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Complete analysis of antibiotics as per pharmacopeia (USP/EP)

The detection of aminoglycosides is not straightforward because they do not have a significant UV-absorbing chromophore. High Performance Ion Chromatography (HPIC) is hence a widely used and recommended technique paired with electrochemical detection to separate aminoglycosides. 

The Pulsed Amperometric Detector (PAD) is ideal for the detection of aminoglycoside antibiotics and their impurities as the analyses requires a powerful detection technique with a broad linear range and extremely low detection. Electrochemical detection has advantages relative to other techniques in that an oxidation potential can be selected for specific analytes while other compounds remain undetected, and derivatization is not required for detection, which simplifies the analysis. 

Discover methods and solutions for complete analysis of antibiotics like Gentamicin, Neomycin, Tobramycin, Kanamycin, Netilmicin, Framycetin, Etimicin etc.) as per recommendations from Pharmacopeia (USP, EP, etc.).


Featured solutions

Dionex™ Integrion™ HPIC™ System

dionex-integrion-hpic-system

High Performance Ion Chromatography (Metal-Free Flow Path Liquid Chromatography) System:

  • Pulse electrochemical detection technology (PAD)
  • Gold as working electrode and Ag-AgCl as reference electrode to provide complete information of electrochemical detection
  • High pressure pump for faster and consistent flow rates

The Thermo Scientific™ Dionex™ Integrion™ HPIC™ System with PAD (Pulse Amperometry Detector) / ECD (Electrochemical Detector) combines flexibility and ease-of-use with high sensitivity and selectivity, bringing a new level of convenience and cost effectiveness to Antibiotic analysis.

Download spec sheet

Ion Chromatography Columns (IC)

dionex-ionpac-IC-columns
Column OptionsRecommended application use

Thermo Scientific™ Dionex™ CarboPac™ PA200 Column

Thermo Scientific™ Dionex™ CarboPac™ PA100 Column
Fast, pH-stable and high-resolution mapping and analysis of charged and neutral oligosaccharides; Thermo Scientific separations based on size, charge, degree of branching, and linkage isomerism
Thermo Scientific™ Dionex™ CarboPac™ PA20 ColumnHigh-resolution separations of mono- and disaccharides, including optimized resolution of glucosamine/ galactose and glucose/mannose peak pairs
Thermo Scientific™ Dionex™ CarboPac™ PA20 Fast Sialic Acid ColumnFast analysis of sialic acids (e.g., N-acetyl- and N-glycolylneuraminic acids) in glycoprotein acid hydrolysates.
Thermo Scientific™ Dionex™ CarboPac™ MA1 ColumnFor sensitive, rugged, and reliable separations of reduced sugars. Achieve baseline resolution of fucose, N-acetyl-(D)-glucosamine, N-acetyl-galactosamine, mannose, glucose, galactose, and neutral oligosaccharides in the same separation.
Thermo Scientific™ Dionex™ CarboPac™ PA10 ColumnSeparation of mono- and disaccharides in drugs and mammalian glycoproteins; separation of sialic acids when sodium acetate is added to the eluent.
Thermo Scientific™ Dionex™ CarboPac™ PA1 ColumnSeparation of mono- and disaccharides including sialic acids, as well as specific oligosaccharides using an isocratic eluent.
Thermo Scientific™ Dionex™ AminoPac™ PA10 ColumnHigh-resolution separation of free amino acids using the Thermo Scientific AAA-Direct Amino Acid Analyzer.
Thermo Scientific™ Dionex™ IonPac™ AmG-3µm C18 columnHigh performance RP (C18) PEEK column for robust ion-pair separation of aminoglycosides.

Microbiology Kits and Reagents

Raw Materials

Maintain high-quality standards with Thermo Scientific Peptones and hydrolysates formulated to meet quality and consistency.

Animal-Derived-Component-Free-vegetable-Peptones

Microbial Identification

For simple, easy to use, reliable and accurate detection.

RapID-ONE-System

Environmental Monitoring

A range of high quality LabServ prepared culture media plates, which are ideal for environmental monitoring.

