Antibiotics Analysis Information

Ensuring the safety of antibiotics

Antibiotics, or antibacterials, are antimicrobial drugs used to treat or prevent bacterial infections. Antibiotics are heralded as the medicinal heroes of the 20th century. However, their effectiveness and accessibility has led to overuse and, in recent years, consequent antibiotic resistance.

Antibiotics are divided into classes  based upon their method of production or bactericidal method of action. They may be produced biologically (fermentation), biologically and chemically (semi-synthetic), or by chemical synthesis alone.

Analysis of biological/natural product antibiotics

E.g., aminoglycosides, macrolides, some tetracylines, lipopeptides and glycopeptides

Antibiotics produced through fermentation processes are less predictable, less controllable and more complex than synthetic antibiotics. For this reason the variability in products derived from fermentation is often greater than products derived by chemical synthesis. The impurity profile of a fermentation product may also be more complex and less predictable than that of a synthetic product.

One of the most common antibiotic groups which is biologically synthesized are the aminoglycosides. An aminoglycoside is a molecule composed of a sugar group and an amino group.

Streptomycin was the first aminoglycoside antibiotic discovered and used in clinical therapy. These antibiotics are now widely used as clinical and veterinary medicines to treat bacterial infections because of their protein synthesis inhibition capability, leading to cell death. However, these antibiotics can have serious side effects and cause varying degrees of toxicity. It is important to develop sensitive and reliable analytical methods to characterize and quantify drug purity and detect minor degradants or impurities. Based on the nature of their production (fermentation), there are often mixtures of related components (congeners, isomers) and fractions which must be monitored and controlled.

As well as being structurally-related, many aminoglycoside antibiotics are actually synthesised from one another. For example, sisomicin is a broad spectrum aminoglycoside isolated from the fermentation broth of Micromonospora. Netilmicin is a semi-synthetic aminoglycoside antibiotic prepared from sisomicin. Both sisomicin and netilmicin are mainly used in the treatment of severe infections, particularly those resistant to gentamicin. Etimicin is semi-synthesized from gentamicin C1a, and so on.

Analysis of aminoglycosides and their related impurities is often achieved by ion-pairing reversed-phase (RP) high performance liquid chromatography (HPLC), based on their hydrophilic and positively charged nature. However, due to the lack of a suitable chromophore, aminoglycosides cannot be detected by ultraviolet (UV). Corona charged aerosol detectors (CAD), evaporative light scattering detectors (ELSD), mass spectrometers (MS), and electrochemical detectors are generally used to detect these compounds without prior derivatization.

ColumnAcclaim RSLC PA2, 2.2 µm Analytical (2.1 x 100 mm)
Mobile Phase A0.025:95:5 HFBA:DI water:acetonitrile
Mobile Phase B0.3:95:5 TFA:DI water:acetonitrile
  • From 0 to 3 min: 1-10% mobile phase B (99-90% mobile phase A)
  • From 3 to 8 min: 10-100% mobile phase B (90-0% mobile phase A)
  • From 8 to 11 min: 100% mobile phase B
  • 4 min of equilibration at 99% mobile phase A before injection
Flow Rate0.45 mL/min
Inj. Volume1.0 µL
Temperature15 °C
DetectionCAD (Corona ultra RS, nebulizer temperature 15 °C, low filter, 60 Hz data collection rate)
  • A) Gentamicin sulfate cream
  • B) Gentamicin sulfate ophthalmic ointment
  • C) Gentamicin sulfate ointment
  • D) Gentamicin sulfate ophthalmic solution
  1. Unretained ions from matrix
  2. Garamine-like compound
  3. Sisomicin
  4. Gentamicin C1a
  5. Gentamicin C2
  6. Gentamicin C2b
  7. Gentamicin C2a
  8. Gentamicin C1

The USP monographs for some aminoglycoside drug substances (and drug products made from them) often involve high pressure anion exchange (HPAE) ion chromatography (IC) assays with integrated pulsed amperometric detection (IPAD).

ColumnDionex CarboPac PA1 guard, 4 x 50 mm
Dionex CarboPac PA1 guard, 4 x 250 mm
Eluent2 mM KDH
Eluent SourceDionex EGC-500 KOH cartridge, with CR-ATC 600 trap column, Dionex high pressure degasser
Flow Rate0.5 mL/min
Column Temp.30 °C
Detector Compart.30 °C
Inj. Volume20 µL
DetectioniPAD, AAA-Direct Au disposable electrode, 0.002" thick gasket
Reference ElectrodepH/Ag/AgC1, pH mode
WaveformAAA-Direct, versus pH, 1.67 Hz
  1. Void volume
  2. System peak
  3. Kanamycin A
  4. Kanamycin B
  5. Tobramycin
  6. Oxygen dip

Analysis of synthetic antibiotics

E.g., sulphonamides, quinolones, oxazolidinones

Chemically synthesised antibiotics often involve a number of intermediate compounds in their preparation, and these may remain as impurities in the final product. Chemical antibiotics are usually assayed by HPLC with UV detection, but where a chromophore is absent in either the active pharmaceutical ingredient (API) or impurities, IC with suppressed conductivity detection is considered the best alternative for selective determination.

Analysis of semi-synthetic antibiotics

E.g., beta-lactams (penicillins, cephalosporins, penems, carbapenems, and monobactams), some tetracylines and glycopeptides

Semi-synthetic antibiotics generally have fewer impurities than their biological counterparts. Impurities may include fermented starting material with related impurities, synthesis by-products, synthesis intermediates, and degradation products.

