Metric) 4 mm

Flow Rate 0.8 to 1.1 mL/min

Max. Pressure 5000 psi

Packing Material Silica, Spherical high purity

Particle Size 3 µm

Pore Size 120 Å

pH 1–9

Length (Metric) 150 mm

Column Construction PEEK

Temperature < 60°C