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.

Reversed-phase HPLC separation of gentamicin on the Thermo Scientific Acclaim AmG C18 column with charged aerosol detection (CAD) demonstrates speed and resolution advantages with optimised solvent composition.
Reversed-phase HPLC separation of gentamicin on the Acclaim AmG C18 LC column with Dionex Corona Veo RS (rapid separation) charged aerosol detection (CAD) demonstrates speed and resolution advantages with optimized solvent composition.
Column Acclaim AmG C18, 3 µm
Dimensions 4.6 x 150 mm
Mobile Phase A 100 mM TFA
Mobile Phase B Acetonitrile
Flow Rate 1 mL/min
Inj. Volume 5 µL
Temperature 30 °C
Detection Corona Veo RS (Filter = 5.0s; Evaporation Temp = 35 °C; Data Rate = 5 Hz; Power Function = 1.00)
Sample Gentamicin (1 mg/mL)
Peaks
  1. C1a
  2. C2
  3. C2b
  4. C2a
  5. C1

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.

Separation of gentamicin in various substances
Separation of gentamicin in A) topical cream extract, B) ophthalmic ointment extract, C) topical ointment extract, and D) diluted ophthalmic solution (25% signal offset) on the Acclaim RSLC PA2 LC column with a HFBA/TFA gradient. The column temperature was maintained at 15 °C, which allowed greater separation between gentamicin C2b and gentamicin C2.
Column Acclaim RSLC PA2, 2.2 µm Analytical (2.1 x 100 mm)
Mobile Phase A 0.025:95:5 HFBA:DI water:acetonitrile
Mobile Phase B 0.3:95:5 TFA:DI water:acetonitrile
Gradient
  • 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 Rate 0.45 mL/min
Inj. Volume 1.0 µL
Temperature 15 °C
Detection CAD (Corona ultra RS, nebulizer temperature 15 °C, low filter, 60 Hz data collection rate)
Sample
  • A) Gentamicin sulfate cream
  • B) Gentamicin sulfate ophthalmic ointment
  • C) Gentamicin sulfate ointment
  • D) Gentamicin sulfate ophthalmic solution
Peaks
  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).

Column Dionex CarboPac PA1 guard, 4 x 50 mm
Dionex CarboPac PA1 guard, 4 x 250 mm
Eluent 2 mM KDH
Eluent Source Dionex EGC-500 KOH cartridge, with CR-ATC 600 trap column, Dionex high pressure degasser
Flow Rate 0.5 mL/min
Column Temp. 30 °C
Detector Compart. 30 °C
Inj. Volume 20 µL
Detection iPAD, AAA-Direct Au disposable electrode, 0.002" thick gasket
Reference Electrode pH/Ag/AgC1, pH mode
Waveform AAA-Direct, versus pH, 1.67 Hz
Peaks
  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.

Column IonPac CG19 Guard, 2 x 50 mm
IonPac CS19 Analytical, 2 x 250 mm
Eluent 7.5mM MSA
Eluent Source EGC-500 MSA with Dionex CR-CTC 500
Flow Rate 0.25 mL/min
Inj. Volume 100 µL
Conc Column Dionex IonPac TCC-ULP1
Detection Suppressed conductivity, Dionex CSRS 300, 2 mm, 7 mA, recycle mode
Peaks
  1. Sodium
  2. Ammonium
  3. Potassium
  4. Morpholine 10
  5. Magnesium
  6. Calcium

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.

Cephalosporins are a members of the β-Lactam antibiotics class, first discovered and isolated from the fungus Cephalosporum Acremonium. The core structure of these antiobitics features a number of hydrogen bond donors and acceptor groups, as well as an acid function. Together these groups contribute towards the polarity of this class of compounds.
Cephalosporins are a members of the β-Lactam antibiotics class, first discovered and isolated from the fungus Cephalosporum acremonium. The core structure of these antiobitics features a number of hydrogen bond donors and acceptor groups, as well as an acid function. Together these groups contribute towards the polarity of this class of compounds.

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.

Separation of a mixture of cephalosporins on a Thermo Scientific Syncronis aQ 3 µm particle column. Good separation was achieved in a total run time of six minutes, with an elution window of 1.8 minutes for all four analytes. A controlled interaction mechanism and selectivity of the Syncronis aQ polar endcapping group is demonstrated.
Separation of a mixture of cephalosporins on a Syncronis aQ LC column with 3 µm particle size. Good separation was achieved in a total run time of six minutes, with an elution window of 1.8 minutes for all four analytes. A controlled interaction mechanism and selectivity of the Syncronis aQ polar endcapping group is demonstrated.
Using the Thermo Scientific Chromeleon Chromatography Data System and simultaneous detection with UV and MS detectors provides complementary information for impurity profiling. The MS component channel with the extracted ion chromatograms of seven antibiotic standards was overlaid to the UV trace. The two traces could be perfectly aligned by taking into account the delay time between the two detectors. This simple comparison reveals the presence of an UV active impurity.
Using the Chromeleon Chromatography Data System and simultaneous detection with UV and MS detectors provides complementary information for impurity profiling. The MS component channel with the extracted ion chromatograms of seven antibiotic standards was overlaid to the UV trace. The two traces could be perfectly aligned by taking into account the delay time between the two detectors. This simple comparison reveals the presence of an UV active impurity.