LabServ-Tryptic-Soya-Agar-TWI-Plates

Aseptic Process Control

Avoid delays and reduce the risk of contamination in the validation of your aseptic manufacturing process.
 

Cold-Filterable-Tryptone-Soya-Broth

Sterility Testing

Formulations designed to meet relevant US, EU and JP pharmacopoeias, as well as FDA and ISO requirements.
 

Microbiology-Testing-Kits-Reagents

Quality Control Organisms

Specific, reproducible numbers of viable microorganisms, derived from authentic, highquality ATCC® cultures, in a safe, ready-to-rehydrate vial.

Quanti-Cult-Plus-Convenience-Set

Chromeleon™ Chromatography Data System (CDS) Software

chromelon-chromatography-data-system

Streamline your laboratory workflow using Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) software. This is the first CDS software which unifies workflows for chromatography and routine quantitative analysis (it provides the full integration of Thermo Scientific™ Dionex™) ion-chromatography instruments. Chromeleon is well capable of delivering instrument control, superior compliance tools, automation, networking, data processing, and more. Run your analyses compliantly in an enterprise environment—from method creation to final reporting.

Dionex™ Integrion™ HPIC™ System

dionex-integrion-hpic-system

High Performance Ion Chromatography (Metal-Free Flow Path Liquid Chromatography) System:

  • Pulse electrochemical detection technology (PAD)
  • Gold as working electrode and Ag-AgCl as reference electrode to provide complete information of electrochemical detection
  • High pressure pump for faster and consistent flow rates

The Thermo Scientific™ Dionex™ Integrion™ HPIC™ System with PAD (Pulse Amperometry Detector) / ECD (Electrochemical Detector) combines flexibility and ease-of-use with high sensitivity and selectivity, bringing a new level of convenience and cost effectiveness to Antibiotic analysis.

Download spec sheet

Ion Chromatography Columns (IC)

dionex-ionpac-IC-columns
Column OptionsRecommended application use

Thermo Scientific™ Dionex™ CarboPac™ PA200 Column

Thermo Scientific™ Dionex™ CarboPac™ PA100 Column
Fast, pH-stable and high-resolution mapping and analysis of charged and neutral oligosaccharides; Thermo Scientific separations based on size, charge, degree of branching, and linkage isomerism
Thermo Scientific™ Dionex™ CarboPac™ PA20 ColumnHigh-resolution separations of mono- and disaccharides, including optimized resolution of glucosamine/ galactose and glucose/mannose peak pairs
Thermo Scientific™ Dionex™ CarboPac™ PA20 Fast Sialic Acid ColumnFast analysis of sialic acids (e.g., N-acetyl- and N-glycolylneuraminic acids) in glycoprotein acid hydrolysates.
Thermo Scientific™ Dionex™ CarboPac™ MA1 ColumnFor sensitive, rugged, and reliable separations of reduced sugars. Achieve baseline resolution of fucose, N-acetyl-(D)-glucosamine, N-acetyl-galactosamine, mannose, glucose, galactose, and neutral oligosaccharides in the same separation.
Thermo Scientific™ Dionex™ CarboPac™ PA10 ColumnSeparation of mono- and disaccharides in drugs and mammalian glycoproteins; separation of sialic acids when sodium acetate is added to the eluent.
Thermo Scientific™ Dionex™ CarboPac™ PA1 ColumnSeparation of mono- and disaccharides including sialic acids, as well as specific oligosaccharides using an isocratic eluent.
Thermo Scientific™ Dionex™ AminoPac™ PA10 ColumnHigh-resolution separation of free amino acids using the Thermo Scientific AAA-Direct Amino Acid Analyzer.
Thermo Scientific™ Dionex™ IonPac™ AmG-3µm C18 columnHigh performance RP (C18) PEEK column for robust ion-pair separation of aminoglycosides.

Microbiology Kits and Reagents

Raw Materials

Maintain high-quality standards with Thermo Scientific Peptones and hydrolysates formulated to meet quality and consistency.

Animal-Derived-Component-Free-vegetable-Peptones

Microbial Identification

For simple, easy to use, reliable and accurate detection.

