Reversed-Phase HPLC Analysis of Aminoglycoside Antibiotics Using Evaporative Light Scattering Detection

1
Reversed-Phase HPLC Analysis ofAminoglycoside
Antibiotics Using Evaporative Light Scattering
Detection

• Laura E. Manis and Melissa J. Wilcox
• Alltech Associates, Inc.
• 2051 Waukegan Road Deerfield, IL 60015
• Phone 1-800-ALLTECH Web Site
  www.alltechweb.com

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Introduction

• The Evaporative Light Scattering Detector (ELSD)
  is becoming more common in todays HPLC
  laboratories. Evaporative light scattering
  detection offers sensitive, universal detection
  of any sample that is less volatile than the
  mobile phase. Unlike UV and RI detectors, the
  ELSDs response is independent of the samples
  optical characteristics. Therefore, any
  non-volatile sample, chromophoric or
  non-chromophoric can be detected, regardless of
  its functional group. Response from an ELSD is
  based on mass concentrations of the analyte and
  the amount of light scattered, which is useful in
  detecting unknown components and impurities. The
  ELSD also maintains a stable baseline over
  gradient runs, making it ideal for gradient
  applications. The ELSD answers the need for
  improved HPLC methods by offering universal and
  sensitive detection for non-volatile samples,
  regardless of optical activity.
• Aminoglycoside (AG) antibiotics are commonly used
  for treating infection caused by gram-negative
  bacteria. A rugged HPLC method is important to
  scientists interested in monitoring or
  researching aminoglycosides. This class of
  antibiotics lacks a significant UV chromophore,
  which makes ELS detection ideal.

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• Other methods of detection for aminoglycosides
  include RI, UV, and MS detection. RI detectors
  are not gradient compatible, less sensitive, and
  susceptible to ambient temperature changes.
  Aminoglycoside antibiotics do not have strong
  chromophores, which makes detection by UV
  difficult and not sensitive. ELSD also offers
  more cost-effective analysis and less complicated
  operation than an MS detector. The ELSD
  overcomes the challenges of gradient analysis,
  optical response, and cost of instrumentation.
• Evaporative Light Scattering Detectors use a
  simple three-step process that produces a signal
  for any non-volatile sample component. This
  unique detection method is the key to the ELSDs
  versatility and performance. Since all particles
  scatter light, all non-volatile sample analytes
  are detected with high sensitivity and accuracy,
  regardless of their functional groups or optical
  properties. All samples are detected with nearly
  equivalent response factors, making concentration
  determination easier when authentic standards are
  not available.

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ELSD Detection Principles Universal, Versatile,
Sensitive
1. Nebulization Inside the nebulizer, the
column effluent passes through a needle,
mixes with nitrogen gas, and forms a
dispersion of droplets. 2. Mobile Phase
Evaporation The droplets pass through a heated
drift tube where the mobile phase evaporates,
leaving a fine mist of dried sample particles in
solvent vapor. 3. Detection The sample
particles pass through a flowcell where they are
hit with a laser light beam. Light scattered by
the sample particles is detected, generating an
electrical signal.
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Objective

• This paper highlights a rugged, ELSD compatible
  HPLC method to analyze four common
  aminoglycosides (AGs) amikacin, neomycin,
  streptomycin, and tobramycin. The study
  investigates analytical column durability in the
  extreme low pH range and the use of buffered
  ion-pair reagents in retaining the antibiotics on
  a C18 column. A comparison of UV and ELSD shows
  the superiority of ELS detection for these
  antibiotics.

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Experimental

• Instrumentation
• Alltech (Deerfield, IL) On-line Vacuum
  Degasser
• Alltech Model 580 Autosampler
• Alltech Model 500 ELSD (Evaporative Light
  Scattering Detector)
• Hitachi (Tokyo, Japan) L-6200A Intelligent
  Pump
• Linear (Thermo Separation Products, San Jose,
  CA) Model 200 UV/Vis Detector
• PE Nelson (Norwalk, CT) Turbochrom EL
  Datastation
• Columns
• Alltech Alltima C18, 5µm, 250 x 4.6mm
• Hamilton (Reno, NV) PRP-1, 5µm, 150 x 4.6mm
• OraChrom (Woburn, MA) Styros 2R/XH, 3µm, 100
  x 4.6mm

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• Reagents and Samples
• 18.2M? water purified by a Millipore (Bedford,
  MA) Elix and Gradient system
• HPLC grade solvents were purchased from
  Burdick and Jackson (Muskegon, MI)
• Antibiotic standards were purchased from
  Sigma (St. Louis, MO)
• Neo-Cinolone cream was obtained from Unicorn
  Laboratories (Hong Kong, China)
• Pentafluoropropionic acid was purchased from
  Sigma (St. Louis, MO)
• Standard Preparation
• 0.02g amikacin, neomycin, streptomycin, and
  tobramycin were weighed individually into 50mL
  volumetric flasks and dissolved with 50mL water
  for a working solution of 0.4mg/mL. A standard
  mixture was prepared with 2.5mL of each
  antibiotic standard. In the standard mixture,
  each antibiotic had a concentration of
  0.1mg/mL.