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.

Chromatographic comparison of clavulanate on the Thermo Scientific Dionex IonPac AS11 column A) without and B) with 4 µg/mL (0.8%) 2-ethylhexanoic acid. This column has high capacity, low surface hydrophobicity and is able to separate a wide range of inorganic and organic anions in complex matrices.
Chromatographic comparison of clavulanate on the Dionex IonPac AS11 column A) without and B) with 4 µg/mL (0.8 %) 2-ethylhexanoic acid. This column has high capacity, low surface hydrophobicity and is able to separate a wide range of inorganic and organic anions in complex matrices.
Column IonPac AG11, AS11, 2 mm
Eluent 3 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 Source EGC II KOH with CR-ATC
Flow Rate 0.25 mL/min
Inj. Volume 5 µL
Temperature 30 °C
Detection Suppressed conductivity, ASRS 300 2 mm, recycle mode,
2 mA suppressor current during 3 mM KOH,
switch to 38 mA at 10.1 min
Samples Clavulanate with and without 4 µg/mL (0.8 %)
2-Ethylhexanoic acid
Peaks 2-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.

Analysis of a sample of cell lysate after tigecycline treatment (A) and the same sample spiked with 0.05 μg/mL tigecycline (B) using an on-line SPE-HPLC-UV method. This is a convenient method to determine trace amounts of tigecycline in tigecycline-treated cells which cannot be determined using a routine HPLC-UV detection method.
Analysis of a sample of cell lysate after tigecycline treatment (A) and the same sample spiked with 0.05 μg/mL tigecycline (B) using an on-line SPE-HPLC-UV method. This is a convenient method to determine trace amounts of tigecycline in tigecycline-treated cells which cannot be determined using a routine HPLC-UV detection method.
Conditions
On-Line SPE
Column Acclaim PolarAdvantage II (PA2) Guard Cartridge, 5 µm, 4.6 x 10 mm (P/N 069699)
Mobile Phase DI water
Flow Rate 1.0 mL/min
Inj. Volume 1500 µL on the on-line SPE cartridge
Separation
Column Acclaim 120, C18, 3 µm Analytical, 3.0 x 150 mm (P/N 063691)
Mobile Phase Phosphate buffer/CH3CN (85:15. v/v)
Flow Rate 0.6 mL/min
Autosampler Temp. 4 °C
Column Temp. 30 °C
Detection UV absorbance at 247 nm

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Analyte
(click title for methods)
References Technique
 Amikacin HPLC, IC
 Amoxicillin IC
 Ampicillin IC
Apramycin HPLC
Arbekacin HPLC
 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
 Cefdinir No references HPLC
 Cefepime (Cephapime) HPLC, IC
 Cefixime No references HPLC
 Cefotaxim (Cefotaxime, Cephotaxime, Cephotaxim) HPLC, IC
 Cefradine (Cephradine) HPLC, IC
 Ceftazidime (Cephtazidime) No references HPLC
 Ceftriaxone No references HPLC
 Chloramphenicol HPLC
Cinchophen (Cinchofen) HPLC
 Cinoxacin HPLC
 Ciprofloxacin HPLC
 Clavulanate (Clavulanic acid; beta lactamase inhibitor) IC
 Cloxacillin HPLC, IC
 Danofloxacin HPLC
 Dihydrostreptomycin HPLC, IC
 Doxorubicin No references HPLC
 Erythromycin HPLC
Etimicin HPLC
 Flucloxacillin No references HPLC
 Fosfomycin No references HPLC
Gentamicin HPLC
 Kanamycin HPLC, IC
 Kanamycin A IC
 Kanamycin B (Bekanamycin) IC
 Lincomycin HPLC, IC
 Linezolid HPLC, IC
 Lomefloxacin HPLC
 Metronidazole No references HPLC
 Minocycline No references HPLC
 Moxifloxacin No references HPLC
 Nalidixic Acid HPLC
 Neomycin (Neamine, Paromamine) HPLC, IC
Netilmicin HPLC
 Nitrofurantoin HPLC
 Norfloxacin No references HPLC
 Ofloxacin HPLC
 Oxolinic Acid HPLC
 Oxytetracycline HPLC
 Paromomycin HPLC, IC
 Penicillin G HPLC, IC
 Penicillin V HPLC, IC
 Polymyxcin No references HPLC
Ribostamycin HPLC
Sisomicin HPLC, IC
 Spectinomycin HPLC
 Streptomycin HPLC, IC
 Streptomycin HPLC, IC
 Streptomycin HPLC, IC
 Streptomycin HPLC, IC
 Sulbactam No references HPLC
 Sulfachloropyridazine HPLC
 Sulfadiazine No references HPLC
 Sulfadimethoxine HPLC
 Sulfamerazine No references HPLC
 Sulfamethazine HPLC
 Sulfamethizole HPLC
 Sulfanilamide HPLC, IC
 Sulfapyridine No references HPLC
 Sulfaquinoxzline No references HPLC
 Sulfathiazole HPLC
 Tetracycline HPLC
 Thiamphenicol No references HPLC
Tigecycline HPLC
 Tobramycin HPLC, IC
 Trimethoprim HPLC, IC
 Tylosin HPLC