RapID-ONE-System

Environmental Monitoring

A range of high quality LabServ prepared culture media plates, which are ideal for environmental monitoring.

LabServ-Tryptic-Soya-Agar-TWI-Plates

Aseptic Process Control

Avoid delays and reduce the risk of contamination in the validation of your aseptic manufacturing process.
 

Cold-Filterable-Tryptone-Soya-Broth

Sterility Testing

Formulations designed to meet relevant US, EU and JP pharmacopoeias, as well as FDA and ISO requirements.
 

Microbiology-Testing-Kits-Reagents

Quality Control Organisms

Specific, reproducible numbers of viable microorganisms, derived from authentic, highquality ATCC® cultures, in a safe, ready-to-rehydrate vial.

Quanti-Cult-Plus-Convenience-Set

Chromeleon™ Chromatography Data System (CDS) Software

chromelon-chromatography-data-system

Streamline your laboratory workflow using Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) software. This is the first CDS software which unifies workflows for chromatography and routine quantitative analysis (it provides the full integration of Thermo Scientific™ Dionex™) ion-chromatography instruments. Chromeleon is well capable of delivering instrument control, superior compliance tools, automation, networking, data processing, and more. Run your analyses compliantly in an enterprise environment—from method creation to final reporting.

Ion Chromatography – Making It Easier To Get Results

UniversalNo need for sample derivatization
FastLittle to no sample preparation needed
SafeMinimal handling of toxic reagents
SimpleSample can simply be reconstituted in water and directly injected
ReliableMaintains sample integrity & stability, no interferences due to labelling reagents
GreenNo generation of hazardous chemical waste

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Speak to a specialist today

We are here to help you learn more about how Thermo Scientific™ Dionex™ Integrion™ HPIC™ System can provide reliable and cost effective solution for antibiotics analysis. Contact us for more information now.

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Antibiotic Analysis Methods

Determination of gentamicin and related impurities in gentamicin sulfate

Gentamicin is water-soluble antibiotic belonging to the group of aminoglycoside antibiotics, produced by fermentation of Micromonospora echinospora (Micromonospora purpurea). Gentamicin sulfate is a prescribed antibiotic having a mixture of closely structurally related four major compounds: gentamicins C1, C1a, C2, and C2a with C2b as a minor component. Not only gentamicin complex but other fermentation impurities and degradation products such as sisomicin may also be present which have antibiotic activity and high renal toxicity. These should be well detected and separated before any clinical usage.  

 

Figure 1. Separation of a system suitability standard (gentamicin 100 μg/mL + sisomicin 20 μg/mL) using a Dionex IonPac AmG-3μm C18 column on a Thermo Scientific™ Dionex™ ICS-5000+HPIC™ system.

Use Dionex™ Integrion™ HPIC™ System having the Dionex IonPac AmG-3μm column which contains a silica-based packing material for reversed-phase chromatography and is packed in a PEEK column body rather than stainless steel. This column was specifically designed for ion-pairing reversed-phase separations of aminoglycoside antibiotics and we used it to execute the methods in the USP and EP Gentamicin Sulfate monographs. Our system is excellent in separating the five congeners (C1, C1a, C2, C2a and C2b). Sisomicin impurity is also detected with good sensitivity. The quality of our analysis such as separation, reproducibility, linearity, and sensitivity is found to meet or exceed the current USP/EP Gentamicin Sulphate monographs performance requirements.

Determination of Etimicin and Netilmicin related impurities in Etimicin sulfate

Etimicin is valuable in the treatment of serious infections caused by gram-negative bacteria and some grampositive bacteria such as those that infect the digestive system or cause perioperative infection. Etimicin is a semi-synthetic aminoglycoside antibiotic prepared from gentamicin C1a by introduction of an ethyl group at the 1-N-position.  Although etimicin is the main product of synthesis, a small of a impurity byproduct such as 3-N-ethyletimicin can also be formed. Differences in antimicrobial potency and toxicity necessitate the limitation and control of the amount of impurities in commercial samples.