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• Mobile Phase Preparation
• 3mL PFPA was added to 1L Methanol for a
  concentration of 0.3 PFPA
• Ammonium Formate buffer was prepared by
  dissolving approximately 2.7g Ammonium Formate
  in 1L purified water. 3mL PFPA was added to
  the buffer.
• Neomycin Sample Preparation
• 1g Neo-Cinolone cream was weighed into a
  beaker. 10mL of chloroform was added to
  dissolve the cream matrix. Then, 10mL of
  purified water was added to dissolve the neomycin
  in the solution. After mixing, the two layers
  separated. The aqueous layer was removed and
  filtered through a 0.45µm syringe filter.
  Filtrate was injected onto the column.

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Figures

• Figure 1
• An increase in pentafluoropropionic acid
  corresponds to an increase in retention time.
  For example, an increase from 0.2 to 0.4
  ion-pair reagent retains neomycin nearly two
  minutes longer on a conventional base-deactivated
  C18 column. Antibiotic standards are typically
  present in the sulfate salt form. Therefore, the
  first peak in the chromatograms is the elution of
  the sulfate in the void volume of the column.

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• Figure 2
• Without pH stability through a buffered mobile
  phase, the conventional base-deactivated C18
  column shows signs of degradation. Retention
  times shift, peak splitting occurs, and peak
  shape deteriorates over time. Pentafluoropropioni
  c acid mobile phases should be buffered to keep
  the pH above 2.0, which will minimize damage to
  silica-based columns.
• Ammonium acetate was used to buffer the
  pentafluoropropionic acid mobile phase at pH 2.7.
  The buffer offered chromatographic stability
  through maintaining the mobile phase pH and
  extending the analytical column life. With the
  ammonium acetate buffer, retention times are
  reproducible, and peak shapes remain symmetrical.
  Additionally, the original gradient method was
  converted to an isocratic method for easier use,
  greater reproducibility, and higher throughput.
  An analytical column heater was also used to
  increase reproducibility of the application.

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• Figure 3
• Aminoglycoside antibiotics lack a significant
  structural chromophore making detection by UV
  difficult. With ELS detection, detector response
  is independent of the samples optical activity,
  making it ideal for the analysis of
  aminoglycoside antibiotics. Without the need to
  derivatize samples, the ELSD offers freedom from
  sample prep and greater sensitivity than UV
  detection.
• Figure 4
• This method is ideal for the analysis of
  Neo-Cinolone cream. Neomycin was extracted from
  the cream through a simple sample prep procedure
  and analyzed by ELSD.
• Figure 5
• This study investigated the use of polymeric
  columns under the method conditions. Selectivity
  was poor with both the Hamilton PRP-1 (150 x
  4.6mm, 5µm) and OraChrom Styros 2R/XH (100 x
  4.6mm, 3µm,). AGs were not retained or separated
  on either column for this comparison.

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Aminoglycoside Antibiotics
Figure 1
0.4 PFPA
0.2 PFPA
1. Streptomycin 2. Amikacin 3. Tobramycin 4.
Neomycin
Column Alltima C18, 5µm, 150 x 4.6mm Mobile
Phase A Pentafluoropropionic Acid in Water B
Methanol Gradient Time 0 10 B
45 65 Flowrate 1.0mL/min Detector 500 ELSD/LTA
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Evidence of Column Degradation Over Time without
Mobile Phase Buffer
Figure 2
1. Streptomycin 2. Amikacin 3. Tobramycin 4.
Neomycin
Column Alltima C18, 5µm, 150 x 4.6mm Mobile
Phase A 0.2 Pentafluoropropionic acid in
Water B Methanol Gradient Time 0 10 B 45
65 Flowrate 1.0mL/min Detector 500 ELSD
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Enhanced Detection Sensitivity Compared to UV
Figure 3
UV (220nm)
500 ELSD
1. Streptomycin 2. Amikacin 3. Tobramycin 4.
Neomycin
Column Alltima C18, 5µm, 250 x 4.6mm Mobile
Phase 0.3 Pentafluoropropionic Acid in
Methanol 0.3 Pentafluoropropionic Acid in
43.7mM Ammonium Formate, pH 2.7
(5545) Flowrate 1.0mL/min
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Neomycin Extracted from New-Cinolone Cream
Figure 4
1. Neomycin
Column Alltima C18, 5µm, 250 x 4.6mm Mobile
Phase 0.3 Pentafluoropropionic Acid in
Methanol 0.3 Pentafluoropropionic Acid in
43.4mM Ammonium Formate, pH 2.6
(5545) Flowrate 1.0mL/min Detector 500
ELSD This analysis was performed under slightly
different conditions resulting in a small
retention time change for neomycin.
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Aminoglycoside Antibiotics Analyzed on Polymeric
Columns
Figure 5
OraChrom Styros, 2R/XH 3µm, 100 x 4.6mm
Hamilton PRP-1 5µm, 150 x 4.6mm
Mobile Phase 0.3 Pentafluoropropionic acid in
Methanol 0.3 Pentafluoropropionic acid in
43.4mM Ammonium Formate, pH 2.6 (5545)
Flowrate 1.0mL/min Detector 500 ELSD
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Results and Discussion