Beta-lactam antibiotics are a class of broad-spectrum antibiotics, consisting of all antibiotic agents that contain a beta-lactam ring in their molecular structures. These antibiotics function through the inhibition of bacterial cell wall synthesis. Following extensive use, some bacterial populations have shown ability to develop resistance to beta-lactams and become more virulent.

While beta-lactam antibiotics are similar to one another in many ways, they may differ in pharmacokinetics, antibacterial activity, and potential to cause serious allergic reactions. Some beta-lactam intermediate compounds and derivatives (from fermentation and/or synthesis) also possess similar sensitization and cross–reactivity properties. Beta-lactam intermediate compounds, such as β-lactam antibiotic API precursors, can undergo molecular changes or purification before use in manufacture. As a result of these changes, the intermediate compounds may develop antigenic characteristics that can produce allergic reactions. Drug manufacturers are required to take steps to control for the risk of cross-contamination and impurities for all beta-lactam products.

Often beta-lactamase inhibitors, such as clavulanate (clauvulanic acid) and sulbactam are co-formulated with beta-lactam antibiotics to increase their effectiveness through the counteraction of bacterial resistance. Impurities associated with the inhibitor as well as the antibiotic therefore need to be controlled and monitored.

ColumnIonPac AG11, AS11, 2 mm
Eluent3 mM KOH from 0 to 10 min,
3 to 60 mM KOH from 10 to 10.1 min,
60 mM KOH from 10.1 to 20.1 min
Eluent SourceEGC II KOH with CR-ATC
Flow Rate0.25 mL/min
Inj. Volume5 µL
Temperature30 °C
DetectionSuppressed conductivity, ASRS 300 2 mm, recycle mode,
2 mA suppressor current during 3 mM KOH,
switch to 38 mA at 10.1 min
SamplesClavulanate with and without 4 µg/mL (0.8 %)
2-Ethylhexanoic acid
Peaks2-Ethylhexanoic acid (4 µg/mL – 0.8 %)

Antibiotics in bioprocess of biopharmaceuticals

Antibiotics are heavily used in the production of biopharmaceuticals. Mammalian cell lines that express biotherapeutic proteins, such as antibodies, must be maintained over several weeks. They are fed with culture media supplemented with various vitamins, growth factors, and antibiotics to avoid contamination and growth failure. Testing of residual antibiotics in the product is required to ensure patient safety.

On-Line SPE
ColumnAcclaim PolarAdvantage II (PA2) Guard Cartridge, 5 µm, 4.6 x 10 mm (P/N 069699)
Mobile PhaseDI water
Flow Rate1.0 mL/min
Inj. Volume1500 µL on the on-line SPE cartridge
ColumnAcclaim 120, C18, 3 µm Analytical, 3.0 x 150 mm (P/N 063691)
Mobile PhasePhosphate buffer/CH3CN (85:15. v/v)
Flow Rate0.6 mL/min
Autosampler Temp.4 °C
Column Temp.30 °C
DetectionUV absorbance at 247 nm

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(click title for methods)
 AmikacinHPLC, IC
 Cefaclor (Cephaclor)HPLC
 Cefadroxil (Cefadroxyl, Cephadroxyl, Cephadroxil)HPLC, IC
 Cefalexin (Cephalexin)HPLC, IC
 Cefaloridine (Cephaloridine)HPLC, IC
Cefalothin (Cephalothin)IC
 Cefamandole (Cephamandole)HPLC
Cefapirin (Cephapirin)IC
 Cefazolin (Cephazolin, Cefazoline, Cephazoline)HPLC, IC
 CefdinirNo referencesHPLC
 Cefepime (Cephapime)HPLC, IC
 CefiximeNo referencesHPLC
 Cefotaxim (Cefotaxime, Cephotaxime, Cephotaxim)HPLC, IC
 Cefradine (Cephradine)HPLC, IC
 Ceftazidime (Cephtazidime)No referencesHPLC
 CeftriaxoneNo referencesHPLC
Cinchophen (Cinchofen)HPLC
 Clavulanate (Clavulanic acid; beta lactamase inhibitor)IC
 CloxacillinHPLC, IC
 DihydrostreptomycinHPLC, IC
 DoxorubicinNo referencesHPLC
 FlucloxacillinNo referencesHPLC
 FosfomycinNo referencesHPLC
 KanamycinHPLC, IC
 Kanamycin AIC
 Kanamycin B (Bekanamycin)IC
 LincomycinHPLC, IC
 LinezolidHPLC, IC
 MetronidazoleNo referencesHPLC
 MinocyclineNo referencesHPLC
 MoxifloxacinNo referencesHPLC
 Nalidixic AcidHPLC
 Neomycin (Neamine, Paromamine)HPLC, IC
 NorfloxacinNo referencesHPLC
 Oxolinic AcidHPLC
 ParomomycinHPLC, IC
 Penicillin GHPLC, IC
 Penicillin VHPLC, IC
 PolymyxcinNo referencesHPLC
SisomicinHPLC, IC
 StreptomycinHPLC, IC
 StreptomycinHPLC, IC
 StreptomycinHPLC, IC
 StreptomycinHPLC, IC
 SulbactamNo referencesHPLC
 SulfadiazineNo referencesHPLC
 SulfamerazineNo referencesHPLC
 SulfanilamideHPLC, IC
 SulfapyridineNo referencesHPLC
 SulfaquinoxzlineNo referencesHPLC
 ThiamphenicolNo referencesHPLC
 TobramycinHPLC, IC
 TrimethoprimHPLC, IC