separation-etimicin-netilmicin

Figure 2. Separation of a system suitability standard (etimicin 25 μg/mL + netilmicin 25 μg/mL) using a Dionex IonPac AmG-3μm C18 column. Netilmicin and etimicin were well separated. Peak resolution between netilmicin and etimicin is 5.43, exceeding the ChP requirement of 4

Determination of Neomycin B and related impurities

Neomycin purified from the fermentation of the actinomycete Streptomyces fradiae is used in a variety of medical applications and administered in the body in various forms (e.g. Neosporin®, NeoDecadron®, PediOtic® Suspension). Neomycin B is the main component of the complex and has the highest antibiotic activity. S. fradiae fermentation broth also contains less active forms of Neomycin: Neomycin A, C, D, E, F and Fradicin as impuritiesOther impurities may also result from chemical degradation during fermentation, manufacture or storage lowering the antibiotic potency. The current United States Pharmacopeia (USP 29, NF 24) and European Pharmacopoeia (EP) compendial method for Neomycin sulfate measures Neomycin B as the primary antibiotic, with Neomycin A, C, D, E, A-LP, and B-LP as impurities. All impurities must be determined and meet specified limit criteria before a manufactured lot may be used clinically.



Separation-Neomycin-B

Figure 3. Separation of Neomycin B and impurities is highly dependent on eluent concentration. Comparison of the resolution of Neomycin B (1 mM, 20000 pmol) and impurities in commercial grade Neomycin sulfate separated using 2.40 mM KOH (chromatogram A) and 2.16 mM KOH (chromatogram B). Neomycin B (peak 20) is injected at a concentration outside its upper limit of detection.

Determination of tobramycin and related impurities

Tobramycin is isolated from the fermentation of the actinomycete Streptomyces tenebrarius. in the isolated product kanamycin A and kanamycin B are also present as impurities from either incomplete isolation or degradation of tobramycin. It is an important antibiotic used in ophthalmic and intravenous treatments to treat bacterial infections by blocking protein synthesis. It is therefore important to assay the tobramycin content and quantify the related impurities of a tobramycin-based antibiotic before any clinical use.





Figure 4. 20 μM tobramycin with trace amounts of kanamycin A and B.

Analysis of the Aminoglycoside Antibiotics Kanamycin and Amikacin

Kanamycin is purified from fermentation of Streptomyces kanamyceticus and is used to treat a wide variety of serious gram-negative-bacterial infections, The main component of purified kanamycin is kanamycin A, and the minor structurally related constituents are kanamycin B, C, and D.

Amikacin is commonly administered parenterally for the treatment of gram-negative infections resistant to kanamycin, gentamicin, or tobramycin. it is synthesized by acylation of the amino group of kanamycin A with L-(-)-γ-amino-α-hydroxybutyric acid (L-HABA). As a result, it is  closely related with kanamycin A and L-HABA and they are expected impurities in commercial amikacin samples.

Figure 5. Chromatograms of (A) resolution solution (kanamycin 0.008 mg/mL and amikacin 0.02 mg/mL), (B) commercial kanamycin A sulfate sample, and (C) commercial amikacin sample.

Determination of gentamicin and related impurities in gentamicin sulfate

Gentamicin is water-soluble antibiotic belonging to the group of aminoglycoside antibiotics, produced by fermentation of Micromonospora echinospora (Micromonospora purpurea). Gentamicin sulfate is a prescribed antibiotic having a mixture of closely structurally related four major compounds: gentamicins C1, C1a, C2, and C2a with C2b as a minor component. Not only gentamicin complex but other fermentation impurities and degradation products such as sisomicin may also be present which have antibiotic activity and high renal toxicity. These should be well detected and separated before any clinical usage.  

 

Figure 1. Separation of a system suitability standard (gentamicin 100 μg/mL + sisomicin 20 μg/mL) using a Dionex IonPac AmG-3μm C18 column on a Thermo Scientific™ Dionex™ ICS-5000+HPIC™ system.