• Aminoglycoside antibiotics are not well retained
  on reversed-phase C18 columns due to their
  hydrophilicity, polarity, and charge.1 To detect
  the antibiotics by reversed-phase HPLC, an
  ion-pair reagent was needed. Alkylsulfonates are
  commonly used as ion-pair reagents for
  aminoglycosides, but are not volatile, and
  therefore not compatible with the ELSD.1
  Trifluoroacetic acid (TFA), a volatile ion-pair
  reagent, has been successful in retaining
  gentamicin in reversed-phase analyses.2 However,
  TFA does not have the same selectivity for other
  aminoglycoside antibiotics and is not effective
  in improving retention.2 Perfluorinated acids
  have been investigated as alternatives to TFA and
  pentafluoropropionic acid (PFPA) has been
  successful in separating AGs2.
• An increase in the concentration of PFPA as
  ion-pair reagent corresponded to an increase in
  the retention time of each antibiotic. As an
  example, Figure 1 shows neomycin eluting at 7.2
  minutes with 0.4 PFPA and eluting at 5.4 minutes
  with only 0.2 PFPA.

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• Adding ion-pair reagent to the aqueous mobile
  phase results in a low pH. Since silica-based
  columns degrade at low pHs, a buffer was added to
  maintain the mobile phase pH at a value greater
  than 2.0. ELS detectors are only compatible with
  volatile buffers and ammonium formate was chosen
  to buffer the acid ion-pair reagent.
• The Hamilton PRP-1, and OraChrom Styros 2R/XH
  polymeric columns were evaluated in the low pH
  range with buffered, ion-pair containing mobile
  phase. Even with an ion-pair reagent, the
  antibiotics were not retained on either of these
  columns. Results were insignificant concerning
  the lifetime of a polymeric column versus a
  silica-based column at these method conditions.
• Quantitative determination of AGs is commonly
  performed by a derivatization technique with
  s-phthalaldehyde (OPA) or 1-fluoro-2,4-dinitrobenz
  ene (FDNB) to improve sensitivity in liquid
  chromatography.1 There is no need to use
  derivatization when using an ELSD. The ELSD will
  detect the non-volatile antibiotics based on mass
  concentration and amount of scattered light.

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Conclusion

• An Alltima C18, 5µm, 250 x 4.6mm column offered
  the best separation of amikacin, neomycin,
  streptomycin, and tobramycin. An increase in the
  concentration of ion-pair reagent correlated to
  an increase in retention time for each
  antibiotic. Overall, a rugged, rapid, isocratic
  HPLC method was developed for the analysis of
  amikacin, neomycin, streptomycin, and tobramycin.
• The analysis time for this method can be reduced
  by using Alltima packing material in a different
  column format with smaller column volume. With a
  Rocket column, separations are typically 70-80
  faster and have equal or better resolution than
  conventional columns. An Alltima Rocket
  column uses 1.5µm or 3µm media and generates high
  efficiency and excellent peak shape. Use an
  Alltima Rocket column to increase sample
  throughput while maintaining or improving peak
  resolution.

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References

• 1. N. Isoherranen and S. Soback.
  Chromatographic Methods for Analysis of
  Aminoglycoside Antibiotics. J. AOAC Int., 82
  (1999) 1017-1045.
• 2. L. McLaughlin and J. Henion. Determination
  of Aminoglycoside Antibiotics by Reversed-phase
  Ion-pair High-performance Liquid Chromatography
  Coupled with Pulsed Amperometry and Ion Spray
  Mass Spectrometry. J. Chromatogr., 591 (1992)
  195-206.

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Acknowledgements

• The authors would like to thank Unicorn
  Laboratories for providing the Neo-Cinolone
  cream.