Use Dionex™ Integrion™ HPIC™ System having the Dionex IonPac AmG-3μm column which contains a silica-based packing material for reversed-phase chromatography and is packed in a PEEK column body rather than stainless steel. This column was specifically designed for ion-pairing reversed-phase separations of aminoglycoside antibiotics and we used it to execute the methods in the USP and EP Gentamicin Sulfate monographs. Our system is excellent in separating the five congeners (C1, C1a, C2, C2a and C2b). Sisomicin impurity is also detected with good sensitivity. The quality of our analysis such as separation, reproducibility, linearity, and sensitivity is found to meet or exceed the current USP/EP Gentamicin Sulphate monographs performance requirements.

Determination of Etimicin and Netilmicin related impurities in Etimicin sulfate

Etimicin is valuable in the treatment of serious infections caused by gram-negative bacteria and some grampositive bacteria such as those that infect the digestive system or cause perioperative infection. Etimicin is a semi-synthetic aminoglycoside antibiotic prepared from gentamicin C1a by introduction of an ethyl group at the 1-N-position.  Although etimicin is the main product of synthesis, a small of a impurity byproduct such as 3-N-ethyletimicin can also be formed. Differences in antimicrobial potency and toxicity necessitate the limitation and control of the amount of impurities in commercial samples.



separation-etimicin-netilmicin

Figure 2. Separation of a system suitability standard (etimicin 25 μg/mL + netilmicin 25 μg/mL) using a Dionex IonPac AmG-3μm C18 column. Netilmicin and etimicin were well separated. Peak resolution between netilmicin and etimicin is 5.43, exceeding the ChP requirement of 4

Determination of Neomycin B and related impurities

Neomycin purified from the fermentation of the actinomycete Streptomyces fradiae is used in a variety of medical applications and administered in the body in various forms (e.g. Neosporin®, NeoDecadron®, PediOtic® Suspension). Neomycin B is the main component of the complex and has the highest antibiotic activity. S. fradiae fermentation broth also contains less active forms of Neomycin: Neomycin A, C, D, E, F and Fradicin as impuritiesOther impurities may also result from chemical degradation during fermentation, manufacture or storage lowering the antibiotic potency. The current United States Pharmacopeia (USP 29, NF 24) and European Pharmacopoeia (EP) compendial method for Neomycin sulfate measures Neomycin B as the primary antibiotic, with Neomycin A, C, D, E, A-LP, and B-LP as impurities. All impurities must be determined and meet specified limit criteria before a manufactured lot may be used clinically.



Separation-Neomycin-B

Figure 3. Separation of Neomycin B and impurities is highly dependent on eluent concentration. Comparison of the resolution of Neomycin B (1 mM, 20000 pmol) and impurities in commercial grade Neomycin sulfate separated using 2.40 mM KOH (chromatogram A) and 2.16 mM KOH (chromatogram B). Neomycin B (peak 20) is injected at a concentration outside its upper limit of detection.

Determination of tobramycin and related impurities

Tobramycin is isolated from the fermentation of the actinomycete Streptomyces tenebrarius. in the isolated product kanamycin A and kanamycin B are also present as impurities from either incomplete isolation or degradation of tobramycin. It is an important antibiotic used in ophthalmic and intravenous treatments to treat bacterial infections by blocking protein synthesis. It is therefore important to assay the tobramycin content and quantify the related impurities of a tobramycin-based antibiotic before any clinical use.





Figure 4. 20 μM tobramycin with trace amounts of kanamycin A and B.

Analysis of the Aminoglycoside Antibiotics Kanamycin and Amikacin

Kanamycin is purified from fermentation of Streptomyces kanamyceticus and is used to treat a wide variety of serious gram-negative-bacterial infections, The main component of purified kanamycin is kanamycin A, and the minor structurally related constituents are kanamycin B, C, and D.

Amikacin is commonly administered parenterally for the treatment of gram-negative infections resistant to kanamycin, gentamicin, or tobramycin. it is synthesized by acylation of the amino group of kanamycin A with L-(-)-γ-amino-α-hydroxybutyric acid (L-HABA). As a result, it is  closely related with kanamycin A and L-HABA and they are expected impurities in commercial amikacin samples.

Figure 5. Chromatograms of (A) resolution solution (kanamycin 0.008 mg/mL and amikacin 0.02 mg/mL), (B) commercial kanamycin A sulfate sample, and (C) commercial amikacin sample.

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